KR102170296B1 - Glycosyltransferase producing high degree of polymerization of isomalto-oligosacharrides and manufacturing method of high degree of polymerization of isomalto-oligosacharrides using the same - Google Patents
Glycosyltransferase producing high degree of polymerization of isomalto-oligosacharrides and manufacturing method of high degree of polymerization of isomalto-oligosacharrides using the same Download PDFInfo
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Abstract
Description
본 발명은 고중합 이소말토올리고당 제조용 당전이효소(TP-DDase), 상기 당전이효소를 코딩하는 염기 서열을 포함하는 재조합 벡터, 상기 재조합 벡터로 형질전환된 형질전환체, 상기 형질전환체를 이용하여 당전이효소를 제조하는 방법 및 상기 당전이효소를 이용하여 이소말토올리고당을 제조하는 방법에 관한 것이다.The present invention uses a glycosyltransferase (TP-DDase) for production of high-polymerization isomaltooligosaccharide, a recombinant vector comprising a nucleotide sequence encoding the glycosyltransferase, a transformant transformed with the recombinant vector, and the transformant The present invention relates to a method of preparing a sugar transfer enzyme and a method of preparing isomaltoligosaccharide using the sugar transfer enzyme.
단맛을 내는 물질인 당류는 당분자의 갯수에 따라 단당류, 이당류, 다당류로 나뉜다. 포도당(glucose)과 과당(fructose)은 분자가 하나로 된 단당류이고, 설탕(sucrose sugar)은 분자가 2개인 이당류, 올리고당(oligosaccharide)은 3개 이상의 분자가 뭉쳐진 다당류에 속한다. 당류는 대부분의 과일, 유제품, 곡류 등에 함유되어 있기 때문에 식품을 통하여 섭취될 수 있으며 에너지를 공급하고, 정신적인 만족감을 주는 기능을 한다. 입자가 작은 단당류일수록 소화와 섭취가 빠르나, 다당류는 소화와 섭취가 어려워 설탕의 3분의 1 수준으로 칼로리가 낮은데다 장내에 공생하는 또는 유리한 미생물의 성장을 자극시켜 개인에 생물학적 효과를 발휘하는 프리바이오틱스로 불리우기도 하며, 체내에서 수용성 식이섬유와 같은 작용을 한다고 알려져 있다.Sugars, which are substances that give off sweet taste, are divided into monosaccharides, disaccharides, and polysaccharides according to the number of sugar molecules. Glucose and fructose are monosaccharides with one molecule, sucrose sugar is a disaccharide with two molecules, and oligosaccharide is a polysaccharide in which three or more molecules are aggregated. Since sugars are contained in most fruits, dairy products, and grains, they can be consumed through food, supply energy, and provide mental satisfaction. Monosaccharides with smaller particles are faster to digest and consume, but polysaccharides have a low calorie level at the level of one-third of sugar because they are difficult to digest and consume, and free of biological effects that exert biological effects on individuals by stimulating the growth of symbiotic or beneficial microorganisms in the intestine. It is also called biotics, and is known to act like water-soluble dietary fiber in the body.
당류 중 많은 양을 차지하는 설탕과 과당의 과잉 섭취는 열량을 높여 비만을 유발하거나, 혈중 중성 지방을 높여 심혈관질환 등 성인병 발생 위험성을 증가시킬 수 있고, 어린이의 경우에는 충치, 과잉행동장애와 같은 질병 발생 위험과 관련이 있는 것으로 보고되고 있는등 유해성 논란이 계속되면서, 건강을 위해 단맛을 내기 위한 설탕의 대체제로 올리고당을 사용하는 소비자가 늘어나고 있다. 국내에서 쉽게 접할 수 있는 올리고당의 예로는 프락토올리고당과 이소말토올리고당이 있다.Excessive intake of sugar and fructose, which occupy a large amount of sugars, can increase calories and cause obesity, or increase blood triglycerides to increase the risk of adult diseases such as cardiovascular disease, and diseases such as tooth decay and hyperactivity disorder in children. As the harmfulness controversy continues, such as being reported to be related to the risk of occurrence, more and more consumers are using oligosaccharides as a substitute for sugar for sweetness for health. Examples of oligosaccharides that can be easily encountered in Korea are fructooligosaccharide and isomaltoligosaccharide.
프락토올리고당(fructo-oligosaccharide)은 설탕의 과당(Fructose) 잔기에 1~3개의 과당이 결합된, 설탕과 유사한 구조를 가진 당류의 혼합물을 말한다. 그 구성 성분은 1-케스토스 (1-Kestose, GF2), 니스토스 (Nystose, GF3), 프락토실 니스토스 (Fructosyl nystose, GF4)이다. 이소말토올리고당(Isomalto-oligosaccharide, IMO)은 포도당이 α-(1,4)- 및/또는 α-(1,6)-글루코시드 결합 형태의 분지결합으로 중합도(degree of polymerization, DP) 2 내지 10 인 당류를 말하는 것으로 이소말토스 (isomaltose), 파노스(panose), 이소말토트리오스(isomaltotriose), 이소말토테트라오스(isomaltotetraose) 및 이소파노스(isopanose)와 같은 기타 고급 분지형 올리고당을 포함하며 장내 유용균으로 대표되는 비피더스균의 증식인자로 알려져 있다. 프락토올리고당과 이소말토올리고당은 모두 다당류인 올리고당에 속하지만 둘의 차이는 올리고당을 구성하는 성분에서 나타난다. 프락토올리고당은 설탕(과당+포도당)을 가공해 포도당을 연결하여 제조하고, 이소말토올리고당은 쌀이나 옥수수 등의 녹말가루(포도당+포도당)를 가공해 포도당을 연결하여 만든 것이다.Fructo-oligosaccharide refers to a mixture of sugars with a structure similar to sugar, in which 1 to 3 fructose are bonded to the fructose residues of sugar. Its constituents are 1-Kestose (GF2), nystose (GF3), and fructosyl nystose (GF4). Isomalto-oligosaccharide (IMO) is a branched bond in the form of α-(1,4)- and/or α-(1,6)-glucoside in which glucose is 2 to 2 degrees of polymerization (DP). Refers to a 10-phosphorus saccharide and includes other higher branched oligosaccharides such as isomaltose, panose, isomaltotriose, isomaltotetraose, and isopanose. It is known as a growth factor of bifidobacteria, which is represented by useful bacteria in the intestine. Both fructooligosaccharide and isomaltooligosaccharide belong to oligosaccharides, which are polysaccharides, but the difference between the two appears in the components constituting the oligosaccharide. Fructoligosaccharide is manufactured by connecting glucose by processing sugar (fructose + glucose), and isomaltooligosaccharide is made by connecting glucose by processing starch powder (glucose + glucose) such as rice or corn.
장내의 비피더스균은 영양, 면역, 노화, 장내 부패물질 억제, 비타민 합성 등 숙주의 건강과 밀접한 관계를 갖고 있다. 비피더스균을 증식시키기 위해 비피더스 균 및 비피더스 생육 촉진물질을 식품에 첨가한 식품이 판매되고 있으며 비피더스의 촉진물질로서 여러 종류의 올리고당이 개발되고 있다. 프락토올리고당은 대부분 난소화성으로 소장에서 분해되지 않고 대장까지 도달하여 장내 서식하는 비피더스 균에 선택적으로 이용됨으로써 증식을 촉진하는데, 과다복용시는 설사를 유발하는 것으로 알려져 있다. 그에 반해 이소말토올리고당은 난소화성과 소화성의 중간 위치에 있으므로 장관에 대한 자극이 약하여 최대 무작용량이 설탕과 유사한 수준인 1.5g/kg 정도로 안전성이 뛰어난 것으로 알려져 있다. 최근에는 국내에도 올리고당 및 비피더스균에 대한 인식이 확대됨에 따라 음료, 유제품, 장류 등 다양한 종류의 식품에 올리고당이 첨가되고 있다. 특히, 중합도가 높은 이소말토올리고당은 건강기능성 효능이 있는 것으로 알려져 있다(Toshiyuki Kaneko et al. Biosci. Biotech. Biochem., 58(12): 2288-2290, 1994).Bifidobacteria in the intestine have a close relationship with the health of the host such as nutrition, immunity, aging, suppression of intestinal decay substances, and synthesis of vitamins. In order to proliferate Bifidobacteria, foods in which Bifidobacteria and a Bifidus growth promoting substance are added to food are on the market, and various types of oligosaccharides are being developed as a promoting substance of Bifidus. Most of the fructooligosaccharides are indigestible and do not decompose in the small intestine, reach the large intestine, and promote proliferation by selectively used for bifidus bacteria living in the intestine, but overdose is known to cause diarrhea. On the other hand, isomaltooligosaccharide is known to be excellent in safety as it is in the middle of indigestibility and digestibility, so irritation to the intestines is weak, and the maximum non-acting amount is similar to that of sugar, about 1.5g/kg. In recent years, as the awareness of oligosaccharides and bifidobacteria has increased in Korea, oligosaccharides have been added to various types of food such as beverages, dairy products, and pastes. In particular, isomaltooligosaccharides with a high degree of polymerization are known to have health functional effects (Toshiyuki Kaneko et al. Biosci. Biotech. Biochem., 58(12): 2288-2290, 1994).
