KR102362873B1 - Tagaturonate epimerase variant and method for producing tagatose from fruxtose using the same - Google Patents

Abstract

본 발명은 써모토가 페트로필라(Thermotoga petrophila) 유래 타카투론산 에피머화 효소의 아미노산 서열로부터, ⅰ) 125번째, 181번째 및 362번째 아미노산이 돌연변이되고, ⅱ) 66번째, 72번째, 129번째 및 140번째 아미노산으로 이루어진 군으로부터 선택된 하나 이상의 아미노산이 추가로 돌연변이되는 것을 특징으로 하는, 타카투론산 에피머화 효소 변이체 및 이를 이용한 과당으로부터 타카토스의 생산방법에 관한 것이다. The present invention is Thermotoga petrophila ( Thermotoga petrophila ) From the amino acid sequence of the derived takaturonic acid epimerase, i) 125th, 181th and 362th amino acids are mutated, ii) 66th, 72nd, 129th and It relates to a takaturonic acid epimerase variant, characterized in that one or more amino acids selected from the group consisting of the 140th amino acid are further mutated, and a method for producing takatose from fructose using the same.

Description

타카투론산 에피머화 효소 변이체 및 이를 이용한 과당으로부터 타카토스의 생산방법{TAGATURONATE EPIMERASE VARIANT AND METHOD FOR PRODUCING TAGATOSE FROM FRUXTOSE USING THE SAME} TAGATURONATE EPIMERASE VARIANT AND METHOD FOR PRODUCING TAGATOSE FROM FRUXTOSE USING THE SAME

본 발명은 과당의 4번 탄소 위치를 에피머화시키는 써모토가 페트로필라(Thermotoga petrophila) 유래 타카투론산 에피머화 효소 변이체 및 이를 이용한 과당으로부터 타카토스의 생산방법에 관한 것이다.The present invention relates to a takaturonic acid epimerase mutant derived from Thermotoga petrophila that epimerizes the 4th carbon position of fructose and a method for producing takatose from fructose using the same.

타가토스는 설탕 당도의 92%로 거의 동일한 당도를 지니고 있으며, 칼로리의 경우 설탕에 비해 2.7배가 낮으며, GI 지수 또한 3으로 매우 낮다. 또한 항산화, 항유전독성, 제2형 당뇨병 치료 보조제 등 다양한 임상적 특성을 지니고 있어 전세계적으로 주목을 받고 있는 희소단당류이다.Tagatose has almost the same sugar content as 92% of the sugar content, 2.7 times lower in calories than sugar, and the GI index is also very low at 3. In addition, it has various clinical properties such as antioxidant, anti-genotoxicity, and type 2 diabetes treatment adjuvant.

이런 이유로 타가토스는 건강 및 식품 보조제로서 주목받고 있으며, 타가토스를 효율적으로 생산할 수 있는 방법을 개발하기 위해 식품 산업 분야에서 많은 연구를 진행하고 있다. 현재 타가토스는 유당 및 갈락토스로부터 생산되고 있으나, 유당의 경우 낙농시장의 변동에 따라 공급가액의 변화폭이 크고 두단계 효소 반응으로 생산되어 수율이 낮고, 갈락토스의 경우 가격이 비싸기 때문에 안정적이고 지속적인 원료 수급에 어려움이 제기되고 있다.For this reason, tagatose is attracting attention as a health and food supplement, and many studies are being conducted in the food industry to develop a method for efficiently producing tagatose. Currently, tagatose is produced from lactose and galactose, but in the case of lactose, the supply price varies greatly according to the fluctuations in the dairy market, and the yield is low because it is produced by a two-step enzymatic reaction. difficulties are being raised.

이러한 문제점들을 해결하기 위해서 유당 및 갈락토스로부터 타가토스를 효율적으로 생산하기 위해서 많은 연구가 되어 왔지만, 사용하는 기질을 변경할 수 없었다. 이론상으로 과당의 4번 탄소 위치에 에피머화시키면 타가토스를 생산할 수 있으나, 현재까지 과당의 4번 탄소 위치를 에피머화 시키는 효소는 발견되지 않았다. 하지만 최근 타가투론산 에피머화 효소가 보고되었으며, 이에 대한 연구는 아직 미흡한 상태이다.In order to solve these problems, many studies have been conducted to efficiently produce tagatose from lactose and galactose, but the substrate used could not be changed. In theory, tagatose can be produced by epimerization at the 4th carbon position of fructose, but no enzyme has been found that epimerizes the 4th carbon position of fructose. However, tagaturonic acid epimerase has recently been reported, and research on it is still insufficient.

따라서 타가투론산 에피머화 효소를 통해 안정적이고 값싼 기질인 과당을 이용하여 타가토스를 생산할 수는 있으나, 과당으로부터 타가토스로의 전환이 거의 일어나지 않고 생산수율이 매우 낮아 현상태로는 산업화에 한계가 있다.Therefore, tagatose can be produced using fructose, a stable and inexpensive substrate, through tagaturonic acid epimerase, but there is almost no conversion from fructose to tagatose and the production yield is very low, so there is a limit to industrialization as it is. .

국내등록특허공보 제10-1940785호 (2019.01.21.)Domestic Registered Patent Publication No. 10-1940785 (2019.01.21.)

본 발명은 종래 공지된 써모토가 페트로필라(Thermotoga petrophila) 유래 타카투론산 에피머화 효소 및 이의 변이체를 이용한 경우에 비해, 과당으로부터 타가토스를 전환하는 비활성을 증가시키기 위한 것으로, 써모토가 페트로필라(Thermotoga petrophila) 유래 타카투론산 에피머화 효소의 아미노산 서열로부터, ⅰ) 125번째, 181번째 및 362번째 아미노산이 돌연변이되고, ⅱ) 66번째, 72번째, 129번째 및 140번째 아미노산으로 이루어진 군으로부터 선택된 하나 이상의 아미노산이 추가로 돌연변이되는 것을 특징으로 하는, 타카투론산 에피머화 효소 변이체 및 이를 이용한 과당으로부터 타카토스의 생산방법 등을 제공하는 것이다. The present invention is to increase the specific activity of converting tagatose from fructose, compared to the case of using the conventionally known Thermotoga petrophila -derived takaturonic acid epimerase and variants thereof, Thermotoga petrophila ( Thermotoga petrophila ) From the amino acid sequence of takaturonic acid epimerase derived, i) the 125th, 181th and 362th amino acids are mutated, ii) 66th, 72th, 129th and 140th amino acids selected from the group consisting of It is to provide a takaturonic acid epimerase variant, characterized in that one or more amino acids are further mutated, and a method for producing takatose from fructose using the same.

그러나, 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

본 발명은 써모토가 페트로필라(Thermotoga petrophila) 유래 타카투론산 에피머화 효소의 아미노산 서열로부터, ⅰ) 125번째, 181번째 및 362번째 아미노산이 돌연변이되고, ⅱ) 66번째, 72번째, 129번째 및 140번째 아미노산으로 이루어진 군으로부터 선택된 하나 이상의 아미노산이 추가로 돌연변이되는 것을 특징으로 하는, 타카투론산 에피머화 효소 변이체를 제공한다. The present invention is Thermotoga petrophila ( Thermotoga petrophila ) From the amino acid sequence of the derived takaturonic acid epimerase, i) 125th, 181th and 362th amino acids are mutated, ii) 66th, 72nd, 129th and At least one amino acid selected from the group consisting of the 140th amino acid is further mutated, it provides a takaturonic acid epimerase variant.

상기 ⅰ)에서 125번째 아미노산인 세린(S)은 아스파트산(D), 글루탐산(E), 라이신(K) 또는 트립토판(W)으로 치환되고, 181번째 아미노산인 트레오닌(T)은 알라닌(A), 아스파트산(D), 페닐알라닌(F), 글라이신(G), 라이신(K), 류신(L), 발린(V) 또는 트립토판(W)으로 치환되며, 362번째 아미노산인 히스티딘(H)은 알라닌(A), 아스파트산(D), 페닐알라닌(F), 류신(L), 트레오닌(T) 또는 트립토판(W)으로 치환되고, 상기 ⅱ)에서 66번째 아미노산인 페닐알라닌(F)은 세린(S)으로 추가로 치환되거나, 72번째 아미노산인 글루탐산(E)은 글라이신(G)으로 추가로 치환되거나, 129번째 아미노산인 아스파라긴(N)은 트레오닌(T), 페닐알라닌(F) 또는 세린(S)으로 추가로 치환되거나, 140번째 아미노산인 류신(L)은 프롤린(P), 라이신(K) 또는 페닐알라닌(F)으로 추가로 치환될 수 있다. In i), serine (S) at the 125th amino acid is substituted with aspartic acid (D), glutamic acid (E), lysine (K) or tryptophan (W), and threonine (T) at the 181st amino acid is alanine (A) ), aspartic acid (D), phenylalanine (F), glycine (G), lysine (K), leucine (L), valine (V) or tryptophan (W), and histidine (H), the 362th amino acid is substituted with alanine (A), aspartic acid (D), phenylalanine (F), leucine (L), threonine (T) or tryptophan (W), and in ii), phenylalanine (F), the 66th amino acid, is serine (S) is further substituted, glutamic acid (E) at the 72nd amino acid is further substituted with glycine (G), or asparagine (N) at the 129th amino acid is threonine (T), phenylalanine (F) or serine (S) ), or leucine (L) at the 140th amino acid may be further substituted with proline (P), lysine (K) or phenylalanine (F).

상기 타카투론산 에피머화 효소의 아미노산 서열로부터, ⅲ) 96번째, 97번째, 98번째, 100번째, 123번째, 124번째, 126번째, 127번째, 128번째, 159번째, 161번째, 162번째, 266번째, 268번째, 270번째, 299번째, 306번째, 307번째, 308번째, 309번째, 339번째, 340번째, 341번째, 342번째, 343번째, 344번째, 346번째, 363번째, 364번째, 365번째, 366번째, 367번째, 368번째, 369번째, 371번째, 403번째, 437번째 및 440번째 아미노산으로 이루어진 군으로부터 선택된 하나 이상의 아미노산이 추가로 돌연변이될 수 있다. From the amino acid sequence of the takaturonic acid epimerase, iii) 96th, 97th, 98th, 100th, 123rd, 124th, 126th, 127th, 128th, 159th, 161th, 162th, 266th, 268th, 270th, 299th, 306th, 307th, 308th, 309th, 339th, 340th, 341th, 342th, 343th, 344th, 346th, 363th, 364th One or more amino acids selected from the group consisting of , 365th, 366th, 367th, 368th, 369th, 371th, 403th, 437th and 440th amino acids may be further mutated.

본 발명의 일 구현예에 따르면, 상기 타카투론산 에피머화 효소 변이체를 암호화하는 유전자를 제공한다. According to one embodiment of the present invention, there is provided a gene encoding the takaturonic acid epimerase variant.

본 발명의 다른 구현예에 따르면, 상기 유전자를 포함하는 재조합 벡터를 제공한다. According to another embodiment of the present invention, there is provided a recombinant vector comprising the gene.

본 발명의 또 다른 구현예에 따르면, 상기 재조합 벡터로 형질전환된 숙주세포를 제공한다. According to another embodiment of the present invention, there is provided a host cell transformed with the recombinant vector.

본 발명의 또 다른 구현예에 따르면, 상기 타카투론산 에피머화 효소 변이체 또는 이를 암호화하는 유전자를 유효성분으로 포함하는 타카토스 생산용 조성물을 제공한다. According to another embodiment of the present invention, there is provided a composition for producing Takatose comprising the takaturonic acid epimerase variant or a gene encoding the same as an active ingredient.

본 발명의 또 다른 구현예에 따르면, 상기 조성물을 과당에 처리하여 에피머화 반응을 수행하는 단계를 포함하는 과당으로부터 타카토스의 생산방법을 제공한다. According to another embodiment of the present invention, there is provided a method for producing Takatose from fructose, comprising the step of performing an epimerization reaction by treating the composition with fructose.

상기 에피머화 반응은 pH 6.5 내지 9.0 및 온도 70℃ 내지 90℃에서 수행될 수 있다. The epimerization reaction may be performed at a pH of 6.5 to 9.0 and a temperature of 70°C to 90°C.

상기 에피머화 반응은 Ni²⁺, Co²⁺, Ca²⁺, Mn²⁺, Mg²⁺, Zn²⁺, Cu²⁺, 및 Fe²⁺ 로 이루어진 군으로부터 선택된 하나 이상의 2가 금속염의 존재 하에 수행될 수 있다. The epimerization reaction is carried out in the presence of one or more divalent metal salts selected from the group consisting of Ni ²⁺ , Co ²⁺ , Ca ²⁺ , Mn ²⁺ , Mg ²⁺ , Zn ²⁺ , Cu ²⁺ , and Fe ²⁺ . can be performed.

본 발명에 따르면, 종래 공지된 써모토가 페트로필라(Thermotoga petrophila) 유래 타카투론산 에피머화 효소 및 이의 변이체를 기반으로 하되 추가 돌연변이시킨 변이체를 이용함으로써, 과당의 4번 탄소 위치를 에피머화시켜 타카토스로 전환하는 비활성을 증가시킬 수 있는바, 최종적으로, 값싸고 안정적으로 수급이 가능한 과당을 이용하여 친환경적인 방법으로 타카토스를 높은 생산성과 높은 수율로 효율적으로 생산할 수 있다. According to the present invention, by using a conventionally known Thermotoga petrophila -derived takaturonic acid epimerase and a variant thereof, but by using an additional mutated variant, the 4th carbon position of fructose is epimerized to taka It is possible to increase the specific activity to convert to toss, and finally, it is possible to efficiently produce Takatos with high productivity and high yield in an eco-friendly way using fructose, which is cheap and can be supplied stably.

도 1은 본 발명의 변이방법에 따른 타가투론산 에피머화 효소 변이체의 제작 과정이다.
도 2는 상동성 모델링의 주형으로 사용하기 위하여, 코넬라 래비리보시(Cohnella laeviribosi) 유래 프럭투론산-타가투론산 에피머화 효소(PDB:6ILB, 회색)와 써모토가 페트로필라(Thermotoga petrophila) 유래 타가투론산 에피머화 효소(노란색)를 중첩시킨 이미지를 나타낸 것이다.
도 3은 유리한 상호작용 개수를 분석하여 나타낸 것으로, (a)는 써모토가 페트로필라(Thermotoga petrophila) 유래 타가투론산 에피머화 효소(야생형)에 과당과 주기질인 프럭투론산을 도킹하여 직접 연관된 잔기들의 유리한 상호작용 개수를 분석하여 S125 변이체를 선별한 것이고, (b)는 (a)에서 선별한 S125 잔기를 여러 아미노산으로 치환하여 각각 치환된 모델에 과당을 도킹하여 직접 연관된 잔기들의 유리한 상호작용 개수를 분석하여 나타냈으며, 추가로 위치 지정 돌연변이법으로 변이체를 만들어 과당에 대해 타가토스로의 전환 활성을 나타내어 S125D 변이체를 선별한 것이다.
도 4는 S125D 변이체의 3차 구조 모델을 분석하여 과당에 대한 전환 비활성을 높이기 위해 개량 타겟 부위를 디자인하였고, 이를 통해 이중부위 포화 돌연변이 라이브러리를 제작하여 과당에 대한 전환 비활성이 가장 높게 증가한 4개의 변이체를 선별하여 나타낸 것이다.
도 5는 선별한 이중 부위 변이체들을 조합하기 위해 포화 돌연변이법을 사용하여 과당에 대한 전환 비활성이 가장 높게 증가한 4개의 변이체를 선별하여 나타낸 것이며, 가장 높은 전환 비활성을 보인 변이체는 S125D/T181A/H362L 변이체임을 나타낸 것이다.
도 6은 선별한 S125D/T181A/H362L 변이체를 기초로 하여 무작위 돌연변이법을 통해 과당에 대한 전환 비활성이 가장 높게 증가한 4개의 변이체를 선별하여 나타낸 것이다.
도 7은 선별한 사중 부위 변이체들을 조합하기 위해 포화 돌연변이법을 사용하여 과당에 대한 전환 비활성이 가장 높게 증가한 4개의 변이체를 선별하여 나타낸 것이며, 가장 높은 전환 활성을 보인 변이체는 S125D/N129T/L140P/T181A/H362L 변이체임을 나타낸 것이다.
도 8은 pH 변화에 따른 S125D/N129T/L140P/T181A/H362L 변이체의 비활성 정도를 보여주는 그래프이다.
도 9는 반응 온도 변화에 따른 S125D/N129T/L140P/T181A/H362L 변이체의 비활성 정도를 보여주는 그래프이다.
도 10은 금속염 종류에 따른 S125D/N129T/L140P/T181A/H362L 변이체의 비활성 정도를 보여주는 그래프이다.
도 11은 야생형 및 과당에 대한 전환활성이 증가하여 선별했던 변이체들의 비활성 비교를 보여주는 그래프이다.
도 12는 고농도의 타가토스 생산을 위해 효소 및 과당의 최적 농도 조건을 나타낸 것으로, (a)는 S125D/N129T/L140P/T181A/H362L 변이체의 효소 농도에 따라 타가토스 생산 변화를 나타낸 그래프이며, (b)는 과당 농도 변화에 따라 타가토스 생산 변화를 나타낸 그래프이다.
도 13은 확립한 최적 조건 하에서 과당으로부터 시간별 타가토스의 생산을 나타낸 그래프이다.1 is a process for producing a tagaturonic acid epimerase mutant according to the mutation method of the present invention.
2 is a fructuronic acid-tagaturonic acid epimerase (PDB: 6ILB, gray) and Thermotoga petrophila derived from Cohnella laeviribosi for use as a template for homology modeling; and Thermotoga petrophila . It shows an image superimposed on the derived tagaturonic acid epimerase (yellow).
Figure 3 shows by analyzing the number of advantageous interactions, (a) is a thermotoga petrophila ( Thermotoga petrophila ) derived tagaturonic acid epimerase (wild type) directly related by docking fructuronic acid and fructose as the main substrate S125 variants were selected by analyzing the number of favorable interactions of residues, and in (b), the S125 residue selected in (a) was substituted with several amino acids, and fructose was docked in each substituted model for favorable interaction of directly related residues. The number was analyzed and shown, and the S125D variant was selected by showing the conversion activity of fructose to tagatose by making a variant by a site-directed mutagenesis method.
4 is an analysis of the tertiary structural model of the S125D mutant to design an improved target site to increase the conversion specific activity to fructose, and through this, a double-site saturation mutant library was produced, 4 variants with the highest conversion specific activity to fructose is selected and shown.
Figure 5 shows the four variants with the highest increase in conversion activity for fructose by using saturation mutagenesis to combine the selected double site variants, and the variant showing the highest conversion activity is S125D/T181A/H362L variant it indicates that
FIG. 6 shows four variants having the highest increase in conversion specific activity for fructose through random mutagenesis based on the selected S125D/T181A/H362L variants.
7 shows the four variants having the highest increase in conversion specific activity for fructose by using saturation mutagenesis to combine the selected quadruple site variants, and the variant showing the highest conversion activity is S125D/N129T/L140P/ It shows that it is a T181A/H362L mutant.
8 is a graph showing the degree of specific activity of the S125D / N129T / L140P / T181A / H362L variant according to the change in pH.
9 is a graph showing the degree of inactivation of the S125D/N129T/L140P/T181A/H362L variant according to the change in reaction temperature.
10 is a graph showing the degree of inactivation of S125D/N129T/L140P/T181A/H362L variants according to the type of metal salt.
11 is a graph showing a comparison of the specific activity of the wild-type and the mutants that were selected with increased conversion activity for fructose.
12 is a graph showing the optimal concentration conditions of enzyme and fructose for the production of high concentration tagatose, (a) is a graph showing the change in tagatose production according to the enzyme concentration of the S125D / N129T / L140P / T181A / H362L variant, ( b) is a graph showing the change in tagatose production according to the change in fructose concentration.
13 is a graph showing the time-dependent production of tagatose from fructose under established optimal conditions.

본 발명자들은 종래 공지된 써모토가 페트로필라(Thermotoga petrophila) 유래 타카투론산 에피머화 효소 및 이의 변이체를 이용한 경우에 비해, 과당으로부터 타가토스를 전환하는 비활성을 증가시키기 위해, 도 1에 나타난 방법을 통해, 본 발명의 타카투론산 에피머화 효소 변이체를 제작하였다. 또한, 이를 기질로서 과당에 처리함으로써 에피머화 반응을 수행하여 타카토스를 높은 생산성과 높은 수율로 효율적으로 생산할 수 있음을 확인하고, 본 발명을 완성하였다.The present inventors compared the conventionally known Thermotoga petrophila -derived takaturonic acid epimerase and its variants, in order to increase the specific activity of converting tagatose from fructose, the method shown in FIG. Through, the takaturonic acid epimerase mutant of the present invention was prepared. In addition, it was confirmed that Takatose can be efficiently produced with high productivity and high yield by performing an epimerization reaction by treating it with fructose as a substrate, thereby completing the present invention.

이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.

타카투론산 에피머화 효소 변이체 및 이를 암호화하는 유전자 Takaturonic acid epimerase variant and gene encoding same

본 발명은 써모토가 페트로필라(Thermotoga petrophila) 유래 타카투론산 에피머화 효소의 아미노산 서열로부터, ⅰ) 125번째, 181번째 및 362번째 아미노산이 돌연변이되고, ⅱ) 66번째, 72번째, 129번째 및 140번째 아미노산으로 이루어진 군으로부터 선택된 하나 이상의 아미노산이 추가로 돌연변이되는 것을 특징으로 하는, 타카투론산 에피머화 효소 변이체를 제공한다. The present invention is Thermotoga petrophila ( Thermotoga petrophila ) From the amino acid sequence of the derived takaturonic acid epimerase, i) 125th, 181th and 362th amino acids are mutated, ii) 66th, 72nd, 129th and At least one amino acid selected from the group consisting of the 140th amino acid is further mutated, it provides a takaturonic acid epimerase variant.

본 발명에 따른 타카투론산 에피머화 효소 변이체는 써모토가 페트로필라(Thermotoga petrophila) 유래 타카투론산 에피머화 효소의 아미노산 서열로부터, ⅰ) 125번째, 181번째 및 362번째 아미노산이 돌연변이되고, ⅱ) 66번째, 72번째, 129번째 및 140번째 아미노산으로 이루어진 군으로부터 선택된 하나 이상의 아미노산이 추가로 돌연변이되는 것을 특징으로 한다. Takaturonic acid epimerase variant according to the present invention from the amino acid sequence of takaturonic acid epimerase derived from Thermotoga petrophila , i) 125th, 181th and 362th amino acids are mutated, and ii) It is characterized in that one or more amino acids selected from the group consisting of the 66th, 72nd, 129th and 140th amino acids are further mutated.

먼저, 상기 타카투론산 에피머화 효소는 야생형(wild-type)으로서, 고온성 미생물인 써모토가 페트로필라(Thermotoga petrophila)로부터 유래한 것으로, 서열번호 1의 아미노산 서열로 이루어진 것뿐만 아니라, 이의 기능적 동등물, 즉, 상기 서열에 하나 이상의 치환, 결손 등의 돌연변이를 유발하여 본 발명의 목적을 달성하는 모든 돌연변이체를 포함하는 것을 의미하며, 서열번호 12의 염기 서열로 이루어진 유전자로부터 발현된 산물일 수 있다. First, the takaturonic acid epimerase is a wild-type, derived from thermotoga petrophila , a thermophilic microorganism, and not only consists of the amino acid sequence of SEQ ID NO: 1, but also its functional Equivalent, that is, it means to include all mutants that achieve the object of the present invention by inducing one or more substitutions, deletions, etc. in the sequence, and is a product expressed from a gene consisting of the nucleotide sequence of SEQ ID NO: 12 can

상기 타카투론산 에피머화 효소 변이체는 상기 타카투론산 에피머화 효소를 돌연변이시킨 것으로, 고온 조건에서 안정적으로 에피머화 반응을 수행할 수 있어, 오염 방지, 기질의 용해 증가, 반응속도 증가 등과 같은 산업적인 이점을 가질 수 있다. 상기 타카투론산 에피머화 효소 변이체의 최적 활성 pH는 6.5 내지 9.0, 바람직하게 8.0 내지 9.0이고, 최적 활성 온도는 70℃ 내지 90℃, 바람직하게, 75℃ 내지 85℃이다. The takaturonic acid epimerase variant is a mutated takaturonic acid epimerase enzyme, and it can stably perform the epimerization reaction under high temperature conditions, thereby preventing contamination, increasing the dissolution of the substrate, increasing the reaction rate, etc. can have an advantage. The optimum active pH of the takaturonic acid epimerase variant is 6.5 to 9.0, preferably 8.0 to 9.0, and the optimum activity temperature is 70°C to 90°C, preferably 75°C to 85°C.

구체적으로, 상기 타카투론산 에피머화 효소 변이체는 써모토가 페트로필라(Thermotoga petrophila) 유래 타카투론산 에피머화 효소의 아미노산 서열로부터, ⅰ) 125번째 아미노산인 세린(S)이 아스파트산(D), 글루탐산(E), 라이신(K) 또는 트립토판(W)(바람직하게, 아스파트산(D))으로 치환되고, 181번째 아미노산인 트레오닌(T)이 알라닌(A), 아스파트산(D), 페닐알라닌(F), 글라이신(G), 라이신(K), 류신(L), 발린(V) 또는 트립토판(W)(바람직하게, 알라닌(A))으로 치환되며, 362번째 아미노산인 히스티딘(H)이 알라닌(A), 아스파트산(D), 페닐알라닌(F), 류신(L), 트레오닌(T) 또는 트립토판(W)(바람직하게, 류신(L))으로 치환되어 돌연변이된 것일 수 있다. Specifically, the takaturonic acid epimerase variant is from the amino acid sequence of takaturonic acid epimerase derived from Thermotoga petrophila , i) serine (S), the 125th amino acid, is aspartic acid (D) , glutamic acid (E), lysine (K) or tryptophan (W) (preferably aspartic acid (D)) is substituted, and the 181th amino acid threonine (T) is alanine (A), aspartic acid (D) , is substituted with phenylalanine (F), glycine (G), lysine (K), leucine (L), valine (V) or tryptophan (W) (preferably alanine (A)), and histidine (H) at position 362 ) may be mutated by being substituted with alanine (A), aspartic acid (D), phenylalanine (F), leucine (L), threonine (T) or tryptophan (W) (preferably, leucine (L)). .

ⅰ)에 추가적으로, ⅱ) 66번째 아미노산인 페닐알라닌(F)은 세린(S)으로 추가로 치환되거나, 72번째 아미노산인 글루탐산(E)은 글라이신(G)으로 추가로 치환되거나, 129번째 아미노산인 아스파라긴(N)이 트레오닌(T), 페닐알라닌(F) 또는 세린(S)(바람직하게, 트레오닌(T))으로 추가로 치환되어 돌연변이되거나, 140번째 아미노산인 류신(L)은 프롤린(P), 라이신(K) 또는 페닐알라닌(F)(바람직하게, 프롤린(P)으로 추가로 치환되어 돌연변이된 것일 수 있다.In addition to i), ii) phenylalanine (F) at the 66th amino acid is further substituted with serine (S), glutamic acid (E) at the 72nd amino acid is further substituted with glycine (G), or asparagine at the 129th amino acid (N) is mutated by being further substituted with threonine (T), phenylalanine (F) or serine (S) (preferably threonine (T)), or leucine (L) at the 140th amino acid is proline (P), lysine (K) or phenylalanine (F) (preferably, it may be mutated by being further substituted with proline (P).

가장 바람직한 일예로, 상기 타카투론산 에피머화 효소 변이체는 써모토가 페트로필라(Thermotoga petrophila) 유래 타카투론산 에피머화 효소의 아미노산 서열로부터, ⅰ) 125번째 아미노산인 세린(S)이 아스파트산(D)으로 치환되고, 181번째 아미노산인 트레오닌(T)이 알라닌(A)으로 치환되며, 362번째 아미노산인 히스티딘(H)이 류신(L)으로 치환되어 돌연변이되면서, ⅰ)에 추가적으로, ⅱ) 129번째 아미노산인 아스파라긴(N)이 트레오닌(T)이 추가로 치환되어 돌연변이되고, 140번째 아미노산인 류신(L)이 프롤린(P)으로 추가로 치환되어 돌연변이된 것(즉, S125D/N129T/L140P/T181A/H362L 변이체)일 수 있다.In a most preferred embodiment, the takaturonic acid epimerase variant is from the amino acid sequence of takaturonic acid epimerase derived from Thermotoga petrophila , i) serine (S), the 125th amino acid, is aspartic acid ( D), threonine (T) at the 181st amino acid is substituted with alanine (A), and histidine (H) at the 362th amino acid is substituted with leucine (L) and mutated. In addition to i), ii) 129 Asparagine (N), which is the second amino acid, is mutated by further substituted with threonine (T), and the 140th amino acid, leucine (L), is further substituted with proline (P) and mutated (ie, S125D/N129T/L140P/ T181A/H362L variant).