건강기능성 효능이 있는 고중합 이소말토올리고당을 합성하기 위해 덱스트란을 기질로 스트렙토코커스 뮤탄스로부터 분리한 덱스트라나아제를 이용하여 중합도가 5 이상인 이소말토올리고당을 합성하는 방법(Bioscience, Biotechnology, and Biochemistry, 1-8. 2018), 전분을 기질로 α-1,6 결합을 연장 시킬 수 있는 Paenibacillus 박테리아로부터 분리한 효소를 사용하여 α-1,6 결합을 가지는 사이클로 이소말토올리고당을 생산하는 방법(Applied Microbiology and Biotechnology February 2017) 등이 보고된 바 있으나, 상기 방법은 중합도가 7 이상이고 α-1,6 결합으로만 이루어진 이소말토올리고당을 생산하는 방법을 개시하고 있지 않다.A method of synthesizing isomaltooligosaccharide with a degree of polymerization of 5 or more using dextranase isolated from Streptococcus mutans using dextran as a substrate to synthesize highly polymerized isomaltooligosaccharide with health functional efficacy (Bioscience, Biotechnology, and Biochemistry, 1-8. 2018), a method of producing isomaltooligosaccharides with α-1,6 bonds using an enzyme isolated from Paenibacillus bacteria that can extend α-1,6 bonds from starch as a substrate ( Applied Microbiology and Biotechnology February 2017) and the like have been reported, but the method does not disclose a method of producing isomaltooligosaccharide consisting of only α-1,6 bonds with a degree of polymerization of 7 or more.
표준품이란 일정한 순도 또는 일정한 생물학적 작용을 갖게 만들어진 물질로서 식품, 의약품 또는 건강기능식품에 대하여 생물학적 또는 이화학적 실험을 할 때 쓰인다. 즉, 표준품이란 특정 식품, 의약품 또는 건강기능식품의 품질관리를 위하여 시험검사시 대조용으로 사용하는 기준 물질이다. 이소말토올리고당의 함량을 정량하기 위하여 필요한 이소말토올리고당 표준품 중 인체에 유익한 중합도가 높은 표준품 이소말토올리고당은 쉽게 구하기 힘들고 현재 중합도가 8 이상인 표준품 이소말토올리고당은 구매가 불가능하다. 일례로 중합도가 7인 이소말토올리고당 표준품은 mg당 425,000원으로 매우 고가이고 시판되는 Transglucosidase 등을 통해 제조된 이소말토올리고당은 대부분 하나의 중합도로 구성된 단일물질로 이루어져 있지 않아서 분석시 여러 개의 표준품을 구입하여 사용하여야 하는 문제점이 있다. 따라서 단일물질로 이루어지고 중합도가 높은 고중합 이소말토올리고당을 제조할 수 있는 방법의 개발이 필요하다. A standard product is a substance made to have a certain purity or a certain biological action, and is used for biological or physicochemical experiments on food, medicine or health functional food. In other words, a standard product is a reference substance used as a reference during test and inspection for quality control of specific foods, medicines or health functional foods. Among the isomaltooligosaccharide standards required to quantify the content of isomaltooligosaccharide, isomaltooligosaccharide, a standard product with a high degree of polymerization that is beneficial to the human body, is difficult to obtain, and isomaltooligosaccharide, a standard product with a polymerization degree of 8 or more, cannot be purchased. For example, a standard product with a polymerization degree of 7 isomaltooligosaccharides is very expensive at 425,000 won per mg, and most of the isomaltooligosaccharides produced through transglucosidase on the market do not consist of a single substance consisting of one polymerization degree, so several standards are purchased during analysis. There is a problem that must be used. Therefore, there is a need to develop a method for producing a highly polymerized isomaltooligosaccharide composed of a single substance and a high degree of polymerization.
이에, 본 발명자들은 써모안아에로박터 써모코프리애 균주에서 분리한 고리형 이소말토올리고당 당전이효소로부터 신규한 당전이효소를 제조하고, 상기 신규한 당전이효소 및 상대적으로 저렴한 기질을 이용하여 α-1,6 결합으로만 이루어진 중합도가 7 내지 10 인 고가의 고중합도의 이소말토올리고당을 제조할 수 있는 방법을 발견하였다. 본 발명에 따른 방법에 의해 제조된 이소말토올리고당은 α-1,6 결합으로만 이루어진 중합도가 긴 구성 당 함량이 높은 바, 고중합 이소말토올리고당 표준품으로서 정량분석시 유용하게 사용될 수 있고, 상기 방법을 이용하여 중합도가 7 이상인 고중합 이소말토올리고당을 생산할 수 있다.Thus, the present inventors prepared a novel sugar transfer enzyme from the cyclic isomaltooligosaccharide sugar transfer enzyme isolated from the thermoan aerobacter thermocoprie strain, and using the novel sugar transfer enzyme and a relatively inexpensive substrate. It has been discovered a method capable of producing an expensive high polymerization degree isomaltooligosaccharide having a polymerization degree of 7 to 10 consisting of only α-1,6 bonds. Isomaltooligosaccharide prepared by the method according to the present invention has a high content of constituent sugars with a long degree of polymerization consisting only of α-1,6 bonds, and can be usefully used in quantitative analysis as a standard product of high polymerization isomaltooligosaccharide. Highly polymerized isomaltooligosaccharide having a degree of polymerization of 7 or more can be produced by using.
본 발명의 목적은 고중합 이소말토올리고당 제조용 당전이효소 및 상기 당전이효소를 이용하여 고중합 이소말토올리고당을 제조하는 방법을 제공하는 것이다.It is an object of the present invention to provide a sugar transfer enzyme for preparing highly polymerized isomaltooligosaccharide and a method for producing highly polymerized isomaltooligosaccharide using the sugar transfer enzyme.
상기 목적을 달성하기 위하여, 본 발명은 서열번호 5의 아미노산 서열로 이루어진 고중합 이소말토올리고당 제조용 당전이효소를 제공한다.In order to achieve the above object, the present invention provides a sugar transfer enzyme for preparing a highly polymerized isomaltoligosaccharide consisting of the amino acid sequence of SEQ ID NO: 5.
또한, 본 발명은 상기 당전이효소를 코딩하는 염기 서열을 포함하는 재조합 벡터를 제공한다.In addition, the present invention provides a recombinant vector comprising the nucleotide sequence encoding the glycotransferase.
또한, 본 발명은 상기 재조합 벡터로 형질전환된 형질전환체를 제공한다.In addition, the present invention provides a transformant transformed with the recombinant vector.
또한, 본 발명은 1) 상기 형질전환체를 배양한 배양물을 제조하는 단계; 및In addition, the present invention comprises the steps of 1) preparing a culture obtained by culturing the transformant; And
2) 상기 배양물로부터 당전이효소를 수득하는 단계;를 포함하는 당전이효소의 제조방법을 제공한다.2) obtaining a sugar transfer enzyme from the culture; it provides a method for producing a sugar transfer enzyme comprising.
또한, 본 발명은 1) 상기 형질전환체를 배양한 배양물을 제조하는 단계; In addition, the present invention comprises the steps of 1) preparing a culture obtained by culturing the transformant;
2) 상기 배양물로부터 당전이효소를 수득하는 단계; 및2) obtaining a sugar transfer enzyme from the culture; And
3) 상기 단계 2)에서 수득한 당전이효소를 기질에 첨가하여 반응시키는 단계; 를 포함하는 이소말토올리고당을 제조하는 방법을 제공한다.3) reacting by adding the sugar transfer enzyme obtained in step 2) to the substrate; It provides a method for producing isomaltooligosaccharide comprising a.
본 발명의 당전이효소 및 상기 당전이효소를 이용한 이소말토올리고당 제조방법은 상대적으로 저렴한 기질을 이용하여 α-1,6 결합으로만 이루어진 중합도가 7 내지 10 인 고가의 고중합도의 이소말토올리고당을 제조할 수 있고 본 발명에 따른 방법에 의해 제조된 이소말토올리고당은 α-1,6 결합으로만 이루어진 중합도가 긴 구성 당 함량이 높은 바, 고중합 이소말토올리고당을 높은 효율로 생산할 수 있고, 정량분석을 위한 이소말토올리고당 표준품으로 유용하게 사용될 수 있다.The sugar transfer enzyme of the present invention and the method for preparing isomaltooligosaccharide using the sugar transfer enzyme use a relatively inexpensive substrate to obtain an expensive high polymerization degree of isomaltooligosaccharide with a degree of polymerization of 7 to 10 consisting only of α-1,6 bonds. Isomaltooligosaccharide prepared by the method according to the present invention has a high content of constituent sugars with a long degree of polymerization consisting only of α-1,6 bonds, and thus highly polymerized isomaltooligosaccharides can be produced with high efficiency, and quantitative It can be usefully used as an isomaltooligosaccharide standard for analysis.
도 1은 써모안아에로박터 써모코프리애 (Thermoanaerobacter thermocopriae) 균주 유래의 당전이효소(TP-DDase)를 암호화하는 염기 서열을 나타내는 모식도이다.
도 2는 당전이효소(TP-DDase)를 암호화하는 염기 서열이 클로닝된 재조합 벡터를 나타내는 이미지이다.
도 3은 당전이효소(TP-DDase)의 SDS-PAGE 분석 결과를 나타낸 도이다.
도 4는 시판 이소말토올리고당, 중합도가 2 내지 7인 이소말토올리고당 표준품 및 중합도가 2 내지 7인 말토올리고당 표준품의 HPAEC-PAD 분석 결과를 나타낸 도이다.
도 5는 말토올리고당 계열 기질과 당전이효소(TP-DDase)를 반응시켜 생성된 생성물의 구성 당 함량을 분석하기 위한 HPAEC-PAD 분석 결과를 나타낸 도이다.
도 6은 이소말토올리고당 계열 기질과 당전이효소(TP-DDase)를 반응시켜 생성된 생성물의 구성 당 함량을 분석하기 위한 HPAEC-PAD 분석 결과를 나타낸 도이다.
도 7은 말토올리고당 계열 기질 및 이소말토올리고당 계열 기질과 당전이효소(TP-DDase)를 반응시켜 생성된 생성물의 구성 당 함량 비율을 나타낸 도이다.
도 8은 말토올리고당 계열 기질 및 이소말토올리고당 계열 기질과 당전이효소(TP-DDase)를 반응시켜 생성된 생성물의 MALDI-TOF/TOF-MS 분석 결과를 나타낸 도이다.1 is a schematic diagram showing a nucleotide sequence encoding a glycotransferase (TP-DDase) derived from a Thermoanaerobacter thermocopriae strain.
2 is an image showing a recombinant vector in which a nucleotide sequence encoding glycotransferase (TP-DDase) is cloned.
3 is a diagram showing the results of SDS-PAGE analysis of glycotransferase (TP-DDase).
4 is a diagram showing the HPAEC-PAD analysis results of commercially available isomaltooligosaccharides, isomaltooligosaccharide standards having a polymerization degree of 2 to 7, and maltooligosaccharide standards having a polymerization degree of 2 to 7;
5 is a diagram showing the results of HPAEC-PAD analysis for analyzing the constituent sugar content of a product produced by reacting a maltooligosaccharide-based substrate and a glycosyltransferase (TP-DDase).
6 is a diagram showing the results of HPAEC-PAD analysis for analyzing the constituent sugar content of a product produced by reacting an isomaltooligosaccharide-based substrate and a glycosyltransferase (TP-DDase).
7 is a diagram showing the ratio of the constituent sugar content of a product produced by reacting a maltooligosaccharide-based substrate and an isomaltooligosaccharide-based substrate and a glycosyltransferase (TP-DDase).
8 is a diagram showing the results of MALDI-TOF/TOF-MS analysis of a product produced by reacting a maltooligosaccharide-based substrate and an isomaltooligosaccharide-based substrate and a glycosyltransferase (TP-DDase).
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 서열번호 5의 아미노산 서열로 이루어진 고중합 이소말토올리고당 제조용 당전이효소를 제공한다.The present invention provides a sugar transfer enzyme for preparing highly polymerized isomaltoligosaccharide consisting of the amino acid sequence of SEQ ID NO: 5.
상기 이소말토올리고당은 중합도가 2인 이소말토오스는 화학식 1, 중합도가 3인 이소말토트리오스는 화학식 2 또는 중합도가 4 내지 10인 이소말토올리고당은 화학식 3의 구조를 갖는 것이 바람직하다.It is preferable that the isomaltooligosaccharide has the structure of the general formula (1) for isomaltose having a degree of polymerization of 2, and the isomaltooligosaccharide of the general formula (2) or the isomaltooligosaccharide having a polymerization degree of 4 to 10 to have a structure of
<화학식 1><
<화학식 2><
<화학식 3><
상기 당전이효소는 덱스트란 덱스트리나아제(Dextran dextrinase)일 수 있다.The glycotransferase may be dextran dextrinase.
상기 고중합 이소말토올리고당은 중합도(Degree of polymerization)가 7 내지 10일 수 있다.The highly polymerized isomaltooligosaccharide may have a degree of polymerization of 7 to 10.
상기 당전이효소는 써모안아에로박터 써모코프리애 (Thermoanaerobacter thermocopriae) 균주 유래일 수 있다.The glycosyltransferase may be derived from a Thermoanaerobacter thermocopriae strain.
당 전이 반응이 일어나는 탄수화물 활성 효소로는 크게 glycosyl transferase(GTase), transglucosidases(TGase), glycoside phosphorylase(GPase) 및 glycoside hydrolases(GHase) 등 네 가지로 분류되며 이 중 glycosyl transferase(GTase)는 당 전이 효율이 좋고 다양한 수용체에 당을 전이할 수 있는 능력이 뛰어나다. Carbohydrate-activating enzymes that undergo sugar transfer reactions are largely classified into four categories: glycosyl transferase (GTase), transglucosidases (TGase), glycoside phosphorylase (GPase), and glycoside hydrolases (GHase). Among them, glycosyl transferase (GTase) is the sugar transfer efficiency. This is good and has excellent ability to transfer sugars to various receptors.
덱스트란 덱스트리나아제(Dextran dextrinase, DDase)는 상기 당전이효소(glycosyl transferase, GTase)의 일종으로 비환원 말단인 α-1,4 결합의 글루코실기 말단을 α-1,6 결합으로 전이한다. Dextran dextrinase (DDase) is a type of glycosyl transferase (GTase) and transfers the glucosyl group end of the non-reducing end of α-1,4 bond to α-1,6 bond. .
상기 α-1,4 결합은 α-D 포도당 분자가 인접한 α-D 포도당 분자 고리와 탄소 1 및 4를 통해 서로 연결된 공유 결합의 유형을 지칭한다. The α-1,4 bond refers to a type of covalent bond in which α-D glucose molecules are connected to each other through an adjacent α-D glucose molecule ring and
상기 α-1,6 결합은 α-D 포도당 분자가 인접한 α-D 포도당 분자 고리와 탄소 1 및 6을 통해 서로 연결된 공유 결합의 유형을 지칭한다. The α-1,6 bond refers to a type of covalent bond in which α-D glucose molecules are connected to each other through an adjacent α-D glucose molecule ring and
본 발명의 이소말토올리고당은 포도당이 α-(1,4)- 및/또는 α-(1,6)-글루코시드의 분지결합으로 이루어진 중합도가 3 내지 10인 다당류를 지칭한다.Isomaltooligosaccharide of the present invention refers to a polysaccharide having a degree of polymerization of 3 to 10 in which glucose is formed by branching bonds of α-(1,4)- and/or α-(1,6)-glucoside.
본 발명의 이소말토올리고당의 분자량은 중량 평균 중합도(DPw) 또는 수 평균 중합도(DPn)로 나타낼 수 있다. The molecular weight of the isomaltooligosaccharide of the present invention can be expressed as a weight average degree of polymerization (DPw) or a number average degree of polymerization (DPn).
본 발명에 있어서, 상기 덱스트란 덱스트리나아제는 당전이효소라고도 할 수 있으며, DDase 또는 TP-DDase라고 기재할 수 있다.In the present invention, the dextran dextrinase may also be referred to as a glycosyltransferase, and may be described as DDase or TP-DDase.
본 발명의 당전이효소(TP-DDase)는 중합도 2 내지 6의 중합도가 낮은 이소말토올리고당 뿐만 아니라 중합도가 7 내지 10인 중합도가 높은 고중합 이소말토올리고당까지 생성할 수 있다.The glycotransferase (TP-DDase) of the present invention can produce not only isomaltooligosaccharides having a polymerization degree of 2 to 6 with a low polymerization degree, but also high polymerization isomaltooligosaccharides having a polymerization degree of 7 to 10.
또한, 상기 당전이효소(TP-DDase)는 '변이체'를 포함하며, '변이체'는 상기 서열번호 5의 아미노산 서열을 갖는 당전이효소(TP-DDase)와 90% 이상의 상동성을 가지는 단백질을 말한다.In addition, the glycotransferase (TP-DDase) includes a'variant', and the'variant' refers to a protein having 90% or more homology with a glycotransferase (TP-DDase) having the amino acid sequence of SEQ ID NO: 5 Say.
"상동성"이란 야생형(wild type) 단백질의 아미노산 서열과의 유사한 정도를 나타내기 위한 것으로서, 본 발명의 당전이효소(TP-DDase)를 코딩하는 아미노산 서열(서열번호 5)과 바람직하게는 90%이상, 보다 바람직하게는 95% 이상, 더욱 바람직하게는 98%이상 동일할 수 있는 아미노산 서열을 포함한다. 이러한 상동성의 비교는 육안으로나 구입이 용이한 비교 프로그램을 이용하여 수행할 수 있다. 시판되는 컴퓨터 프로그램은 2개 이상의 서열간의 상동성을 백분율(%)로 계산할 수 있으며, 상동성(%)은 인접한 서열에 대해 계산될 수 있다.The term "homology" is intended to indicate the degree of similarity with the amino acid sequence of a wild type protein, and is preferably 90 with the amino acid sequence encoding the glycotransferase (TP-DDase) of the present invention (SEQ ID NO: 5). % Or more, more preferably 95% or more, and even more preferably 98% or more of the same amino acid sequence. The comparison of homology can be performed with the naked eye or using a comparison program that is easy to purchase. Commercially available computer programs can calculate homology between two or more sequences as a percentage (%), and homology (%) can be calculated for adjacent sequences.