한편, 상기 타카투론산 에피머화 효소 변이체는 ⅰ) 및 ⅱ)에 추가적으로, 96번째, 97번째, 98번째, 100번째, 123번째, 124번째, 126번째, 127번째, 128번째, 159번째, 161번째, 162번째, 266번째, 268번째, 270번째, 299번째, 306번째, 307번째, 308번째, 309번째, 339번째, 340번째, 341번째, 342번째, 343번째, 344번째, 346번째, 363번째, 364번째, 365번째, 366번째, 367번째, 368번째, 369번째, 371번째, 403번째, 437번째 및 440번째 아미노산으로 이루어진 군으로부터 선택된 하나 이상의 아미노산이 추가로 돌연변이될 수 있는데, 구체적으로, 96번째 아미노산인 글라이신(G)이 알라닌(A), 아스파트산(D), 페닐알라닌(F), 라이신(K), 류신(L), 아스파라긴(N), 트레오닌(T) 또는 트립토판(W)으로 추가로 치환되어 돌연변이되거나; 97번째 아미노산인 트레오닌(T)이 알라닌(A), 아스파트산(D), 페닐알라닌(F), 글라이신(G), 라이신(K), 류신(L), 발린(V) 또는 트립토판(W)으로 추가로 치환되어 돌연변이되거나; 98번째 아미노산인 글라이신(G)이 알라닌(A)으로 추가로 치환되어 돌연변이되거나; 100번째 아미노산인 알지닌(R)이 알라닌(A), 아스파트산(D), 페닐알라닌(F), 글라이신(G), 라이신(K), 류신(L), 아스파라긴(N), 발린(V) 또는 트립토판(W)으로 추가로 치환되어 돌연변이되거나; 123번째 아미노산인 글루타민(Q)이 알라닌(A) 또는 아스파라긴(N)으로 추가로 치환되어 돌연변이되거나; 124번째 아미노산인 글루타민(Q)이 알라닌(A)으로 추가로 치환되어 돌연변이되거나; 126번째 아미노산인 발린(V)이 알라닌(A), 아스파트산(D), 글루탐산(E), 페닐알라닌(F), 글라이신(G), 라이신(K), 류신(L), 아스파라긴(N), 트레오닌(T), 발린(V) 또는 트립토판(W)으로 추가로 치환되어 돌연변이되거나; 127번째 아미노산인 알지닌(R)이 알라닌(A), 아스파트산(D), 글루탐산(E), 페닐알라닌(F), 글라이신(G), 라이신(K), 류신(L), 아스파라긴(N), 트레오닌(T), 발린(V) 또는 트립토판(W)으로 추가로 치환되어 돌연변이되거나; 128번째 아미노산인 글루탐산(E)이 알라닌(A), 페닐알라닌(F), 글라이신(G), 라이신(K), 류신(L), 트레오닌(T), 발린(V) 또는 트립토판(W)으로 추가로 치환되어 돌연변이되거나; 159번째 아미노산인 아스파트산(D)은 알라닌(A), 페닐알라닌(F), 글라이신(G), 라이신(K), 류신(L), 트레오닌(T), 발린(V) 또는 트립토판(W)으로 추가로 치환되어 돌연변이되거나; 161번째 아미노산인 아스파트산(D)이 알라닌(A), 페닐알라닌(F), 글라이신(G), 라이신(K), 류신(L), 트레오닌(T), 발린(V) 또는 트립토판(W)으로 추가로 치환되어 돌연변이되거나; 162번째 아미노산인 히스티딘(H)이 알라닌(A)으로 추가로 치환되어 돌연변이되거나; 266번째 아미노산인 글루탐산(E)이 알라닌(A)으로 추가로 치환되어 돌연변이되거나; 268번째 아미노산인 세린(S)이 알라닌(A), 아스파트산(D), 글루탐산(E), 페닐알라닌(F), 글라이신(G), 라이신(K), 류신(L), 아스파라긴(N), 트레오닌(T), 발린(V) 또는 트립토판(W)으로 추가로 치환되어 돌연변이되거나; 270번째 아미노산인 아스파트산(D)이 알라닌(A), 글루탐산(E), 페닐알라닌(F), 글라이신(G), 라이신(K), 류신(L), 트레오닌(T) 또는 발린(V)으로 추가로 치환되어 돌연변이되거나; 299번째 아미노산인 알라닌(A)이 아스파트산(D), 페닐알라닌(F) 또는 트립토판(W)으로 추가로 치환되어 돌연변이되거나; 306번째 아미노산인 트립토판(W)이 알라닌(A), 아스파트산(D), 글루탐산(E), 페닐알라닌(F), 글라이신(G), 라이신(K), 류신(L), 아스파라긴(N), 트레오닌(T), 발린(V) 또는 트립토판(W)으로 추가로 치환되어 돌연변이되거나; 307번째 아미노산인 글루탐산(E)이 알라닌(A), 페닐알라닌(F), 글라이신(G), 라이신(K), 류신(L), 트레오닌(T), 발린(V) 또는 트립토판(W)으로 추가로 치환되어 돌연변이되거나; 308번째 아미노산인 라이신(K)이 알라닌(A), 아스파트산(D), 페닐알라닌(F), 글라이신(G), 류신(L), 아스파라긴(N), 트레오닌(T) 또는 트립토판(W)으로 추가로 치환되어 돌연변이되거나; 309번째 아미노산인 글라이신(G)이 알라닌(A), 아스파트산(D), 페닐알라닌(F), 라이신(K), 류신(L), 아스파라긴(N), 트레오닌(T) 또는 트립토판(W)으로 추가로 치환되어 돌연변이되거나; 339번째 아미노산인 세린(S)이 알라닌(A), 아스파트산(D), 글루탐산(E), 페닐알라닌(F), 글라이신(G), 라이신(K), 류신(L), 아스파라긴(N), 트레오닌(T), 발린(V) 또는 트립토판(W)으로 추가로 치환되어 돌연변이되거나; 340번째 아미노산인 류신(L)이 알라닌(A), 아스파트산(D), 페닐알라닌(F), 글라이신(G), 라이신(K), 아스파라긴(N), 트레오닌(T) 또는 트립토판(W)으로 추가로 치환되어 돌연변이되거나; 341번째 아미노산인 히스티딘(H)이 알라닌(A)으로 추가로 치환되어 돌연변이되거나; 342번째 아미노산인 세린(S)이 알라닌(A), 아스파트산(D), 페닐알라닌(F), 글라이신(G), 라이신(K), 류신(L), 발린(V) 또는 트립토판(W)으로 추가로 치환되어 돌연변이되거나; 343번째 아미노산인 글라이신(G)이 알라닌(A), 아스파트산(D), 페닐알라닌(F), 라이신(K), 류신(L), 아스파라긴(N), 트레오닌(T) 또는 트립토판(W)으로 추가로 치환되어 돌연변이되거나; 344번째 아미노산인 세린(S)이 알라닌(A), 아스파트산(D), 페닐알라닌(F), 글라이신(G), 라이신(K), 류신(L), 발린(V) 또는 트립토판(W)으로 추가로 치환되어 돌연변이되거나; 346번째 아미노산인 라이신(K)이 알라닌(A), 아스파트산(D), 페닐알라닌(F), 글라이신(G), 류신(L), 아스파라긴(N), 트레오닌(T) 또는 트립토판(W)으로 추가로 치환되어 돌연변이되거나; 363번째 아미노산인 발린(V)이 알라닌(A), 아스파트산(D), 글루탐산(E), 페닐알라닌(F), 글라이신(G), 라이신(K), 류신(L), 아스파라긴(N), 트레오닌(T), 발린(V) 또는 트립토판(W)으로 추가로 치환되어 돌연변이되거나; 364번째 아미노산인 라이신(K)이 알라닌(A), 아스파트산(D), 페닐알라닌(F), 글라이신(G), 류신(L), 아스파라긴(N), 트레오닌(T) 또는 트립토판(W)으로 추가로 치환되어 돌연변이되거나; 365번째 아미노산인 트레오닌(T)이 알라닌(A), 아스파트산(D), 페닐알라닌(F), 글라이신(G), 라이신(K), 류신(L), 아스파라긴(N) 또는 트립토판(W)으로 추가로 치환되어 돌연변이되거나; 366번째 아미노산인 알라닌(A)이 아스파트산(D) 또는 페닐알라닌(F)으로 추가로 치환되어 돌연변이되거나; 367번째 아미노산인 글라이신(G)이 알라닌(A), 아스파트산(D), 페닐알라닌(F), 라이신(K), 류신(L), 아스파라긴(N), 트레오닌(T) 또는 트립토판(W)으로 추가로 치환되어 돌연변이되거나; 368번째 아미노산인 트레오닌(T)이 알라닌(A), 아스파트산(D), 페닐알라닌(F), 글라이신(G), 라이신(K), 류신(L), 발린(V) 또는 트립토판(W)으로 추가로 치환되어 돌연변이되거나; 369번째 아미노산인 세린(S)이 알라닌(A), 아스파트산(D), 글루탐산(E), 페닐알라닌(F), 글라이신(G), 라이신(K), 류신(L), 아스파라긴(N), 트레오닌(T), 발린(V) 또는 트립토판(W)으로 추가로 치환되어 돌연변이되거나; 371번째 아미노산인 류신(L)이 알라닌(A), 아스파트산(D), 페닐알라닌(F), 글라이신(G), 라이신(K), 트레오닌(T) 또는 트립토판(W)으로 추가로 치환되어 돌연변이되거나; 403번째 아미노산인 티로신(Y)이 알라닌(A), 아스파트산(D), 글라이신(G), 라이신(K), 류신(L), 트레오닌(T), 발린(V) 또는 트립토판(W)으로 추가로 치환되어 돌연변이되거나; 437번째 아미노산인 히스티딘(H)이 알라닌(A), 아스파트산(D), 페닐알라닌(F), 글라이신(G), 류신(L), 트레오닌(T) 또는 트립토판(W)으로 추가로 치환되어 돌연변이되거나; 440번째 아미노산인 티로신(Y)이 알라닌(A)으로 추가로 치환되어 돌연변이된 것일 수 있다. On the other hand, the takaturonic acid epimerase variant is in addition to i) and ii), 96th, 97th, 98th, 100th, 123th, 124th, 126th, 127th, 128th, 159th, 161 th, 162th, 266th, 268th, 270th, 299th, 306th, 307th, 308th, 309th, 339th, 340th, 341th, 342th, 343th, 344th, 346th, One or more amino acids selected from the group consisting of 363th, 364th, 365th, 366th, 367th, 368th, 369th, 371th, 403th, 437th and 440th amino acids may be further mutated, specifically where the 96th amino acid, glycine (G), is alanine (A), aspartic acid (D), phenylalanine (F), lysine (K), leucine (L), asparagine (N), threonine (T) or tryptophan ( W) is further substituted with mutated; The 97th amino acid, threonine (T), is alanine (A), aspartic acid (D), phenylalanine (F), glycine (G), lysine (K), leucine (L), valine (V), or tryptophan (W). or mutated by being further substituted with; The 98th amino acid, glycine (G) is further substituted with alanine (A) to be mutated; Arginine (R), the 100th amino acid, is alanine (A), aspartic acid (D), phenylalanine (F), glycine (G), lysine (K), leucine (L), asparagine (N), valine (V) ) or further substituted with tryptophan (W) to be mutated; Glutamine (Q) at the 123rd amino acid is further substituted with alanine (A) or asparagine (N) to be mutated; Glutamine (Q) at the 124th amino acid is further substituted with alanine (A) to be mutated; The 126th amino acid, valine (V), is alanine (A), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), lysine (K), leucine (L), asparagine (N) , mutated by further substitution with threonine (T), valine (V) or tryptophan (W); Arginine (R), the 127th amino acid, is alanine (A), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), lysine (K), leucine (L), asparagine (N). ), threonine (T), valine (V) or tryptophan (W) is further substituted to be mutated; Glutamic acid (E) at the 128th amino acid is added as alanine (A), phenylalanine (F), glycine (G), lysine (K), leucine (L), threonine (T), valine (V) or tryptophan (W) is mutated by being substituted with; The 159th amino acid, aspartic acid (D), contains alanine (A), phenylalanine (F), glycine (G), lysine (K), leucine (L), threonine (T), valine (V), or tryptophan (W). or mutated by being further substituted with; The 161st amino acid, aspartic acid (D), is alanine (A), phenylalanine (F), glycine (G), lysine (K), leucine (L), threonine (T), valine (V) or tryptophan (W). or mutated by being further substituted with; histidine (H) at amino acid position 162 is further substituted with alanine (A) to be mutated; Glutamic acid (E) at the 266th amino acid is further substituted with alanine (A) to be mutated; Serine (S), the 268th amino acid, is alanine (A), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), lysine (K), leucine (L), asparagine (N) , mutated by further substitution with threonine (T), valine (V) or tryptophan (W); The 270th amino acid, aspartic acid (D), is converted to alanine (A), glutamic acid (E), phenylalanine (F), glycine (G), lysine (K), leucine (L), threonine (T), or valine (V). or mutated by being further substituted with; alanine (A) at position 299 is further substituted with aspartic acid (D), phenylalanine (F) or tryptophan (W) to be mutated; The 306th amino acid tryptophan (W) is alanine (A), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), lysine (K), leucine (L), asparagine (N) , mutated by further substitution with threonine (T), valine (V) or tryptophan (W); The 307th amino acid, glutamic acid (E), is added as alanine (A), phenylalanine (F), glycine (G), lysine (K), leucine (L), threonine (T), valine (V) or tryptophan (W). is mutated by being substituted with; 308 amino acid lysine (K) is alanine (A), aspartic acid (D), phenylalanine (F), glycine (G), leucine (L), asparagine (N), threonine (T) or tryptophan (W) or mutated by being further substituted with; The 309th amino acid, glycine (G), is alanine (A), aspartic acid (D), phenylalanine (F), lysine (K), leucine (L), asparagine (N), threonine (T) or tryptophan (W). or mutated by being further substituted with; The 339th amino acid, serine (S), is alanine (A), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), lysine (K), leucine (L), asparagine (N) , mutated by further substitution with threonine (T), valine (V) or tryptophan (W); The 340th amino acid, leucine (L), is converted to alanine (A), aspartic acid (D), phenylalanine (F), glycine (G), lysine (K), asparagine (N), threonine (T) or tryptophan (W). or mutated by being further substituted with; histidine (H) at position 341 is further substituted with alanine (A) to be mutated; The 342th amino acid, serine (S), is alanine (A), aspartic acid (D), phenylalanine (F), glycine (G), lysine (K), leucine (L), valine (V), or tryptophan (W). or mutated by being further substituted with; The 343th amino acid, glycine (G), is alanine (A), aspartic acid (D), phenylalanine (F), lysine (K), leucine (L), asparagine (N), threonine (T) or tryptophan (W). or mutated by being further substituted with; The 344th amino acid, serine (S), is alanine (A), aspartic acid (D), phenylalanine (F), glycine (G), lysine (K), leucine (L), valine (V) or tryptophan (W). or mutated by being further substituted with; The 346th amino acid, lysine (K), is converted to alanine (A), aspartic acid (D), phenylalanine (F), glycine (G), leucine (L), asparagine (N), threonine (T), or tryptophan (W). or mutated by being further substituted with; 363 amino acid valine (V) is alanine (A), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), lysine (K), leucine (L), asparagine (N) , mutated by further substitution with threonine (T), valine (V) or tryptophan (W); 364 amino acid lysine (K) is alanine (A), aspartic acid (D), phenylalanine (F), glycine (G), leucine (L), asparagine (N), threonine (T) or tryptophan (W) or mutated by being further substituted with; The 365th amino acid, threonine (T), is alanine (A), aspartic acid (D), phenylalanine (F), glycine (G), lysine (K), leucine (L), asparagine (N) or tryptophan (W). or mutated by being further substituted with; alanine (A) at amino acid position 366 is further substituted with aspartic acid (D) or phenylalanine (F) to be mutated; Glycine (G) at position 367 is alanine (A), aspartic acid (D), phenylalanine (F), lysine (K), leucine (L), asparagine (N), threonine (T) or tryptophan (W). or mutated by being further substituted with; The 368th amino acid, threonine (T), is alanine (A), aspartic acid (D), phenylalanine (F), glycine (G), lysine (K), leucine (L), valine (V), or tryptophan (W). or mutated by being further substituted with; The 369th amino acid, serine (S), is alanine (A), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), lysine (K), leucine (L), asparagine (N) , mutated by further substitution with threonine (T), valine (V) or tryptophan (W); 371 amino acid leucine (L) is further substituted with alanine (A), aspartic acid (D), phenylalanine (F), glycine (G), lysine (K), threonine (T) or tryptophan (W) mutated; The 403 amino acid tyrosine (Y) is alanine (A), aspartic acid (D), glycine (G), lysine (K), leucine (L), threonine (T), valine (V) or tryptophan (W) or mutated by being further substituted with; Histidine (H) at position 437 is further substituted with alanine (A), aspartic acid (D), phenylalanine (F), glycine (G), leucine (L), threonine (T) or tryptophan (W) mutated; Tyrosine (Y) at the 440th amino acid may be further substituted with alanine (A) and mutated.

뿐만 아니라, 본 발명은 상기 타카투론산 에피머화 효소 변이체를 암호화하는 유전자; 이를 포함하는 재조합 벡터; 및 이로 형질전환된 숙주세포를 제공한다. In addition, the present invention provides a gene encoding the takaturonic acid epimerase variant; Recombinant vectors containing them; And it provides a transformed host cell.

상기 타카투론산 에피머화 효소 변이체를 암호화하는 유전자는 타카투론산 에피머화 효소 변이체를 암호화하는 것으로, 바람직한 일예로, 상기 타카투론산 에피머화 효소 변이체가 각각 F66S /S125D/T181A/H362L, E72G/S125D/T181A/H362L, S125D/N129F/T181A/H362L, S125D/L140P/T181A/H362L, S125D/N129S/L140F/T181A/H362L, S125D/N129T/L140K/T181A/H362L, S125D/N129T/L140F/T181A/H362L 또는 S125D/N129T/L140P/T181A/H362L 변이체인 경우, 상기 타카투론산 에피머화 효소 변이체를 암호화하는 유전자는 각각 서열번호 15, 서열번호 16, 서열번호 17, 서열번호 18, 서열번호 19, 서열번호 20, 서열번호 21 또는 서열번호 22의 염기 서열로 이루어진 것뿐만 아니라, 이의 기능적 동등물, 즉, 상기 서열에 하나 이상의 치환, 결손 등의 돌연변이를 유발하여 본 발명의 목적을 달성하는 모든 돌연변이체를 포함하는 것을 의미한다. The gene encoding the takaturonic acid epimerase variant encodes the takaturonic acid epimerase variant, and in a preferred embodiment, the takaturonic acid epimerase variant is F66S /S125D / T181A / H362L, E72G / S125D, respectively. /T181A/H362L, S125D/N129F/T181A/H362L, S125D/L140P/T181A/H362L, S125D/N129S/L140F/T181A/H362L, S125D/N129T/L140K/T181A/H362L, S125D/N129T/L140F/N129T/L140F or S125D/N129T/L140P/T181A/H362L variant, the gene encoding the takaturonic acid epimerase variant is SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22, as well as functional equivalents thereof, that is, all mutants that achieve the object of the present invention by inducing mutations such as one or more substitutions, deletions, etc. in the sequence means to include

타카토스 생산용 조성물Composition for production of Takatos

본 발명은 상기 타카투론산 에피머화 효소 변이체 또는 이를 암호화하는 유전자를 유효성분으로 포함하는 타카토스 생산용 조성물을 제공한다. The present invention provides a composition for producing takatose comprising the takaturonic acid epimerase variant or a gene encoding the same as an active ingredient.

본 발명에 따른 타카토스 생산용 조성물은 상기 타카투론산 에피머화 효소 변이체 또는 이를 암호화하는 유전자를 유효성분으로 포함하는 것으로, 상기 타카투론산 에피머화 효소 변이체 또는 이를 암호화하는 유전자를 포함하는 전세포를 포함할 수 있다. The composition for producing takatos according to the present invention comprises the mutant takaturonic acid epimerase or a gene encoding the same as an active ingredient, and whole cells comprising the mutant takaturonic acid epimerase or a gene encoding the same may include

상기 타카투론산 에피머화 효소 변이체 또는 이를 암호화하는 유전자의 구체적인 내용에 대해서는 전술한 바와 같다. The specific details of the takaturonic acid epimerase variant or the gene encoding the same are as described above.

구체적으로, 상기 타카투론산 에피머화 효소 변이체 또는 이를 암호화하는 유전자를 포함하는 전세포는 상기 타카투론산 에피머화 효소 변이체를 암호화하는 유전자, 바람직한 일예로, 서열번호 15, 서열번호 16, 서열번호 17, 서열번호 18, 서열번호 19, 서열번호 20, 서열번호 21 또는 서열번호 22의 염기 서열로 이루어진 유전자를 포함하는 재조합 발현 벡터로 형질전환된 형질전환 미생물을 배양하고, 이를 수득하여 사용하는 것이 바람직하다.Specifically, the whole cell comprising the takaturonic acid epimerase variant or a gene encoding the same is a gene encoding the takaturonic acid epimerase variant, in a preferred example, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 , SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22 culturing the transformed microorganism transformed with a recombinant expression vector comprising a gene consisting of the base sequence, it is preferable to obtain and use it do.

상기 재조합 발현 벡터로서, 유전자 재조합을 위하여 당업계에서 사용되고 있는 플라스미드 벡터라면 어느 벡터를 사용해도 무방하고, 구체적으로 pET-29b 벡터를 사용하는 것이 보다 바람직하나, 이에 한정되지 않는다.As the recombinant expression vector, any vector may be used as long as it is a plasmid vector used in the art for gene recombination. Specifically, it is more preferable to use a pET-29b vector, but is not limited thereto.

상기 형질전환 미생물로서, 재조합 벡터로 형질전환하여 목적하는 단백질을 과발현하는 시스템으로 당업계에 사용되고 있는 미생물이라면 어느 미생물을 사용해도 무방하고, 구체적으로 대장균 ER 2566을 사용하는 것이 보다 바람직하나, 이에 한정되지 않는다.As the transforming microorganism, any microorganism may be used as long as it is a microorganism used in the art as a system for overexpressing a target protein by transformation with a recombinant vector, and specifically, E. coli ER 2566 is more preferably used, but limited thereto doesn't happen

상기 전세포는 ⅰ) 상기 미생물의 배양액을 원심분리하여 1차 전세포를 회수하는 단계; ⅱ) 상기 회수한 전세포를 생리식염수(saline solution)으로 세척하는 단계; ⅲ) 상기 세척된 전세포를 2차 원심분리하여 상등액을 제거하고 전세포를 얻는 단계; 및 ⅳ) 상기 2차로 회수한 전세포를 다시 한번 생리식염수로 세척하는 단계를 포함하여 수득될 수 있다. 구체적으로, ⅰ) 단계에서 전세포의 회수는 원심분리기 등 당업계 공지된 기기를 사용하여 13,000 rpm 내외의 범위에서 수행될 수 있고, ⅱ) 단계에서 전세포의 세척은 0.85% 이하의 염화나트륨 용액으로 수행하는 것이 적당하다.The whole cells are obtained by: i) centrifuging the culture solution of the microorganism to recover primary whole cells; ii) washing the recovered whole cells with saline solution; iii) second centrifuging the washed whole cells to remove the supernatant to obtain whole cells; and iv) washing the secondarily recovered whole cells with physiological saline once again. Specifically, the recovery of whole cells in step i) can be performed at around 13,000 rpm using a device known in the art, such as a centrifuge, and washing of whole cells in step ii) is performed with 0.85% or less sodium chloride solution. It is appropriate to perform

이때, 상기 조성물 내 상기 타카투론산 에피머화 효소 변이체 또는 이를 암호화하는 유전자를 포함하는 전세포의 농도는 1 mg/ml 내지 50 mg/ml인 것이 바람직하고, 10 mg/ml 내지 50 mg/ml인 것이 보다 바람직하나, 이에 한정되지 않는다. 상기 타카투론산 에피머화 효소 변이체 또는 이를 암호화하는 유전자는 이러한 농도를 유지함으로써, 타카토스의 생산 농도를 향상시킬 수 있다.In this case, the concentration of the whole cell containing the takaturonic acid epimerase variant or the gene encoding it in the composition is preferably 1 mg/ml to 50 mg/ml, and 10 mg/ml to 50 mg/ml is more preferable, but is not limited thereto. The takaturonic acid epimerase variant or a gene encoding the same can improve the production concentration of takatose by maintaining this concentration.

한편, 상기 조성물은 과당에 처리하기 위한 것일 수 있다. 상기 조성물에 대하여, 상기 과당의 농도는 50 g/L 내지 1,000 g/L인 것이 바람직하고, 400 g/L 내지 1,000 g/L인 것이 보다 바람직하나, 이에 한정되지 않는다. 마찬가지로, 상기 과당은 이러한 농도를 유지함으로써, 타카토스의 생산 농도를 향상시킬 수 있다.Meanwhile, the composition may be for treatment with fructose. With respect to the composition, the concentration of fructose is preferably 50 g/L to 1,000 g/L, more preferably 400 g/L to 1,000 g/L, but is not limited thereto. Likewise, the fructose can improve the production concentration of Takatose by maintaining this concentration.

과당으로부터 타카토스 생산방법How to produce Takatose from fructose

본 발명은 상기 조성물을 과당에 처리하여 에피머화 반응을 수행하는 단계를 포함하는 과당으로부터 타카토스의 생산방법을 제공한다. The present invention provides a method for producing Takatose from fructose, comprising the step of performing an epimerization reaction by treating the composition with fructose.

본 발명에 따른 과당으로부터 타가토스의 생산방법은 조성물을 과당에 처리하여 에피머화 반응을 수행하는 단계를 포함한다. The method for producing tagatose from fructose according to the present invention includes the step of performing an epimerization reaction by treating the composition with fructose.

상기 조성물은 상기 타카투론산 에피머화 효소 변이체 또는 이를 암호화하는 유전자를 유효성분으로 포함하는 것으로, 구체적인 내용에 대해서는 전술한 바와 같다. The composition is to include the takaturonic acid epimerase variant or a gene encoding the same as an active ingredient, the specific details are as described above.

상기 조성물에 대하여, 상기 과당의 농도는 50 g/L 내지 1,000 g/L인 것이 바람직하고, 400 g/L 내지 1,000 g/L인 것이 보다 바람직하나, 이에 한정되지 않는다. 마찬가지로, 상기 과당은 이러한 농도를 유지함으로써, 타카토스의 생산 농도를 향상시킬 수 있다.With respect to the composition, the concentration of fructose is preferably 50 g/L to 1,000 g/L, more preferably 400 g/L to 1,000 g/L, but is not limited thereto. Likewise, the fructose can improve the production concentration of Takatose by maintaining this concentration.

상기 에피머화 반응은 pH 6.5 내지 9.0 및 온도 70℃ 내지 90℃에서 수행되는 것이 바람직하고, pH 8.0 내지 9.0 및 온도 75℃내지 85℃에서 수행되는 것이 보다 바람직하나, 이에 한정되지 않는다. 이러한 pH 조건을 유지하기 위해서 반응용매로 HEPES, EPPS, CHES 또는 PIPES 완충용액을 사용할 수 있다. 이러한 pH 조건을 유지함으로써, 사용되는 효소를 최적으로 활성화시킬 수 있어, 최종적으로 과당을 높은 생산성과 높은 수율로 제조할 수 있다. 또한, 상기 에피머화 반응은 20분 이상, 바람직하게, 1시간 이상 수행되는 것이 바람직하나, 이에 한정되지 않는다.The epimerization reaction is preferably carried out at a pH of 6.5 to 9.0 and a temperature of 70 °C to 90 °C, and more preferably carried out at a pH of 8.0 to 9.0 and a temperature of 75 °C to 85 °C, but is not limited thereto. In order to maintain these pH conditions, HEPES, EPPS, CHES or PIPES buffer may be used as a reaction solvent. By maintaining such a pH condition, the enzyme used can be optimally activated, and fructose can be finally produced with high productivity and high yield. In addition, the epimerization reaction is preferably carried out for 20 minutes or more, preferably for 1 hour or more, but is not limited thereto.

한편, 상기 에피머화 반응은 2가 금속염의 존재 하에 수행될 수 있고, 이때, 2가 금속염은 Ni²⁺, Co²⁺, Ca²⁺, Mn²⁺, Mg²⁺, Zn²⁺, Cu²⁺, 및 Fe²⁺ 로 이루어진 군으로부터 선택된 하나 이상일 수 있고, Ni²⁺인 것이 바람직하나, 이에 한정되지 않는다. 구체적으로, 상기 에피머화 반응시, NiSO₄, CoSO₄, CaSO₄, MnSO₄, MgSO₄, ZnSO₄, CuSO₄ 및 FeSO₄로 이루어진 군으로부터 선택된 하나 이상의 2가 금속염을 1.0 mM 내지 5 mM로 첨가할 수 있다. 이로써, 사용되는 효소를 최적으로 활성화시킬 수 있어, 최종적으로 타가토스를 높은 생산성과 높은 수율로 제조할 수 있다.Meanwhile, the epimerization reaction may be performed in the presence of a divalent metal salt, wherein the divalent metal salt is Ni ²⁺ , Co ²⁺ , Ca ²⁺ , Mn ²⁺ , Mg ²⁺ , Zn ²⁺ , Cu ² It may be at least one selected from the group consisting of ⁺ , and Fe ²⁺ , and Ni ²⁺ is preferable, but is not limited thereto. Specifically, during the epimerization reaction, at least one divalent metal salt selected from the group consisting of NiSO ₄ , CoSO ₄ , CaSO ₄ , MnSO ₄ , MgSO ₄ , ZnSO ₄ , CuSO ₄ and FeSO ₄ is added at 1.0 mM to 5 mM. can do. Accordingly, the enzyme used can be optimally activated, and finally tagatose can be produced with high productivity and high yield.

상기한 바와 같이, 본 발명에 따르면, 종래 공지된 써모토가 페트로필라(Thermotoga petrophila) 유래 타카투론산 에피머화 효소 및 이의 변이체를 기반으로 하되 추가 돌연변이시킨 변이체를 이용함으로써, 과당의 4번 탄소 위치를 에피머화시켜 타카토스로 전환하는 비활성을 증가시킬 수 있는바, 최종적으로, 값싸고 안정적으로 수급이 가능한 과당을 이용하여 친환경적인 방법으로 타카토스를 높은 생산성과 높은 수율로 효율적으로 생산할 수 있다. As described above, according to the present invention, by using a conventionally known Thermotoga petrophila -derived takaturonic acid epimerase and variants thereof, but further mutated variants are used, the 4th carbon position of fructose can be epimerized to increase the specific activity of converting to Takatose. Finally, it is possible to efficiently produce Takatose with high productivity and high yield in an eco-friendly way using fructose, which can be supplied and supplied inexpensively and stably.

이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시계는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

실시예 1. 개량 타겟 위치 디자인 및 분석Example 1. Improved target location design and analysis

써모토가 페트로필라(Thermotoga petrophila) 유래 타가투론산 에피머화 효소(이하, 야생형이라 함)의 아미노산과 상동성을 보유하는 상동유전자의 활성부위 3차 구조 모델을 기초로 상동성 모델을 제작하였으며, 상동성 모델에 다양한 포즈로 과당을 도킹하여 과당과 유리한 상호작용 개수를 분석하여 1차적으로 변이체를 선정하였다. 그 후, 변이체에 대한 3차 구조 모델을 기초로 예측된 과당 결합 위치 주변 아미노산 잔기들을 2차 선정 후, 이들에 대한 활성 부위 돌연변이(active site mutagenesis) 분석 후 재설계된 활성부위 구조 및 과당 간 도킹모델 분석 결과를 기반으로 과당에 대한 에피머화 전환반응의 비활성 향상을 위해 개량 타겟 부위를 디자인하였다. 이를 상세하게 설명하면 하기와 같다.Thermotoga petrophila ( Thermotoga petrophila ) A homology model was prepared based on the active site tertiary structure model of the homologous gene possessing homology with amino acids of tagaturonic acid epimerase (hereinafter, referred to as wild type), By docking fructose in various poses to the homology model, the number of favorable interactions with fructose was analyzed to select variants primarily. After that, after secondary selection of amino acid residues around the fructose binding site predicted based on the tertiary structural model for the variant, active site mutagenesis analysis for them, the redesigned active site structure and fructose-to-fructose docking model Based on the analysis results, an improved target site was designed to improve the specific activity of the epimerization conversion reaction for fructose. This will be described in detail as follows.

1-1. 효소 3차 구조 모델 분석1-1. Enzyme tertiary structure model analysis

단백질 데이터 뱅크(Protein Data Bank) 데이터베이스 내에 야생형과 동일한 효소인 코넬라 래비리보시(Cohnella laeviribosi) 유래 프럭투론산-타가투론산 에피머화 효소의 단백질 구조(PDB:6ILB)가 보고되어 있으며, 이를 기초로 야생형과 아미노산 서열 상동성(Identity)은 약 40%로 확인하였고, 이를 통해 상동성 모델을 제작하였다(도 2 참조).The protein structure (PDB: 6ILB) of the fructuronic acid-tagaturonic acid epimerase derived from Cohnella laeviribosi , the same enzyme as the wild-type, has been reported in the Protein Data Bank database, and based on this As a result, the amino acid sequence homology with the wild type was confirmed to be about 40%, and a homology model was prepared through this (see FIG. 2).

1-2. 타가투론산 에피머화 효소의 제조1-2. Preparation of tagaturonic acid epimerase

내열성 미생물인 써모토가 페트로필라(Thermotoga petrophila, strain RKU-1 / ATCC BAA-488 / DSM 13995)의 genomic DNA(서열번호 12)를 주형으로, 하기의 표 1과 같이 프라이머를 첨가하여 PCR 반응을 수행하였다.Thermotoga petrophila ( Thermotoga petrophila , strain RKU-1 / ATCC BAA-488 / DSM 13995), a thermostable microorganism, genomic DNA (SEQ ID NO: 12) as a template, primers were added as shown in Table 1 below to conduct a PCR reaction. carried out.

[표 1][Table 1]

PCR 반응 조건은 하기의 표 2 및 표 3과 같다. PCR 반응을 통하여 증폭된 유전자를 pET-29b 벡터에 삽입하였고, 대장균(Escherichia coli) ER2566에 형질전환하였으며, 이로부터 플라스미드를 분리하여 삽입된 유전자의 염기 서열을 확인한 후 단백질 발현용 균주인 대장균(Escherichia coli) ER2566에 다시 형질전환하여 타가투론산 에피머화 효소를 생산하는데 이용하였다. 타가투론산 에피머화 효소를 생산하기 위해서 대장균(Escherichia coli) ER2566-pET29b-TP(Thermotoga petrophila)를 100 mg/ml 농도의 카나마이신을 포함한 LB 배지에서 배양온도 37℃, 교반속도 180 rpm으로 2시간 배양하였다. 배양 후, 600 nm에서 미생물의 광학밀도(optical density)를 측정하여 그 값이 0.4 내지 0.8 범위에 들어오면, 0.25 mM IPTG(Isopropyl β-D-1-thiogalactopyranoside)를 첨가하여 동일 조건에서 하룻밤 동안 더 배양하여, 단백질 발현을 유도하였다.PCR reaction conditions are shown in Tables 2 and 3 below. The gene amplified through the PCR reaction was inserted into the pET-29b vector, transformed into Escherichia coli ER2566, and the plasmid was isolated from this to confirm the nucleotide sequence of the inserted gene, and then the protein expression strain Escherichia coli ) ER2566 was transformed again and used to produce tagaturonic acid epimerase. In order to produce tagaturonic acid epimerase, Escherichia coli ER2566-pET29b-TP ( Thermotoga petrophila ) was cultured in LB medium containing kanamycin at a concentration of 100 mg/ml at a culture temperature of 37° C. and agitation speed of 180 rpm for 2 hours. did After incubation, measure the optical density of the microorganism at 600 nm and when the value is in the range of 0.4 to 0.8, 0.25 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside) is added and overnight under the same conditions. Incubated to induce protein expression.