또한, 본 발명의 당전이효소(TP-DDase) 또는 이의 상동체는 효소 활성을 가지는 한 아미노산 서열 변이체를 포함한다. 서열 변이체란 천연의 아미노산 서열과 하나 이상의 아미노산 잔기가 상이한 서열을 가지는 단백질을 의미한다. 변이체 단백질은 야생형 단백질과 동일한 생물학적 활성을 나타내나, 단백질의 특성이 변형된 변이체일 수 있다. 바람직하게는 아미노산 서열상의 변이와 수식으로 단백질의 열, pH 등에 대한 구조적 안정성이 증대될 수 있다. 예를 들어, 천연의 단백질이 효소 활성을 보이지 않는 강산성에서도 효소 활성을 나타낼 수 있고, 저온이나 고온에서도 효소 활성을 나타낼 수 있다. 또한 아미노산 서열상의 변이와 수식으로 당전이효소의 기질 특이성을 강화되거나 효소와 반응하는 기질의 종류가 확대된 변이체일 수 있다.In addition, the glycotransferase (TP-DDase) or a homologue thereof of the present invention includes an amino acid sequence variant having enzymatic activity. Sequence variant refers to a protein having a sequence different from a natural amino acid sequence and one or more amino acid residues. The variant protein exhibits the same biological activity as the wild-type protein, but may be a variant whose properties are modified. Preferably, the structural stability of the protein against heat, pH, etc. may be increased by mutations and modifications on the amino acid sequence. For example, a natural protein may exhibit enzyme activity even in strong acidity that does not exhibit enzyme activity, and may exhibit enzyme activity even at low or high temperatures. In addition, it may be a variant in which the substrate specificity of the glycosyltransferase is enhanced or the type of substrate reacting with the enzyme is expanded by mutations and modifications in the amino acid sequence.
또한, 본 발명은 서열번호 5의 아미노산 서열을 코딩하는 염기 서열로 이루어진 당전이효소(TP-DDase)의 유전자를 포함하는 재조합 벡터를 제공한다.In addition, the present invention provides a recombinant vector comprising a glycotransferase (TP-DDase) gene consisting of a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 5.
본 발명의 당전이효소(TP-DDase)는 서열번호 5로 나타내는 아미노산 서열 또는 이를 암호화하는 뉴클레오타이드 서열을 이용하여 유전공학적 방법을 통해 생산되어질 수 있다. 본 발명에서 상기 재조합 벡터는 상기 당전이효소(TP-DDase) 유전자의 효율적인 발현을 위해 발현벡터를 사용하는 것이 바람직하다. The glycotransferase (TP-DDase) of the present invention may be produced through a genetic engineering method using an amino acid sequence represented by SEQ ID NO: 5 or a nucleotide sequence encoding it. In the present invention, the recombinant vector is preferably an expression vector for efficient expression of the glycotransferase (TP-DDase) gene.
본 발명에서 "발현벡터"란 적당한 숙주세포에서 목적 펩타이드를 발현할 수 있는 재조합 벡터로서, 유전자 삽입물이 발현되도록 작동하게 연결된 필수적인 조절 요소를 포함하는 유전자 제작물을 말한다. 본 발명의 발현벡터는 적합한 발현 벡터가 일반적으로 가지고 있는 요소로서 프로모터, 오퍼레이터, 개시코돈 같은 발현조절 요소들을 포함한다. 개시 코돈 및 종결 코돈은 일반적으로 폴리펩타이드를 암호화하는 뉴클레오티드 서열의 일부로 간주되며, 유전자 제작물이 투여되었을 때 개체에서 반드시 작용을 나타내야 하며 코딩 서열과 인프레임(in frame)에 있어야 한다. 벡터의 프로모터는 구성적 또는 유도성일 수 있다. 또한, 세포 배양액으로부터 단백질의 분리를 촉진하기 위하여 융합 폴리펩타이드의 배출을 위한 시그널 서열을 포함할 수 있다. 특이적인 개시 시그널은 또한 삽입된 핵산 서열의 효율적인 번역에 필요할 수도 있다. 이들 시그널은 ATG 개시코돈 및 인접한 서열들을 포함한다. 어떤 경우에는, ATG 개시 코돈을 포함할 수 있는 외인성 번역 조절 시그널이 제공되어야 한다. 이들 외인성 번역 조절 시그널들 및 개시 코돈들은 다양한 천연 및 합성 공급원일 수 있다. 발현 효율은 적당한 전사 또는 번역 강화 인자의 도입에 의하여 증가될 수 있다.In the present invention, "expression vector" refers to a recombinant vector capable of expressing a target peptide in a suitable host cell, and refers to a gene construct comprising essential regulatory elements operatively linked to express a gene insert. The expression vector of the present invention is an element generally possessed by a suitable expression vector and includes expression control elements such as a promoter, an operator, and an initiation codon. The start and stop codons are generally considered to be part of the nucleotide sequence encoding the polypeptide, and must exhibit an action in the individual when the gene construct is administered and must be in frame with the coding sequence. The promoter of the vector can be constitutive or inducible. In addition, a signal sequence for release of the fusion polypeptide may be included in order to facilitate the separation of the protein from the cell culture medium. Specific initiation signals may also be required for efficient translation of the inserted nucleic acid sequence. These signals include the ATG start codon and adjacent sequences. In some cases, an exogenous translational control signal should be provided that may include the ATG initiation codon. These exogenous translational control signals and initiation codons can be of a variety of natural and synthetic sources. Expression efficiency can be increased by introduction of appropriate transcriptional or translation enhancing factors.
발현벡터는 통상의 모든 발현벡터를 다 사용할 수 있다. 예를 들어, 플라스미드 DNA, 파아지 DNA 등이 사용될 수 있다. 플라스미드 DNA의 구체적인 예로는 pET28a, pET 같은 상업적인 플라스미드를 포함한다. 본 발명에 사용될 수 있는 플라스미드의 다른 예로는 대장균 유래 플라스미드(pET28a, pET, pGEX, pQE, pDEST 및 pCOLD), 바실러스 서브틸리스(Bacillus subtilis)-유래 플라스미드(pUB110 및 pTP5) 및 효모-유래 플라스미드(YEp13, YEp24 및 YCp50)가 있다. 파아지 DNA의 구체적인 예로는 λ-파아지(Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11 및 λZAP)가 있다. 또한, 레트로바이러스(retrovirus), 아데노바이러스(adenovirus) 또는 백시니아 바이러스(vaccinia virus)와 같은 동물 바이러스, 배큘로바이러스(baculovirus)와 같은 곤충 바이러스가 또한 사용될 수 있고, 상기 당전이효소(TP-DDase) 유전자의 효율적인 발현을 위해서는 상기 대장균 유래 플라스미드(pET28a, pET, pGEX, pQE, pDEST 및 pCOLD)를 사용하는 것이 바람직하다. 이러한 발현벡터는 숙주 세포에 따라서 단백질의 발현량과 수식 등이 다르게 나타나므로,목적에 가장 적합한 숙주세포를 선택하여 사용하면 된다.As the expression vector, all conventional expression vectors can be used. For example, plasmid DNA, phage DNA, and the like can be used. Specific examples of plasmid DNA include commercial plasmids such as pET28a and pET. Other examples of plasmids that can be used in the present invention include E. coli-derived plasmids (pET28a, pET, pGEX, pQE, pDEST and pCOLD), Bacillus subtilis-derived plasmids (pUB110 and pTP5), and yeast-derived plasmids ( YEp13, YEp24 and YCp50). Specific examples of phage DNA include λ-phage (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, and λZAP). In addition, animal viruses such as retrovirus, adenovirus or vaccinia virus, insect viruses such as baculovirus may also be used, and the glycotransferase (TP-DDase) ) It is preferable to use the E. coli-derived plasmids (pET28a, pET, pGEX, pQE, pDEST and pCOLD) for efficient expression of the gene. These expression vectors have different expression levels and expressions of proteins depending on the host cell, so you can select and use the most suitable host cell for the purpose.
또한, 본 발명은 상기 당전이효소(TP-DDase)를 암호화하는 염기 서열이 도입된 재조합 벡터로 형질전환된 형질전환체를 제공한다.In addition, the present invention provides a transformant transformed with a recombinant vector into which the nucleotide sequence encoding the glycotransferase (TP-DDase) has been introduced.
본 발명의 형질전환체는 본 발명의 재조합벡터를, 상기 재조합벡터를 제작할 때에 사용한 발현벡터에 적합한 숙주 속에 도입함으로써 얻게 된다. 숙주의 종류로는 에셰리키아(Esherichia)속, 슈도모나스(Pseudomonas)속, 랄스토니아(Ralstonia)속, 알칼리게네스(Alcaligenes)속, 코마모나스(Comamonas)속, 버크홀데리아(Burkholderia)속, 아그로박테리움(Agrobacterium)속, 플라보박테리움(Flabobacterium)속, 비브리오(Vibrio)속, 엔테로박터(Enterobacter)속, 리조비움(Rhizobium)속, 글루코노박터(Gluconobacter)속, 아시네토박터(Acinetobacter)속, 라셀라(Moraxella)속, 트로조모나스(Nitrosomonas)속, 아에로모나스(Aeromonas)속, 파라코커스(Paracoccus)속, 바실루스(Bacillus)속, 클로스트리디움(Clostridium)속, 락토바실루스(Lactobacillus)속, 코리네박테리움(Corynebacterium)속, 아르트로박터(Arthrobacter)속, 아크로모박터(Achromobacter)속, 미크로코커스(Micrococcus)속, 마이코박테리움(Mycobacterium)속, 스트렙토코커스 (Streptococcus)속, 스트렙토마이세스(Streptomyces)속, 악티노마이세스(Actinomyces)속, 노카르디아(Nocardia)속, 메틸로박테리움(Methylobacterium)속 등의 각종 세균을 사용할 수 있다. 또, 상기 세균 이외에, 사카로마이세스(Saccharomyces)속, 칸디다(Candida)속 등의 효모, 또한 각종 곰팡이 등을 숙주로 사용할 수 있다. 본 발명에서는 BL21(DE3), Rosetta(DE3), Rosetta2(DE3), ArcticExpress(DE3), STAR(DE3), C41(DE3) 및 C43(DE3)로 이루어진 군에서 선택되는 하나의 대장균을 숙주로 이용하는 것이 보다 바람직하다. 대장균 등의 세균을 숙주로서 사용하는 경우는, 본 발명의 재조합 벡터는, 그 자신이 숙주 속에서 자율 복제가능 한 동시에, 프로모터, 당전이효소 유전자를 함유하는 DNA 및 전사종결서열 등의 발현에 필요한 구성을 갖는 것이 바람직하다. 세균에의 재조합 DNA의 도입방법으로서는, 염화칼슘법이나 일렉트로포레이션법(Method Enzymol., 194, 182-187(1990)), 스페로플라스트법(Proc. atl.Acad. Sci.USA, 84, 1929-1933(1978)), 아세트산리튬법(J. Bacteriol., 153, 63-168(1983)) 등이 이용 가능하다.The transformant of the present invention is obtained by introducing the recombinant vector of the present invention into a host suitable for the expression vector used when constructing the recombinant vector. Types of hosts include Esherichia, Pseudomonas, Ralstonia, Alcaligenes, Comamonas, Burkholderia, Agrobacterium genus, Flavobacterium genus, Vibrio genus, Enterobacter genus, Rhizobium genus, Gluconobacter genus, Acinetobacter genus ), Moraxella, Nitrosomonas, Aeromonas, Paracoccus, Bacillus, Clostridium, Lactobacillus (Lactobacillus) genus, Corynebacterium genus, Arthrobacter genus, Achromobacter genus, Micrococcus genus, Mycobacterium genus, Streptococcus ), Streptomyces, Actinomyces, Nocardia, and methylobacterium. In addition to the above bacteria, yeasts such as Saccharomyces genus and Candida genus, and various fungi can be used as hosts. In the present invention, using one E. coli selected from the group consisting of BL21 (DE3), Rosetta (DE3), Rosetta2 (DE3), ArcticExpress (DE3), STAR (DE3), C41 (DE3) and C43 (DE3) as a host It is more preferable. When bacteria such as E. coli are used as a host, the recombinant vector of the present invention is capable of autonomous replication in the host, and is necessary for expression of a promoter, DNA containing a glycotransferase gene, and transcription termination sequence. It is desirable to have a configuration. As a method for introducing recombinant DNA into bacteria, the calcium chloride method, the electroporation method (Method Enzymol., 194, 182-187 (1990)) and the spheroplast method (Proc. atl. Acad. Sci. USA, 84, 1929) -1933 (1978)), lithium acetate method (J. Bacteriol., 153, 63-168 (1983)) and the like are available.
또한, 재조합벡터는 발현의 억제 또는 증폭, 또는 유도를 위한 각종의 기능을 가진 발현억제용의 단편이나, 형질전환체의 선택을 위한 마커나 항생물질에 대한 내성유전자 또는 균체 밖으로의 분비를 목적으로 한 시그널을 코딩하는 유전자 등을 포함하는 것도 가능하다.In addition, the recombinant vector is a fragment for inhibiting expression having various functions for inhibiting, amplifying, or inducing expression, or for the purpose of secretion of a resistant gene for a marker or antibiotic for selection of a transformant, or for secretion outside the cells. It is also possible to include a gene or the like encoding a signal.
또한, 본 발명은 1) 상기 형질전환체를 배양한 배양물을 제조하는 단계; 및In addition, the present invention comprises the steps of 1) preparing a culture obtained by culturing the transformant; And
2) 상기 배양물로부터 당전이효소(TP-DDase)를 수득하는 단계;를 포함하는 당전이효소(TP-DDase)의 제조방법을 제공한다.2) obtaining a sugar transfer enzyme (TP-DDase) from the culture; it provides a method for producing a sugar transfer enzyme (TP-DDase) comprising.
본 발명의 당전이효소(TP-DDase)의 제조는 상기 형질전환체를 배양한 배양물(배양균체 또는 배양상층액) 속에 유전자 산물인 당전이효소(TP-DDase)를 생성, 축적시킨 후, 상기 배양물로부터 당전이효소(TP-DDase)를 취득함으로써 행하여진다.In the preparation of the glycosyltransferase (TP-DDase) of the present invention, a glycosyl transferase (TP-DDase), which is a gene product, is generated and accumulated in a culture (cultured cells or culture supernatant) in which the transformant is cultured, This is carried out by obtaining a glycosyltransferase (TP-DDase) from the culture.
본 발명의 형질전환체를 배양하는 방법은, 숙주의 배양에 사용되는 통상의 방법을 사용하면 된다.As a method of culturing the transformant of the present invention, a conventional method used for culturing a host may be used.
또 배양방법은, 배치(batch)식, 유동배치식, 연속배양, 리액터형식 등 통상의 미생물의 배양에 사용하는 어떠한 방법도 사용할 수 있다. 대장균 등의 세균을 숙주로 해서 얻게 된 형질전환체의 배지로서는, 완전배지 또는 합성배지, 예를 들면 LB배지, M9배지 등을 들 수 있다. 또, 배양온도는 상기 언급한 적정 온도의 범위에서 배양함으로써 당전이효소를 균체 내에 축적시키고 회수할 수 있다.In addition, as the cultivation method, any method used for cultivation of ordinary microorganisms, such as a batch type, a flow batch type, a continuous culture type, and a reactor type, can be used. Examples of the medium for transformants obtained using bacteria such as E. coli as a host include complete medium or synthetic medium such as LB medium and M9 medium. In addition, the cultivation temperature can be cultivated in the range of the above-mentioned appropriate temperature to accumulate and recover the glycotransferase in the cells.
미생물의 증식에 필요한 탄소원은 예를 들면 글루코스, 프럭토스, 슈크로스, 말토스, 갈락토스, 전분 등의 당류; 에탄올, 프로판올, 부탄올 등의 저급알콜류;글리세롤 등의 다가알콜류; 아세트산, 시트르산, 숙신산, 타르타르산, 락트산, 글루콘산 등의 유기산; 프로피온산, 부탄산, 펜탄산, 헥산산, 헵탄산, 옥탄산, 노난산, 데칸산, 운데칸산, 도데칸산 등의 지방산 등을 이용할 수 있다.Carbon sources necessary for the growth of microorganisms include, for example, sugars such as glucose, fructose, sucrose, maltose, galactose, and starch; Lower alcohols such as ethanol, propanol and butanol; polyhydric alcohols such as glycerol; Organic acids such as acetic acid, citric acid, succinic acid, tartaric acid, lactic acid, and gluconic acid; Fatty acids, such as propionic acid, butanoic acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, and dodecanoic acid, can be used.
질소원으로서는, 예를 들면 암모니아, 염화암모늄, 황산암모늄, 인산암모늄등의 암모늄염 외에, 펩톤, 고기즙, 효모엑기스, 맥아엑기스, 카제인분해물, 옥수수 침지액 등의 천연물유래의 것을 들 수 있다. 또, 무기물로서는, 예를 들면 인산 제1칼륨,인산 제2칼륨, 인산마그네슘, 황산마그네슘, 염화나트륨 등을 들 수 있다. 또한, 배양액에, 카나마이신, 암피실린, 테트라사이클린, 클로람페니콜, 스트렙토마이신 등의 항생물질을 첨가할 수 있다.Examples of the nitrogen source include ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, and ammonium phosphate, as well as those derived from natural products such as peptone, meat juice, yeast extract, malt extract, casein decomposition product, and corn steep liquor. Further, examples of the inorganic substance include first potassium phosphate, second potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, and the like. In addition, antibiotics such as kanamycin, ampicillin, tetracycline, chloramphenicol, and streptomycin may be added to the culture solution.