[표 2][Table 2]

[표 3][Table 3]

1-3. 타가투론산 에피머화 효소 및 변이체의 정제1-3. Purification of tagaturonic acid epimerase and variants

미생물 내에 발현된 타가투론산 에피머화 효소 및 변이체의 활성을 측정하기 위하여, 하기와 같은 방법을 이용하여 단백질 정제를 수행하였다. 단백질 발현이 완료된 배양액을 8,000 rpm에서 10분간 원심분리를 하여 균체를 수거한 후, 10 mM 이미다졸(imidazole)과 300 mM NaCl이 포함되어있는 50 mM NaH₂PO₄(pH 8.0) 완충 용액에 재현탁 하였다. 현탁된 균체를 세포파쇄기(sonicator)를 이용하여 파쇄하였으며, 이를 13,000 rpm에서 10분간 원심분리하여 상등액을 회수하였다. 회수된 상등액을 충진제로서 Ni-NTA 수지가 충진되어 있는 컬럼에 통액하고, 20 mM 이미다졸 및 300 mM NaCl을 함유하는 50 mM NaH₂PO₄(pH8.0) 완충 용액을 컬럼안의 충진제가 차지하는 부피의 10배 부피로 흘려주어 비특이적으로 결합될 수 있는 단백질을 제거하였다. 최종 250 mM 이미다졸 및 300 mM NaCl이 포함된 50 mM NaH2PO4 (pH 8.0) 완충 용액을 흘려주어 타가투론산 에피머화 효소를 용출 정제하였으며, 정제 효소 내의 이미다졸을 제거하기 위하여 50 mM NaH₂PO₄ (pH 8.0) 완충 용액을 수회 흘려주어 그 농도가 0.01 mM 이하가 되도록 하였다. 정제된 효소는 bovine serum albumin을 이용하여 단백질 농도를 정량하였다.In order to measure the activity of tagaturonic acid epimerase and variants expressed in microorganisms, protein purification was performed using the following method. After collecting the cells by centrifugation at 8,000 rpm for 10 minutes at 8,000 rpm, the culture medium in which protein expression is completed is reproduced in 50 mM NaH ₂ PO ₄ (pH 8.0) buffer solution containing 10 mM imidazole and 300 mM NaCl. it was cloudy The suspended cells were disrupted using a sonicator, and the supernatant was recovered by centrifugation at 13,000 rpm for 10 minutes. The recovered supernatant is passed through a column filled with Ni-NTA resin as a filler, and a 50 mM NaH ₂ PO ₄ (pH 8.0) buffer solution containing 20 mM imidazole and 300 mM NaCl is added to the volume of the filler in the column. Proteins that can be non-specifically bound were removed by flowing in a volume 10 times the Finally, a 50 mM NaH2PO4 (pH 8.0) buffer solution containing 250 mM imidazole and 300 mM NaCl was flowed to elute and purify tagaturonic acid epimerase. In order to remove imidazole in the purified enzyme, 50 mM NaH ₂ PO ₄ (pH 8.0) buffer solution was flowed several times so that the concentration was 0.01 mM or less. The protein concentration of the purified enzyme was quantified using bovine serum albumin.

1-4. 도킹 결합 분석1-4. Docking binding analysis

상동성 모델 제작 후 과당과 프럭투론산의 도킹 시뮬레이션을 통해 과당과 프럭투론산에 대한 유리한 상호작용 개수를 분석하였다. 도 3(a)를 참조하면 분석된 아미노산 잔기들 중에서 125번 세린(S)은 야생형의 주 기질인 프럭투론산일 때 결합에 전혀 관여하지 않는 것으로 확인되지만, 과당이 도킹되었을 때 결합에 관여를 하는 것으로 확인되어 상동성 모델을 125번 세린(S)에 대해 다른 여러 아미노산으로 치환하여 제작하였다. 그 후 제작된 상동성 모델에 과당을 다시 도킹하여 분석하였다. 그 결과 도 3(b)에서 참고하면 S125D 변이체일 때 야생형에 비해 과당에 대한 유리한 상호작용 개수가 약 7배 증가한 것을 확인할 수 있었다.After making the homology model, the number of favorable interactions between fructose and fructuronic acid was analyzed through the docking simulation of fructose and fructuronic acid. Referring to FIG. 3(a), among the analyzed amino acid residues, serine 125 (S) was found not to be involved in binding at all when it was fructuronic acid, the main substrate of the wild type, but was not involved in binding when fructose was docked. It was confirmed that the homology model was produced by substituting several other amino acids for serine 125 (S). After that, fructose was docked again to the produced homology model and analyzed. As a result, referring to FIG. 3(b), it was confirmed that the number of favorable interactions with fructose in the S125D mutant was increased by about 7 times compared to the wild type.

1-5. 과당으로부터 타가토스로의 전환 비활성 비교를 통한 변이체의 선별1-5. Selection of variants by comparison of the conversion activity from fructose to tagatose

실시예 1-4에서 얻어진 S125D 변이체 및 야생형의 비활성을 비교 측정하기 위해서 실시예 1-3의 단백질 정제법으로 정제하여 정제된 효소를 10 mM 과당, 1.5 mM NiSO₄, 100 mM 인산염 완충용액 (pH 8.0)에 넣어 반응온도 60℃에서 1시간 동안 반응을 수행하였다. 그 후 반응산물을 Bio-LC 분석법(컬럼 PA1, 컬럼 분석 온도 30℃, 이동상 H₂O 및 200 mM NaCl, 유속 1 ml/min)을 이용하여 야생형 대비 전환 비활성이 약 16배 증가한 S125D 변이체를 선별하였다(도 3(b) 참조).In order to compare and measure the specific activity of the S125D mutant and wild-type obtained in Example 1-4, the purified enzyme was purified by the protein purification method of Example 1-3, 10 mM fructose, 1.5 mM NiSO ₄ , 100 mM phosphate buffer (pH). 8.0), and the reaction was performed at a reaction temperature of 60° C. for 1 hour. Thereafter, the reaction product was subjected to Bio-LC analysis (column PA1, column analysis temperature 30° C., mobile phase H ₂ O and 200 mM NaCl, flow rate 1 ml/min) to select the S125D mutant with about 16-fold increase in conversion specific activity compared to the wild type. (see Fig. 3(b)).

실시예 2. 활성 부위 돌연변이법을 이용한 변이체 제작 및 활성 개량 변이체 선별Example 2. Production of variants using active site mutagenesis and selection of active-improved variants

실시예 1에서 디자인한 활성 부위(야생형 타가투론산 에피머화 효소의 96, 97, 98, 100, 123, 124, 126, 127, 128, 159, 161, 162, 181, 266, 268, 270, 299, 306, 307, 308, 309, 339, 340, 341, 342, 343, 344, 346, 362, 363, 364, 365, 366, 367, 368, 369, 371, 403, 437, 440번 위치의 아미노산 잔기) 40곳을 활성 부위 돌연변이를 수행하여 비활성이 많이 향상된 T181과 H362를 선정하였다. 선정한 이중부위의 포화돌연변이 라이브러리(double-site saturation mutagenesis library)를 제작하고, 활성이 개량되는 변이 부위 및 아미노산들을 스크리닝 선별하였다. 선발된 개량 부위의 정보들을 통합하여 다중 변이체를 제작 후 과당 4번탄소 에피머화 전환반응의 비활성이 향상된 변이체를 개발하였다.The active site designed in Example 1 (96, 97, 98, 100, 123, 124, 126, 127, 128, 159, 161, 162, 181, 266, 268, 270, 299 of wild-type tagaturonic acid epimerase) , 306, 307, 308, 309, 339, 340, 341, 342, 343, 344, 346, 362, 363, 364, 365, 366, 367, 368, 369, 371, 403, 437, amino acid at position 440 Residues) 40 places were subjected to active site mutation to select T181 and H362, which had significantly improved specific activity. A double-site saturation mutagenesis library of the selected double-site was prepared, and mutation sites and amino acids whose activity was improved were screened and selected. After integrating the information of the selected improvement sites to make multiple variants, a variant with improved specific activity of fructose carbon 4 epimerization conversion reaction was developed.

2-1. 활성 부위 돌연변이(active site mutagenesis) 및 도킹 결합 분석2-1. Active site mutagenesis and docking binding assay

앞서 서술한 타가투론산 에피머화 S125 변이체의 활성부위를 3차 구조 모델 분석으로 선정된 아미노산들을 위치 지정 돌연변이법(site-directed mutagenesis)으로 여러 아미노산으로 치환 변이하여, 이러한 재조합 변이체들을 대장균에서 생산한 후 각 변이부위들의 특성을 분석하였다. 활성 부위 돌연변이 분석 후 재설계된 활성부위 구조 및 과당 간 도킹 시뮬레이션을 통해 기능적으로 중요할 것으로 예측되는 아미노산들을 선별하여 과당 4번 탄소 에피머화 전환반응의 활성 향상을 위해 개량 타겟 부위를 디자인하였다. 활성 부위 돌연변이와 앞서 서술한 보고된 프럭투론산-타가투론산 에피머화 효소 단백질 구조를 통해 활성이 완전 소실되는 아미노산 부위 [촉매금속이온 결합 잔기 및 탈양성자화/양성자화(deprotonation/protonation) 관여 촉매 잔기]는 활성 개량을 위한 타겟 부위에서 배제하였다.The active site of the above-described tagaturonic acid epimerization S125 mutant was substituted with several amino acids by site-directed mutagenesis for the amino acids selected by tertiary structural model analysis, and these recombinant mutants were produced in E. coli. Afterwards, the characteristics of each mutation site were analyzed. After active site mutation analysis, amino acids predicted to be functionally important were selected through the redesigned active site structure and fructose-to-fructose docking simulation, and an improved target site was designed to improve the activity of fructose carbon 4 epimerization conversion reaction. An amino acid site in which activity is completely lost through active site mutation and the previously reported fructuronic acid-tagaturonic acid epimerase protein structure [catalyst metal ion binding residue and deprotonation/protonation involvement catalyst residues] were excluded from the target site for activity improvement.

2-2. 포화 돌연변이(saturation mutagenesis)2-2. Saturation mutagenesis

앞서 활성 부위 돌연변이로 총 40곳의 S125D를 기초로 한 이중부위 포화돌연변이 라이브러리를 제작하였고, 표4를 참조하여 과당에 대해 비활성이 가장 높았던 2개의 변이체인 S125D/T181D 변이체 및 S125D/H362W 변이체를 선별하였다. 야생형 효소 유전자 야생형의 대장균 ER2566 발현을 위해 제작된 재조합 발현벡터(pET-29b의 NdeI 및 XhoI 제한효소 부위에 야생형을 도입하고 야생형의 C-말단에 6xHis-tag이 결합한 재조합효소를 발현함)에서 S125D로 치환된 재조합 발현벡터(서열번호 13)를 변이주 라이브러리 제작을 위한 포화 돌연변이법의 주형(template)으로 사용하였다. 변이분포 다양성 및 변이체 수율 등을 고려하여 위치 지정 돌연변이법(site directed mutagenesis)을 사용하였다. 상세하게는, 각각의 변위 부위의 치환할 염기 3bp(각각 NNK)로 하여 혼합 프라이머를 제작 이용하였다. PCR 조건은 95℃에서 30초간 변성 후, 95℃ 30초 변성, 55℃ 1분 어닐링, 72℃ 10분 신장을 18 회 반복한 후, 72℃에서 60분간 신장반응을 수행하였다. 변이 부위별로 포화 돌연변이 라이브러리를 제작 후 라이브러리별 변이주를 무작위 선발하고 염기 서열을 분석하여 아미노산 변이 분포를 평가하였다.Previously, a total of 40 S125D-based double-site saturation mutation libraries were prepared by active site mutation. Referring to Table 4, S125D/T181D mutant and S125D/H362W mutant, the two variants with the highest specific activity to fructose, were selected. did Wild-type enzyme gene S125D in a recombinant expression vector (introduced wild-type into the NdeI and XhoI restriction enzyme sites of pET-29b, and expressing a recombinase with 6xHis-tag bound to the C-terminus of the wild-type) constructed for wild-type E. coli ER2566 expression. The recombinant expression vector (SEQ ID NO: 13) substituted with was used as a template for saturation mutagenesis for the production of a mutant library. Site directed mutagenesis was used in consideration of mutation distribution diversity and mutant yield. In detail, a mixed primer was prepared and used with 3 bp of the base to be substituted in each displacement site (each NNK). PCR conditions were repeated 18 times of denaturation at 95° C. for 30 seconds, denaturation at 95° C. for 30 seconds, annealing at 55° C. for 1 minute, and extension at 72° C. for 10 minutes, followed by extension at 72° C. for 60 minutes. After a saturated mutation library was prepared for each mutation site, mutant strains for each library were randomly selected and the nucleotide sequence was analyzed to evaluate the distribution of amino acid mutations.

[표 4][Table 4]

2-3. 활성 개량 변이체 스크리닝 및 다중 변이체 제작2-3. Screening of active improved variants and production of multiple variants

제작된 포화 돌연변이 라이브러리에서 활성 개량 변이체를 대량으로 스크리닝하기 위해 과당을 특이적으로 정량화할 수 있는 발색 측정법을 이용하였다. 상세하게는 과당 측정법을 이용하여 타가토스를 기질로 한 효소 반응 완료액에 0.1 M의 potassium ferricyanide와 D-fructose dehydrogenase를 넣어 5분간 반응 후, ferric sulfate-SDS solution을 넣어 20분간 반응을 멈추고 660 nm에서 측정하여 OD 값으로 비교 분석하였다. S125D 변이체와 상대활성 비교시 총 6000주 중에서 비활성이 증가된 타가투론산 에피머화 효소 변이체 4개를 선별하였다(도 4 참조).A colorimetric assay capable of specifically quantifying fructose was used in order to large-scale screening of activity-improved mutants in the prepared saturated mutant library. In detail, 0.1 M potassium ferricyanide and D-fructose dehydrogenase were added to the completed enzyme reaction solution using tagatose as a substrate using the fructose measurement method, reacted for 5 minutes, ferric sulfate-SDS solution was added, the reaction was stopped for 20 minutes, and the reaction was stopped at 660 nm. was measured and analyzed by comparison with OD values. When comparing the relative activity with the S125D variant, four tagaturonic acid epimerase variants with increased specific activity among a total of 6000 strains were selected (see FIG. 4 ).

2-4. 과당으로부터 타가토스로의 전환활성 비교를 통한 변이체의 선별2-4. Selection of variants through comparison of conversion activity from fructose to tagatose

실시예 2-2에서 얻어진 변이체와 S125D 변이체의 비활성을 비교 측정하기 위해서 실시예 1-3의 단백질 정제법으로 정제하여 정제된 효소를 10 mM 과당, 1.5 mM NiSO₄, 100 mM 인산염 완충용액 (pH 8.0)에 넣어 반응온도 60℃에서 1시간 동안 반응을 수행하였다. 그 후 반응산물을 Bio-LC 분석법(컬럼 PA1, 컬럼 분석 온도 30℃, 이동상 H₂O 및 200mM NaCl, 유속 1 ml/min)을 이용하여 S125D 대비 전환 비활성이 약 3.8배 증가한 S125D/T181A/H362L 변이체를 선별하였다(도 5 참조).In order to compare and measure the specific activity of the variant obtained in Example 2-2 and the S125D variant, the enzyme purified by the protein purification method of Example 1-3 was purified with 10 mM fructose, 1.5 mM NiSO ₄ , 100 mM phosphate buffer (pH). 8.0), and the reaction was performed at a reaction temperature of 60° C. for 1 hour. After that, the reaction product was analyzed using Bio-LC analysis method (column PA1, column analysis temperature 30° C., mobile phase H ₂ O and 200 mM NaCl, flow rate 1 ml/min) to S125D/T181A/H362L, which had an approximately 3.8-fold increase in conversion specific activity compared to S125D Variants were selected (see Figure 5).

실시예 3. 무작위 돌연변이법을 이용한 변이체 제작 및 활성 개량 변이체 선별Example 3. Production of variants using random mutagenesis and selection of active-improved variants

실시예 2에서 활성 부위 주변 3개의 위치가 돌연변이된 S125D/T181A/H362L 변이체를 기초로 하여 추가로 활성 부위 외의 효소 전체에서 돌연변이를 일으키기 위해 무작위 돌연변이(random mutagenesis)를 일으켰다. 이를 통해 포화 돌연변이 라이브러리를 제작하고, 비활성이 개량되는 변이 부위 및 아미노산들을 스크리닝 선별하였고, 이러한 다중 변이체를 과당의 4번 탄소 에피머화 전환 반응의 비활성이 향상된 변이체를 최종적으로 개발하였다.In Example 2, based on the S125D/T181A/H362L mutant in which three positions around the active site were mutated, random mutagenesis was performed to further induce mutations in the entire enzyme other than the active site. Through this, a saturation mutation library was prepared, mutation sites and amino acids with improved specific activity were screened and selected, and a mutant with improved specific activity of fructose carbon 4 epimerization conversion reaction of fructose was finally developed.

3-1. 무작위 돌연변이 (random mutagenesis) 및 DNA shuffling3-1. Random mutagenesis and DNA shuffling

실시예 2에서 선별한 S125D/T181A/H362L 변이체를 기초로 하여 무작위 돌연변이를 수행하였다. 구체적으로, ClonTech사의 Diversify random mutagenesis kit을 이용하여 S125D/T181A/H362L 변이체에 대해 무작위 돌연변이를 유도하였고, 아래 표 5 및6의 조건의 PCR 반응을 통하여 증폭된 유전자를 pET-29b 벡터에 삽입하여 대장균(Escherichia coli) ER2566에 형질전환시켰다.Random mutation was performed based on the S125D/T181A/H362L mutant selected in Example 2. Specifically, ClonTech's Diversify random mutagenesis kit was used to induce random mutations in the S125D/T181A/H362L mutant, and the amplified gene through the PCR reaction under the conditions of Tables 5 and 6 below was inserted into the pET-29b vector in E. coli ( Escherichia coli ) Transformed into ER2566.

[표 5][Table 5]

[표 6][Table 6]

무작위 돌연변이를 통하여 확보한 변이주 라이브러리로부터 추가로 DNA Shuffling을 진행하였다. 구체적으로, JBS사의 DNA-Shuffling kit를 이용하여 무작위 돌연변이를 통하여 확보한 변이주에 대해 DNA-shuffling을 유도하였고, 증폭된 유전자를 pET-29b 벡터에 삽입하여 대장균(Escherichia coli) ER2566에 형질전환 시켰다. 이후 단계는 실시예 1-2와 동일하게 실시하였다. 무작위 돌연변이 및 DNA shuffling을 통하여 확보한 변이주 라이브러리에서 활성개량 변이체를 대량으로 스크리닝하기 위해 실시예 2-2와 동일하게 총 6000주를 타가토스를 기질로 한 효소 반응 완료액을 과당 측정법으로 이용하여 활성을 측정하였다. 그 결과 S125D/T181A/H362L 변이체과 상대활성 비교시 총 6000주 중에서 비활성이 증가된 타가투론산 에피머화 효소 변이체 4개를 선별하였다.DNA shuffling was further performed from the mutant library obtained through random mutation. Specifically, DNA-shuffling was induced for a mutant obtained through random mutation using JBS' DNA-Shuffling kit, and the amplified gene was inserted into the pET-29b vector and transformed into Escherichia coli ER2566. Subsequent steps were carried out in the same manner as in Example 1-2. In order to screen a large amount of activity-improved mutants from the mutant library obtained through random mutation and DNA shuffling, a total of 6000 strains of tagatose as a substrate were used as a fructose measurement method in the same manner as in Example 2-2. was measured. As a result, when comparing the relative activity with the S125D/T181A/H362L variant, 4 tagaturonic acid epimerase variants with increased specific activity among a total of 6000 strains were selected.

3-2. 과당으로부터 타가토스로의 전환3-2. Conversion from fructose to tagatose

실시예 3-1에서 얻어진 변이체와 S125D/T181A/H362L 변이체의 비활성을 비교 측정하기 위해서 실시예 1-3의 단백질 정제법으로 정제하여 정제된 효소를 10 mM 과당, 1.5 mM NiSO₄, 100 mM 인산염 완충용액 (pH 8.0)에 넣어 반응온도 60℃에서 1시간 동안 반응을 수행하였다. 그 후 반응산물을 Bio-LC 분석법(컬럼 PA1, 컬럼 분석 온도 30℃, 이동상 H₂O 및 200mM NaCl, 유속 1 ml/min)을 이용하여 S125D/T181A/H362L 변이체 대비 전환 비활성이 각각 약 1.8, 2.0 배 증가한 S125D/N129F/T181A/H362L 변이체 및 S125D/L140P/T181A/H362L 변이체를 선별하였다(도 6 참조).In order to compare and measure the specific activity of the variant obtained in Example 3-1 and the S125D/T181A/H362L variant, the enzyme purified by the protein purification method of Example 1-3 was purified by 10 mM fructose, 1.5 mM NiSO ₄ , 100 mM phosphate. It was put into a buffer solution (pH 8.0) and the reaction was carried out at a reaction temperature of 60° C. for 1 hour. Thereafter, the reaction product was analyzed by Bio-LC analysis (column PA1, column analysis temperature 30° C., mobile phase H ₂ O and 200 mM NaCl, flow rate 1 ml/min) to have a conversion specific activity of about 1.8, respectively, compared to S125D/T181A/H362L variants, S125D/N129F/T181A/H362L variants and S125D/L140P/T181A/H362L variants, which increased 2.0-fold, were selected (see FIG. 6 ).

3-3. 포화 돌연변이(saturation mutagenesis)3-3. Saturation mutagenesis

실시예 3-2에서 선별한 2개 변이체들을 조합하기 위해서 S125D/T181A/H362L 변이체를 기초로하여 각 N129, L140의 위치에 대해 실시예 2-1과 동일하게 포화 돌연변이법을 사용하였다. 포화 돌연변이를 통하여 확보한 변이주 라이브러리에서 활성개량 변이체를 대량으로 스크리닝하기 위해 실시예 2-2와 동일하게 총 6000주를 타가토스를 기질로 한 효소 반응 완료액을 과당 측정법으로 이용하여 활성을 측정하였다. 그 결과 S125D/T181A/H362L 변이체와 상대활성 비교 시 총 6000주 중에서 활성이 증가된 타가투론산 에피머화 효소 변이체 4개를 선별하였다.In order to combine the two variants selected in Example 3-2, the saturation mutagenesis method was used in the same manner as in Example 2-1 for the positions of N129 and L140 based on the S125D/T181A/H362L variant. In order to screen a large amount of activity-improved mutants from the mutant library obtained through saturation mutation, the activity was measured by using the complete enzyme reaction solution with tagatose as a substrate for a total of 6000 strains as in Example 2-2 as a fructose measurement method. . As a result, when comparing the relative activity with the S125D/T181A/H362L variant, four tagaturonic acid epimerase variants with increased activity were selected out of a total of 6000 strains.

3-4. 과당으로부터 타가토스로의 전환활성 비교를 통한 변이체의 선별3-4. Selection of variants through comparison of conversion activity from fructose to tagatose

실시예 3-3에서 얻어진 변이체와 S125D/T181A/H362L 변이체의 비활성을 비교 측정하기 위해서 실시예 1-3의 단백질 정제법으로 정제하여 정제된 효소를 10 mM 과당, 1.5 mM NiSO₄, 100 mM 인산염 완충용액 (pH 8.0)에 넣어 반응온도 60℃에서 1시간 동안 반응을 수행하였다. 그 후 반응산물을 Bio-LC 분석법(컬럼 PA1, 컬럼 분석 온도 30℃, 이동상 H₂O 및 200mM NaCl, 유속 1 ml/min)을 이용하여 S125D/T181A/H362L 변이체 대비 전환 비활성이 각각 약 2.5 배 증가한 S125D/N129T/L140P/T181A/H362L 변이체를 최종 선별하였다(도 7 참조).In order to compare and measure the specific activity of the variant obtained in Example 3-3 and the S125D/T181A/H362L variant, the enzyme purified by the protein purification method of Example 1-3 was purified by 10 mM fructose, 1.5 mM NiSO ₄ , 100 mM phosphate. It was put into a buffer solution (pH 8.0) and the reaction was carried out at a reaction temperature of 60° C. for 1 hour. Thereafter, the reaction product was analyzed by Bio-LC analysis (column PA1, column analysis temperature 30° C., mobile phase H ₂ O and 200 mM NaCl, flow rate 1 ml/min) to approximately 2.5 times the conversion specific activity compared to the S125D/T181A/H362L variants, respectively. The increased S125D/N129T/L140P/T181A/H362L variants were finally selected (see FIG. 7 ).

실시예 4. 타가투론산 에피머화 효소 변이체 이용 과당으로부터 타가토스로의 최적 조건 확립Example 4. Establishment of optimal conditions from fructose to tagatose using tagaturonic acid epimerase variants

타가투론산 에피머화 효소 변이체를 사용하여 타가토스를 생산하기 위해 아래에 기재된 것과 같은 방법으로 온도, pH, 금속특이성에 따른 최적 조건을 확립하였다.Optimal conditions according to temperature, pH, and metal specificity were established in the same manner as described below to produce tagatose using the tagaturonic acid epimerase variant.

4-1. 타가투론산 에피머화 효소 변이체의 pH 변화에 따른 최적 활성 조건4-1. Optimal activity conditions according to pH change of tagaturonic acid epimerase mutant

반응 pH 변화에 따른 효소 활성의 최적 pH를 확인하기 위해 실시예 3에서 선별한 S125D/N129T/L140P/T181A/H362L 변이체를 실시예 1-3의 단백질 정제법으로 정제하여 정제된 효소를 10 mM 과당, 1.5 mM NiSO₄를 pH 6.5에서 pH 9.0 범위에서 사용되는 CHES, EPPS, HEPPES 또는 PIPES 완충용액에 넣어 반응온도 60℃에서 30분 동안 반응을 수행하였다. 그 후 반응산물을 Bio-LC 분석법(컬럼 PA1, 컬럼 분석 온도 30℃, 이동상 H₂O 및 200 mM NaCl, 유속 1 ml/min)을 이용하여 최적 pH를 확인하였다. 그 결과, 최적 pH는 8.5 (EPPS완충용액)이었다(도 8 참조).In order to confirm the optimal pH of the enzyme activity according to the change in reaction pH, the S125D/N129T/L140P/T181A/H362L mutant selected in Example 3 was purified by the protein purification method of Example 1-3, and the purified enzyme was purified by 10 mM fructose. , 1.5 mM NiSO ₄ was put into CHES, EPPS, HEPPES or PIPES buffers used in the pH 6.5 to pH 9.0 range, and the reaction was carried out at a reaction temperature of 60° C. for 30 minutes. Thereafter, the optimal pH of the reaction product was confirmed using Bio-LC analysis (column PA1, column analysis temperature 30° C., mobile phase H ₂ O and 200 mM NaCl, flow rate 1 ml/min). As a result, the optimal pH was 8.5 (EPPS buffer solution) (see FIG. 8).

4-2. 타가투론산 에피머화 효소 변이체의 온도 변화에 따른 최적 활성 조건4-2. Optimal activity conditions according to temperature change of tagaturonic acid epimerase mutant

반응 온도 변화에 따른 효소 활성의 최적 온도를 확인하기 위해 실시예 3에서 선별한 S125D/N129T/L140P/T181A/H362L 변이체를 실시예 1-3의 단백질 정제법으로 정제하여 정제된 효소를 10 mM 과당, 1.5 mM NiSO₄를 100 mM EPPS (pH 8.5) 완충용액에 넣어 반응온도 70℃에서 90℃ 범위에서 30분 동안 반응을 수행하였다. 그 후 반응산물을 Bio-LC 분석법(컬럼 PA1, 컬럼 분석 온도 30℃, 이동상 H₂O 및 200mM NaCl, 유속 1 ml/min)을 이용하여 최적 온도를 확인하였다. 그 결과, 최적 온도는 80℃이었다(도 9 참조).In order to confirm the optimum temperature of enzyme activity according to the change in reaction temperature, the S125D/N129T/L140P/T181A/H362L mutant selected in Example 3 was purified by the protein purification method of Example 1-3, and the purified enzyme was purified by 10 mM fructose. , 1.5 mM NiSO ₄ was put in 100 mM EPPS (pH 8.5) buffer, and the reaction was performed at a reaction temperature of 70 ° C. to 90 ° C. for 30 minutes. After that, the optimal temperature was confirmed for the reaction product using Bio-LC analysis (column PA1, column analysis temperature 30° C., mobile phase H ₂ O and 200 mM NaCl, flow rate 1 ml/min). As a result, the optimum temperature was 80°C (see FIG. 9).

4-3. 타가투론산 에피머화 효소 변이체의 금속 특이성에 따른 최적 활성 조건4-3. Optimal activity conditions according to metal specificity of tagaturonic acid epimerase variants

금속염 종류에 따른 효소 활성의 변화를 확인하기 위해 실시예 3에서 선별한 S125D/N129T/L140P/T181A/H362L 변이체를 실시예 1-3의 단백질 정제법으로 정제하여 정제된 효소를 10 mM 과당과 함께 100 mM EPPS(pH 8.5) 완충용액에 넣어 반응온도 80℃에서 30분 동안 반응을 수행하였다. 이 때, 반응에는 각각 상이한 8종의 금속염이 1.5 mM로 첨가되었다. 그 후 반응산물을 Bio-LC 분석법(컬럼 PA1, 컬럼 분석 온도 30℃, 이동상 H₂O 및 200mM NaCl, 유속 1 ml/min)을 이용하여 최적 금속염을 선별하였다. 그 결과는 Ni²⁺>Co²⁺>Ca²⁺>Mn²⁺>Mg²⁺=Zn²⁺>Cu²⁺=Fe²⁺ 순으로 효소 활성에 있어 차이가 있었다(도 10 참조).In order to confirm the change in enzyme activity according to the type of metal salt, the S125D/N129T/L140P/T181A/H362L mutant selected in Example 3 was purified by the protein purification method of Example 1-3, and the purified enzyme was purified with 10 mM fructose. It was put into 100 mM EPPS (pH 8.5) buffer, and the reaction was performed at a reaction temperature of 80° C. for 30 minutes. At this time, 1.5 mM of each of 8 different metal salts was added to the reaction. After that, the optimal metal salt was selected for the reaction product using Bio-LC analysis (column PA1, column analysis temperature 30° C., mobile phase H ₂ O and 200 mM NaCl, flow rate 1 ml/min). As a result, there was a difference in enzyme activity in the order of Ni ²⁺ >Co ²⁺ >Ca ²⁺ >Mn ²⁺ >Mg ²⁺ =Zn ²⁺ >Cu ²⁺ =Fe ²⁺ (see FIG. 10 ).

4-4. 타가투론산 에피머화 효소 야생형 및 변이체들의 전환 활성 비교4-4. Comparison of conversion activity of tagaturonic acid epimerase wild-type and mutants

실시예 4-1, 4-2 및 4-3에서 확립한 최적조건에서의 야생형 및 변이체들의 과당으로부터 타가토스의 전환 활성 비교를 실시하였다. 야생형과 S125D 변이체, S125D/H362W 변이체, S125D/T181D 변이체, S125D/T181A/H362L 변이체, S125D/N129T/T181A/H362L 변이체, S125D/L140K/T181A/H362L 변이체, S125D/N129T/L140P/T181A/H362L 변이체들을 실시예 1-3의 단백질 정제법으로 정제하여 정제된 효소를 각각 4, 2, 0.4, 0.4, 0.1, 0.05, 0.05, 0.025 mg/ml의 농도로 10 mM 과당, 1.5 mM NiSO₄를 100 mM EPPS(pH 8.5) 완충용액에 넣어 반응온도 80℃에서 30분간 반응을 수행하였다. 그 후 반응산물을 Bio-LC 분석법(컬럼 PA1, 컬럼 분석 온도 30℃, 이동상 H₂O 및 200mM NaCl, 유속 1 ml/min)을 이용하여 야생형 및 변이체들의 전환 활성을 비교하였다. 그 결과, 야생형으로부터 최종 선별된 변이체의 전환 비활성은 총 188배 증가한 것으로 확인되었다(도 11 참조).The conversion activity of tagatose from fructose in the wild-type and mutants under the optimal conditions established in Examples 4-1, 4-2 and 4-3 was compared. Wild type and S125D mutant, S125D/H362W mutant, S125D/T181D mutant, S125D/T181A/H362L mutant, S125D/N129T/T181A/H362L mutant, S125D/L140K/T181A/H362L mutant, S125D/L140K/T181A/H362L mutant, S125D/L140K/T181A/H362L mutant, S125D/N129T/L125D/N129T/L125D/N129T/ The purified enzymes were purified by the protein purification method of Examples 1-3, respectively, at a concentration of 4, 2, 0.4, 0.4, 0.1, 0.05, 0.05, 0.025 mg/ml, 10 mM fructose, 1.5 mM NiSO ₄ 100 mM It was put in EPPS (pH 8.5) buffer solution and the reaction was performed at a reaction temperature of 80 °C for 30 minutes. Thereafter, the conversion activity of wild-type and mutants was compared with the reaction product using Bio-LC analysis (column PA1, column analysis temperature 30° C., mobile phase H ₂ O and 200 mM NaCl, flow rate 1 ml/min). As a result, it was confirmed that the conversion specific activity of the mutant finally selected from the wild-type increased by a total of 188-fold (see FIG. 11 ).

실시예 5. 타가투론산 에피머화 효소 변이체 이용 과당으로부터 타가토스로의 생산Example 5. Production of tagatose from fructose using tagaturonic acid epimerase variant

타가투론산 에피머화 효소 변이체를 사용하여 과당으로부터 타가토스를 생산하기 위해 실시예 4에서 확인한 최적 조건으로 효소 및 과당의 최적 농도를 확립하고, 타가토스를 생산하였다.In order to produce tagatose from fructose using the tagaturonic acid epimerase variant, optimal concentrations of the enzyme and fructose were established under the optimal conditions confirmed in Example 4, and tagatose was produced.