형질전환한 미생물을 배양하는 경우는 프로모터의 종류에 적합한 유도물질을 배지에 첨가하여 목적하는 단백질을 생산할 수 있다. 유도물질의 예로, 이소프로필-β-D-티오갈락토피라노시드(IPTG), 테트라사이클린, 인돌아크릴산(IAA) 등을 유도물질로 사용할 수 있다. 당전이효소의 취득 및 정제는 얻게 되는 배양물 중으로부터 균체 또는 상층액을 원심 회수하여, 균체파쇄, 추출, 친화성크로마토그래피, 양이온 또는 음이온교환크로마토그래피, 겔 여과 등을 단독으로 또는 적당히 조합함으로써 행할 수 있다. 얻게 된 정제물질이 목적의 효소인 것의 확인은, 통상의 방법, 예를 들면 SDS-폴리아크릴아미드겔 전기영동(SDS-PAGE), 웨스턴블랏팅(western blotting) 등에 의해 행할 수 있다.In the case of culturing the transformed microorganism, the desired protein can be produced by adding an inducer suitable for the type of promoter to the medium. As an example of the inducer, isopropyl-β-D-thiogalactopyranoside (IPTG), tetracycline, indoleacrylic acid (IAA), and the like may be used as the inducer. Acquisition and purification of the glycotransferase is carried out by centrifugal recovery of the cells or supernatant from the culture to be obtained, and cell disruption, extraction, affinity chromatography, cation or anion exchange chromatography, gel filtration, etc., alone or in an appropriate combination. Can be done. Confirmation that the obtained purified material is the target enzyme can be performed by a conventional method, for example, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, or the like.
또한, 숙주로서 미생물을 사용한 형질전환체의 배양, 형질전환체에 의한 당전이효소(TP-DDase)의 생산과 균체 내에의 축적, 및 균체로부터의 당전이효소(TP-DDase)의 회수는 상기의 방법에 한정되는 것은 아니다.In addition, cultivation of a transformant using a microorganism as a host, production of glycotransferase (TP-DDase) by the transformant and accumulation in the cells, and recovery of glycotransferase (TP-DDase) from the cells are described above. It is not limited to the method of.
또한, 본 발명은 1) 상기 형질전환체를 배양한 배양물을 제조하는 단계; In addition, the present invention comprises the steps of 1) preparing a culture obtained by culturing the transformant;
2) 상기 배양물로부터 당전이효소(TP-DDase)를 수득하는 단계; 및2) obtaining a glycosyltransferase (TP-DDase) from the culture; And
3) 상기 단계 2)에서 수득한 당전이효소(TP-DDase)를 기질에 첨가하여 반응시키는 단계; 를 포함하는 이소말토올리고당을 제조하는 방법을 제공한다.3) reacting by adding the sugar transfer enzyme (TP-DDase) obtained in step 2) to the substrate; It provides a method for producing isomaltooligosaccharide comprising a.
상기 기질은 말토올리고당 또는 이소말토올리고당일 수 있으며, 바람직하게는 중합도가 2 내지 5인 이소말토올리고당일 수 있다.The substrate may be maltooligosaccharide or isomaltooligosaccharide, preferably isomaltooligosaccharide having a degree of polymerization of 2 to 5.
상기 단계 3)의 반응은 당전이효소에 의해 수행되는 반응을 지칭한다. 반응 용액은 일반적으로 기질인 말토올리고당 또는 이소말토올리고당, 물, 임의의 다른 성분을 포함하는 용액 중 적어도 하나 이상을 포함하고 여기에 활성 당전이효소를 더 포함하는 용액을 지칭한다. 물, 기질로서 말토올리고당 또는 이소말토올리고당 및 당전이효소를 접촉시키는 단계가 수행되는 곳은 반응 용액이다. 상기 반응에서는 당전이효소의 활성을 통해 말토올리고당 또는 이소말토올리고당의 α-1,4 결합이 α-1,6 결합으로 전환되는 반응이 일어난다.The reaction of step 3) refers to a reaction performed by a sugar transfer enzyme. The reaction solution generally refers to a solution containing at least one or more of a solution containing maltooligosaccharide or isomaltooligosaccharide, water, or any other component as a substrate, and further comprising an active sugar transfer enzyme thereto. The reaction solution is where the step of contacting water, maltooligosaccharide or isomaltooligosaccharide as a substrate and a glycosyltransferase is performed. In the above reaction, a reaction in which the α-1,4 bond of maltooligosaccharide or isomaltooligosaccharide is converted into an α-1,6 bond occurs through the activity of the glycosyltransferase.
상기 단계 3)의 기질과 당전이효소의 반응은 20 내지 60℃에서 이루어지는 것일 수 있고, 바람직하게는 30 내지 50℃에서 이루어지는 것일 수 있다.The reaction between the substrate and the sugar transfer enzyme in step 3) may be performed at 20 to 60°C, preferably at 30 to 50°C.
상기 단계 3)의 기질과 당전이효소의 반응은 2 내지 10시간 동안 이루어지는 것일 수 있고, 바람직하게는 3 내지 9시간 동안 이루어지는 것일 수 있으며, 더 바람직하게는 4 내지 8시간 동안 이루어지는 것일 수 있다.The reaction of the substrate and the sugar transfer enzyme in step 3) may be performed for 2 to 10 hours, preferably for 3 to 9 hours, and more preferably for 4 to 8 hours.
상기 단계 3)의 당전이효소는 20 내지 80㎕ 첨가될 수 있고, 바람직하게는 30 내지 70㎕ 첨가될 수 있으며, 더 바람직하게는 40 내지 60㎕ 첨가될 수 있다.The sugar transfer enzyme of step 3) may be added 20 to 80 µl, preferably 30 to 70 µl, and more preferably 40 to 60 µl.
상기 말토올리고당 또는 이소말토올리고당의 농도는 2 내지 10mM 일 수 있고, 바람직하게는 3 내지 8mM일 수 있으며, 더 바람직하게는 4 내지 6mM 일 수 있다.The concentration of the maltooligosaccharide or isomaltooligosaccharide may be 2 to 10mM, preferably 3 to 8mM, and more preferably 4 to 6mM.
상기 방법으로 생성된 이소말토올리고당은 중합도(Degree of polymerization)가 7 내지 10인 이소말토올리고당일 수 있다.The isomaltooligosaccharide produced by the above method may be an isomaltooligosaccharide having a degree of polymerization of 7 to 10.
상기 방법으로 생성된 이소말토올리고당은 α-1,6 결합으로만 구성된 이소말토올리고당 일 수 있다.The isomaltooligosaccharide produced by the above method may be an isomaltooligosaccharide composed of only α-1,6 bonds.
상기 단계 3)에서 얻은 생성물은 중합도가 7 내지 10인 이소말토올리고당을 전체 중량에 대하여 0.2 내지 14.5 중량 % 포함하는 것일 수 있고, 바람직하게는 3 내지 14 중량 % 포함하는 것일 수 있으며, 더 바람직하게는 6 내지 14% 포함하는 것일 수 있다.The product obtained in step 3) may contain 0.2 to 14.5% by weight of isomaltooligosaccharide with a degree of polymerization of 7 to 10 based on the total weight, and preferably may contain 3 to 14% by weight, more preferably May be containing 6 to 14%.
본 발명의 구체적인 실시예에서, 본 발명자들은 써모안아에로박터 써모코프리애 균주로부터 분리한 고리형 이소말토올리고당 당전이 효소(cyclo-isomaltooligosaccharide glucanotransferase, CITase)를 제한효소 EcoRI 및 XhoI로 절단한 2463bp의 당전이효소(TP-DDase)를 코딩하는 염기 서열을 포함한 재조합 벡터를 제작하고(도 1 및 도 2 참조), 상기 재조합 벡터를 대장균에 도입하여 제작한 형질전환체에서 당전이효소(TP-DDase)를 생산하였다. 상기 당전이효소를 말토올리고당 계열 기질 및 이소말토올리고당 계열 기질과 반응시킨 결과, α-1,6 결합으로만 이루어지고, 중합도가 7 이상인 구성 당의 중합도가 긴 이소말토올리고당을 생성할 수 있는 것을 확인하였다(도 6 내지 도 8 참조).In a specific embodiment of the present invention, the
따라서, 본 발명의 당전이효소 및 상기 당전이효소를 이용한 이소말토올리고당 제조방법은 상대적으로 저렴한 기질을 이용하여 α-1,6 결합으로만 이루어진 중합도가 7 내지 10 인 고가의 고중합도의 이소말토올리고당을 제조할 수 있으므로, 본 발명에 따른 방법에 의해 제조된 이소말토올리고당은 α-1,6 결합으로만 이루어지고, 중합도가 긴 구성 당 함량이 높은 바, 이소말토올리고당 표준품으로 유용하게 사용될 수 있다.Therefore, the sugar transfer enzyme of the present invention and the method for producing isomaltoligosaccharide using the sugar transfer enzyme are expensive, high polymerization degree of isomalto, consisting of only α-1,6 bonds using a relatively inexpensive substrate. Since oligosaccharides can be prepared, isomaltooligosaccharides prepared by the method according to the present invention consist only of α-1,6 bonds, and have a high content of constituent sugars with a long polymerization degree, so they can be usefully used as a standard product of isomaltooligosaccharides. have.
이하, 본 발명을 실시예 및 실험예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail by examples and experimental examples.
단, 하기 실시예 및 실험예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예 및 실험예에 의해 한정되는 것은 아니다. However, the following examples and experimental examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following examples and experimental examples.
<실시예 1> 당전이효소(DDase)를 암호화하는 서열을 포함하는 재조합 발현 벡터 및 형질전환체의 제조<Example 1> Preparation of a recombinant expression vector and transformant containing a sequence encoding glycotransferase (DDase)
<1-1> 당전이효소(DDase)를 암호화하는 염기 서열의 증폭<1-1> Amplification of nucleotide sequence encoding glycotransferase (DDase)
당전이효소(TP-DDase) 단백질을 생산하기 위해 상기 효소를 암호화하는 염기 서열을 증폭하였다. In order to produce a glycotransferase (TP-DDase) protein, the nucleotide sequence encoding the enzyme was amplified.