5-1. 타가투론산 에피머화 효소 변이체의 효소 농도 변화에 따른 최적 효소 농도 조건5-1. Optimal enzyme concentration conditions according to changes in the enzyme concentration of the tagaturonic acid epimerase variant

효소 농도에 따른 타가토스 생산의 최적 효소 농도를 확인하기 위해 실시예 3에서 선별한 S125D/N129T/L140P/T181A/H362L 변이체를 실시예 1-3의 단백질 정제법으로 정제하여 최대 20 mg/ml 범위의 정제된 효소를 600 g/L 과당, 1.5 mM NiSO₄를 100 mM EPPS(pH 8.5) 완충용액에 넣어 반응온도 80℃에서 10 분 동안 반응을 수행하였다. 그 후 반응산물을 Bio-LC 분석법(컬럼 PA1, 컬럼 분석 온도 30℃, 이동상 H₂O 및 200 mM NaCl, 유속 1 ml/min을 이용하여 변이체의 최적 효소 농도를 확립하였다. 그 결과, 타가토스 생산의 최적 효소 농도는 18 mg/ml이었다(도 12 (a) 참조).In order to confirm the optimal enzyme concentration for tagatose production according to the enzyme concentration, the S125D/N129T/L140P/T181A/H362L mutant selected in Example 3 was purified by the protein purification method of Example 1-3, and the maximum range of 20 mg/ml of purified enzyme 600 g/L fructose, 1.5 mM NiSO ₄ was put in 100 mM EPPS (pH 8.5) buffer, and the reaction was performed at a reaction temperature of 80° C. for 10 minutes. The reaction product was then analyzed using Bio-LC analysis (column PA1, column analysis temperature 30° C., mobile phase H ₂ O and 200 mM NaCl, flow rate 1 ml/min to establish the optimal enzyme concentration of the mutant. As a result, tagatose The optimal enzyme concentration for production was 18 mg/ml (see Fig. 12 (a)).

5-2. 타가투론산 에피머화 효소 변이체의 과당 농도 변화에 따른 최적 과당 농도 조건5-2. Optimal fructose concentration conditions according to changes in fructose concentration of tagaturonic acid epimerase variants

과당 농도에 따른 고농도 타가토스 생산의 최적 과당 농도를 확인하기 위해 실시예 3에서 선별한 S125D/N129T/L140P/T181A/H362L 변이체를 실시예 1-3의 단백질 정제법으로 정제하여 정제된 효소를 50 g/L에서 750 g/L 범위의 과당, 1.5 mM NiSO₄를 100 mM EPPS(pH 8.5) 완충용액에 넣어 반응온도 80℃에서 10 분 동안 반응을 수행하였다. 그 후 반응산물을 Bio-LC 분석법(컬럼 PA1, 컬럼 분석 온도 30℃, 이동상 H₂O 및 200 mM NaCl, 유속 1 ml/min)을 이용하여 최적 과당 농도를 확립하였다. 그 결과, 타가토스 생산의 최적 과당 농도는 700 g/L이었다(도 12 (b) 참조).S125D/N129T/L140P/T181A/H362L mutant selected in Example 3 to confirm the optimal fructose concentration for high-concentration tagatose production according to the fructose concentration was purified by the protein purification method of Example 1-3, and the purified enzyme was 50 Fructose in the range of g/L to 750 g/L, 1.5 mM NiSO ₄ was put in 100 mM EPPS (pH 8.5) buffer, and the reaction was performed at a reaction temperature of 80° C. for 10 minutes. Thereafter, the optimal fructose concentration was established for the reaction product using Bio-LC analysis (column PA1, column analysis temperature 30° C., mobile phase H ₂ O and 200 mM NaCl, flow rate 1 ml/min). As a result, the optimal fructose concentration for tagatose production was 700 g/L (see Fig. 12 (b)).

5-3. 타가투론산 에피머화 효소 변이체를 통한 과당으로부터 타가토스의 생산5-3. Production of tagatose from fructose via tagaturonic acid epimerase variants

실시예 3에서 선별한 S125D/N129T/L140P/T181A/H362L 변이체를 실시예 1-3의 단백질 정제법으로 정제하여 앞서 확립한 최적조건으로 과당으로부터 타가토스의 고농도 생산을 진행하였다. 고농도의 타가토스 생산을 위하여 앞서 확립한 최적 조건인 700 g/L 과당, 18 mg/ml의 S125D/N129T/L140P/T181A/H362L 변이체, 1.5 mM NiSO₄를 100 mM EPPS(pH 8.5)에 넣어 반응온도 80℃에서 총 2시간 동안 반응을 수행하였다. 시간 별 타가토스의 생산 상태를 확인하기 위하여, 일정 시간 반응한 용액에 2 M 염산 용액을 반응 부피의 10%에 해당되는 부피를 첨가하여 반응 종료하고, 타가토스 생산량을 Bio-LC 분석법(컬럼 PA1, 컬럼 분석 온도 30℃, 이동상 H₂O 및 200mM NaCl, 유속 1 ml/min)을 이용하여 확인하였다. 그 결과, 700 g/L의 과당으로부터 타가토스로 약 30% 전환율을 보였으며 최종적으로 213 g/L의 타가토스를 생산하였다(도 13 참조).The S125D/N129T/L140P/T181A/H362L mutant selected in Example 3 was purified by the protein purification method of Example 1-3, and a high concentration of tagatose was produced from fructose under the optimal conditions established above. 700 g/L fructose, 18 mg/ml of S125D/N129T/L140P/T181A/H362L variant, 1.5 mM NiSO ₄ , which are the optimal conditions previously established for the production of high-concentration tagatose, reacted with 100 mM EPPS (pH 8.5) The reaction was carried out at a temperature of 80° C. for a total of 2 hours. In order to check the production status of tagatose by time, 2 M hydrochloric acid solution was added to the solution reacted for a certain time with a volume corresponding to 10% of the reaction volume to terminate the reaction, and the production of tagatose was measured by Bio-LC analysis (column PA1). , column analysis temperature 30° C., mobile phase H ₂ O and 200 mM NaCl, flow rate 1 ml/min) was used. As a result, it showed about 30% conversion from 700 g/L of fructose to tagatose, and finally 213 g/L of tagatose was produced (see FIG. 13).

이상으로 본 발명 내용의 특정부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시 양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 것은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As the specific parts of the present invention have been described in detail above, for those of ordinary skill in the art, it is clear that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. will be. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> Konkuk University Industrial Cooperation Corp <120> TAGATURONATE EPIMERASE VARIANT EMZYME AND METHOD FOR PRODUCING TAGATOSE FROM FRUXTOSE USING THE SAME <130> 2020-I147 <160> 22 <170> KoPatentIn 3.0 <210> 1 <211> 482 <212> PRT <213> Unknown <220> <223> Thermotoga petrophila <400> 1 Met Val Leu Lys Val Phe Lys Asp His Phe Gly Arg Gly Tyr Glu Val 1 5 10 15 Tyr Glu Lys Ser Tyr Arg Glu Lys Asp Ser Leu Ser Phe Phe Leu Thr 20 25 30 Lys Gly Glu Glu Gly Lys Ile Leu Val Val Ala Gly Glu Lys Ala Pro 35 40 45 Glu Gly Leu Ser Phe Phe Lys Lys Gln Arg Val Glu Gly Val Ser Phe 50 55 60 Phe Phe Cys Glu Arg Asn His Glu Asn Leu Glu Val Leu Arg Lys Tyr 65 70 75 80 Phe Pro Asp Leu Lys Pro Val Arg Ala Gly Leu Arg Ala Ser Phe Gly 85 90 95 Thr Gly Asp Arg Leu Gly Ile Thr Thr Pro Ala His Val Arg Ala Leu 100 105 110 Lys Asp Ser Gly Leu Phe Pro Ile Phe Ala Gln Gln Ser Val Arg Glu 115 120 125 Asn Glu Arg Thr Gly Arg Thr Trp Arg Asp Val Leu Asp Asp Ala Thr 130 135 140 Trp Gly Val Phe Gln Glu Gly Tyr Ser Glu Gly Phe Gly Ala Asp Ala 145 150 155 160 Asp His Val Lys Arg Pro Glu Asp Leu Val Ser Ala Ala Arg Glu Gly 165 170 175 Phe Thr Met Phe Thr Ile Asp Pro Ser Asp His Val Arg Asn Leu Ser 180 185 190 Lys Leu Thr Glu Lys Glu Arg Asn Glu Lys Phe Glu Glu Ile Leu Arg 195 200 205 Lys Glu Arg Ile Asp Arg Ile Tyr Leu Gly Lys Lys Tyr Ser Val Leu 210 215 220 Gly Glu Lys Ile Glu Phe Asp Glu Lys Asn Leu Arg Asp Ala Ala Leu 225 230 235 240 Val Tyr Tyr Asp Ala Ile Ala His Val Asp Met Met Tyr Gln Ile Leu 245 250 255 Lys Asp Glu Thr Pro Asp Phe Asp Phe Glu Val Ser Val Asp Glu Thr 260 265 270 Glu Thr Pro Thr Ser Pro Leu Phe His Ile Phe Val Val Glu Glu Leu 275 280 285 Arg Arg Arg Gly Val Glu Phe Thr Asn Leu Ala Leu Arg Phe Ile Gly 290 295 300 Glu Trp Glu Lys Gly Ile Asp Tyr Lys Gly Asp Leu Ala Gln Phe Glu 305 310 315 320 Arg Glu Ile Lys Met His Ala Glu Ile Ala Arg Met Phe Glu Gly Tyr 325 330 335 Lys Ile Ser Leu His Ser Gly Ser Asp Lys Phe Ser Val Tyr Pro Ala 340 345 350 Phe Ala Ser Ala Thr Gly Gly Leu Phe His Val Lys Thr Ala Gly Thr 355 360 365 Ser Tyr Leu Glu Ala Val Lys Val Ile Ser Met Val Asn Pro Glu Leu 370 375 380 Phe Arg Glu Ile Tyr Arg Cys Ala Leu Asp His Phe Glu Glu Asp Arg 385 390 395 400 Lys Ser Tyr His Ile Ser Ala Asp Leu Ser Lys Val Pro Glu Val Glu 405 410 415 Lys Val Lys Asp Glu Asp Leu Pro Gly Leu Phe Glu Asp Ile Asn Val 420 425 430 Arg Gln Leu Ile His Val Thr Tyr Gly Ser Val Leu Lys Asp Ala Ser 435 440 445 Leu Lys Glu Arg Leu Phe Lys Thr Leu Glu Gln Asn Glu Glu Leu Phe 450 455 460 Tyr Glu Thr Val Ala Lys His Ile Lys Arg His Val Asp Leu Leu Lys 465 470 475 480 Gly Glx <210> 2 <211> 482 <212> PRT <213> Artificial Sequence <220> <223> S125D mutant of tagaturonate epimerase <400> 2 Met Val Leu Lys Val Phe Lys Asp His Phe Gly Arg Gly Tyr Glu Val 1 5 10 15 Tyr Glu Lys Ser Tyr Arg Glu Lys Asp Ser Leu Ser Phe Phe Leu Thr 20 25 30 Lys Gly Glu Glu Gly Lys Ile Leu Val Val Ala Gly Glu Lys Ala Pro 35 40 45 Glu Gly Leu Ser Phe Phe Lys Lys Gln Arg Val Glu Gly Val Ser Phe 50 55 60 Phe Phe Cys Glu Arg Asn His Glu Asn Leu Glu Val Leu Arg Lys Tyr 65 70 75 80 Phe Pro Asp Leu Lys Pro Val Arg Ala Gly Leu Arg Ala Ser Phe Gly 85 90 95 Thr Gly Asp Arg Leu Gly Ile Thr Thr Pro Ala His Val Arg Ala Leu 100 105 110 Lys Asp Ser Gly Leu Phe Pro Ile Phe Ala Gln Gln Asp Val Arg Glu 115 120 125 Thr Glu Arg Thr Gly Arg Thr Trp Arg Asp Val Pro Asp Asp Ala Thr 130 135 140 Trp Gly Val Phe Gln Glu Gly Tyr Ser Glu Gly Phe Gly Ala Asp Ala 145 150 155 160 Asp His Val Lys Arg Pro Glu Asp Leu Val Ser Ala Ala Arg Glu Gly 165 170 175 Phe Thr Met Phe Ala Ile Asp Pro Ser Asp His Val Arg Asn Leu Ser 180 185 190 Lys Leu Thr Glu Lys Glu Arg Asn Glu Lys Phe Glu Glu Ile Leu Arg 195 200 205 Lys Glu Arg Ile Asp Arg Ile Tyr Leu Gly Lys Lys Tyr Ser Val Leu 210 215 220 Gly Glu Lys Ile Glu Phe Asp Glu Lys Asn Leu Arg Asp Ala Ala Leu 225 230 235 240 Val Tyr Tyr Asp Ala Ile Ala His Val Asp Met Met Tyr Gln Ile Leu 245 250 255 Lys Asp Glu Thr Pro Asp Phe Asp Phe Glu Val Ser Val Asp Glu Thr 260 265 270 Glu Thr Pro Thr Ser Pro Leu Phe His Ile Phe Val Val Glu Glu Leu 275 280 285 Arg Arg Arg Gly Val Glu Phe Thr Asn Leu Ala Leu Arg Phe Ile Gly 290 295 300 Glu Trp Glu Lys Gly Ile Asp Tyr Lys Gly Asp Leu Ala Gln Phe Glu 305 310 315 320 Arg Glu Ile Lys Met His Ala Glu Ile Ala Arg Met Phe Glu Gly Tyr 325 330 335 Lys Ile Ser Leu His Ser Gly Ser Asp Lys Phe Ser Val Tyr Pro Ala 340 345 350 Phe Ala Ser Ala Thr Gly Gly Leu Phe Leu Val Lys Thr Ala Gly Thr 355 360 365 Ser Tyr Leu Glu Ala Val Lys Val Ile Ser Met Val Asn Pro Glu Leu 370 375 380 Phe Arg Glu Ile Tyr Arg Cys Ala Leu Asp His Phe Glu Glu Asp Arg 385 390 395 400 Lys Ser Tyr His Ile Ser Ala Asp Leu Ser Lys Val Pro Glu Val Glu 405 410 415 Lys Val Lys Asp Glu Asp Leu Pro Gly Leu Phe Glu Asp Ile Asn Val 420 425 430 Arg Gln Leu Ile His Val Thr Tyr Gly Ser Val Leu Lys Asp Ala Ser 435 440 445 Leu Lys Glu Arg Leu Phe Lys Thr Leu Glu Gln Asn Glu Glu Leu Phe 450 455 460 Tyr Glu Thr Val Ala Lys His Ile Lys Arg His Val Asp Leu Leu Lys 465 470 475 480 Gly Glx <210> 3 <211> 482 <212> PRT <213> Artificial Sequence <220> <223> S125D/T181A/H362L mutant of tagaturonate epimerase <400> 3 Met Val Leu Lys Val Phe Lys Asp His Phe Gly Arg Gly Tyr Glu Val 1 5 10 15 Tyr Glu Lys Ser Tyr Arg Glu Lys Asp Ser Leu Ser Phe Phe Leu Thr 20 25 30 Lys Gly Glu Glu Gly Lys Ile Leu Val Val Ala Gly Glu Lys Ala Pro 35 40 45 Glu Gly Leu Ser Phe Phe Lys Lys Gln Arg Val Glu Gly Val Ser Phe 50 55 60 Phe Phe Cys Glu Arg Asn His Glu Asn Leu Glu Val Leu Arg Lys Tyr 65 70 75 80 Phe Pro Asp Leu Lys Pro Val Arg Ala Gly Leu Arg Ala Ser Phe Gly 85 90 95 Thr Gly Asp Arg Leu Gly Ile Thr Thr Pro Ala His Val Arg Ala Leu 100 105 110 Lys Asp Ser Gly Leu Phe Pro Ile Phe Ala Gln Gln Asp Val Arg Glu 115 120 125 Asn Glu Arg Thr Gly Arg Thr Trp Arg Asp Val Leu Asp Asp Ala Thr 130 135 140 Trp Gly Val Phe Gln Glu Gly Tyr Ser Glu Gly Phe Gly Ala Asp Ala 145 150 155 160 Asp His Val Lys Arg Pro Glu Asp Leu Val Ser Ala Ala Arg Glu Gly 165 170 175 Phe Thr Met Phe Ala Ile Asp Pro Ser Asp His Val Arg Asn Leu Ser 180 185 190 Lys Leu Thr Glu Lys Glu Arg Asn Glu Lys Phe Glu Glu Ile Leu Arg 195 200 205 Lys Glu Arg Ile Asp Arg Ile Tyr Leu Gly Lys Lys Tyr Ser Val Leu 210 215 220 Gly Glu Lys Ile Glu Phe Asp Glu Lys Asn Leu Arg Asp Ala Ala Leu 225 230 235 240 Val Tyr Tyr Asp Ala Ile Ala His Val Asp Met Met Tyr Gln Ile Leu 245 250 255 Lys Asp Glu Thr Pro Asp Phe Asp Phe Glu Val Ser Val Asp Glu Thr 260 265 270 Glu Thr Pro Thr Ser Pro Leu Phe His Ile Phe Val Val Glu Glu Leu 275 280 285 Arg Arg Arg Gly Val Glu Phe Thr Asn Leu Ala Leu Arg Phe Ile Gly 290 295 300 Glu Trp Glu Lys Gly Ile Asp Tyr Lys Gly Asp Leu Ala Gln Phe Glu 305 310 315 320 Arg Glu Ile Lys Met His Ala Glu Ile Ala Arg Met Phe Glu Gly Tyr 325 330 335 Lys Ile Ser Leu His Ser Gly Ser Asp Lys Phe Ser Val Tyr Pro Ala 340 345 350 Phe Ala Ser Ala Thr Gly Gly Leu Phe Leu Val Lys Thr Ala Gly Thr 355 360 365 Ser Tyr Leu Glu Ala Val Lys Val Ile Ser Met Val Asn Pro Glu Leu 370 375 380 Phe Arg Glu Ile Tyr Arg Cys Ala Leu Asp His Phe Glu Glu Asp Arg 385 390 395 400 Lys Ser Tyr His Ile Ser Ala Asp Leu Ser Lys Val Pro Glu Val Glu 405 410 415 Lys Val Lys Asp Glu Asp Leu Pro Gly Leu Phe Glu Asp Ile Asn Val 420 425 430 Arg Gln Leu Ile His Val Thr Tyr Gly Ser Val Leu Lys Asp Ala Ser 435 440 445 Leu Lys Glu Arg Leu Phe Lys Thr Leu Glu Gln Asn Glu Glu Leu Phe 450 455 460 Tyr Glu Thr Val Ala Lys His Ile Lys Arg His Val Asp Leu Leu Lys 465 470 475 480 Gly Glx <210> 4 <211> 482 <212> PRT <213> Artificial Sequence <220> <223> F66S/S125D/T181A/H362L mutant of tagaturonate epimerase <400> 4 Met Val Leu Lys Val Phe Lys Asp His Phe Gly Arg Gly Tyr Glu Val 1 5 10 15 Tyr Glu Lys Ser Tyr Arg Glu Lys Asp Ser Leu Ser Phe Phe Leu Thr 20 25 30 Lys Gly Glu Glu Gly Lys Ile Leu Val Val Ala Gly Glu Lys Ala Pro 35 40 45 Glu Gly Leu Ser Phe Phe Lys Lys Gln Arg Val Glu Gly Val Ser Phe 50 55 60 Phe Ser Cys Glu Arg Asn His Glu Asn Leu Glu Val Leu Arg Lys Tyr 65 70 75 80 Phe Pro Asp Leu Lys Pro Val Arg Ala Gly Leu Arg Ala Ser Phe Gly 85 90 95 Thr Gly Asp Arg Leu Gly Ile Thr Thr Pro Ala His Val Arg Ala Leu 100 105 110 Lys Asp Ser Gly Leu Phe Pro Ile Phe Ala Gln Gln Asp Val Arg Glu 115 120 125 Asn Glu Arg Thr Gly Arg Thr Trp Arg Asp Val Leu Asp Asp Ala Thr 130 135 140 Trp Gly Val Phe Gln Glu Gly Tyr Ser Glu Gly Phe Gly Ala Asp Ala 145 150 155 160 Asp His Val Lys Arg Pro Glu Asp Leu Val Ser Ala Ala Arg Glu Gly 165 170 175 Phe Thr Met Phe Ala Ile Asp Pro Ser Asp His Val Arg Asn Leu Ser 180 185 190 Lys Leu Thr Glu Lys Glu Arg Asn Glu Lys Phe Glu Glu Ile Leu Arg 195 200 205 Lys Glu Arg Ile Asp Arg Ile Tyr Leu Gly Lys Lys Tyr Ser Val Leu 210 215 220 Gly Glu Lys Ile Glu Phe Asp Glu Lys Asn Leu Arg Asp Ala Ala Leu 225 230 235 240 Val Tyr Tyr Asp Ala Ile Ala His Val Asp Met Met Tyr Gln Ile Leu 245 250 255 Lys Asp Glu Thr Pro Asp Phe Asp Phe Glu Val Ser Val Asp Glu Thr 260 265 270 Glu Thr Pro Thr Ser Pro Leu Phe His Ile Phe Val Val Glu Glu Leu 275 280 285 Arg Arg Arg Gly Val Glu Phe Thr Asn Leu Ala Leu Arg Phe Ile Gly 290 295 300 Glu Trp Glu Lys Gly Ile Asp Tyr Lys Gly Asp Leu Ala Gln Phe Glu 305 310 315 320 Arg Glu Ile Lys Met His Ala Glu Ile Ala Arg Met Phe Glu Gly Tyr 325 330 335 Lys Ile Ser Leu His Ser Gly Ser Asp Lys Phe Ser Val Tyr Pro Ala 340 345 350 Phe Ala Ser Ala Thr Gly Gly Leu Phe Leu Val Lys Thr Ala Gly Thr 355 360 365 Ser Tyr Leu Glu Ala Val Lys Val Ile Ser Met Val Asn Pro Glu Leu 370 375 380 Phe Arg Glu Ile Tyr Arg Cys Ala Leu Asp His Phe Glu Glu Asp Arg 385 390 395 400 Lys Ser Tyr His Ile Ser Ala Asp Leu Ser Lys Val Pro Glu Val Glu 405 410 415 Lys Val Lys Asp Glu Asp Leu Pro Gly Leu Phe Glu Asp Ile Asn Val 420 425 430 Arg Gln Leu Ile His Val Thr Tyr Gly Ser Val Leu Lys Asp Ala Ser 435 440 445 Leu Lys Glu Arg Leu Phe Lys Thr Leu Glu Gln Asn Glu Glu Leu Phe 450 455 460 Tyr Glu Thr Val Ala Lys His Ile Lys Arg His Val Asp Leu Leu Lys 465 470 475 480 Gly Glx <210> 5 <211> 482 <212> PRT <213> Artificial Sequence <220> <223> E72G/S125D/T181A/H362L mutant of tagaturonate epimerase <400> 5 Met Val Leu Lys Val Phe Lys Asp His Phe Gly Arg Gly Tyr Glu Val 1 5 10 15 Tyr Glu Lys Ser Tyr Arg Glu Lys Asp Ser Leu Ser Phe Phe Leu Thr 20 25 30 Lys Gly Glu Glu Gly Lys Ile Leu Val Val Ala Gly Glu Lys Ala Pro 35 40 45 Glu Gly Leu Ser Phe Phe Lys Lys Gln Arg Val Glu Gly Val Ser Phe 50 55 60 Phe Phe Cys Glu Arg Asn His Gly Asn Leu Glu Val Leu Arg Lys Tyr 65 70 75 80 Phe Pro Asp Leu Lys Pro Val Arg Ala Gly Leu Arg Ala Ser Phe Gly 85 90 95 Thr Gly Asp Arg Leu Gly Ile Thr Thr Pro Ala His Val Arg Ala Leu 100 105 110 Lys Asp Ser Gly Leu Phe Pro Ile Phe Ala Gln Gln Asp Val Arg Glu 115 120 125 Asn Glu Arg Thr Gly Arg Thr Trp Arg Asp Val Leu Asp Asp Ala Thr 130 135 140 Trp Gly Val Phe Gln Glu Gly Tyr Ser Glu Gly Phe Gly Ala Asp Ala 145 150 155 160 Asp His Val Lys Arg Pro Glu Asp Leu Val Ser Ala Ala Arg Glu Gly 165 170 175 Phe Thr Met Phe Ala Ile Asp Pro Ser Asp His Val Arg Asn Leu Ser 180 185 190 Lys Leu Thr Glu Lys Glu Arg Asn Glu Lys Phe Glu Glu Ile Leu Arg 195 200 205 Lys Glu Arg Ile Asp Arg Ile Tyr Leu Gly Lys Lys Tyr Ser Val Leu 210 215 220 Gly Glu Lys Ile Glu Phe Asp Glu Lys Asn Leu Arg Asp Ala Ala Leu 225 230 235 240 Val Tyr Tyr Asp Ala Ile Ala His Val Asp Met Met Tyr Gln Ile Leu 245 250 255 Lys Asp Glu Thr Pro Asp Phe Asp Phe Glu Val Ser Val Asp Glu Thr 260 265 270 Glu Thr Pro Thr Ser Pro Leu Phe His Ile Phe Val Val Glu Glu Leu 275 280 285 Arg Arg Arg Gly Val Glu Phe Thr Asn Leu Ala Leu Arg Phe Ile Gly 290 295 300 Glu Trp Glu Lys Gly Ile Asp Tyr Lys Gly Asp Leu Ala Gln Phe Glu 305 310 315 320 Arg Glu Ile Lys Met His Ala Glu Ile Ala Arg Met Phe Glu Gly Tyr 325 330 335 Lys Ile Ser Leu His Ser Gly Ser Asp Lys Phe Ser Val Tyr Pro Ala 340 345 350 Phe Ala Ser Ala Thr Gly Gly Leu Phe Leu Val Lys Thr Ala Gly Thr 355 360 365 Ser Tyr Leu Glu Ala Val Lys Val Ile Ser Met Val Asn Pro Glu Leu 370 375 380 Phe Arg Glu Ile Tyr Arg Cys Ala Leu Asp His Phe Glu Glu Asp Arg 385 390 395 400 Lys Ser Tyr His Ile Ser Ala Asp Leu Ser Lys Val Pro Glu Val Glu 405 410 415 Lys Val Lys Asp Glu Asp Leu Pro Gly Leu Phe Glu Asp Ile Asn Val 420 425 430 Arg Gln Leu Ile His Val Thr Tyr Gly Ser Val Leu Lys Asp Ala Ser 435 440 445 Leu Lys Glu Arg Leu Phe Lys Thr Leu Glu Gln Asn Glu Glu Leu Phe 450 455 460 Tyr Glu Thr Val Ala Lys His Ile Lys Arg His Val Asp Leu Leu Lys 465 470 475 480 Gly Glx <210> 6 <211> 482 <212> PRT <213> Artificial Sequence <220> <223> S125D/N129F/T181A/H362L mutant of tagaturonate epimerase <400> 6 Met Val Leu Lys Val Phe Lys Asp His Phe Gly Arg Gly Tyr Glu Val 1 5 10 15 Tyr Glu Lys Ser Tyr Arg Glu Lys Asp Ser Leu Ser Phe Phe Leu Thr 20 25 30 Lys Gly Glu Glu Gly Lys Ile Leu Val Val Ala Gly Glu Lys Ala Pro 35 40 45 Glu Gly Leu Ser Phe Phe Lys Lys Gln Arg Val Glu Gly Val Ser Phe 50 55 60 Phe Phe Cys Glu Arg Asn His Glu Asn Leu Glu Val Leu Arg Lys Tyr 65 70 75 80 Phe Pro Asp Leu Lys Pro Val Arg Ala Gly Leu Arg Ala Ser Phe Gly 85 90 95 Thr Gly Asp Arg Leu Gly Ile Thr Thr Pro Ala His Val Arg Ala Leu 100 105 110 Lys Asp Ser Gly Leu Phe Pro Ile Phe Ala Gln Gln Asp Val Arg Glu 115 120 125 Phe Glu Arg Thr Gly Arg Thr Trp Arg Asp Val Leu Asp Asp Ala Thr 130 135 140 Trp Gly Val Phe Gln Glu Gly Tyr Ser Glu Gly Phe Gly Ala Asp Ala 145 150 155 160 Asp His Val Lys Arg Pro Glu Asp Leu Val Ser Ala Ala Arg Glu Gly 165 170 175 Phe Thr Met Phe Ala Ile Asp Pro Ser Asp His Val Arg Asn Leu Ser 180 185 190 Lys Leu Thr Glu Lys Glu Arg Asn Glu Lys Phe Glu Glu Ile Leu Arg 195 200 205 Lys Glu Arg Ile Asp Arg Ile Tyr Leu Gly Lys Lys Tyr Ser Val Leu 210 215 220 Gly Glu Lys Ile Glu Phe Asp Glu Lys Asn Leu Arg Asp Ala Ala Leu 225 230 235 240 Val Tyr Tyr Asp Ala Ile Ala His Val Asp Met Met Tyr Gln Ile Leu 245 250 255 Lys Asp Glu Thr Pro Asp Phe Asp Phe Glu Val Ser Val Asp Glu Thr 260 265 270 Glu Thr Pro Thr Ser Pro Leu Phe His Ile Phe Val Val Glu Glu Leu 275 280 285 Arg Arg Arg Gly Val Glu Phe Thr Asn Leu Ala Leu Arg Phe Ile Gly 290 295 300 Glu Trp Glu Lys Gly Ile Asp Tyr Lys Gly Asp Leu Ala Gln Phe Glu 305 310 315 320 Arg Glu Ile Lys Met His Ala Glu Ile Ala Arg Met Phe Glu Gly Tyr 325 330 335 Lys Ile Ser Leu His Ser Gly Ser Asp Lys Phe Ser Val Tyr Pro Ala 340 345 350 Phe Ala Ser Ala Thr Gly Gly Leu Phe Leu Val Lys Thr Ala Gly Thr 355 360 365 Ser Tyr Leu Glu Ala Val Lys Val Ile Ser Met Val Asn Pro Glu Leu 370 375 380 Phe Arg Glu Ile Tyr Arg Cys Ala Leu Asp His Phe Glu Glu Asp Arg 385 390 395 400 Lys Ser Tyr His Ile Ser Ala Asp Leu Ser Lys Val Pro Glu Val Glu 405 410 415 Lys Val Lys Asp Glu Asp Leu Pro Gly Leu Phe Glu Asp Ile Asn Val 420 425 430 Arg Gln Leu Ile His Val Thr Tyr Gly Ser Val Leu Lys Asp Ala Ser 435 440 445 Leu Lys Glu Arg Leu Phe Lys Thr Leu Glu Gln Asn Glu Glu Leu Phe 450 455 460 Tyr Glu Thr Val Ala Lys His Ile Lys Arg His Val Asp Leu Leu Lys 465 470 475 480 Gly Glx <210> 7 <211> 482 <212> PRT <213> Artificial Sequence <220> <223> S125D/L140P/T181A/H362L mutant of tagaturonate epimerase <400> 7 Met Val Leu Lys Val Phe Lys Asp His Phe Gly Arg Gly Tyr Glu Val 1 5 10 15 Tyr Glu Lys Ser Tyr Arg Glu Lys Asp Ser Leu Ser Phe Phe Leu Thr 20 25 30 Lys Gly Glu Glu Gly Lys Ile Leu Val Val Ala Gly Glu Lys Ala Pro 35 40 45 Glu Gly Leu Ser Phe Phe Lys Lys Gln Arg Val Glu Gly Val Ser Phe 50 55 60 Phe Phe Cys Glu Arg Asn His Glu Asn Leu Glu Val Leu Arg Lys Tyr 65 70 75 80 Phe Pro Asp Leu Lys Pro Val Arg Ala Gly Leu Arg Ala Ser Phe Gly 85 90 95 Thr Gly Asp Arg Leu Gly Ile Thr Thr Pro Ala His Val Arg Ala Leu 100 105 110 Lys Asp Ser Gly Leu Phe Pro Ile Phe Ala Gln Gln Asp Val Arg Glu 115 120 125 Asn Glu Arg Thr Gly Arg Thr Trp Arg Asp Val Pro Asp Asp Ala Thr 130 135 140 Trp Gly Val Phe Gln Glu Gly Tyr Ser Glu Gly Phe Gly Ala Asp Ala 145 150 155 160 Asp His Val Lys Arg Pro Glu Asp Leu Val Ser Ala Ala Arg Glu Gly 165 170 175 Phe Thr Met Phe Ala Ile Asp Pro Ser Asp His Val Arg Asn Leu Ser 180 185 190 Lys Leu Thr Glu Lys Glu Arg Asn Glu Lys Phe Glu Glu Ile Leu Arg 195 200 205 Lys Glu Arg Ile Asp Arg Ile Tyr Leu Gly Lys Lys Tyr Ser Val Leu 210 215 220 Gly Glu Lys Ile Glu Phe Asp Glu Lys Asn Leu Arg Asp Ala Ala Leu 225 230 235 240 Val Tyr Tyr Asp Ala Ile Ala His Val Asp Met Met Tyr Gln Ile Leu 245 250 255 Lys Asp Glu Thr Pro Asp Phe Asp Phe Glu Val Ser Val Asp Glu Thr 260 265 270 Glu Thr Pro Thr Ser Pro Leu Phe His Ile Phe Val Val Glu Glu Leu 275 280 285 Arg Arg Arg Gly Val Glu Phe Thr Asn Leu Ala Leu Arg Phe Ile Gly 290 295 300 Glu Trp Glu Lys Gly Ile Asp Tyr Lys Gly Asp Leu Ala Gln Phe Glu 305 310 315 320 Arg Glu Ile Lys Met His Ala Glu Ile Ala Arg Met Phe Glu Gly Tyr 325 330 335 Lys Ile Ser Leu His Ser Gly Ser Asp Lys Phe Ser Val Tyr Pro Ala 340 345 350 Phe Ala Ser Ala Thr Gly Gly Leu Phe Leu Val Lys Thr Ala Gly Thr 355 360 365 Ser Tyr Leu Glu Ala Val Lys Val Ile Ser Met Val Asn Pro Glu Leu 370 375 380 Phe Arg Glu Ile Tyr Arg Cys Ala Leu Asp His Phe Glu Glu Asp Arg 385 390 395 400 Lys Ser Tyr His Ile Ser Ala Asp Leu Ser Lys Val Pro Glu Val Glu 405 410 415 Lys Val Lys Asp Glu Asp Leu Pro Gly Leu Phe Glu Asp Ile Asn Val 420 425 430 Arg Gln Leu Ile His Val Thr Tyr Gly Ser Val Leu Lys Asp Ala Ser 435 440 445 Leu Lys Glu Arg Leu Phe Lys Thr Leu Glu Gln Asn Glu Glu Leu Phe 450 455 460 Tyr Glu Thr Val Ala Lys His Ile Lys Arg His Val Asp Leu Leu Lys 465 470 475 480 Gly Glx <210> 8 <211> 482 <212> PRT <213> Artificial Sequence <220> <223> S125D/N129S/L140F/T181A/H362L mutant of tagaturonate epimerase <400> 8 Met Val Leu Lys Val Phe Lys Asp His Phe Gly Arg Gly Tyr Glu Val 1 5 10 15 Tyr Glu Lys Ser Tyr Arg Glu Lys Asp Ser Leu Ser Phe Phe Leu Thr 20 25 30 Lys Gly Glu Glu Gly Lys Ile Leu Val Val Ala Gly Glu Lys Ala Pro 35 40 45 Glu Gly Leu Ser Phe Phe Lys Lys Gln Arg Val Glu Gly Val Ser Phe 50 55 60 Phe Phe Cys Glu Arg Asn His Glu Asn Leu Glu Val Leu Arg Lys Tyr 65 70 75 80 Phe Pro Asp Leu Lys Pro Val Arg Ala Gly Leu Arg Ala Ser Phe Gly 85 90 95 Thr Gly Asp Arg Leu Gly Ile Thr Thr Pro Ala His Val Arg Ala Leu 100 105 110 Lys Asp Ser Gly Leu Phe Pro Ile Phe Ala Gln Gln Asp Val Arg Glu 115 120 125 Ser Glu Arg Thr Gly Arg Thr Trp Arg Asp Val Phe Asp Asp Ala Thr 130 135 140 Trp Gly Val Phe Gln Glu Gly Tyr Ser Glu Gly Phe Gly Ala Asp Ala 145 150 155 160 Asp His Val Lys Arg Pro Glu Asp Leu Val Ser Ala Ala Arg Glu Gly 165 170 175 Phe Thr Met Phe Ala Ile Asp Pro Ser Asp His Val Arg Asn Leu Ser 180 185 190 Lys Leu Thr Glu Lys Glu Arg Asn Glu Lys Phe Glu Glu Ile Leu Arg 195 200 205 Lys Glu Arg Ile Asp Arg Ile Tyr Leu Gly Lys Lys Tyr Ser Val Leu 210 215 220 Gly Glu Lys Ile Glu Phe Asp Glu Lys Asn Leu Arg Asp Ala Ala Leu 225 230 235 240 Val Tyr Tyr Asp Ala Ile Ala His Val Asp Met Met Tyr Gln Ile Leu 245 250 255 Lys Asp Glu Thr Pro Asp Phe Asp Phe Glu Val Ser Val Asp Glu Thr 260 265 270 Glu Thr Pro Thr Ser Pro Leu Phe His Ile Phe Val Val Glu Glu Leu 275 280 285 Arg Arg Arg Gly Val Glu Phe Thr Asn Leu Ala Leu Arg Phe Ile Gly 290 295 300 Glu Trp Glu Lys Gly Ile Asp Tyr Lys Gly Asp Leu Ala Gln Phe Glu 305 310 315 320 Arg Glu Ile Lys Met His Ala Glu Ile Ala Arg Met Phe Glu Gly Tyr 325 330 335 Lys Ile Ser Leu His Ser Gly Ser Asp Lys Phe Ser Val Tyr Pro Ala 340 345 350 Phe Ala Ser Ala Thr Gly Gly Leu Phe Leu Val Lys Thr Ala Gly Thr 355 360 365 Ser Tyr Leu Glu Ala Val Lys Val Ile Ser Met Val Asn Pro Glu Leu 370 375 380 Phe Arg Glu Ile Tyr Arg Cys Ala Leu Asp His Phe Glu Glu Asp Arg 385 390 395 400 Lys Ser Tyr His Ile Ser Ala Asp Leu Ser Lys Val Pro Glu Val Glu 405 410 415 Lys Val Lys Asp Glu Asp Leu Pro Gly Leu Phe Glu Asp Ile Asn Val 420 425 430 Arg Gln Leu Ile His Val Thr Tyr Gly Ser Val Leu Lys Asp Ala Ser 435 440 445 Leu Lys Glu Arg Leu Phe Lys Thr Leu Glu Gln Asn Glu Glu Leu Phe 450 455 460 Tyr Glu Thr Val Ala Lys His Ile Lys Arg His Val Asp Leu Leu Lys 465 470 475 480 Gly Glx <210> 9 <211> 482 <212> PRT <213> Artificial Sequence <220> <223> S125D/N129T/L140K/T181A/H362L mutant of tagaturonate epimerase <400> 9 Met Val Leu Lys Val Phe Lys Asp His Phe Gly Arg Gly Tyr Glu Val 1 5 10 15 Tyr Glu Lys Ser Tyr Arg Glu Lys Asp Ser Leu Ser Phe Phe Leu Thr 20 25 30 Lys Gly Glu Glu Gly Lys Ile Leu Val Val Ala Gly Glu Lys Ala Pro 35 40 45 Glu Gly Leu Ser Phe Phe Lys Lys Gln Arg Val Glu Gly Val Ser Phe 50 55 60 Phe Phe Cys Glu Arg Asn His Glu Asn Leu Glu Val Leu Arg Lys Tyr 65 70 75 80 Phe Pro Asp Leu Lys Pro Val Arg Ala Gly Leu Arg Ala Ser Phe Gly 85 90 95 Thr Gly Asp Arg Leu Gly Ile Thr Thr Pro Ala His Val Arg Ala Leu 100 105 110 Lys Asp Ser Gly Leu Phe Pro Ile Phe Ala Gln Gln Asp Val Arg Glu 115 120 125 Thr Glu Arg Thr Gly Arg Thr Trp Arg Asp Val Lys Asp Asp Ala Thr 130 135 140 Trp Gly Val Phe Gln Glu Gly Tyr Ser Glu Gly Phe Gly Ala Asp Ala 145 150 155 160 Asp His Val Lys Arg Pro Glu Asp Leu Val Ser Ala Ala Arg Glu Gly 165 170 175 Phe Thr Met Phe Ala Ile Asp Pro Ser Asp His Val Arg Asn Leu Ser 180 185 190 Lys Leu Thr Glu Lys Glu Arg Asn Glu Lys Phe Glu Glu Ile Leu Arg 195 200 205 Lys Glu Arg Ile Asp Arg Ile Tyr Leu Gly Lys Lys Tyr Ser Val Leu 210 215 220 Gly Glu Lys Ile Glu Phe Asp Glu Lys Asn Leu Arg Asp Ala Ala Leu 225 230 235 240 Val Tyr Tyr Asp Ala Ile Ala His Val Asp Met Met Tyr Gln Ile Leu 245 250 255 Lys Asp Glu Thr Pro Asp Phe Asp Phe Glu Val Ser Val Asp Glu Thr 260 265 270 Glu Thr Pro Thr Ser Pro Leu Phe His Ile Phe Val Val Glu Glu Leu 275 280 285 Arg Arg Arg Gly Val Glu Phe Thr Asn Leu Ala Leu Arg Phe Ile Gly 290 295 300 Glu Trp Glu Lys Gly Ile Asp Tyr Lys Gly Asp Leu Ala Gln Phe Glu 305 310 315 320 Arg Glu Ile Lys Met His Ala Glu Ile Ala Arg Met Phe Glu Gly Tyr 325 330 335 Lys Ile Ser Leu His Ser Gly Ser Asp Lys Phe Ser Val Tyr Pro Ala 340 345 350 Phe Ala Ser Ala Thr Gly Gly Leu Phe Leu Val Lys Thr Ala Gly Thr 355 360 365 Ser Tyr Leu Glu Ala Val Lys Val Ile Ser Met Val Asn Pro Glu Leu 370 375 380 Phe Arg Glu Ile Tyr Arg Cys Ala Leu Asp His Phe Glu Glu Asp Arg 385 390 395 400 Lys Ser Tyr His Ile Ser Ala Asp Leu Ser Lys Val Pro Glu Val Glu 405 410 415 Lys Val Lys Asp Glu Asp Leu Pro Gly Leu Phe Glu Asp Ile Asn Val 420 425 430 Arg Gln Leu Ile His Val Thr Tyr Gly Ser Val Leu Lys Asp Ala Ser 435 440 445 Leu Lys Glu Arg Leu Phe Lys Thr Leu Glu Gln Asn Glu Glu Leu Phe 450 455 460 Tyr Glu Thr Val Ala Lys His Ile Lys Arg His Val Asp Leu Leu Lys 465 470 475 480 Gly Glx <210> 10 <211> 482 <212> PRT <213> Artificial Sequence <220> <223> S125D/N129T/L140F/T181A/H362L mutant of tagaturonate epimerase <400> 10 Met Val Leu Lys Val Phe Lys Asp His Phe Gly Arg Gly Tyr Glu Val 1 5 10 15 Tyr Glu Lys Ser Tyr Arg Glu Lys Asp Ser Leu Ser Phe Phe Leu Thr 20 25 30 Lys Gly Glu Glu Gly Lys Ile Leu Val Val Ala Gly Glu Lys Ala Pro 35 40 45 Glu Gly Leu Ser Phe Phe Lys Lys Gln Arg Val Glu Gly Val Ser Phe 50 55 60 Phe Phe Cys Glu Arg Asn His Glu Asn Leu Glu Val Leu Arg Lys Tyr 65 70 75 80 Phe Pro Asp Leu Lys Pro Val Arg Ala Gly Leu Arg Ala Ser Phe Gly 85 90 95 Thr Gly Asp Arg Leu Gly Ile Thr Thr Pro Ala His Val Arg Ala Leu 100 105 110 Lys Asp Ser Gly Leu Phe Pro Ile Phe Ala Gln Gln Asp Val Arg Glu 115 120 125 Thr Glu Arg Thr Gly Arg Thr Trp Arg Asp Val Phe Asp Asp Ala Thr 130 135 140 Trp Gly Val Phe Gln Glu Gly Tyr Ser Glu Gly Phe Gly Ala Asp Ala 145 150 155 160 Asp His Val Lys Arg Pro Glu Asp Leu Val Ser Ala Ala Arg Glu Gly 165 170 175 Phe Thr Met Phe Ala Ile Asp Pro Ser Asp His Val Arg Asn Leu Ser 180 185 190 Lys Leu Thr Glu Lys Glu Arg Asn Glu Lys Phe Glu Glu Ile Leu Arg 195 200 205 Lys Glu Arg Ile Asp Arg Ile Tyr Leu Gly Lys Lys Tyr Ser Val Leu 210 215 220 Gly Glu Lys Ile Glu Phe Asp Glu Lys Asn Leu Arg Asp Ala Ala Leu 225 230 235 240 Val Tyr Tyr Asp Ala Ile Ala His Val Asp Met Met Tyr Gln Ile Leu 245 250 255 Lys Asp Glu Thr Pro Asp Phe Asp Phe Glu Val Ser Val Asp Glu Thr 260 265 270 Glu Thr Pro Thr Ser Pro Leu Phe His Ile Phe Val Val Glu Glu Leu 275 280 285 Arg Arg Arg Gly Val Glu Phe Thr Asn Leu Ala Leu Arg Phe Ile Gly 290 295 300 Glu Trp Glu Lys Gly Ile Asp Tyr Lys Gly Asp Leu Ala Gln Phe Glu 305 310 315 320 Arg Glu Ile Lys Met His Ala Glu Ile Ala Arg Met Phe Glu Gly Tyr 325 330 335 Lys Ile Ser Leu His Ser Gly Ser Asp Lys Phe Ser Val Tyr Pro Ala 340 345 350 Phe Ala Ser Ala Thr Gly Gly Leu Phe Leu Val Lys Thr Ala Gly Thr 355 360 365 Ser Tyr Leu Glu Ala Val Lys Val Ile Ser Met Val Asn Pro Glu Leu 370 375 380 Phe Arg Glu Ile Tyr Arg Cys Ala Leu Asp His Phe Glu Glu Asp Arg 385 390 395 400 Lys Ser Tyr His Ile Ser Ala Asp Leu Ser Lys Val Pro Glu Val Glu 405 410 415 Lys Val Lys Asp Glu Asp Leu Pro Gly Leu Phe Glu Asp Ile Asn Val 420 425 430 Arg Gln Leu Ile His Val Thr Tyr Gly Ser Val Leu Lys Asp Ala Ser 435 440 445 Leu Lys Glu Arg Leu Phe Lys Thr Leu Glu Gln Asn Glu Glu Leu Phe 450 455 460 Tyr Glu Thr Val Ala Lys His Ile Lys Arg His Val Asp Leu Leu Lys 465 470 475 480 Gly Glx <210> 11 <211> 482 <212> PRT <213> Artificial Sequence <220> <223> S125D/N129T/L140P/T181A/H362L mutant of tagaturonate epimerase <400> 11 Met Val Leu Lys Val Phe Lys Asp His Phe Gly Arg Gly Tyr Glu Val 1 5 10 15 Tyr Glu Lys Ser Tyr Arg Glu Lys Asp Ser Leu Ser Phe Phe Leu Thr 20 25 30 Lys Gly Glu Glu Gly Lys Ile Leu Val Val Ala Gly Glu Lys Ala Pro 35 40 45 Glu Gly Leu Ser Phe Phe Lys Lys Gln Arg Val Glu Gly Val Ser Phe 50 55 60 Phe Phe Cys Glu Arg Asn His Glu Asn Leu Glu Val Leu Arg Lys Tyr 65 70 75 80 Phe Pro Asp Leu Lys Pro Val Arg Ala Gly Leu Arg Ala Ser Phe Gly 85 90 95 Thr Gly Asp Arg Leu Gly Ile Thr Thr Pro Ala His Val Arg Ala Leu 100 105 110 Lys Asp Ser Gly Leu Phe Pro Ile Phe Ala Gln Gln Asp Val Arg Glu 115 120 125 Thr Glu Arg Thr Gly Arg Thr Trp Arg Asp Val Pro Asp Asp Ala Thr 130 135 140 Trp Gly Val Phe Gln Glu Gly Tyr Ser Glu Gly Phe Gly Ala Asp Ala 145 150 155 160 Asp His Val Lys Arg Pro Glu Asp Leu Val Ser Ala Ala Arg Glu Gly 165 170 175 Phe Thr Met Phe Ala Ile Asp Pro Ser Asp His Val Arg Asn Leu Ser 180 185 190 Lys Leu Thr Glu Lys Glu Arg Asn Glu Lys Phe Glu Glu Ile Leu Arg 195 200 205 Lys Glu Arg Ile Asp Arg Ile Tyr Leu Gly Lys Lys Tyr Ser Val Leu 210 215 220 Gly Glu Lys Ile Glu Phe Asp Glu Lys Asn Leu Arg Asp Ala Ala Leu 225 230 235 240 Val Tyr Tyr Asp Ala Ile Ala His Val Asp Met Met Tyr Gln Ile Leu 245 250 255 Lys Asp Glu Thr Pro Asp Phe Asp Phe Glu Val Ser Val Asp Glu Thr 260 265 270 Glu Thr Pro Thr Ser Pro Leu Phe His Ile Phe Val Val Glu Glu Leu 275 280 285 Arg Arg Arg Gly Val Glu Phe Thr Asn Leu Ala Leu Arg Phe Ile Gly 290 295 300 Glu Trp Glu Lys Gly Ile Asp Tyr Lys Gly Asp Leu Ala Gln Phe Glu 305 310 315 320 Arg Glu Ile Lys Met His Ala Glu Ile Ala Arg Met Phe Glu Gly Tyr 325 330 335 Lys Ile Ser Leu His Ser Gly Ser Asp Lys Phe Ser Val Tyr Pro Ala 340 345 350 Phe Ala Ser Ala Thr Gly Gly Leu Phe Leu Val Lys Thr Ala Gly Thr 355 360 365 Ser Tyr Leu Glu Ala Val Lys Val Ile Ser Met Val Asn Pro Glu Leu 370 375 380 Phe Arg Glu Ile Tyr Arg Cys Ala Leu Asp His Phe Glu Glu Asp Arg 385 390 395 400 Lys Ser Tyr His Ile Ser Ala Asp Leu Ser Lys Val Pro Glu Val Glu 405 410 415 Lys Val Lys Asp Glu Asp Leu Pro Gly Leu Phe Glu Asp Ile Asn Val 420 425 430 Arg Gln Leu Ile His Val Thr Tyr Gly Ser Val Leu Lys Asp Ala Ser 435 440 445 Leu Lys Glu Arg Leu Phe Lys Thr Leu Glu Gln Asn Glu Glu Leu Phe 450 455 460 Tyr Glu Thr Val Ala Lys His Ile Lys Arg His Val Asp Leu Leu Lys 465 470 475 480 Gly Glx <210> 12 <211> 1446 <212> DNA <213> Unknown <220> <223> Thermotoga petrophila <400> 12 atggtcttga aagtgttcaa agatcacttt ggaaggggat acgaagttta cgaaaagtct 60 tatagagaaa aggattctct ctctttcttc ttgacaaagg gagaggaagg aaaaattctg 120 gtagtggctg gagaaaaggc acctgagggt ctgtcgtttt tcaaaaaaca gcgggtggag 180 ggtgtttcgt tctttttctg tgagagaaat catgagaact tggaagttct cagaaaatac 240 tttccagatc tcaaaccagt tcgagcggga ttgagagcgt cttttggaac aggtgacaga 300 ctcggtatca ccacaccggc tcacgtgagg gcgttgaagg attcagggct ttttcccatc 360 tttgcgcagc agtcggtgag ggagaacgag agaacgggaa ggacctggag agatgtgctg 420 gacgatgcca catggggagt tttccaggag ggatacagtg agggattcgg agcagacgcc 480 gatcacgtga agcggccgga ggatcttgtt tcggctgcaa gggaaggttt caccatgttc 540 acaatcgatc cttcggatca tgtgaggaat ctttcaaaac ttacagaaaa ggaaagaaat 600 gagaaattcg aagagattct gagaaaggaa aggatcgaca ggatctatct cggtaagaaa 660 tactctgttc tcggtgagaa gatcgaattc gatgagaaga atctcagaga tgcggcgctc 720 gtgtattacg atgcgattgc ccacgtggat atgatgtatc aaattttgaa agacgaaacc 780 ccggatttcg acttcgaagt gtcagttgac gaaacagaaa ctcccacgag tcctctcttc 840 cacattttcg ttgtggaaga actcagacga agaggtgtgg agttcaccaa tcttgccctg 900 agattcatcg gcgaatggga aaagggaata gattacaagg gggatcttgc acagttcgag 960 agagaaatca aaatgcacgc agaaatcgca aggatgttcg aaggatacaa aatatcactc 1020 cactctggaa gcgacaaatt ttccgtgtat cctgcttttg cttccgcgac aggaggcctt 1080 ttccacgtga agacagccgg aacgagttat cttgaggcgg tgaaggtcat atccatggtc 1140 aacccggagc tcttccggga gatctacagg tgtgctctcg atcactttga ggaagacaga 1200 aagtcctatc acatatctgc ggatctgtcg aaagttccgg aagtagagaa agtgaaagat 1260 gaagatcttc caggtctttt tgaagacatc aacgtgagac agttgatcca tgtcacctat 1320 ggctctgttc tgaaagatgc atctttgaaa gaacggctgt ttaagacgct tgaacaaaat 1380 gaggaactct tctacgagac cgtggcaaaa catataaaaa ggcacgtaga cctgttgaag 1440 gggtga 1446 <210> 13 <211> 1446 <212> DNA <213> Artificial Sequence <220> <223> S125D mutant of tagaturonate epimerase <400> 13 atggtcttga aagtgttcaa agatcacttt ggaaggggat acgaagttta cgaaaagtct 60 tatagagaaa aggattctct ctctttcttc ttgacaaagg gagaggaagg aaaaattctg 120 gtagtggctg gagaaaaggc acctgagggt ctgtcgtttt tcaaaaaaca gcgggtggag 180 ggtgtttcgt tctttagctg tgagagaaat catgagaact tggaagttct cagaaaatac 240 tttccagatc tcaaaccagt tcgagcggga ttgagagcgt cttttggaac aggtgacaga 300 ctcggtatca ccacaccggc tcacgtgagg gcgttgaagg attcagggct ttttcccatc 360 tttgcgcagc aggatgtgag ggagaacgag agaacgggaa ggacctggag agatgtgctg 420 gacgatgcca catggggagt tttccaggag ggatacagtg agggattcgg agcagacgcc 480 gatcacgtga agcggccgga ggatcttgtt tcggctgcaa gggaaggttt caccatgttc 540 gcaatcgatc cttcggatca tgtgaggaat ctttcaaaac ttacagaaaa ggaaagaaat 600 gagaaattcg aagagattct gagaaaggaa aggatcgaca ggatctatct cggtaagaaa 660 tactctgttc tcggtgagaa gatcgaattc gatgagaaga atctcagaga tgcggcgctc 720 gtgtattacg atgcgattgc ccacgtggat atgatgtatc aaattttgaa agacgaaacc 780 ccggatttcg acttcgaagt gtcagttgac gaaacagaaa ctcccacgag tcctctcttc 840 cacattttcg ttgtggaaga actcagacga agaggtgtgg agttcaccaa tcttgccctg 900 agattcatcg gcgaatggga aaagggaata gattacaagg gggatcttgc acagttcgag 960 agagaaatca aaatgcacgc agaaatcgca aggatgttcg aaggatacaa aatatcactc 1020 cactctggaa gcgacaaatt ttccgtgtat cctgcttttg cttccgcgac aggaggcctt 1080 ttcctcgtga agacagccgg aacgagttat cttgaggcgg tgaaggtcat atccatggtc 1140 aacccggagc tcttccggga gatctacagg tgtgctctcg atcactttga ggaagacaga 1200 aagtcctatc acatatctgc ggatctgtcg aaagttccgg aagtagagaa agtgaaagat 1260 gaagatcttc caggtctttt tgaagacatc aacgtgagac agttgatcca tgtcacctat 1320 ggctctgttc tgaaagatgc atctttgaaa gaacggctgt ttaagacgct tgaacaaaat 1380 gaggaactct tctacgagac cgtggcaaaa catataaaaa ggcacgtaga cctgttgaag 1440 gggtga 1446 <210> 14 <211> 1446 <212> DNA <213> Artificial Sequence <220> <223> S125D/T181A/H362L mutant of tagaturonate epimerase <400> 14 atggtcttga aagtgttcaa agatcacttt ggaaggggat acgaagttta cgaaaagtct 60 tatagagaaa aggattctct ctctttcttc ttgacaaagg gagaggaagg aaaaattctg 120 gtagtggctg gagaaaaggc acctgagggt ctgtcgtttt tcaaaaaaca gcgggtggag 180 ggtgtttcgt tctttttctg tgagagaaat catgagaact tggaagttct cagaaaatac 240 tttccagatc tcaaaccagt tcgagcggga ttgagagcgt cttttggaac aggtgacaga 300 ctcggtatca ccacaccggc tcacgtgagg gcgttgaagg attcagggct ttttcccatc 360 tttgcgcagc aggatgtgag ggagaacgag