구체적으로, 써모아나에로박터 써모코프리애(Thermoanaerobacter thermocopriae) 균주(RIKEN, Tokyo, Japan)로부터 분리한 DNA를 주형으로 사용하여, 제한효소 사이트(EcoRI, XhoI)를 포함하는 하기 표 1의 염기 서열을 가지는 프라이머 쌍을 이용하여 중합효소 연쇄반응(PCR)을 실시하였다.Specifically, using the DNA isolated from the thermoanaerobacter thermocopriae (Thermoanaerobacter thermocopriae) strain (RIKEN, Tokyo, Japan) as a template, the base of Table 1 containing restriction enzyme sites (EcoRI, XhoI) Polymerase chain reaction (PCR) was performed using a primer pair having a sequence.
그 결과, 전체 유전자(고리형 이소말토올리고당 당전이효소, cyclo-isomaltooligosacharride glucanotransferase, TPCITase ; 4680bp)(서열번호 1) 중 C-말단의 2463bp(서열번호 2)를 포함하는 TP-DDase를 암호화하는 염기서열의 PCR 산물을 수득하였다(도 1).As a result, a base encoding TP-DDase containing 2463 bp (SEQ ID NO: 2) of the C-terminus of the entire gene (cyclic isomaltooligosaccharide glycotransferase, cyclo-isomaltooligosacharride glucanotransferase, TPCITase; 4680 bp) (SEQ ID NO: 1) A PCR product of the sequence was obtained (Fig. 1).
<1-2> 재조합 벡터 및 형질전환체의 제조<1-2> Preparation of recombinant vector and transformant
당전이효소(TP-DDase)의 클로닝을 위해 상기 중합효소연쇄반응(PCR)을 통해 대량으로 수득한 PCR 산물(TP-DDase)을 발현벡터인 pGEX-6p-1(Amersham, Buckinghamshire, UK)의 EcoRI와 XhoI 제한효소 부위에 삽입하여 재조합 벡터(pGEX-6p-1-TP-DDase)를 제작하였다.For the cloning of glycotransferase (TP-DDase), the PCR product (TP-DDase) obtained in large quantities through the polymerase chain reaction (PCR) was used as an expression vector pGEX-6p-1 (Amersham, Buckinghamshire, UK). Recombinant vector (pGEX-6p-1-TP-DDase) was constructed by inserting into EcoRI and XhoI restriction sites.
제작한 재조합 벡터를 통상적인 형질전환 방법에 이하여 대장균 BL21(DE3) 균주에 형질전환하였다. 제작된 형질전환체의 당전이효소(TP-DDase) 활성을 분석한 후, 가장 높은 활성을 보이는 형질전환체를 분리하였으며, 상기 도입된 당전이효소(TP-DDase)를 암호화하는 염기 서열을 분석하여 서열번호 5의 아미노산 서열을 획득하였다.The prepared recombinant vector was transformed into E. coli BL21 (DE3) strain according to a conventional transformation method. After analyzing the glycotransferase (TP-DDase) activity of the prepared transformant, the transformant showing the highest activity was isolated, and the nucleotide sequence encoding the introduced glycotransferase (TP-DDase) was analyzed. Thus, the amino acid sequence of SEQ ID NO: 5 was obtained.
<실시예 2> 당전이효소(TP-DDase)의 제조 및 회수<Example 2> Preparation and recovery of sugar transferase (TP-DDase)
상기 실시예 1-2에서 제작한 당전이효소(TP-DDase)를 암호화하는 염기 서열이 도입된 형질전환체를 50㎍/㎖의 카나마이신을 포함하는 500㎖의 LB(Difco, Sparks, MD, USA) 배지가 함유된 플라스크에 도말하고 당전이효소를 암호화하는 염기서열을 포함하는 형질전환체의 콜로니를 수득하여 37℃에서 전배양하였다. 배양시 배양액의 흡광도(optical density, OD)가 600nm에서 0.5가 되었을 때, 단백질 발현 유도제인 0.1mM의 IPTG(isoprophyl-β-D-1-thiogalactopyranoside)를 첨가하고, 18℃에서 21시간 동안 150rev/min으로 교반하면서 배양하였다. 상기 배양된 세포는 10000rpm, 4℃ 조건에서 10분간 원심분리하여 회수하였고, 25㎖의 lysis 완충용액로 현탁하여 초음파분쇄기(sonicator)를 이용하여 세포를 파쇄하였다. 세포 파쇄물을 12,000rpm에서 30분간 원심분리한 후 수득한 상층액을 DEAE-650M 컬럼에 로딩하여 정제하였다. 단백질용 흡착제는 20mM sodium phosphate buffer(pH 7.4)를 사용하였으며, 컬럼에 흡착되지 않은 단백질을 취하여 정제를 완료하였다. 최종적으로 정제된 단백질의 순도는 10% Tris-Glycine SDS-PAGE Gel 분석으로 확인하였다. 상기 분석 SDS-PAGE 전기영동 분석 결과를 도 3에 나타내었고, TP-DDase 예상 크기인 92.4kDa의 재조합 단백질이 성공적으로 생산됨을 확인할 수 있었다.500 ml of LB (Difco, Sparks, MD, USA) containing 50 µg/ml kanamycin of the transformant into which the nucleotide sequence encoding the glycotransferase (TP-DDase) prepared in Example 1-2 was introduced. ) A flask containing a medium was plated to obtain a colony of transformants containing a nucleotide sequence encoding a glycosyltransferase, and pre-cultured at 37°C. When the optical density (OD) of the culture medium during cultivation became 0.5 at 600 nm, 0.1 mM isoprophyl-β-D-1-thiogalactopyranoside (IPTG), which is a protein expression inducing agent, was added, and 150 rev/during at 18° C. for 21 hours. Incubated with stirring at min. The cultured cells were recovered by centrifugation for 10 minutes at 10000 rpm and 4° C., suspended in 25 ml of lysis buffer, and disrupted using a sonicator. After centrifuging the cell lysate at 12,000 rpm for 30 minutes, the obtained supernatant was loaded onto a DEAE-650M column and purified. As an adsorbent for protein, a 20 mM sodium phosphate buffer (pH 7.4) was used, and purification was completed by taking the protein not adsorbed to the column. The purity of the finally purified protein was confirmed by 10% Tris-Glycine SDS-PAGE Gel analysis. The analysis result of the SDS-PAGE electrophoresis analysis is shown in FIG. 3, and it was confirmed that a recombinant protein of 92.4 kDa, which is an expected TP-DDase size, was successfully produced.
<실시예 3> HPAEC-PAD(High Performance Anion Exchange Chromatography with pulsed amperometric detection) 분석을 이용한 당전이효소(TP-DDase)에 의한 생성물 분석<Example 3> Product analysis by glycotransferase (TP-DDase) using HPAEC-PAD (High Performance Anion Exchange Chromatography with pulsed amperometric detection) analysis
기질로 말토올리고당 계열 및 이소말토올리고당 계열을 사용한 경우 생성물의 구성 당 함량을 분석하기 위해 HPAEC-PAD 분석을 수행하였다. When using maltooligosaccharide series and isomaltooligosaccharide series as substrates, HPAEC-PAD analysis was performed to analyze the constituent sugar content of the product.
<3-1> 당전이효소(TP-DDase)와 기질의 반응<3-1> Reaction of glycotransferase (TP-DDase) and substrate
먼저, 말토올리고당 계열의 기질인 5mM 농도의 말토오스(maltose, M2), 말토트리오스(maltotriose, M3), 말토테트라오스(maltotetraose, M4) 또는 말토펜타오스(maltopentaose, M5) 및 이소말토올리고당 계열의 기질인 5mM 농도의 이소말토오스(isomaltose, IM2), 이소말토트리오스(isomaltotriose, IM3), 이소말토테트라오스(isomaltotetraose, IM4) 및 이소말토펜타오스(isomaltopentaose, IM5)(TRC, 캐나다)가 각가 함유된 80mM의 B-R 완충용액(pH 5.5) 50㎕에 상기 실시예 2에서 제조한 당전이효소(TP-DDase) 50㎕를 첨가하고, 40℃에서 6시간 동안 반응시켰다. 이후 100℃에서 5분간 가열하여 반응을 정지시킨 후 HPAEC-PAD 분석을 수행하였다.First, maltose (M2), maltotriose (M3), maltotetraose (M4), or maltopentaose (M5) and isomaltooligosaccharides at a concentration of 5 mM, which are substrates of the maltooligosaccharide family. Substrates of 5 mM concentration of isomaltose (IM2), isomaltotriose (IM3), isomaltotetraose (IM4), and isomaltopentaose (IM5) (TRC, Canada) are each contained. 50 μl of the glycotransferase (TP-DDase) prepared in Example 2 was added to 50 μl of the 80 mM BR buffer solution (pH 5.5), and reacted at 40° C. for 6 hours. After heating at 100° C. for 5 minutes to stop the reaction, HPAEC-PAD analysis was performed.