agaacgggaa ggacctggag agatgtgctg 420 gacgatgcca catggggagt tttccaggag ggatacagtg agggattcgg agcagacgcc 480 gatcacgtga agcggccgga ggatcttgtt tcggctgcaa gggaaggttt caccatgttc 540 gcaatcgatc cttcggatca tgtgaggaat ctttcaaaac ttacagaaaa ggaaagaaat 600 gagaaattcg aagagattct gagaaaggaa aggatcgaca ggatctatct cggtaagaaa 660 tactctgttc tcggtgagaa gatcgaattc gatgagaaga atctcagaga tgcggcgctc 720 gtgtattacg atgcgattgc ccacgtggat atgatgtatc aaattttgaa agacgaaacc 780 ccggatttcg acttcgaagt gtcagttgac gaaacagaaa ctcccacgag tcctctcttc 840 cacattttcg ttgtggaaga actcagacga agaggtgtgg agttcaccaa tcttgccctg 900 agattcatcg gcgaatggga aaagggaata gattacaagg gggatcttgc acagttcgag 960 agagaaatca aaatgcacgc agaaatcgca aggatgttcg aaggatacaa aatatcactc 1020 cactctggaa gcgacaaatt ttccgtgtat cctgcttttg cttccgcgac aggaggcctt 1080 ttcctcgtga agacagccgg aacgagttat cttgaggcgg tgaaggtcat atccatggtc 1140 aacccggagc tcttccggga gatctacagg tgtgctctcg atcactttga ggaagacaga 1200 aagtcctatc acatatctgc ggatctgtcg aaagttccgg aagtagagaa agtgaaagat 1260 gaagatcttc caggtctttt tgaagacatc aacgtgagac agttgatcca tgtcacctat 1320 ggctctgttc tgaaagatgc atctttgaaa gaacggctgt ttaagacgct tgaacaaaat 1380 gaggaactct tctacgagac cgtggcaaaa catataaaaa ggcacgtaga cctgttgaag 1440 gggtga 1446 <210> 15 <211> 1446 <212> DNA <213> Artificial Sequence <220> <223> F66S/S125D/T181A/H362L mutant of tagaturonate epimerase <400> 15 atggtcttga aagtgttcaa agatcacttt ggaaggggat acgaagttta cgaaaagtct 60 tatagagaaa aggattctct ctctttcttc ttgacaaagg gagaggaagg aaaaattctg 120 gtagtggctg gagaaaaggc acctgagggt ctgtcgtttt tcaaaaaaca gcgggtggag 180 ggtgtttcgt tctttagctg tgagagaaat catgagaact tggaagttct cagaaaatac 240 tttccagatc tcaaaccagt tcgagcggga ttgagagcgt cttttggaac aggtgacaga 300 ctcggtatca ccacaccggc tcacgtgagg gcgttgaagg attcagggct ttttcccatc 360 tttgcgcagc aggatgtgag ggagaacgag agaacgggaa ggacctggag agatgtgctg 420 gacgatgcca catggggagt tttccaggag ggatacagtg agggattcgg agcagacgcc 480 gatcacgtga agcggccgga ggatcttgtt tcggctgcaa gggaaggttt caccatgttc 540 gcaatcgatc cttcggatca tgtgaggaat ctttcaaaac ttacagaaaa ggaaagaaat 600 gagaaattcg aagagattct gagaaaggaa aggatcgaca ggatctatct cggtaagaaa 660 tactctgttc tcggtgagaa gatcgaattc gatgagaaga atctcagaga tgcggcgctc 720 gtgtattacg atgcgattgc ccacgtggat atgatgtatc aaattttgaa agacgaaacc 780 ccggatttcg acttcgaagt gtcagttgac gaaacagaaa ctcccacgag tcctctcttc 840 cacattttcg ttgtggaaga actcagacga agaggtgtgg agttcaccaa tcttgccctg 900 agattcatcg gcgaatggga aaagggaata gattacaagg gggatcttgc acagttcgag 960 agagaaatca aaatgcacgc agaaatcgca aggatgttcg aaggatacaa aatatcactc 1020 cactctggaa gcgacaaatt ttccgtgtat cctgcttttg cttccgcgac aggaggcctt 1080 ttcctcgtga agacagccgg aacgagttat cttgaggcgg tgaaggtcat atccatggtc 1140 aacccggagc tcttccggga gatctacagg tgtgctctcg atcactttga ggaagacaga 1200 aagtcctatc acatatctgc ggatctgtcg aaagttccgg aagtagagaa agtgaaagat 1260 gaagatcttc caggtctttt tgaagacatc aacgtgagac agttgatcca tgtcacctat 1320 ggctctgttc tgaaagatgc atctttgaaa gaacggctgt ttaagacgct tgaacaaaat 1380 gaggaactct tctacgagac cgtggcaaaa catataaaaa ggcacgtaga cctgttgaag 1440 gggtga 1446 <210> 16 <211> 1446 <212> DNA <213> Artificial Sequence <220> <223> E72G/S125D/T181A/H362L mutant of tagaturonate epimerase <400> 16 atggtcttga aagtgttcaa agatcacttt ggaaggggat acgaagttta cgaaaagtct 60 tatagagaaa aggattctct ctctttcttc ttgacaaagg gagaggaagg aaaaattctg 120 gtagtggctg gagaaaaggc acctgagggt ctgtcgtttt tcaaaaaaca gcgggtggag 180 ggtgtttcgt tctttttctg tgagagaaat catgggaact tggaagttct cagaaaatac 240 tttccagatc tcaaaccagt tcgagcggga ttgagagcgt cttttggaac aggtgacaga 300 ctcggtatca ccacaccggc tcacgtgagg gcgttgaagg attcagggct ttttcccatc 360 tttgcgcagc aggatgtgag ggagaacgag agaacgggaa ggacctggag agatgtgctg 420 gacgatgcca catggggagt tttccaggag ggatacagtg agggattcgg agcagacgcc 480 gatcacgtga agcggccgga ggatcttgtt tcggctgcaa gggaaggttt caccatgttc 540 gcaatcgatc cttcggatca tgtgaggaat ctttcaaaac ttacagaaaa ggaaagaaat 600 gagaaattcg aagagattct gagaaaggaa aggatcgaca ggatctatct cggtaagaaa 660 tactctgttc tcggtgagaa gatcgaattc gatgagaaga atctcagaga tgcggcgctc 720 gtgtattacg atgcgattgc ccacgtggat atgatgtatc aaattttgaa agacgaaacc 780 ccggatttcg acttcgaagt gtcagttgac gaaacagaaa ctcccacgag tcctctcttc 840 cacattttcg ttgtggaaga actcagacga agaggtgtgg agttcaccaa tcttgccctg 900 agattcatcg gcgaatggga aaagggaata gattacaagg gggatcttgc acagttcgag 960 agagaaatca aaatgcacgc agaaatcgca aggatgttcg aaggatacaa aatatcactc 1020 cactctggaa gcgacaaatt ttccgtgtat cctgcttttg cttccgcgac aggaggcctt 1080 ttcctcgtga agacagccgg aacgagttat cttgaggcgg tgaaggtcat atccatggtc 1140 aacccggagc tcttccggga gatctacagg tgtgctctcg atcactttga ggaagacaga 1200 aagtcctatc acatatctgc ggatctgtcg aaagttccgg aagtagagaa agtgaaagat 1260 gaagatcttc caggtctttt tgaagacatc aacgtgagac agttgatcca tgtcacctat 1320 ggctctgttc tgaaagatgc atctttgaaa gaacggctgt ttaagacgct tgaacaaaat 1380 gaggaactct tctacgagac cgtggcaaaa catataaaaa ggcacgtaga cctgttgaag 1440 gggtga 1446 <210> 17 <211> 1446 <212> DNA <213> Artificial Sequence <220> <223> S125D/N129F/T181A/H362L mutant of tagaturonate epimerase <400> 17 atggtcttga aagtgttcaa agatcacttt ggaaggggat acgaagttta cgaaaagtct 60 tatagagaaa aggattctct ctctttcttc ttgacaaagg gagaggaagg aaaaattctg 120 gtagtggctg gagaaaaggc acctgagggt ctgtcgtttt tcaaaaaaca gcgggtggag 180 ggtgtttcgt tctttttctg tgagagaaat catgagaact tggaagttct cagaaaatac 240 tttccagatc tcaaaccagt tcgagcggga ttgagagcgt cttttggaac aggtgacaga 300 ctcggtatca ccacaccggc tcacgtgagg gcgttgaagg attcagggct ttttcccatc 360 tttgcgcagc aggatgtgag ggagttcgag agaacgggaa ggacctggag agatgtgctg 420 gacgatgcca catggggagt tttccaggag ggatacagtg agggattcgg agcagacgcc 480 gatcacgtga agcggccgga ggatcttgtt tcggctgcaa gggaaggttt caccatgttc 540 gcaatcgatc cttcggatca tgtgaggaat ctttcaaaac ttacagaaaa ggaaagaaat 600 gagaaattcg aagagattct gagaaaggaa aggatcgaca ggatctatct cggtaagaaa 660 tactctgttc tcggtgagaa gatcgaattc gatgagaaga atctcagaga tgcggcgctc 720 gtgtattacg atgcgattgc ccacgtggat atgatgtatc aaattttgaa agacgaaacc 780 ccggatttcg acttcgaagt gtcagttgac gaaacagaaa ctcccacgag tcctctcttc 840 cacattttcg ttgtggaaga actcagacga agaggtgtgg agttcaccaa tcttgccctg 900 agattcatcg gcgaatggga aaagggaata gattacaagg gggatcttgc acagttcgag 960 agagaaatca aaatgcacgc agaaatcgca aggatgttcg aaggatacaa aatatcactc 1020 cactctggaa gcgacaaatt ttccgtgtat cctgcttttg cttccgcgac aggaggcctt 1080 ttcctcgtga agacagccgg aacgagttat cttgaggcgg tgaaggtcat atccatggtc 1140 aacccggagc tcttccggga gatctacagg tgtgctctcg atcactttga ggaagacaga 1200 aagtcctatc acatatctgc ggatctgtcg aaagttccgg aagtagagaa agtgaaagat 1260 gaagatcttc caggtctttt tgaagacatc aacgtgagac agttgatcca tgtcacctat 1320 ggctctgttc tgaaagatgc atctttgaaa gaacggctgt ttaagacgct tgaacaaaat 1380 gaggaactct tctacgagac cgtggcaaaa catataaaaa ggcacgtaga cctgttgaag 1440 gggtga 1446 <210> 18 <211> 1446 <212> DNA <213> Artificial Sequence <220> <223> S125D/L140P/T181A/H362L mutant of tagaturonate epimerase <400> 18 atggtcttga aagtgttcaa agatcacttt ggaaggggat acgaagttta cgaaaagtct 60 tatagagaaa aggattctct ctctttcttc ttgacaaagg gagaggaagg aaaaattctg 120 gtagtggctg gagaaaaggc acctgagggt ctgtcgtttt tcaaaaaaca gcgggtggag 180 ggtgtttcgt tctttttctg tgagagaaat catgagaact tggaagttct cagaaaatac 240 tttccagatc tcaaaccagt tcgagcggga ttgagagcgt cttttggaac aggtgacaga 300 ctcggtatca ccacaccggc tcacgtgagg gcgttgaagg attcagggct ttttcccatc 360 tttgcgcagc aggatgtgag ggagaacgag agaacgggaa ggacctggag agatgtgccg 420 gacgatgcca catggggagt tttccaggag ggatacagtg agggattcgg agcagacgcc 480 gatcacgtga agcggccgga ggatcttgtt tcggctgcaa gggaaggttt caccatgttc 540 gcaatcgatc cttcggatca tgtgaggaat ctttcaaaac ttacagaaaa ggaaagaaat 600 gagaaattcg aagagattct gagaaaggaa aggatcgaca ggatctatct cggtaagaaa 660 tactctgttc tcggtgagaa gatcgaattc gatgagaaga atctcagaga tgcggcgctc 720 gtgtattacg atgcgattgc ccacgtggat atgatgtatc aaattttgaa agacgaaacc 780 ccggatttcg acttcgaagt gtcagttgac gaaacagaaa ctcccacgag tcctctcttc 840 cacattttcg ttgtggaaga actcagacga agaggtgtgg agttcaccaa tcttgccctg 900 agattcatcg gcgaatggga aaagggaata gattacaagg gggatcttgc acagttcgag 960 agagaaatca aaatgcacgc agaaatcgca aggatgttcg aaggatacaa aatatcactc 1020 cactctggaa gcgacaaatt ttccgtgtat cctgcttttg cttccgcgac aggaggcctt 1080 ttcctcgtga agacagccgg aacgagttat cttgaggcgg tgaaggtcat atccatggtc 1140 aacccggagc tcttccggga gatctacagg tgtgctctcg atcactttga ggaagacaga 1200 aagtcctatc acatatctgc ggatctgtcg aaagttccgg aagtagagaa agtgaaagat 1260 gaagatcttc caggtctttt tgaagacatc aacgtgagac agttgatcca tgtcacctat 1320 ggctctgttc tgaaagatgc atctttgaaa gaacggctgt ttaagacgct tgaacaaaat 1380 gaggaactct tctacgagac cgtggcaaaa catataaaaa ggcacgtaga cctgttgaag 1440 gggtga 1446 <210> 19 <211> 1446 <212> DNA <213> Artificial Sequence <220> <223> S125D/N129S/L140F/T181A/H362L mutant of tagaturonate epimerase <400> 19 atggtcttga aagtgttcaa agatcacttt ggaaggggat acgaagttta cgaaaagtct 60 tatagagaaa aggattctct ctctttcttc ttgacaaagg gagaggaagg aaaaattctg 120 gtagtggctg gagaaaaggc acctgagggt ctgtcgtttt tcaaaaaaca gcgggtggag 180 ggtgtttcgt tctttttctg tgagagaaat catgagaact tggaagttct cagaaaatac 240 tttccagatc tcaaaccagt tcgagcggga ttgagagcgt cttttggaac aggtgacaga 300 ctcggtatca ccacaccggc tcacgtgagg gcgttgaagg attcagggct ttttcccatc 360 tttgcgcagc aggatgtgag ggagagcgag agaacgggaa ggacctggag agatgtgttc 420 gacgatgcca catggggagt tttccaggag ggatacagtg agggattcgg agcagacgcc 480 gatcacgtga agcggccgga ggatcttgtt tcggctgcaa gggaaggttt caccatgttc 540 gcaatcgatc cttcggatca tgtgaggaat ctttcaaaac ttacagaaaa ggaaagaaat 600 gagaaattcg aagagattct gagaaaggaa aggatcgaca ggatctatct cggtaagaaa 660 tactctgttc tcggtgagaa gatcgaattc gatgagaaga atctcagaga tgcggcgctc 720 gtgtattacg atgcgattgc ccacgtggat atgatgtatc aaattttgaa agacgaaacc 780 ccggatttcg acttcgaagt gtcagttgac gaaacagaaa ctcccacgag tcctctcttc 840 cacattttcg ttgtggaaga actcagacga agaggtgtgg agttcaccaa tcttgccctg 900 agattcatcg gcgaatggga aaagggaata gattacaagg gggatcttgc acagttcgag 960 agagaaatca aaatgcacgc agaaatcgca aggatgttcg aaggatacaa aatatcactc 1020 cactctggaa gcgacaaatt ttccgtgtat cctgcttttg cttccgcgac aggaggcctt 1080 ttcctcgtga agacagccgg aacgagttat cttgaggcgg tgaaggtcat atccatggtc 1140 aacccggagc tcttccggga gatctacagg tgtgctctcg atcactttga ggaagacaga 1200 aagtcctatc acatatctgc ggatctgtcg aaagttccgg aagtagagaa agtgaaagat 1260 gaagatcttc caggtctttt tgaagacatc aacgtgagac agttgatcca tgtcacctat 1320 ggctctgttc tgaaagatgc atctttgaaa gaacggctgt ttaagacgct tgaacaaaat 1380 gaggaactct tctacgagac cgtggcaaaa catataaaaa ggcacgtaga cctgttgaag 1440 gggtga 1446 <210> 20 <211> 1446 <212> DNA <213> Artificial Sequence <220> <223> S125D/N129T/L140K/T181A/H362L mutant of tagaturonate epimerase <400> 20 atggtcttga aagtgttcaa agatcacttt ggaaggggat acgaagttta cgaaaagtct 60 tatagagaaa aggattctct ctctttcttc ttgacaaagg gagaggaagg aaaaattctg 120 gtagtggctg gagaaaaggc acctgagggt ctgtcgtttt tcaaaaaaca gcgggtggag 180 ggtgtttcgt tctttttctg tgagagaaat catgagaact tggaagttct cagaaaatac 240 tttccagatc tcaaaccagt tcgagcggga ttgagagcgt cttttggaac aggtgacaga 300 ctcggtatca ccacaccggc tcacgtgagg gcgttgaagg attcagggct ttttcccatc 360 tttgcgcagc aggatgtgag ggagaccgag agaacgggaa ggacctggag agatgtgaag 420 gacgatgcca catggggagt tttccaggag ggatacagtg agggattcgg agcagacgcc 480 gatcacgtga agcggccgga ggatcttgtt tcggctgcaa gggaaggttt caccatgttc 540 gcaatcgatc cttcggatca tgtgaggaat ctttcaaaac ttacagaaaa ggaaagaaat 600 gagaaattcg aagagattct gagaaaggaa aggatcgaca ggatctatct cggtaagaaa 660 tactctgttc tcggtgagaa gatcgaattc gatgagaaga atctcagaga tgcggcgctc 720 gtgtattacg atgcgattgc ccacgtggat atgatgtatc aaattttgaa agacgaaacc 780 ccggatttcg acttcgaagt gtcagttgac gaaacagaaa ctcccacgag tcctctcttc 840 cacattttcg ttgtggaaga actcagacga agaggtgtgg agttcaccaa tcttgccctg 900 agattcatcg gcgaatggga aaagggaata gattacaagg gggatcttgc acagttcgag 960 agagaaatca aaatgcacgc agaaatcgca aggatgttcg aaggatacaa aatatcactc 1020 cactctggaa gcgacaaatt ttccgtgtat cctgcttttg cttccgcgac aggaggcctt 1080 ttcctcgtga agacagccgg aacgagttat cttgaggcgg tgaaggtcat atccatggtc 1140 aacccggagc tcttccggga gatctacagg tgtgctctcg atcactttga ggaagacaga 1200 aagtcctatc acatatctgc ggatctgtcg aaagttccgg aagtagagaa agtgaaagat 1260 gaagatcttc caggtctttt tgaagacatc aacgtgagac agttgatcca tgtcacctat 1320 ggctctgttc tgaaagatgc atctttgaaa gaacggctgt ttaagacgct tgaacaaaat 1380 gaggaactct tctacgagac cgtggcaaaa catataaaaa ggcacgtaga cctgttgaag 1440 gggtga 1446 <210> 21 <211> 1446 <212> DNA <213> Artificial Sequence <220> <223> S125D/N129T/L140F/T181A/H362L mutant of tagaturonate epimerase <400> 21 atggtcttga aagtgttcaa agatcacttt ggaaggggat acgaagttta cgaaaagtct 60 tatagagaaa aggattctct ctctttcttc ttgacaaagg gagaggaagg aaaaattctg 120 gtagtggctg gagaaaaggc acctgagggt ctgtcgtttt tcaaaaaaca gcgggtggag 180 ggtgtttcgt tctttttctg tgagagaaat catgagaact tggaagttct cagaaaatac 240 tttccagatc tcaaaccagt tcgagcggga ttgagagcgt cttttggaac aggtgacaga 300 ctcggtatca ccacaccggc tcacgtgagg gcgttgaagg attcagggct ttttcccatc 360 tttgcgcagc aggatgtgag ggagaccgag agaacgggaa ggacctggag agatgtgttc 420 gacgatgcca catggggagt tttccaggag ggatacagtg agggattcgg agcagacgcc 480 gatcacgtga agcggccgga ggatcttgtt tcggctgcaa gggaaggttt caccatgttc 540 gcaatcgatc cttcggatca tgtgaggaat ctttcaaaac ttacagaaaa ggaaagaaat 600 gagaaattcg aagagattct gagaaaggaa aggatcgaca ggatctatct cggtaagaaa 660 tactctgttc tcggtgagaa gatcgaattc gatgagaaga atctcagaga tgcggcgctc 720 gtgtattacg atgcgattgc ccacgtggat atgatgtatc aaattttgaa agacgaaacc 780 ccggatttcg acttcgaagt gtcagttgac gaaacagaaa ctcccacgag tcctctcttc 840 cacattttcg ttgtggaaga actcagacga agaggtgtgg agttcaccaa tcttgccctg 900 agattcatcg gcgaatggga aaagggaata gattacaagg gggatcttgc acagttcgag 960 agagaaatca aaatgcacgc agaaatcgca aggatgttcg aaggatacaa aatatcactc 1020 cactctggaa gcgacaaatt ttccgtgtat cctgcttttg cttccgcgac aggaggcctt 1080 ttcctcgtga agacagccgg aacgagttat cttgaggcgg tgaaggtcat atccatggtc 1140 aacccggagc tcttccggga gatctacagg tgtgctctcg atcactttga ggaagacaga 1200 aagtcctatc acatatctgc ggatctgtcg aaagttccgg aagtagagaa agtgaaagat 1260 gaagatcttc caggtctttt tgaagacatc aacgtgagac agttgatcca tgtcacctat 1320 ggctctgttc tgaaagatgc atctttgaaa gaacggctgt ttaagacgct tgaacaaaat 1380 gaggaactct tctacgagac cgtggcaaaa catataaaaa ggcacgtaga cctgttgaag 1440 gggtga 1446 <210> 22 <211> 1446 <212> DNA <213> Artificial Sequence <220> <223> S125D/N129T/L140P/T181A/H362L mutant of tagaturonate epimerase <400> 22 atggtcttga aagtgttcaa agatcacttt ggaaggggat acgaagttta cgaaaagtct 60 tatagagaaa aggattctct ctctttcttc ttgacaaagg gagaggaagg aaaaattctg 120 gtagtggctg gagaaaaggc acctgagggt ctgtcgtttt tcaaaaaaca gcgggtggag 180 ggtgtttcgt tctttttctg tgagagaaat catgagaact tggaagttct cagaaaatac 240 tttccagatc tcaaaccagt tcgagcggga ttgagagcgt cttttggaac aggtgacaga 300 ctcggtatca ccacaccggc tcacgtgagg gcgttgaagg attcagggct ttttcccatc 360 tttgcgcagc aggatgtgag ggagaccgag agaacgggaa ggacctggag agatgtgccg 420 gacgatgcca catggggagt tttccaggag ggatacagtg agggattcgg agcagacgcc 480 gatcacgtga agcggccgga ggatcttgtt tcggctgcaa gggaaggttt caccatgttc 540 gcaatcgatc cttcggatca tgtgaggaat ctttcaaaac ttacagaaaa ggaaagaaat 600 gagaaattcg aagagattct gagaaaggaa aggatcgaca ggatctatct cggtaagaaa 660 tactctgttc tcggtgagaa gatcgaattc gatgagaaga atctcagaga tgcggcgctc 720 gtgtattacg atgcgattgc ccacgtggat atgatgtatc aaattttgaa agacgaaacc 780 ccggatttcg acttcgaagt gtcagttgac gaaacagaaa ctcccacgag tcctctcttc 840 cacattttcg ttgtggaaga actcagacga agaggtgtgg agttcaccaa tcttgccctg 900 agattcatcg gcgaatggga aaagggaata gattacaagg gggatcttgc acagttcgag 960 agagaaatca aaatgcacgc agaaatcgca aggatgttcg aaggatacaa aatatcactc 1020 cactctggaa gcgacaaatt ttccgtgtat cctgcttttg cttccgcgac aggaggcctt 1080 ttcctcgtga agacagccgg aacgagttat cttgaggcgg tgaaggtcat atccatggtc 1140 aacccggagc tcttccggga gatctacagg tgtgctctcg atcactttga ggaagacaga 1200 aagtcctatc acatatctgc ggatctgtcg aaagttccgg aagtagagaa agtgaaagat 1260 gaagatcttc caggtctttt tgaagacatc aacgtgagac agttgatcca tgtcacctat 1320 ggctctgttc tgaaagatgc atctttgaaa gaacggctgt ttaagacgct tgaacaaaat 1380 gaggaactct tctacgagac cgtggcaaaa catataaaaa ggcacgtaga cctgttgaag 1440 gggtga 1446 <110> Konkuk University Industrial Cooperation Corp <120> TAGATURONATE EPIMERASE VARIANT EMZYME AND METHOD FOR PRODUCING TAGATOSE FROM FRUXTOSE USING THE SAME <130> 2020-I147 <160> 22 <170> KoPatentIn 3.0 <210> 1 <211> 482 <212 > PRT <213> Unknown <220> <223> Thermotoga petrophila <400> 1 Met Val Leu Lys Val Phe Lys Asp His Phe Gly Arg Gly Tyr Glu Val 1 5 10 15 Tyr Glu Lys Ser Tyr Arg Glu Lys Asp Ser Leu Ser Phe Phe Leu Thr 20 25 30 Lys Gly Glu Glu Gly Lys Ile Leu Val Val Ala Gly Glu Lys Ala Pro 35 40 45 Glu Gly Leu Ser Phe Phe Lys Lys Gln Arg Val Glu Gly Val Ser Phe 50 55 60 Phe Phe Cys Glu Arg Asn His Glu Asn Leu Glu Val Leu Arg Lys Tyr 65 70 75 80 Phe Pro Asp Leu Lys Pro Val Arg Ala Gly Leu Arg Ala Ser Phe Gly 85 90 95 Thr Gly Asp Arg Leu Gly Ile Thr Thr Pro Ala His Val Arg Ala Leu 100 105 110 Lys Asp Ser Gly Leu Phe Pro Ile Phe Ala Gln Gln Ser Val Arg Glu 115 120 125 Asn Glu Arg Thr Gly Arg Thr Trp Arg Asp Val Leu Asp Asp Ala Thr 130 135 140 Trp Gly Val Phe Gln Glu Gly Tyr Ser Glu Gly Phe Gly Ala Asp Ala 145 150 155 160 Asp His Val Lys Arg Pro Glu Asp Leu Val Ser Ala Ala Arg Glu Gly 165 170 175 Phe Thr Met Phe Thr Ile Asp Pro Ser Asp His Val Arg Asn Leu Ser 180 185 190 Lys Leu Thr Glu Lys Glu Arg Asn Glu Lys Phe Glu Glu Ile Leu Arg 195 200 205 Lys Glu Arg Ile Asp Arg Ile Tyr Leu Gly Lys Lys Tyr Ser Val Leu 210 215 220 Gly Glu Lys Ile Glu Phe Asp Glu Lys Asn Leu Arg Asp Ala Ala Leu 225 230 235 240 Val Tyr Tyr Asp Ala Ile Ala His Val Asp Met Met Tyr Gln Ile Leu 245 250 255 Lys Asp Glu Thr Pro Asp Phe Asp Phe Glu Val Ser Val Asp Glu Thr 260 265 270 Glu Thr Pro Thr Ser Pro Leu Phe His Ile Phe Val Val Glu Glu Leu 275 280 285 Arg Arg Arg Gly Val Glu Phe Thr Asn Leu Ala Leu Arg Phe Ile Gly 290 295 300 Glu Trp Glu Lys Gly Ile Asp Tyr Lys Gly Asp Leu Ala Gln Phe Glu 305 310 315 320 Arg Glu Ile Lys Met His Ala Glu Ile Ala Arg Met Phe Glu Gly Tyr 325 330 335 Lys Ile Ser Leu His Ser Gly Ser Asp Lys Phe Ser Val Tyr Pro Ala 340 345 350 Phe Ala Ser Ala Thr Gly Gly Leu Phe His Val Lys Thr Ala Gly Thr 355 360 365 Ser Tyr Leu Glu Ala Val Lys Val Ile Ser Met Val Asn Pro Glu Leu 370 375 380 Phe Arg Glu Ile Tyr Arg Cys Ala Leu Asp His Phe Glu Glu Asp Arg 385 390 395 400 Lys Ser Tyr His Ile Ser Ala Asp Leu Ser Lys Val Pro Glu Val Glu 405 410 415 Lys Val Lys Asp Glu Asp Leu Pro Gly Leu Phe Glu Asp Ile Asn Val 420 425 430 Arg Gln Leu Ile His Val Thr Tyr Gly Ser Val Leu Lys Asp Ala Ser 435 440 445 Leu Lys Glu Arg Leu Phe Lys Thr Leu Glu Gln Asn Glu Glu Leu Phe 450 455 460 Tyr Glu Thr Val Ala Lys His Ile Lys Arg His Val Asp Leu Leu Lys 465 470 475 480 Gly Glx <210> 2 <211> 482 <212> PRT <213> Artificial Sequence <220> <223> S125D mutant of tagaturonate epimerase <400> 2 Met Val Leu Lys Val Phe Lys Asp His Phe Gly Arg Gly Tyr Glu Val 1 5 10 15 Tyr Glu Lys Ser Tyr Arg Glu Lys Asp Ser Leu Ser Phe Phe Leu Thr 20 25 30 Lys Gly Glu Glu Gly Lys Ile Leu Val Val Ala Gly Glu Lys Ala Pro 35 40 45 Glu Gly Leu Ser Phe Phe Lys Lys Gln Arg Val Glu Gly Val Ser Phe 50 55 60 Phe Phe Cys Glu Arg Asn His Glu Asn Leu Glu Val Leu Arg Lys Tyr 65 70 75 80 Phe Pro Asp Leu Lys Pro Val Arg Ala Gly Leu Arg Ala Ser Phe Gly 85 90 95 Thr Gly Asp Arg Leu Gly Ile Thr Thr Pro Ala His Val Arg Ala Leu 100 105 110 Lys Asp Ser Gly Leu Phe Pro Ile Phe Ala Gln Gln Asp Val Arg Glu 115 120 125 Thr Glu Arg Thr Gly Arg Thr Trp Arg Asp Val Pro Asp Asp Ala Thr 130 135 140 Trp Gly Val Phe Gln Glu Gly Tyr Ser Glu Gly Phe Gly Ala Asp Ala 145 150 155 160 Asp His Val Lys Arg Pro Glu Asp Leu Val Ser Ala Ala Arg Glu Gly 165 170 175 Phe Thr Met Phe Ala Ile Asp Pro Ser Asp His Val Arg Asn Leu Ser 180 185 190 Lys Leu Thr Glu Lys Glu Arg Asn Glu Lys Phe Glu Glu Ile Leu Arg 195 200 205 Lys Glu Arg Ile Asp Arg Ile Tyr Leu Gly Lys Lys Tyr Ser Val Leu 210 215 220 Gly Glu Lys Ile Glu Phe Asp Glu Lys Asn Leu Arg Asp Ala Ala Leu 225 230 235 240 Val Tyr Tyr Asp Ala Ile Ala His Val Asp Met Met Tyr Gln Ile Leu 245 250 255 Lys Asp Glu Thr Pro Asp Phe Asp Phe Glu Val Ser Val Asp Glu Thr 260 265 270 Glu Thr Pro Thr Ser Pro Leu Phe His Ile Phe Val Val Glu Glu Leu 275 280 285 Arg Arg Arg Gly Val Glu Phe Thr Asn Leu Ala Leu Arg Phe Ile Gly 290 295 300 Glu Trp Glu Lys Gly Ile Asp Tyr Lys Gly Asp Leu Ala Gln Phe Glu 305 310 315 320 Arg Glu Ile Lys Met His Ala Glu Ile Ala Arg Met Phe Glu Gly Tyr 325 330 335 Lys Ile Ser Leu His Ser Gly Ser Asp Lys Phe Ser Val Tyr Pro Ala 340 345 350 Phe Ala Ser Ala Thr Gly Gly Leu Phe Leu Val Lys Thr Ala Gly Thr 355 360 365 Ser Tyr Leu Glu Ala Val Lys Val Ile Ser Met Val Asn Pro Glu Leu 370 375 380 Phe Arg Glu Ile Tyr Arg Cys Ala Leu Asp His Phe Glu Glu Asp Arg 385 390 395 400 Lys Ser Tyr His Ile Ser Ala Asp Leu Ser Lys Val Pro Glu Val Glu 405 410 415 Lys Val Lys Asp Glu Asp Leu Pro Gly Leu Phe Glu Asp Ile Asn Val 420 425 430 Arg Gln Leu Ile His Val Thr Tyr Gly Ser Val Leu Lys Asp Ala Ser 435 440 445 Leu Lys Glu Arg Leu Phe Lys Thr Leu Glu Gln Asn Glu Glu Leu Phe 450 455 460 Tyr Glu Thr Val Ala Lys His Ile Lys Arg His Val Asp Leu Leu Lys 465 470 475 480 Gly Glx <210> 3 <211> 482 <212> PRT <213> Artificial Sequence <220> <223> S125D/T181A/H362L mutant of tagaturonate epimerase <400> 3 Met Val Leu Lys Val Phe Lys Asp His Phe Gly Arg Gly Tyr Glu Val 1 5 10 15 Tyr Glu Lys Ser Tyr Arg Glu Lys Asp Ser Leu Ser Phe Phe Leu Thr 20 25 30 Lys Gly Glu Glu Gly Lys Ile Leu Val Val Ala Gly Glu Lys Ala Pro 35 40 45 Glu Gly Leu Ser Phe Phe Lys Lys Gln Arg Val Glu Gly Val Ser Phe 50 55 60 Phe Phe Cys Glu Arg Asn His Glu Asn Leu Glu Val Leu Arg Lys Tyr 65 70 75 80 Phe Pro Asp Leu Lys Pro Val Arg Ala Gly Leu Arg Ala Ser Phe Gly 85 90 95 Thr Gly Asp Arg Leu Gly Ile Thr Thr Pro Ala His Val Arg Ala Leu 100 105 110 Lys Asp Ser Gly Leu Phe Pro Ile Phe Ala Gln Gln Asp Val Arg Glu 115 120 125 Asn Glu Arg Thr Gly Arg Thr Trp Arg Asp Val Leu Asp Asp Ala Thr 130 135 140 Trp Gly Val Phe Gln Glu Gly Tyr Ser Glu Gly Phe Gly Ala Asp Ala 145 150 155 160 Asp His Val Lys Arg Pro Glu Asp Leu Val Ser Ala Ala Arg Glu Gly 165 170 175 Phe Thr Met Phe Ala Ile Asp Pro Ser Asp His Val Arg Asn Leu Ser 180 185 190 Lys Leu Thr Glu Lys Glu Arg Asn Glu L ys Phe Glu Glu Ile Leu Arg 195 200 205 Lys Glu Arg Ile Asp Arg Ile Tyr Leu Gly Lys Lys Tyr Ser Val Leu 210 215 220 Gly Glu Lys Ile Glu Phe Asp Glu Lys Asn Leu Arg Asp Ala Ala Leu 225 230 235 240 Val Tyr Tyr Asp Ala Ile Ala His Val Asp Met Met Tyr Gln Ile Leu 245 250 255 Lys Asp Glu Thr Pro Asp Phe Asp Phe Glu Val Ser Val Asp Glu Thr 260 265 270 Glu Thr Pro Thr Ser Pro Leu Phe His Ile Phe Val Val Glu Glu Leu 275 280 285 Arg Arg Arg Gly Val Glu Phe Thr Asn Leu Ala Leu Arg Phe Ile Gly 290 295 300 Glu Trp Glu Lys Gly Ile Asp Tyr Lys Gly Asp Leu Ala Gln Phe Glu 305 310 315 320 Arg Glu Ile Lys Met His Ala Glu Ile Ala Arg Met Phe Glu Gly Tyr 325 330 335 Lys Ile Ser Leu His Ser G ly Ser Asp Lys Phe Ser Val Tyr Pro Ala 340 345 350 Phe Ala Ser Ala Thr Gly Gly Leu Phe Leu Val Lys Thr Ala Gly Thr 355 360 365 Ser Tyr Leu Glu Ala Val Lys Val Ile Ser Met Val Asn Pro Glu Leu 370 375 380 Phe Arg Glu Ile Tyr Arg Cys Ala Leu Asp His Phe Glu Glu Asp Arg 385 390 395 400 Lys Ser Tyr His Ile Ser Ala Asp Leu Ser Lys Val Pro Glu Val Glu 405 410 415 Lys Val Lys Asp Glu Asp Leu Pro Gly Leu Phe Glu Asp Ile Asn Val 420 425 430 Arg Gln Leu Ile His Val Thr Tyr Gly Ser Val Leu Lys Asp Ala Ser 435 440 445 Leu Lys Glu Arg Leu Phe Lys Thr Leu Glu Gln Asn Glu Glu Leu Phe 450 455 460 Tyr Glu Thr Val Ala Lys His Ile Lys Arg His Val Asp Leu Leu Lys 465 470 475 480 Gly Glx <210> 4 <211> 482 <212> PRT <213> Artificial Sequence <220> <223> F66S/S125D/T181A/H362L mutant of tagaturonate epimerase <400> 4 Met Val Leu Lys Val Phe Lys Asp His Phe Gly Arg Gly Tyr Glu Val 1 5 10 15 Tyr Glu Lys Ser Tyr Arg Glu Lys Asp Ser Leu Ser Phe Phe Leu Thr 20 25 30 Lys Gly Glu Glu Gly Lys Ile Leu Val Val Ala Gly Glu Lys Ala Pro 35 40 45 Glu Gly Leu Ser Phe Phe Lys Lys Gln Arg Val Glu Gly Val Ser Phe 50 55 60 Phe Ser Cys Glu Arg Asn His Glu Asn Leu Glu Val Leu Arg Lys Tyr 65 70 75 80 Phe Pro Asp Leu Lys Pro Val Arg Ala Gly Leu Arg Ala Ser Phe Gly 85 90 95 Thr Gly Asp Arg Leu Gly Ile Thr Thr Pro Ala His Val Arg Ala Leu 100 105 110 Lys Asp Ser Gly Leu Phe Pro Ile Phe Ala Gln Gln Asp Val Arg Glu 115 120 125 Asn Glu Arg Thr Gly Arg Thr Trp Arg Asp Val Leu Asp Asp Ala Thr 130 135 140 Trp Gly Val Phe Gln Glu Gly Tyr Ser Glu Gly Phe Gly Ala Asp Ala 145 150 155 160 Asp His Val Lys Arg Pro Glu Asp Leu Val Ser Ala Ala Arg Glu Gly 165 170 175 Phe Thr Met Phe Ala Ile Asp Pro Ser Asp His Val Arg Asn Leu Ser 180 185 190 Lys Leu Thr Glu Lys Glu Arg Asn Glu Lys Phe Glu Glu Ile Leu Arg 195 200 205 Lys Glu Arg Ile Asp Arg Ile Tyr Leu Gly Lys Lys Tyr Ser Val Leu 210 215 220 Gly Glu Lys Ile Glu Phe Asp Glu Lys Asn Leu Arg Asp Ala Ala Leu 225 230 235 240 Val Tyr Tyr Asp Ala Ile Ala His Val Asp Met Met Tyr Gln Ile Leu 245 250 255 Lys Asp Glu Thr Pro Asp Phe Asp Phe Glu Val Ser Val Asp Glu Thr 260 265 270 Glu Thr Pro Thr Ser Pro Leu Phe His Ile Phe Val Val Glu Glu Leu 275 280 285 Arg Arg Arg Gly Val Glu Phe Thr Asn Leu Ala Leu Arg Phe Ile Gly 290 295 300 Glu Trp Glu Lys Gly Ile Asp Tyr Lys Gly Asp Leu Ala Gln Phe Glu 305 310 315 320 Arg Glu Ile Lys Met His Ala Glu Ile Ala Arg Met Phe Glu Gly Tyr 325 330 335 Lys Ile Ser Leu His Ser Gly Ser Asp Lys Phe Ser Val Tyr Pro Ala 340 345 350 Phe Ala Ser Ala Thr Gly Gly Leu Phe Leu Val Lys Thr Ala Gly Thr 355 360 365 Ser Tyr Leu Glu Ala Val Lys Val Ile Ser Met Val Asn Pro Glu Leu 370 375 380 Phe Arg Glu Ile Tyr Arg Cys Ala Leu Asp His Phe Glu Glu Asp Arg 385 390 395 400 Lys Ser Tyr His Ile Ser Ala Asp Leu Ser Lys Val Pro Glu Val Glu 405 410 415 Lys Val Lys Asp Glu Asp Leu Pro Gly Leu Phe Glu Asp Ile Asn Val 420 425 430 Arg Gln Leu Ile His Val Thr Tyr Gly Ser Val Leu Lys Asp Ala Ser 435 440 445 Leu Lys Glu Arg Leu Phe Lys Thr Leu Glu Gln Asn Glu Glu Leu Phe 450 455 460 Tyr Glu Thr Val Ala Lys His Ile Lys Arg His Val Asp Leu Leu Lys 465 470 475 480 Gly Glx <210> 5 <211> 482 <212> PRT <213> Artificial Sequence <220> <223> E72G/S125D/T181A/H362L mutant of tagaturonate epimerase <400> 5 Met Val Leu Lys Val Phe Lys Asp His Phe Gly Arg Gly Tyr Glu Val 1 5 10 15 Tyr Glu Lys Ser Tyr Arg Glu Lys Asp Ser Leu Ser Phe Phe Leu Thr 20 25 30 Lys Gly Glu Glu Gly Lys Ile Leu Val Val Ala Gly Glu Lys Ala Pro 35 40 45 Glu Gly Leu Ser Phe Phe Lys Lys Gln Arg Val Glu Gly Val Ser Phe 50 55 60 Phe Phe Cys Glu Arg Asn His Gly Asn Leu Glu Val Leu Arg Lys Tyr 65 70 75 80 Phe Pro Asp Leu Lys Pro Val Arg Ala Gly Leu Arg Ala Ser Phe Gly 85 90 95 Thr Gly Asp Arg Leu Gly Ile Thr Thr Pro Ala His Val Arg Ala Leu 100 105 110 Lys Asp Ser Gly Leu Phe P ro Ile Phe Ala Gln Gln Asp Val Arg Glu 115 120 125 Asn Glu Arg Thr Gly Arg Thr Trp Arg Asp Val Leu Asp Asp Ala Thr 130 135 140 Trp Gly Val Phe Gln Glu Gly Tyr Ser Glu Gly Phe Gly Ala Asp Ala 145 150 155 160 Asp His Val Lys Arg Pro Glu Asp Leu Val Ser Ala Ala Arg Glu Gly 165 170 175 Phe Thr Met Phe Ala Ile Asp Pro Ser Asp His Val Arg Asn Leu Ser 180 185 190 Lys Leu Thr Glu Lys Glu Arg Asn Glu Lys Phe Glu Glu Ile Leu Arg 195 200 205 Lys Glu Arg Ile Asp Arg Ile Tyr Leu Gly Lys Lys Tyr Ser Val Leu 210 215 220 Gly Glu Lys Ile Glu Phe Asp Glu Lys Asn Leu Arg Asp Ala Ala Leu 225 230 235 240 Val Tyr Tyr Asp Ala Ile Ala His Val Asp Met Met Tyr Gln Ile Leu 245 250 255 Lys Asp Glu T hr Pro Asp Phe Asp Phe Glu Val Ser Val Asp Glu Thr 260 265 270 Glu Thr Pro Thr Ser Pro Leu Phe His Ile Phe Val Val Glu Glu Leu 275 280 285 Arg Arg Arg Gly Val Glu Phe Thr Asn Leu Ala Leu Arg Phe Ile Gly 290 295 300 Glu Trp Glu Lys Gly Ile Asp Tyr Lys Gly Asp Leu Ala Gln Phe Glu 305 310 315 320 Arg Glu Ile Lys Met His Ala Glu Ile Ala Arg Met Phe Glu Gly Tyr 325 330 335 Lys Ile Ser Leu His Ser Gly Ser Asp Lys Phe Ser Val Tyr Pro Ala 340 345 350 Phe Ala Ser Ala Thr Gly Gly Leu Phe Leu Val Lys Thr Ala Gly Thr 355 360 365 Ser Tyr Leu Glu Ala Val Lys Val Ile Ser Met Val Asn Pro Glu Leu 370 375 380 Phe Arg Glu Ile Tyr Arg Cys Ala Leu Asp His Phe Glu Glu Asp Arg 385 390 395 400 L ys Ser Tyr His Ile Ser Ala Asp Leu Ser Lys Val Pro Glu Val Glu 405 410 415 Lys Val Lys Asp Glu Asp Leu Pro Gly Leu Phe Glu Asp Ile Asn Val 420 425 430 Arg Gln Leu Ile His Val Thr Tyr Gly Ser Val Leu Lys Asp Ala Ser 435 440 445 Leu Lys Glu Arg Leu Phe Lys Thr Leu Glu Gln Asn Glu Glu Leu Phe 450 455 460 Tyr Glu Thr Val Ala Lys His Ile Lys Arg His Val Asp Leu Leu Lys 465 470 475 480 Gly Glx <210> 6 <211> 482 <212> PRT <213> Artificial Sequence <220> <223> S125D/N129F/T181A/H362L mutant of tagaturonate epimerase <400> 6 Met Val Leu Lys Val Phe Lys Asp His Phe Gly Arg Gly Tyr Glu Val 1 5 10 15 Tyr Glu Lys Ser Tyr Arg Glu Lys Asp Ser Leu Ser Phe Phe Leu Thr 20 25 30 Lys Gly Glu Glu Gly Lys Ile Leu Val Val Ala Gly Glu Lys Ala Pro 35 40 45 Glu Gly Leu Ser Phe Phe Lys Lys Gln Arg Val Glu Gly Val Ser Phe 50 55 60 Phe Phe Cys Glu Arg Asn His Glu Asn Leu Glu Val Leu Arg Lys Tyr 65 70 75 80 Phe Pro Asp Leu Lys Pro Val Arg Ala Gly Leu Arg Ala Ser Phe Gly 85 90 95 Thr Gly Asp Arg Leu Gly Ile Thr Thr Pro Ala His Val Arg Ala L eu 100 105 110 Lys Asp Ser Gly Leu Phe Pro Ile Phe Ala Gln Gln Asp Val Arg Glu 115 120 125 Phe Glu Arg Thr Gly Arg Thr Trp Arg Asp Val Leu Asp Asp Ala Thr 130 135 140 Trp Gly Val Phe Gln Glu Gly Tyr Ser Glu Gly Phe Gly Ala Asp Ala 145 150 155 160 Asp His Val Lys Arg Pro Glu Asp Leu Val Ser Ala Ala Arg Glu Gly 165 170 175 Phe Thr Met Phe Ala Ile Asp Pro Ser Asp His Val Arg Asn Leu Ser 180 185 190 Lys Leu Thr Glu Lys Glu Arg Asn Glu Lys Phe Glu Glu Ile Leu Arg 195 200 205 Lys Glu Arg Ile Asp Arg Ile Tyr Leu Gly Lys Lys Tyr Ser Val Leu 210 215 220 Gly Glu Lys Ile Glu Phe Asp Glu Lys Asn Leu Arg Asp Ala Ala Leu 225 230 235 240 Val Tyr Tyr Asp Ala Ile Ala His Val Asp Met Met T yr Gln Ile Leu 245 250 255 Lys Asp Glu Thr Pro Asp Phe Asp Phe Glu Val Ser Val Asp Glu Thr 260 265 270 Glu Thr Pro Thr Ser Pro Leu Phe His Ile Phe Val Val Glu Glu Leu 275 280 285 Arg Arg Arg Gly Val Glu Phe Thr Asn Leu Ala Leu Arg Phe Ile Gly 290 295 300 Glu Trp Glu Lys Gly Ile Asp Tyr Lys Gly Asp Leu Ala Gln Phe Glu 305 310 315 320 Arg Glu Ile Lys Met His Ala Glu Ile Ala Arg Met Phe Glu Gly Tyr 325 330 335 Lys Ile Ser Leu His Ser Gly Ser Asp Lys Phe Ser Val Tyr Pro Ala 340 345 350 Phe Ala Ser Ala Thr Gly Gly Leu Phe Leu Val Lys Thr Ala Gly Thr 355 360 365 Ser Tyr Leu Glu Ala Val Lys Val Ile Ser Met Val Asn Pro Glu Leu 370 375 380 Phe Arg Glu Ile Tyr Arg Cys Ala Leu Asp His Phe Glu Glu A sp Arg 385 390 395 400 Lys Ser Tyr His Ile Ser Ala Asp Leu Ser Lys Val Pro Glu Val Glu 405 410 415 Lys Val Lys Asp Glu Asp Leu Pro Gly Leu Phe Glu Asp Ile Asn Val 420 425 430 Arg Gln Leu Ile His Val Thr Tyr Gly Ser Val Leu Lys Asp Ala Ser 435 440 445 Leu Lys Glu Arg Leu Phe Lys Thr Leu Glu Gln Asn Glu Glu Leu Phe 450 455 460 Tyr Glu Thr Val Ala Lys His Ile Lys Arg His Val Asp Leu Leu Lys 465 470 475 480 Gly Glx <210> 7 <211> 482 <212> PRT <213> Artificial Sequence <220> <223> S125D/L140P/T181A/H362L mutant of tagaturonate epimerase <400> 7 Met Val Leu Lys Val Phe Lys Asp His Phe Gly Arg Gly Tyr Glu Val 1 5 10 15 Tyr Glu Lys Ser Tyr Arg Glu Lys Asp Ser Leu Ser Phe Phe Leu Thr 20 25 30 Lys Gly Glu Glu Gly Lys Ile Leu Val Val Ala Gly Glu Lys Ala Pro 35 40 45 Glu Gly Leu Ser Phe Phe Lys Lys Gln Arg Val Glu Gly Val Ser Phe 50 55 60 Phe Phe Cys Glu Arg Asn His Glu Asn Leu Glu Val Leu Arg Lys Tyr 65 70 75 80 Phe Pro Asp Leu Lys Pro Val Arg Ala Gly Leu Arg Ala Ser Phe Gly 85 90 95 Thr Gly Asp Arg Leu Gly Ile Thr Thr Pro Ala His Val Arg Ala Leu 100 105 110 Lys Asp Ser Gly Leu Phe Pro Ile Phe Ala Gln Gln Asp Val Arg Glu 115 120 125 Asn Glu Arg Thr Gly Arg Thr Trp Arg Asp Val Pro Asp Asp Ala Thr 130 135 140 Trp Gly Val Phe Gln Glu Gly Tyr Ser Glu Gly Phe Gly Ala Asp Ala 145 150 155 160 Asp His Val Lys Arg Pro Glu Asp Leu Val Ser Ala Ala Arg Glu Gly 165 170 175 Phe Thr Met Phe Ala Ile Asp Pro Ser Asp His Val Arg Asn Leu Ser 180 185 190 Lys Leu Thr Glu Lys Glu Arg Asn Glu Lys Phe Glu Glu Ile Leu Arg 195 200 20 5 Lys Glu Arg Ile Asp Arg Ile Tyr Leu Gly Lys Lys Tyr Ser Val Leu 210 215 220 Gly Glu Lys Ile Glu Phe Asp Glu Lys Asn Leu Arg Asp Ala Ala Leu 225 230 235 240 Val Tyr Tyr Asp Ala Ile Ala His Val Asp Met Met Tyr Gln Ile Leu 245 250 255 Lys Asp Glu Thr Pro Asp Phe Asp Phe Glu Val Ser Val Asp Glu Thr 260 265 270 Glu Thr Pro Thr Ser Pro Leu Phe His Ile Phe Val Val Glu Glu Leu 275 280 285 Arg Arg Arg Gly Val Glu Phe Thr Asn Leu Ala Leu Arg Phe Ile Gly 290 295 300 Glu Trp Glu Lys Gly Ile Asp Tyr Lys Gly Asp Leu Ala Gln Phe Glu 305 310 315 320 Arg Glu Ile Lys Met His Ala Glu Ile Ala Arg Met Phe Glu Gly Tyr 325 330 335 Lys Ile Ser Leu His Ser Gly Ser Asp Lys Phe Ser Val Tyr Pro Ala 340 345 350 Phe Ala Ser Ala Thr Gly Gly Leu Phe Leu Val Lys Thr Ala Gly Thr 355 360 365 Ser Tyr Leu Glu Ala Val Lys Val Ile Ser Met Val Asn Pro Glu Leu 370 375 380 Phe Arg Glu Ile Tyr Arg Cys Ala Leu Asp His Phe Glu Glu Asp Arg 385 390 395 400 Lys Ser Tyr His Ile Ser Ala Asp Leu Ser Lys Val Pro Glu Val Glu 405 410 415 Lys Val Lys Asp Glu Asp Leu Pro Gly Leu Phe Glu Asp Ile Asn Val 420 425 430 Arg Gln Leu Ile His Val Thr Tyr Gly Ser Val Leu Lys Asp Ala Ser 435 440 445 Leu Lys Glu Arg Leu Phe Lys Thr Leu Glu Gln Asn Glu Glu Leu Phe 450 455 460 Tyr Glu Thr Val Ala Lys His Ile Lys Arg His Val Asp Leu Leu Lys 465 470 475 480 Gly Glx <210> 8 <211> 482 <212> PRT <213> Artificial Sequence <220> <223> S125D/N1 29S/L140F/T181A/H362L mutant of tagaturonate epimerase <400> 8 Met Val Leu Lys Val Phe Lys Asp His Phe Gly Arg Gly Tyr Glu Val 1 5 10 15 Tyr Glu Lys Ser Tyr Arg Glu Lys Asp Ser Leu Ser Phe Phe Leu Thr 20 25 30 Lys Gly Glu Glu Gly Lys Ile Leu Val Val Ala Gly Glu Lys Ala Pro 35 40 45 Glu Gly Leu Ser Phe Phe Lys Lys Gln Arg Val Glu Gly Val Ser Phe 50 55 60 Phe Phe Cys Glu Arg Asn His Glu Asn Leu Glu Val Leu Arg Lys Tyr 65 70 75 80 Phe Pro Asp Leu Lys Pro Val Arg Ala Gly Leu Arg Ala Ser Phe Gly 85 90 95 Thr Gly Asp Arg Leu Gly Ile Thr Thr Pro Ala His Val Arg Ala Leu 100 105 110 Lys Asp Ser Gly Leu Phe Pro Ile Phe Ala Gln Gln Asp Val Arg Glu 115 120 125 Ser Glu Arg Thr Gly Arg Thr Trp Arg Asp Val Phe Asp Asp Ala Thr 130 135 140 Trp Gly Val Phe Gln Glu Gly Tyr Ser Glu Gly Phe Gly Ala Asp Ala 145 150 155 160 Asp His Val Lys Arg Pro Glu A sp Leu Val Ser Ala Ala Arg Glu Gly 165 170 175 Phe Thr Met Phe Ala Ile Asp Pro Ser Asp His Val Arg Asn Leu Ser 180 185 190 Lys Leu Thr Glu Lys Glu Arg Asn Glu Lys Phe Glu Glu Ile Leu Arg 195 200 205 Lys Glu Arg Ile Asp Arg Ile Tyr Leu Gly Lys Lys Tyr Ser Val Leu 210 215 220 Gly Glu Lys Ile Glu Phe Asp Glu Lys Asn Leu Arg Asp Ala Ala Leu 225 230 235 240 Val Tyr Tyr Asp Ala Ile Ala His Val Asp Met Met Tyr Gln Ile Leu 245 250 255 Lys Asp Glu Thr Pro Asp Phe Asp Phe Glu Val Ser Val Asp Glu Thr 260 265 270 Glu Thr Pro Thr Ser Pro Leu Phe His Ile Phe Val Val Glu Glu Leu 275 280 285 Arg Arg Arg Gly Val Glu Phe Thr Asn Leu Ala Leu Arg Phe Ile Gly 290 295 300 Glu Trp Glu Lys Gly Ile Asp Tyr Lys G ly Asp Leu Ala Gln Phe Glu 305 310 315 320 Arg Glu Ile Lys Met His Ala Glu Ile Ala Arg Met Phe Glu Gly Tyr 325 330 335 Lys Ile Ser Leu His Ser Gly Ser Asp Lys Phe Ser Val Tyr Pro Ala 340 345 350 Phe Ala Ser Ala Thr Gly Gly Leu Phe Leu Val Lys Thr Ala Gly Thr 355 360 365 Ser Tyr Leu Glu Ala Val Lys Val Ile Ser Met Val Asn Pro Glu Leu 370 375 380 Phe Arg Glu Ile Tyr Arg Cys Ala Leu Asp His Phe Glu Glu Asp Arg 385 390 395 400 Lys Ser Tyr His Ile Ser Ala Asp Leu Ser Lys Val Pro Glu Val Glu 405 410 415 Lys Val Lys Asp Glu Asp Leu Pro Gly Leu Phe Glu Asp Ile Asn Val 420 425 430 Arg Gln Leu Ile His Val Thr Tyr Gly Ser Val Leu Lys Asp Ala Ser 435 440 445 Leu Lys Glu Arg Leu Phe L ys Thr Leu Glu Gln Asn Glu Glu Leu Phe 450 455 460 Tyr Glu Thr Val Ala Lys His Ile Lys Arg His Val Asp Leu Leu Lys 465 470 475 480 Gly Glx <210> 9 <211> 482 <212> PRT <213> Artificial Sequence <220> <223> S125D/N129T/L140K/T181A/H362L mutant of tagaturonate epimerase <400> 9 Met Val Leu Lys Val Phe Lys Asp His Phe Gly Arg Gly Tyr Glu Val 1 5 10 15 Tyr Glu Lys Ser Tyr Arg Glu Lys Asp Ser Leu Ser Phe Phe Leu Thr 20 25 30 Lys Gly Glu Glu Gly Lys Ile Leu Val Val Ala Gly Glu Lys Ala Pro 35 40 45 Glu Gly Leu Ser Phe Phe Lys Lys Gln Arg Val Glu Gly Val Ser Phe 50 55 60 Phe Phe Cys Glu Arg Asn His Glu Asn Leu Glu Val Leu Arg Lys Tyr 65 70 75 80 Phe Pro Asp Leu Lys Pro Val Arg Ala Gly Leu Arg Ala Ser Phe Gly 85 90 95 Thr Gly Asp Arg Leu Gly Ile Thr Thr Pro Ala His Val Arg Ala Leu 100 105 110 Lys Asp Ser Gly Leu Phe Pro Ile Phe Ala Gln Gln Asp Val Arg Glu 115 120 125 Thr Glu Arg Thr Gly Arg Thr Trp Arg Asp Val Lys Asp Asp Ala Thr 130 135 140 Trp Gly Val Phe Gln Glu Gly Tyr Ser Glu Gly Phe Gly Ala Asp Ala 145 150 155 160 Asp His Val Lys Arg Pro Glu Asp Leu Val Ser Ala Ala Arg Glu Gly 165 170 175 Phe Thr Met Phe Ala Ile Asp Pro Ser Asp His Val Arg Asn Leu Ser 180 185 190 Lys Leu Thr Glu Lys Glu Arg Asn Glu Lys Phe Glu Glu Ile Leu Arg 195 200 205 Lys Glu Arg Ile Asp Arg Ile Tyr Leu Gly Lys Lys Tyr Ser Val Leu 210 215 220 Gly Glu Lys Ile Glu Phe Asp Glu Lys Asn Leu Arg Asp Ala Ala Leu 225 230 235 240 Val Tyr Tyr Asp Ala Ile Ala His Val Asp Met Met Tyr Gln Ile Leu 245 250 255 Lys Asp Glu Thr Pro Asp Phe Asp Phe Glu Val Ser Val Asp Glu Thr 260 265 270 Glu Thr Pro Thr Ser Pro Leu Phe His Ile Phe Val Val Glu Glu Leu 275 280 285 Arg Arg Arg Gly Val Glu Phe Thr Asn Leu Ala Leu Arg Phe Ile Gly 290 295 300 Glu Trp Glu Lys Gly Ile Asp Tyr Lys Gly Asp Leu Ala Gln Phe Glu 305 310 315 320 Arg Glu Ile Lys Met His Ala Glu Ile Ala Arg Met Phe Glu Gly Tyr 325 330 335 Lys Ile Ser Leu His Ser Gly Ser Asp Lys Phe Ser Val Tyr Pro Ala 340 345 350 Phe Ala Ser Ala Thr Gly Gly Leu Phe Leu Val Lys Thr Ala Gly Thr 355 360 365 Ser Tyr Leu Glu Ala Val Lys Val Ile Ser Met Val Asn Pro Glu Leu 370 375 380 Phe Arg Glu Ile Tyr Arg Cys Ala Leu Asp His Phe Glu Glu Asp Arg 385 390 395 400 Lys Ser Tyr His Ile Ser Ala Asp Leu Ser Lys Val Pro Glu Val Glu 405 410 415 Lys Val Lys Asp Glu Asp Leu Pro Gly Leu Phe Glu Asp Ile Asn Val 420 425 430 Arg Gln Leu Ile His Val Thr Tyr Gly Ser Val Leu Lys Asp Ala Ser 435 440 445 Leu Lys Glu Arg Leu Phe Lys Thr Leu Glu Gln Asn Glu Glu Leu Phe 450 455 460 Tyr Glu Thr Val Ala Lys His Ile Lys Arg His Val Asp Leu Leu Lys 465 470 475 480 Gly Glx <210> 10 <211> 482 <212> PRT <213> Artificial Sequence <220> <223> S125D/N129T/L140F/T181A/H362L mutant of tagaturonate epimerase <400> 10 Met Val Leu Lys Val Phe Lys Asp His Phe Gly Arg Gly Tyr Glu Val 1 5 10 15 Tyr Glu Lys Ser Tyr Arg Glu Lys Asp Ser Leu Ser Phe Phe Leu Thr 20 25 30 Lys Gly Glu Glu Gly Lys Ile Leu Val Val Ala Gly Glu Lys Ala Pro 35 40 45 Glu Gly Leu Ser Phe Phe Lys Lys Gln Arg Val Glu Gly Val Ser Phe 50 55 60 Phe Phe Cys Glu Arg Asn His Glu Asn Leu Gl u Val Leu Arg Lys Tyr 65 70 75 80 Phe Pro Asp Leu Lys Pro Val Arg Ala Gly Leu Arg Ala Ser Phe Gly 85 90 95 Thr Gly Asp Arg Leu Gly Ile Thr Thr Pro Ala His Val Arg Ala Leu 100 105 110 Lys Asp Ser Gly Leu Phe Pro Ile Phe Ala Gln Gln Asp Val Arg Glu 115 120 125 Thr Glu Arg Thr Gly Arg Thr Trp Arg Asp Val Phe Asp Asp Ala Thr 130 135 140 Trp Gly Val Phe Gln Glu Gly Tyr Ser Glu Gly Phe Gly Ala Asp Ala 145 150 155 160 Asp His Val Lys Arg Pro Glu Asp Leu Val Ser Ala Ala Arg Glu Gly 165 170 175 Phe Thr Met Phe Ala Ile Asp Pro Ser Asp His Val Arg Asn Leu Ser 180 185 190 Lys Leu Thr Glu Lys Glu Arg Asn Glu Lys Phe Glu Glu Ile Leu Arg 195 200 205 Lys Glu Arg Ile Asp Arg Ile Tyr Leu Gly Lys Lys Tyr Ser Val Leu 210 215 220 Gly Glu L ys Ile Glu Phe Asp Glu Lys Asn Leu Arg Asp Ala Ala Leu 225 230 235 240 Val Tyr Tyr Asp Ala Ile Ala His Val Asp Met Met Tyr Gln Ile Leu 245 250 255 Lys Asp Glu Thr Pro Asp Phe Asp Phe Glu Val Ser Val Asp Glu Thr 260 265 270 Glu Thr Pro Thr Ser Pro Leu Phe His Ile Phe Val Val Glu Glu Leu 275 280 285 Arg Arg Arg Gly Val Glu Phe Thr Asn Leu Ala Leu Arg Phe Ile Gly 290 295 300 Glu Trp Glu Lys Gly Ile Asp Tyr Lys Gly Asp Leu Ala Gln Phe Glu 305 310 315 320 Arg Glu Ile Lys Met His Ala Glu Ile Ala Arg Met Phe Glu Gly Tyr 325 330 335 Lys Ile Ser Leu His Ser Gly Ser Asp Lys Phe Ser Val Tyr Pro Ala 340 345 350 Phe Ala Ser Ala Thr Gly Gly Leu Phe Leu Val Lys Thr Ala Gly Thr 355 360 3 65 Ser Tyr Leu Glu Ala Val Lys Val Ile Ser Met Val Asn Pro Glu Leu 370 375 380 Phe Arg Glu Ile Tyr Arg Cys Ala Leu Asp His Phe Glu Glu Asp Arg 385 390 395 400 Lys Ser Tyr His Ile Ser Ala Asp Leu Ser Lys Val Pro Glu Val Glu 405 410 415 Lys Val Lys Asp Glu Asp Leu Pro Gly Leu Phe Glu Asp Ile Asn Val 420 425 430 Arg Gln Leu Ile His Val Thr Tyr Gly Ser Val Leu Lys Asp Ala Ser 435 440 445 Leu Lys Glu Arg Leu Phe Lys Thr Leu Glu Gln Asn Glu Glu Leu Phe 450 455 460 Tyr Glu Thr Val Ala Lys His Ile Lys Arg His Val Asp Leu Leu Lys 465 470 475 480 Gly Glx <210 > 11 <211> 482 <212> PRT <213> Artificial Sequence <220> <223> S125D/N129T/L140P/T181A/H362L mutant of tagaturonate epimerase <400> 11 Met Val Leu Lys Val Phe Lys Asp His Phe Gly Arg Gly Tyr Glu Val 1 5 10 15 Tyr Glu Lys Ser Tyr Arg Glu Lys Asp Ser Leu Ser Phe Phe Leu Thr 20 25 30 Lys Gly Glu Glu Gly Lys Ile Leu Val Val Ala Gly Glu Lys Ala Pro 35 40 45 Glu Gly Leu Ser Phe Phe Lys Lys Gln Arg Val Glu Gly Val Ser Phe 50 55 60 Phe Phe Cys Glu Arg Asn His Glu Asn Leu Glu Val Leu Arg Lys Tyr 65 70 75 80 Phe Pro Asp Leu Lys Pro Val Arg Ala Gly Leu Arg Ala Ser Phe Gly 85 90 95 Thr Gly Asp Arg Leu Gly Ile Thr Thr Pro Ala His Val Arg Ala Leu 100 105 110 Lys Asp Ser Gly Leu Phe Pro Ile Phe Ala Gln Gln Asp Val Arg Glu 115 120 125 Thr Glu Arg Thr Gly Arg Thr Trp Arg Asp Val Pro Asp Asp Ala Thr 130 135 140 Trp Gly Val Phe Gln Glu Gly Tyr Ser Glu Gly Phe Gly Ala Asp Ala 145 150 155 160 Asp His Val Lys Arg Pro Glu Asp Leu Val Ser Ala Ala Arg Glu Gly 165 170 175 Phe Thr Met Phe Ala Ile Asp Pro Ser Asp His Val Arg Asn Leu Ser 180 185 190 Lys Leu Thr Glu Lys Glu Arg Asn Glu Lys Phe Glu Glu Ile Leu Arg 195 200 205 Lys Glu Arg Ile Asp Arg Ile Tyr Leu Gly Lys Lys Tyr Ser Val L eu 210 215 220 Gly Glu Lys Ile Glu Phe Asp Glu Lys Asn Leu Arg Asp Ala Ala Leu 225 230 235 240 Val Tyr Tyr Asp Ala Ile Ala His Val Asp Met Met Tyr Gln Ile Leu 245 250 255 Lys Asp Glu Thr Pro Asp Phe Asp Phe Glu Val Ser Val Asp Glu Thr 260 265 270 Glu Thr Pro Thr Ser Pro Leu Phe His Ile Phe Val Val Glu Glu Leu 275 280 285 Arg Arg Arg Gly Val Glu Phe Thr Asn Leu Ala Leu Arg Phe Ile Gly 290 295 300 Glu Trp Glu Lys Gly Ile Asp Tyr Lys Gly Asp Leu Ala Gln Phe Glu 305 310 315 320 Arg Glu Ile Lys Met His Ala Glu Ile Ala Arg Met Phe Glu Gly Tyr 325 330 335 Lys Ile Ser Leu His Ser Gly Ser Asp Lys Phe Ser Val Tyr Pro Ala 340 345 350 Phe Ala Ser Ala Thr Gly Gly Leu Phe Leu Val Lys T hr Ala Gly Thr 355 360 365 Ser Tyr Leu Glu Ala Val Lys Val Ile Ser Met Val Asn Pro Glu Leu 370 375 380 Phe Arg Glu Ile Tyr Arg Cys Ala Leu Asp His Phe Glu Glu Asp Arg 385 390 395 400 Lys Ser Tyr His Ile Ser Ala Asp Leu Ser Lys Val Pro Glu Val Glu 405 410 415 Lys Val Lys Asp Glu Asp Leu Pro Gly Leu Phe Glu Asp Ile Asn Val 420 425 430 Arg Gln Leu Ile His Val Thr Tyr Gly Ser Val Leu Lys Asp Ala Ser 435 440 445 Leu Lys Glu Arg Leu Phe Lys Thr Leu Glu Gln Asn Glu Glu Leu Phe 450 455 460 Tyr Glu Thr Val Ala Lys His Ile Lys Arg His Val Asp Leu Leu Lys 465 470 475 480 Gly Glx <210> 12 <211 > 1446 <212> DNA <213> Unknown <220> <223> Thermotoga petrophila <400> 12 atggtcttga aagtgttcaa agatcacttt ggaaggggat acgaag ttta cgaaaagtct 60 tatagagaaa aggattctct ctctttcttc ttgacaaagg gagaggaagg aaaaattctg 120 gtagtggctg gagaaaaggc acctgagggt ctgtcgtttt tcaaaaaaca gcgggtggag 180 ggtgtttcgt tctttttctg tgagagaaat catgagaact tggaagttct cagaaaatac 240 tttccagatc tcaaaccagt tcgagcggga ttgagagcgt cttttggaac aggtgacaga 300 ctcggtatca ccacaccggc tcacgtgagg gcgttgaagg attcagggct ttttcccatc 360 tttgcgcagc agtcggtgag ggagaacgag agaacgggaa ggacctggag agatgtgctg 420 gacgatgcca catggggagt tttccaggag ggatacagtg agggattcgg agcagacgcc 480 gatcacgtga agcggccgga ggatcttgtt tcggctgcaa gggaaggttt caccatgttc 540 acaatcgatc cttcggatca tgtgaggaat ctttcaaaac ttacagaaaa ggaaagaaat 600 gagaaattcg aagagattct gagaaaggaa aggatcgaca ggatctatct cggtaagaaa 660 tactctgttc tcggtgagaa gatcgaattc gatgagaaga atctcagaga tgcggcgctc 720 gtgtattacg atgcgattgc ccacgtggat atgatgtatc aaattttgaa agacgaaacc 780 ccggatttcg acttcgaagt gtcagttgac gaaacagaaa ctcccacgag tcctctcttc 840 cacattttcg ttgtggaaga actcagacga agaggtgtgg agttcaccaa tcttgccctg 900 a gattcatcg gcgaatggga aaagggaata gattacaagg gggatcttgc acagttcgag 960 agagaaatca aaatgcacgc agaaatcgca aggatgttcg aaggatacaa aatatcactc 1020 cactctggaa gcgacaaatt ttccgtgtat cctgcttttg cttccgcgac aggaggcctt 1080 ttccacgtga agacagccgg aacgagttat cttgaggcgg tgaaggtcat atccatggtc 1140 aacccggagc tcttccggga gatctacagg tgtgctctcg atcactttga ggaagacaga 1200 aagtcctatc acatatctgc ggatctgtcg aaagttccgg aagtagagaa agtgaaagat 1260 gaagatcttc caggtctttt tgaagacatc aacgtgagac agttgatcca tgtcacctat 1320 ggctctgttc tgaaagatgc atctttgaaa gaacggctgt ttaagacgct tgaacaaaat 1380 gaggaactct tctacgagac cgtggcaaaa catataaaaa ggcacgtaga cctgttgaag 1440 cctgtga 1446 <210> 13 <tag211> 1446 t mutmer <gt a tc t c t t ggtga 1446 <210> 13 <tag211> 1446 t mut aga <gt a c t c t a t gg t a artificial t t a t aga < gt a t a t t g t na c t t o c t t o t aga < gt 223 ggaaggggat acgaagttta cgaaaagtct 60 tatagagaaa aggattctct ctctttcttc ttgacaaagg gagaggaagg aaaaattctg 120 gtagtggctg gagaaaaggc acctgagggt ctgtcgttttt tct ggtagtgtt gc gg ggtaaaca t agaaat catgagaact tggaagttct cagaaaatac 240 tttccagatc tcaaaccagt tcgagcggga ttgagagcgt cttttggaac aggtgacaga 300 ctcggtatca ccacaccggc tcacgtgagg gcgttgaagg attcagggct ttttcccatc 360 tttgcgcagc aggatgtgag ggagaacgag agaacgggaa ggacctggag agatgtgctg 420 gacgatgcca catggggagt tttccaggag ggatacagtg agggattcgg agcagacgcc 480 gatcacgtga agcggccgga ggatcttgtt tcggctgcaa gggaaggttt caccatgttc 540 gcaatcgatc cttcggatca tgtgaggaat ctttcaaaac ttacagaaaa ggaaagaaat 600 gagaaattcg aagagattct gagaaaggaa aggatcgaca ggatctatct cggtaagaaa 660 tactctgttc tcggtgagaa gatcgaattc gatgagaaga atctcagaga tgcggcgctc 720 gtgtattacg atgcgattgc ccacgtggat atgatgtatc aaattttgaa agacgaaacc 780 ccggatttcg acttcgaagt gtcagttgac gaaacagaaa ctcccacgag tcctctcttc 840 cacattttcg ttgtggaaga actcagacga agaggtgtgg agttcaccaa tcttgccctg 900 agattcatcg gcgaatggga aaagggaata gattacaagg gggatcttgc acagttcgag 960 agagaaatca aaatgcacgc agaaatcgca aggatgttcg aaggatacaa aatatcactc 1020 cactctggaa gcgacaaatt ttccgtgtat cctgcttttg c ttccgcgac aggaggcctt 1080 ttcctcgtga agacagccgg aacgagttat cttgaggcgg tgaaggtcat atccatggtc 1140 aacccggagc tcttccggga gatctacagg tgtgctctcg atcactttga ggaagacaga 1200 aagtcctatc acatatctgc ggatctgtcg aaagttccgg aagtagagaa agtgaaagat 1260 gaagatcttc caggtctttt tgaagacatc aacgtgagac agttgatcca tgtcacctat 1320 ggctctgttc tgaaagatgc atctttgaaa gaacggctgt ttaagacgct tgaacaaaat 1380 gaggaactct tctacgagac cgtggcaaaa catataaaaa ggcacgtaga cctgttgaag 1440 gggtga 1446 <210> 14 <211> 1446 <212> DNA <213> Artificial Sequence <220> <223> S125D/T181A/H362L mutant of tagaturonate epimerase <400> 14 atggtcttga aagtgttcaa agatcacttt ggaaggggat acgagtagtta ggta ggt ggt ggt ggt ggt gatta ggt gatta gatta gatta accatta gatta gatta accatta ttagagaaag agg aggatta ctgtcgtttt tcaaaaaaca gcgggtggag 180 ggtgtttcgt tctttttctg tgagagaaat catgagaact tggaagttct cagaaaatac 240 tttccagatc tcaaaccagt tcgagcggga ttgagagcgt cttttggaac aggtgacaga 300 ctcggtatca ccacaccggc tcacgtgagg gcgttgaagg attcagggct tttt cccatc 360 tttgcgcagc aggatgtgag ggagaacgag agaacgggaa ggacctggag agatgtgctg 420 gacgatgcca catggggagt tttccaggag ggatacagtg agggattcgg agcagacgcc 480 gatcacgtga agcggccgga ggatcttgtt tcggctgcaa gggaaggttt caccatgttc 540 gcaatcgatc cttcggatca tgtgaggaat ctttcaaaac ttacagaaaa ggaaagaaat 600 gagaaattcg aagagattct gagaaaggaa aggatcgaca ggatctatct cggtaagaaa 660 tactctgttc tcggtgagaa gatcgaattc gatgagaaga atctcagaga tgcggcgctc 720 gtgtattacg atgcgattgc ccacgtggat atgatgtatc aaattttgaa agacgaaacc 780 ccggatttcg acttcgaagt gtcagttgac gaaacagaaa ctcccacgag tcctctcttc 840 cacattttcg ttgtggaaga actcagacga agaggtgtgg agttcaccaa tcttgccctg 900 agattcatcg gcgaatggga aaagggaata gattacaagg gggatcttgc acagttcgag 960 agagaaatca aaatgcacgc agaaatcgca aggatgttcg aaggatacaa aatatcactc 1020 cactctggaa gcgacaaatt ttccgtgtat cctgcttttg cttccgcgac aggaggcctt 1080 ttcctcgtga agacagccgg aacgagttat cttgaggcgg tgaaggtcat atccatggtc 1140 aacccggagc tcttccggga gatctacagg tgtgctctcg atcactttga ggaagacaga 1200 aagtc ctatc acatatctgc ggatctgtcg aaagttccgg aagtagagaa agtgaaagat 1260 gaagatcttc caggtctttt tgaagacatc aacgtgagac agttgatcca tgtcacctat 1320 ggctctgttc tgaaagatgc atctttgaaa gaacggctgt ttaagacgct tgaacaaaat 1380 gaggaactct tctacgagac cgtggcaaaa catataaaaa ggcacgtaga cctgttgaag 1440 gggtga 1446 <210> 15 <211> 1446 <212> DNA <213> Artificial Sequence <220> <223> F66S / S125D / T181A / H362L mutant of tagaturonate epimerase <400> 15 atggtcttga aagtgttcaa agatcacttt ggaaggggat acgaagttta cgaaaagtct 60 tatagagaaa aggattctct ctctttcttc ttgacaaagg gagaggaagg aaaaattctg 120 gtagtggctg gagaaaaggc acctgagggt ctgtcgtttt tcaaaaaaca gcgggtggag 180 ggtgtttcgt tctttagctg tgagagaaat catgagaact tggaagttct cagaaaatac 240 tttccagatc tcaaaccagt tcgagcggga ttgagagcgt cttttggaac aggtgacaga 300 ctcggtatca ccacaccggc tcacgtgagg gcgttgaagg attcagggct ttttcccatc 360 tttgcgcagc aggatgtgag ggagaacgag agaacgggaa ggacctggag agatgtgctg 420 gacgatgcca catggggagt tttccaggag ggatacagtg agggattcgg agcagacgcc 480 gatcacgtga agcggc cgga ggatcttgtt tcggctgcaa gggaaggttt caccatgttc 540 gcaatcgatc cttcggatca tgtgaggaat ctttcaaaac ttacagaaaa ggaaagaaat 600 gagaaattcg aagagattct gagaaaggaa aggatcgaca ggatctatct cggtaagaaa 660 tactctgttc tcggtgagaa gatcgaattc gatgagaaga atctcagaga tgcggcgctc 720 gtgtattacg atgcgattgc ccacgtggat atgatgtatc aaattttgaa agacgaaacc 780 ccggatttcg acttcgaagt gtcagttgac gaaacagaaa ctcccacgag tcctctcttc 840 cacattttcg ttgtggaaga actcagacga agaggtgtgg agttcaccaa tcttgccctg 900 agattcatcg gcgaatggga aaagggaata gattacaagg gggatcttgc acagttcgag 960 agagaaatca aaatgcacgc agaaatcgca aggatgttcg aaggatacaa aatatcactc 1020 cactctggaa gcgacaaatt ttccgtgtat cctgcttttg cttccgcgac aggaggcctt 1080 ttcctcgtga agacagccgg aacgagttat cttgaggcgg tgaaggtcat atccatggtc 1140 aacccggagc tcttccggga gatctacagg tgtgctctcg atcactttga ggaagacaga 1200 aagtcctatc acatatctgc ggatctgtcg aaagttccgg aagtagagaa agtgaaagat 1260 gaagatcttc caggtctttt tgaagacatc aacgtgagac agttgatcca tgtcacctat 1320 ggctctgttc tgaaagatgc atctttgaa a gaacggctgt ttaagacgct tgaacaaaat 1380 gaggaactct tctacgagac cgtggcaaaa catataaaaa ggcacgtaga cctgttgaag 1440 gggtga 1446 <210> 16 <211> 1446 <212> DNA <213> Artificial Sequence <220> <223> E72G/HaturonSate125D/T181G/HaturonSate125D/T400A/ > 16 atggtcttga aagtgttcaa agatcacttt ggaaggggat acgaagttta cgaaaagtct 60 tatagagaaa aggattctct ctctttcttc ttgacaaagg gagaggaagg aaaaattctg 120 gtagtggctg gagaaaaggc acctgagggt ctgtcgtttt tcaaaaaaca gcgggtggag 180 ggtgtttcgt tctttttctg tgagagaaat catgggaact tggaagttct cagaaaatac 240 tttccagatc tcaaaccagt tcgagcggga ttgagagcgt cttttggaac aggtgacaga 300 ctcggtatca ccacaccggc tcacgtgagg gcgttgaagg attcagggct ttttcccatc 360 tttgcgcagc aggatgtgag ggagaacgag agaacgggaa ggacctggag agatgtgctg 420 gacgatgcca catggggagt tttccaggag ggatacagtg agggattcgg agcagacgcc 480 gatcacgtga agcggccgga ggatcttgtt tcggctgcaa gggaaggttt caccatgttc 540 gcaatcgatc cttcggatca tgtgaggaat ctttcaaaac ttacagaaaa ggaaagaaat 600 gagaaattcg aagagattct gagaaaggaa aggatcgaca g gatctatct cggtaagaaa 660 tactctgttc tcggtgagaa gatcgaattc gatgagaaga atctcagaga tgcggcgctc 720 gtgtattacg atgcgattgc ccacgtggat atgatgtatc aaattttgaa agacgaaacc 780 ccggatttcg acttcgaagt gtcagttgac gaaacagaaa ctcccacgag tcctctcttc 840 cacattttcg ttgtggaaga actcagacga agaggtgtgg agttcaccaa tcttgccctg 900 agattcatcg gcgaatggga aaagggaata gattacaagg gggatcttgc acagttcgag 960 agagaaatca aaatgcacgc agaaatcgca aggatgttcg aaggatacaa aatatcactc 1020 cactctggaa gcgacaaatt ttccgtgtat cctgcttttg cttccgcgac aggaggcctt 1080 ttcctcgtga agacagccgg aacgagttat cttgaggcgg tgaaggtcat atccatggtc 1140 aacccggagc tcttccggga gatctacagg tgtgctctcg atcactttga ggaagacaga 1200 aagtcctatc acatatctgc ggatctgtcg aaagttccgg aagtagagaa agtgaaagat 1260 gaagatcttc caggtctttt tgaagacatc aacgtgagac agttgatcca tgtcacctat 1320 ggctctgttc tgaaagatgc atctttgaaa gaacggctgt ttaagacgct tgaacaaaat 1380 gaggaactct tctacgagac cgtggcaaaa catataaaaa ggcacgtaga cctgttgaag 1440 gggtga 1446 <210> 17 <211> 1446 <212> DNA <213> Artificia l Sequence <220> <223> S125D / N129F / T181A / H362L mutant of tagaturonate epimerase <400> 17 atggtcttga aagtgttcaa agatcacttt ggaaggggat acgaagttta cgaaaagtct 60 tatagagaaa aggattctct ctctttcttc ttgacaaagg gagaggaagg aaaaattctg 120 gtagtggctg gagaaaaggc acctgagggt ctgtcgtttt tcaaaaaaca gcgggtggag 180 ggtgtttcgt tctttttctg tgagagaaat catgagaact tggaagttct cagaaaatac 240 tttccagatc tcaaaccagt tcgagcggga ttgagagcgt cttttggaac aggtgacaga 300 ctcggtatca ccacaccggc tcacgtgagg gcgttgaagg attcagggct ttttcccatc 360 tttgcgcagc aggatgtgag ggagttcgag agaacgggaa ggacctggag agatgtgctg 420 gacgatgcca catggggagt tttccaggag ggatacagtg agggattcgg agcagacgcc 480 gatcacgtga agcggccgga ggatcttgtt tcggctgcaa gggaaggttt caccatgttc 540 gcaatcgatc cttcggatca tgtgaggaat ctttcaaaac ttacagaaaa ggaaagaaat 600 gagaaattcg aagagattct gagaaaggaa aggatcgaca ggatctatct cggtaagaaa 660 tactctgttc tcggtgagaa gatcgaattc gatgagaaga atctcagaga tgcggcgctc 720 gtgtattacg atgcgattgc ccacgtggat atgatgtatc aaattttgaa agacgaaacc 780 cc ggatttcg acttcgaagt gtcagttgac gaaacagaaa ctcccacgag tcctctcttc 840 cacattttcg ttgtggaaga actcagacga agaggtgtgg agttcaccaa tcttgccctg 900 agattcatcg gcgaatggga aaagggaata gattacaagg gggatcttgc acagttcgag 960 agagaaatca aaatgcacgc agaaatcgca aggatgttcg aaggatacaa aatatcactc 1020 cactctggaa gcgacaaatt ttccgtgtat cctgcttttg cttccgcgac aggaggcctt 1080 ttcctcgtga agacagccgg aacgagttat cttgaggcgg tgaaggtcat atccatggtc 1140 aacccggagc tcttccggga gatctacagg tgtgctctcg atcactttga ggaagacaga 1200 aagtcctatc acatatctgc ggatctgtcg aaagttccgg aagtagagaa agtgaaagat 1260 gaagatcttc caggtctttt tgaagacatc aacgtgagac agttgatcca tgtcacctat 1320 ggctctgttc tgaaagatgc atctttgaaa gaacggctgt ttaagacgct tgaacaaaat 1380 gaggaactct tctacgagac cgtggcaaaa catataaaaa ggcacgtaga cctgttgaag 1440 gggtga 1446 <210> 18 <211> 1446 <212> DNA <213> Artificial Sequence <220> < 223> S125D/L140P/T181A/H362L mutant of tagaturonate epimerase <400> 18 atggtcttga aagtgttcaa agatcacttt ggaaggggat acgaagttta cgaaaagtct 60 tatagagaa a aggattctct ctctttcttc ttgacaaagg gagaggaagg aaaaattctg 120 gtagtggctg gagaaaaggc acctgagggt ctgtcgtttt tcaaaaaaca gcgggtggag 180 ggtgtttcgt tctttttctg tgagagaaat catgagaact tggaagttct cagaaaatac 240 tttccagatc tcaaaccagt tcgagcggga ttgagagcgt cttttggaac aggtgacaga 300 ctcggtatca ccacaccggc tcacgtgagg gcgttgaagg attcagggct ttttcccatc 360 tttgcgcagc aggatgtgag ggagaacgag agaacgggaa ggacctggag agatgtgccg 420 gacgatgcca catggggagt tttccaggag ggatacagtg agggattcgg agcagacgcc 480 gatcacgtga agcggccgga ggatcttgtt tcggctgcaa gggaaggttt caccatgttc 540 gcaatcgatc cttcggatca tgtgaggaat ctttcaaaac ttacagaaaa ggaaagaaat 600 gagaaattcg aagagattct gagaaaggaa aggatcgaca ggatctatct cggtaagaaa 660 tactctgttc tcggtgagaa gatcgaattc gatgagaaga atctcagaga tgcggcgctc 720 gtgtattacg atgcgattgc ccacgtggat atgatgtatc aaattttgaa agacgaaacc 780 ccggatttcg acttcgaagt gtcagttgac gaaacagaaa ctcccacgag tcctctcttc 840 cacattttcg ttgtggaaga actcagacga agaggtgtgg agttcaccaa tcttgccctg 900 agattcatcg gcgaatggga aaaggga ata gattacaagg gggatcttgc acagttcgag 960 agagaaatca aaatgcacgc agaaatcgca aggatgttcg aaggatacaa aatatcactc 1020 cactctggaa gcgacaaatt ttccgtgtat cctgcttttg cttccgcgac aggaggcctt 1080 ttcctcgtga agacagccgg aacgagttat cttgaggcgg tgaaggtcat atccatggtc 1140 aacccggagc tcttccggga gatctacagg tgtgctctcg atcactttga ggaagacaga 1200 aagtcctatc acatatctgc ggatctgtcg aaagttccgg aagtagagaa agtgaaagat 1260 gaagatcttc caggtctttt tgaagacatc aacgtgagac agttgatcca tgtcacctat 1320 ggctctgttc tgaaagatgc atctttgaaa gaacggctgt ttaagacgct tgaacaaaat 1380 gaggaactct tctacgagac cgtggcaaaa catataaaaa ggcacgtaga cctgttgaag 1440 gggtga 1446 <210> 19 <211> 1446 <212> DNA < 213> Artificial Sequence <220> <223> S125D / N129S / L140F / T181A / H362L mutant of tagaturonate epimerase <400> 19 atggtcttga aagtgttcaa agatcacttt ggaaggggat acgaagttta cgaaaagtct 60 tatagagaaa aggattctct ctctttcttc ttgacaaagg gagaggaagg aaaaattctg 120 gtagtggctg gagaaaaggc acctgagggt ctgtcgtttt tcaaaaaaca gcgggtggag 180 ggtgtttcgt tctttttctg tgagagaaat catgagaact tggaagttct cagaaaatac 240 tttccagatc tcaaaccagt tcgagcggga ttgagagcgt cttttggaac aggtgacaga 300 ctcggtatca ccacaccggc tcacgtgagg gcgttgaagg attcagggct ttttcccatc 360 tttgcgcagc aggatgtgag ggagagcgag agaacgggaa ggacctggag agatgtgttc 420 gacg atgcca catggggagt tttccaggag ggatacagtg agggattcgg agcagacgcc 480 gatcacgtga agcggccgga ggatcttgtt tcggctgcaa gggaaggttt caccatgttc 540 gcaatcgatc cttcggatca tgtgaggaat ctttcaaaac ttacagaaaa ggaaagaaat 600 gagaaattcg aagagattct gagaaaggaa aggatcgaca ggatctatct cggtaagaaa 660 tactctgttc tcggtgagaa gatcgaattc gatgagaaga atctcagaga tgcggcgctc 720 gtgtattacg atgcgattgc ccacgtggat atgatgtatc aaattttgaa agacgaaacc 780 ccggatttcg acttcgaagt gtcagttgac gaaacagaaa ctcccacgag tcctctcttc 840 cacattttcg ttgtggaaga actcagacga agaggtgtgg agttcaccaa tcttgccctg 900 agattcatcg gcgaatggga aaagggaata gattacaagg gggatcttgc acagttcgag 960 agagaaatca aaatgcacgc agaaatcgca aggatgttcg aaggatacaa aatatcactc 1020 cactctggaa gcgacaaatt ttccgtgtat cctgcttttg cttccgcgac aggaggcctt 1080 ttcctcgtga agacagccgg aacgagttat cttgaggcgg tgaaggtcat atccatggtc 1140 aacccggagc tcttccggga gatctacagg tgtgctctcg atcactttga ggaagacaga 1200 aagtcctatc acatatctgc ggatctgtcg aaagttccgg aagtagagaa agtgaaagat 1260 gaagatcttc caggtctt tt tgaagacatc aacgtgagac agttgatcca tgtcacctat 1320 ggctctgttc tgaaagatgc atctttgaaa gaacggctgt ttaagacgct tgaacaaaat 1380 gaggaactct tctacgagac cgtggcaaaa catataaaaa ggcacgtaga cctgttgaag 1440 gggtga 1446 <210> 20 <211> 1446 <212> DNA <213> Artificial Sequence <220> <223> S125D / N129T / L140K / T181A / H362L mutant of tagaturonate epimerase <400> 20 atggtcttga aagtgttcaa agatcacttt ggaaggggat acgaagttta cgaaaagtct 60 tatagagaaa aggattctct ctctttcttc ttgacaaagg gagaggaagg aaaaattctg 120 gtagtggctg gagaaaaggc acctgagggt ctgtcgtttt tcaaaaaaca gcgggtggag 180 ggtgtttcgt tctttttctg tgagagaaat catgagaact tggaagttct cagaaaatac 240 tttccagatc tcaaaccagt tcgagcggga ttgagagcgt cttttggaac aggtgacaga 300 ctcggtatca ccacaccggc tcacgtgagg gcgttgaagg attcagggct ttttcccatc 360 tttgcgcagc aggatgtgag ggagaccgag agaacgggaa ggacctggag agatgtgaag 420 gacgatgcca catggggagt tttccaggag ggatacagtg agggattcgg agcagacgcc 480 gatcacgtga agcggccgga ggatcttgtt tcggctgcaa gggaaggttt caccatgttc 540 gcaatcgatc cttcggatca tgt gaggaat ctttcaaaac ttacagaaaa ggaaagaaat 600 gagaaattcg aagagattct gagaaaggaa aggatcgaca ggatctatct cggtaagaaa 660 tactctgttc tcggtgagaa gatcgaattc gatgagaaga atctcagaga tgcggcgctc 720 gtgtattacg atgcgattgc ccacgtggat atgatgtatc aaattttgaa agacgaaacc 780 ccggatttcg acttcgaagt gtcagttgac gaaacagaaa ctcccacgag tcctctcttc 840 cacattttcg ttgtggaaga actcagacga agaggtgtgg agttcaccaa tcttgccctg 900 agattcatcg gcgaatggga aaagggaata gattacaagg gggatcttgc acagttcgag 960 agagaaatca aaatgcacgc agaaatcgca aggatgttcg aaggatacaa aatatcactc 1020 cactctggaa gcgacaaatt ttccgtgtat cctgcttttg cttccgcgac aggaggcctt 1080 ttcctcgtga agacagccgg aacgagttat cttgaggcgg tgaaggtcat atccatggtc 1140 aacccggagc tcttccggga gatctacagg tgtgctctcg atcactttga ggaagacaga 1200 aagtcctatc acatatctgc ggatctgtcg aaagttccgg aagtagagaa agtgaaagat 1260 gaagatcttc caggtctttt tgaagacatc aacgtgagac agttgatcca tgtcacctat 1320 ggctctgttc tgaaagatgc atctttgaaa gaacggctgt ttaagacgct tgaacaaaat 1380 gaggaactct tctacgagac cgtggcaaaa catat aaaaa ggcacgtaga cctgttgaag 1440 gggtga 1446 <210> 21 <211> 1446 <212> DNA <213> Artificial Sequence <220> <223> S125D/N129T/L140F/T181A/H362L mutant of tagaturonate epimerase <400> 21 atggtcttga aagtgttcaa acgaagttta cgaaaagtct 60 tatagagaaa aggattctct ctctttcttc ttgacaaagg gagaggaagg aaaaattctg 120 gtagtggctg gagaaaaggc acctgagggt ctgtcgtttt tcaaaaaaca gcgggtggag 180 ggtgtttcgt tctttttctg tgagagaaat catgagaact tggaagttct cagaaaatac 240 tttccagatc tcaaaccagt tcgagcggga ttgagagcgt cttttggaac aggtgacaga 300 ctcggtatca ccacaccggc tcacgtgagg gcgttgaagg attcagggct ttttcccatc 360 tttgcgcagc aggatgtgag ggagaccgag agaacgggaa ggacctggag agatgtgttc 420 gacgatgcca catggggagt tttccaggag ggatacagtg agggattcgg agcagacgcc 480 gatcacgtga agcggccgga ggatcttgtt tcggctgcaa gggaaggttt caccatgttc 540 gcaatcgatc cttcggatca tgtgaggaat ctttcaaaac ttacagaaaa ggaaagaaat 600 gagaaattcg aagagattct gagaaaggaa aggatcgaca ggatctatct cggtaagaaa 660 tactctgttc tcggtgagaa gatcgaattc at gatgagaaga ctcagaga tgcggcgctc 720 gtgtattacg atgcgattgc ccacgtggat atgatgtatc aaattttgaa agacgaaacc 780 ccggatttcg acttcgaagt gtcagttgac gaaacagaaa ctcccacgag tcctctcttc 840 cacattttcg ttgtggaaga actcagacga agaggtgtgg agttcaccaa tcttgccctg 900 agattcatcg gcgaatggga aaagggaata gattacaagg gggatcttgc acagttcgag 960 agagaaatca aaatgcacgc agaaatcgca aggatgttcg aaggatacaa aatatcactc 1020 cactctggaa gcgacaaatt ttccgtgtat cctgcttttg cttccgcgac aggaggcctt 1080 ttcctcgtga agacagccgg aacgagttat cttgaggcgg tgaaggtcat atccatggtc 1140 aacccggagc tcttccggga gatctacagg tgtgctctcg atcactttga ggaagacaga 1200 aagtcctatc acatatctgc ggatctgtcg aaagttccgg aagtagagaa agtgaaagat 1260 gaagatcttc caggtctttt tgaagacatc aacgtgagac agttgatcca tgtcacctat 1320 ggctctgttc tgaaagatgc atctttgaaa gaacggctgt ttaagacgct tgaacaaaat 1380 gaggaactct tctacgagac cgtggcaaaa catataaaaa ggcacgtaga cctgttgaag 1440 gggtga 1446 <210> 22 <211> 1446 <212> DNA <213> Artificial Sequence <220> <223> S125D/N129T/L140P/T181A/H362L mutant of tagaturo nate epimerase <400> 22 atggtcttga aagtgttcaa agatcacttt ggaaggggat acgaagttta cgaaaagtct 60 tatagagaaa aggattctct ctctttcttc ttgacaaagg gagaggaagg aaaaattctg 120 gtagtggctg gagaaaaggc acctgagggt ctgtcgtttt tcaaaaaaca gcgggtggag 180 ggtgtttcgt tctttttctg tgagagaaat catgagaact tggaagttct cagaaaatac 240 tttccagatc tcaaaccagt tcgagcggga ttgagagcgt cttttggaac aggtgacaga 300 ctcggtatca ccacaccggc tcacgtgagg gcgttgaagg attcagggct ttttcccatc 360 tttgcgcagc aggatgtgag ggagaccgag agaacgggaa ggacctggag agatgtgccg 420 gacgatgcca catggggagt tttccaggag ggatacagtg agggattcgg agcagacgcc 480 gatcacgtga agcggccgga ggatcttgtt tcggctgcaa gggaaggttt caccatgttc 540 gcaatcgatc cttcggatca tgtgaggaat ctttcaaaac ttacagaaaa ggaaagaaat 600 gagaaattcg aagagattct gagaaaggaa aggatcgaca ggatctatct cggtaagaaa 660 tactctgttc tcggtgagaa gatcgaattc gatgagaaga atctcagaga tgcggcgctc 720 gtgtattacg atgcgattgc ccacgtggat atgatgtatc aaattttgaa agacgaaacc 780 ccggatttcg acttcgaagt gtcagttgac gaaacagaaa ctccccacgag tcctctcttc 8 40 cacattttcg ttgtggaaga actcagacga agaggtgtgg agttcaccaa tcttgccctg 900 agattcatcg gcgaatggga aaagggaata gattacaagg gggatcttgc acagttcgag 960 agagaaatca aaatgcacgc agaaatcgca aggatgttcg aaggatacaa aatatcactc 1020 cactctggaa gcgacaaatt ttccgtgtat cctgcttttg cttccgcgac aggaggcctt 1080 ttcctcgtga agacagccgg aacgagttat cttgaggcgg tgaaggtcat atccatggtc 1140 aacccggagc tcttccggga gatctacagg tgtgctctcg atcactttga ggaagacaga 1200 aagtcctatc acatatctgc ggatctgtcg aaagttccgg aagtagagaa agtgaaagat 1260 gaagatcttc caggtctttt tgaagacatc aacgtgagac agttgatcca tgtcacctat 1320 ggctctgttc tgaaagatgc atctttgaaa gaacggctgt ttaagacgct tgaacaaaat tgaacaaaat 1380 gaggaacta gtggcata 1440 gaggaacta gtggcata

Claims (10)

써모토가 페트로필라(Thermotoga petrophila) 유래 타카투론산 에피머화 효소의 서열번호 1의 아미노산 서열로부터,
ⅰ) 125번째, 181번째 및 362번째 아미노산이 돌연변이되고,
ⅱ) 66번째, 72번째, 129번째 및 140번째 아미노산으로 이루어진 군으로부터 선택된 하나 이상의 아미노산이 추가로 돌연변이되는 것을 특징으로 하는, 타카투론산 에피머화 효소 변이체.
From the amino acid sequence of SEQ ID NO: 1 of takaturonic acid epimerase derived from Thermotoga petrophila ,
i) the 125th, 181st and 362th amino acids are mutated;
ii) at least one amino acid selected from the group consisting of the 66th, 72nd, 129th and 140th amino acids are further mutated, takaturonic acid epimerase variants.
제1항에 있어서,
상기 ⅰ)에서 125번째 아미노산인 세린(S)은 아스파트산(D), 글루탐산(E), 라이신(K) 또는 트립토판(W)으로 치환되고, 181번째 아미노산인 트레오닌(T)은 알라닌(A), 아스파트산(D), 페닐알라닌(F), 글라이신(G), 라이신(K), 류신(L), 발린(V) 또는 트립토판(W)으로 치환되며, 362번째 아미노산인 히스티딘(H)은 알라닌(A), 아스파트산(D), 페닐알라닌(F), 류신(L), 트레오닌(T) 또는 트립토판(W)으로 치환되고,
상기 ⅱ)에서 66번째 아미노산인 페닐알라닌(F)은 세린(S)으로 추가로 치환되거나, 72번째 아미노산인 글루탐산(E)은 글라이신(G)으로 추가로 치환되거나, 129번째 아미노산인 아스파라긴(N)은 트레오닌(T), 페닐알라닌(F) 또는 세린(S)으로 추가로 치환되거나, 140번째 아미노산인 류신(L)은 프롤린(P), 라이신(K) 또는 페닐알라닌(F)으로 추가로 치환된 것을 특징으로 하는, 타카투론산 에피머화 효소 변이체.
According to claim 1,
In i), serine (S) at the 125th amino acid is substituted with aspartic acid (D), glutamic acid (E), lysine (K) or tryptophan (W), and threonine (T) at the 181st amino acid is alanine (A) ), aspartic acid (D), phenylalanine (F), glycine (G), lysine (K), leucine (L), valine (V) or tryptophan (W), and histidine (H), the 362th amino acid is substituted with alanine (A), aspartic acid (D), phenylalanine (F), leucine (L), threonine (T) or tryptophan (W),
In ii), phenylalanine (F) at the 66th amino acid is further substituted with serine (S), glutamic acid (E) at the 72nd amino acid is further substituted with glycine (G), or asparagine (N) at the 129th amino acid is further substituted with threonine (T), phenylalanine (F) or serine (S), or leucine (L) at the 140th amino acid is further substituted with proline (P), lysine (K) or phenylalanine (F) Characterized, takaturonic acid epimerase variant.
제1항에 있어서,
상기 타카투론산 에피머화 효소의 서열번호 1의 아미노산 서열로부터,
ⅲ) 96번째, 97번째, 98번째, 100번째, 123번째, 124번째, 126번째, 127번째, 128번째, 159번째, 161번째, 162번째, 266번째, 268번째, 270번째, 299번째, 306번째, 307번째, 308번째, 309번째, 339번째, 340번째, 341번째, 342번째, 343번째, 344번째, 346번째, 363번째, 364번째, 365번째, 366번째, 367번째, 368번째, 369번째, 371번째, 403번째, 437번째 및 440번째 아미노산으로 이루어진 군으로부터 선택된 하나 이상의 아미노산이 추가로 돌연변이되는 것을 특징으로 하는, 타카투론산 에피머화 효소 변이체.
According to claim 1,
From the amino acid sequence of SEQ ID NO: 1 of the takaturonic acid epimerase,
iii) 96th, 97th, 98th, 100th, 123th, 124th, 126th, 127th, 128th, 159th, 161th, 162th, 266th, 268th, 270th, 299th, 306th, 307th, 308th, 309th, 339th, 340th, 341th, 342th, 343th, 344th, 346th, 363th, 364th, 365th, 366th, 367th, 368th , 369th, 371th, 403th, one or more amino acids selected from the group consisting of 437th and 440th amino acids are further mutated, takaturonic acid epimerase variants.
제1항 내지 제3항 중 어느 한 항에 따른 타카투론산 에피머화 효소 변이체를 암호화하는 유전자.
A gene encoding a takaturonic acid epimerase variant according to any one of claims 1 to 3.
제4항에 따른 유전자를 포함하는 재조합 벡터.
A recombinant vector comprising the gene according to claim 4.
제5항의 재조합 벡터로 형질전환된 숙주세포.
A host cell transformed with the recombinant vector of claim 5 .
제1항 내지 제3항 중 어느 한 항에 따른 타카투론산 에피머화 효소 변이체 또는 이를 암호화하는 유전자를 유효성분으로 포함하는 타카토스 생산용 조성물.
[Claim 4] A composition for producing Takatose comprising the takaturonic acid epimerase variant or a gene encoding the same according to any one of claims 1 to 3 as an active ingredient.
제7항에 따른 조성물을 과당에 처리하여 에피머화 반응을 수행하는 단계를 포함하는 과당으로부터 타카토스의 생산방법.
A method for producing Takatose from fructose, comprising the step of performing an epimerization reaction by treating the composition according to claim 7 with fructose.
제8항에 있어서,
상기 에피머화 반응은 pH 6.5 내지 9.0 및 온도 70℃ 내지 90℃에서 수행되는 것을 특징으로 하는, 과당으로부터 타카토스의 생산방법.
9. The method of claim 8,
The epimerization reaction is a method for producing takatose from fructose, characterized in that it is carried out at a pH of 6.5 to 9.0 and a temperature of 70 to 90 °C.
제8항에 있어서,
상기 에피머화 반응은 Ni²⁺, Co²⁺, Ca²⁺, Mn²⁺, Mg²⁺ 및 Zn²⁺로 이루어진 군으로부터 선택된 하나 이상의 2가 금속염의 존재 하에 수행되는 것을 특징으로 하는, 과당으로부터 타카토스의 생산방법. 9. The method of claim 8,
The epimerization reaction is carried out in the presence of at least one divalent metal salt selected from the group consisting of Ni ²⁺ , Co ²⁺ , Ca ²⁺ , Mn ²⁺ , Mg ²⁺ and Zn ²⁺ , from fructose The production method of Takatos.

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