<3-2> HPAEC-PAD 분석<3-2> HPAEC-PAD analysis
분석 기기로는 DIONEX ICS-5000+ SP(Thermo Fisher Scientific, 미국) 및 DIONEX ICS-5000+ DC(Thermo Fisher Scientific)를 사용하였고, 하기 표 2의 분석 조건에 따라 HPAEC-PAD 분석을 수행하였다.As analysis instruments, DIONEX ICS-5000+ SP (Thermo Fisher Scientific, USA) and DIONEX ICS-5000+ DC (Thermo Fisher Scientific) were used, and HPAEC-PAD analysis was performed according to the analysis conditions shown in Table 2 below.
구체적으로, 도 4에 나타낸 바와 같이 시판 이소말토올리고당(Megazyme Co. Copenhagen, Denmark), 중합도가 2 내지 7인 이소말토올리고당 표준품(Carbosynth, Compton, Berkshire, UK) 및 중합도가 2 내지 7인 말토올리고당 표준품(Tokyo chemical industry, Tokyo, Japan)의 HPAEC-PAD 분석을 수행하였다.Specifically, as shown in Figure 4 commercially available isomaltooligosaccharide (Megazyme Co. Copenhagen, Denmark), isomaltooligosaccharide standard product with a polymerization degree of 2 to 7 (Carbosynth, Compton, Berkshire, UK) and maltooligosaccharide having a polymerization degree of 2 to 7 HPAEC-PAD analysis of standard products (Tokyo chemical industry, Tokyo, Japan) was performed.
그 결과, 하기 표 3의 머무름 시간(retention time, RT)를 확인하여 말토올리고당 및 이소말토올리고당을 기질로 사용한 당전이효소(TP-DDase)에 의한 생성물의 이소말토올리고당 구성 당 분석을 위한 표준물질로 이용하였다.As a result, by checking the retention time (RT) in Table 3 below, a standard material for analysis of the composition of the product by the glycotransferase (TP-DDase) using maltooligosaccharide and isomaltooligosaccharide as a substrate Was used as.
<3-3> 말토올리고당 계열 기질의 생성물 분석<3-3> Product analysis of maltooligosaccharide-based substrate
말토올리고당 계열 기질과 당전이효소(TP-DDase)를 반응시킨 경우의 생성물의 머무름 시간을 상기 실시예 3-2에서 수득한 이소말토올리고당 표준품의 머무름 시간과 대조하여 구성 당의 중합도를 분석하였다. The degree of polymerization of the constituent sugar was analyzed by comparing the retention time of the product when the maltooligosaccharide-based substrate was reacted with the glycosyltransferase (TP-DDase) with the retention time of the isomaltooligosaccharide standard obtained in Example 3-2.
그 결과, 도 5 및 도 7에 나타난 바와 같이 말토올리고당의 α-1,4 결합이 가수분해되어 포도당이 생성되었고, 비환원말단으로의 당 전이 반응을 통해 α-1,6 결합이 생성되어 다양한 중합도를 가진 이소말토올리고당이 생성되었다. 말토오스, 말토트리오스, 말토테트라오스 및 말토펜타오스 기질을 당전이효소(TP-DDase)와 반응시킨 경우, 하기 표 4에 나타낸 바와 같이 중합도가 6 이하인 이소말토올리고당은 전체 생성물의 38 내지 40%, 중합도 7 이상인 이소말토올리고당은 2.1 내지 6.3% 비율을 차지하였다. 또한, 생성물 중 이소말토올리고당의 비율이 가장 높은 기질은 말토테트라오스(maltotetraose, M4)였다. 말토올리고당 계열 기질을 사용한 경우 중합도가 6 이상인 이소말토올리고당 생성시 이중, 삼중 피크가 발견되어 이는 순수한 α-1,6 결합으로 이루어진 이소말토올리고당이 아닌 α-1,4 결합이 동시에 존재하는 이소말토올리고당인 것으로 확인되었다.As a result, as shown in FIGS. 5 and 7, α-1,4 bonds of maltooligosaccharide were hydrolyzed to generate glucose, and α-1,6 bonds were generated through the sugar transfer reaction to the non-reducing terminal. Isomaltooligosaccharides with a degree of polymerization were produced. When maltose, maltotriose, maltotetraose, and maltopentase substrates are reacted with glycotransferase (TP-DDase), as shown in Table 4 below, isomaltooligosaccharide having a degree of polymerization of 6 or less is 38 to 40% of the total product. , Isomaltooligosaccharide having a degree of polymerization of 7 or more accounted for 2.1 to 6.3%. In addition, the substrate with the highest ratio of isomaltooligosaccharide in the product was maltotetraose (M4). When a maltooligosaccharide-based substrate is used, double and triple peaks are found when isomaltooligosaccharides with a degree of polymerization of 6 or more are generated, which is not an isomaltooligosaccharide consisting of pure α-1,6 bonds, but isomaltoe where α-1,4 bonds are present at the same time. It was confirmed to be an oligosaccharide.
<3-3> 이소말토올리고당 계열 기질의 생성물 분석<3-3> Product Analysis of Isomaltooligosaccharide Substrate
이소말토올리고당 계열 기질과 당전이효소(TP-DDase)를 반응시킨 경우의 생성물을 상기 실시예 3-2의 방법과 동일하게 분석하였다.A product obtained by reacting an isomaltooligosaccharide-based substrate with a glycosyltransferase (TP-DDase) was analyzed in the same manner as in Example 3-2.
그 결과, 도 6 및 도 7에 나타난 바와 같이 이소말토올리고당의 α-1,4 결합이 가수분해되어 포도당이 생성되었고, 비환원말단으로의 당 전이 반응을 통해 α-1,6 결합이 생성되어 높은 중합도를 가진 이소말토올리고당이 생성되었다. 이소말토오스, 이소말토트리오스, 이소말토테트라오스 및 이소말토펜타오스 기질을 당전이효소(TP-DDase)와 반응시킨 경우, 하기 표 5에 나타난 바와 같이 중합도가 6 이하인 이소말토올리고당은 전체 생성물의 47 내지 61%, 중합도 7 이상인 이소말토올리고당은 0.2 내지 14% 비율을 차지하였다. 또한, 생성물 중 이소말토올리고당의 비율이 가장 높은 기질은 이소말토펜토오스(isomaltopentose)였다. 이소말토올리고당 계열 기질을 사용한 경우 중합도가 7 이상인 이소말토올리고당 생성시 단일 피크가 관찰되어 순수한 α-1,6 결합으로 이루어진 이소말토올리고당을 생성하는 것으로 확인되었다.As a result, as shown in FIGS. 6 and 7, α-1,4 bonds of isomaltooligosaccharide were hydrolyzed to generate glucose, and α-1,6 bonds were generated through a sugar transfer reaction to the non-reducing terminal. Isomaltooligosaccharides with a high degree of polymerization were produced. When isomaltose, isomaltotriose, isomaltotetraose, and isomaltopentaose substrates are reacted with glycotransferase (TP-DDase), isomaltooligosaccharides having a degree of polymerization of 6 or less as shown in Table 5 below Isomaltooligosaccharides having a degree of polymerization of 47 to 61% and a polymerization degree of 7 or higher accounted for 0.2 to 14%. In addition, the substrate with the highest ratio of isomaltooligosaccharide in the product was isomaltopentose. When an isomaltooligosaccharide-based substrate was used, a single peak was observed when isomaltooligosaccharide having a degree of polymerization of 7 or more was generated, and it was confirmed that isomaltooligosaccharide consisting of pure α-1,6 bonds was produced.
따라서 상기 결과를 통해 이소말토올리고당을 기질로 이용하였을 때 중합도가 7 이상인 이소말토올리고당을 높은 함량으로 생산할 수 있음을 확인할 수 있다.Therefore, through the above results, it can be seen that when isomaltooligosaccharide is used as a substrate, isomaltooligosaccharide having a degree of polymerization of 7 or more can be produced in a high content.
<실시예 4> MALDI-TOF/TOF-MS(Matrix assisted laser desorption- ionization time of flight mass spectrometry) 분석을 통한 구성 당 분석<Example 4> Analysis per composition through MALDI-TOF/TOF-MS (Matrix assisted laser desorption-ionization time of flight mass spectrometry) analysis
표준물질을 구할 수 없는 중합도가 8이상인 이소말토올리고당은 생성된 이소말토올리고당의 분자량 및 중합도 확인을 위해 MALDI-TOF/TOF-MS(Matrix assisted laser desorption- ionization time of flight mass spectrometry) 질량 분석을 수행하였다.For Isomaltooligosaccharides with a degree of polymerization of 8 or higher for which a standard substance cannot be obtained, MALDI-TOF/TOF-MS (Matrix assisted laser desorption-ionization time of flight mass spectrometry) mass spectrometry is performed to confirm the molecular weight and degree of polymerization of the produced isomaltooligosaccharide. I did.
구체적으로, 상기 실시예 3-1과 같이 기질과 당전이효소(TP-DDase)를 반응시킨 후, 반응이 완료된 2㎕의 생성물을 2㎕의 매트릭스 용액(α-Cyano-4-hydroxycinnamic acid in 0.1% TFA/ACN (1:1, v/v)(SA matrix) 및 2,5-Dihydroxybenzoic acid in 0.1% TFA/ACN (1:1, v/v)와 10mM의 NaCl이 7:3의 부피비로 혼합된 DHB matrix로 구성)과 혼합하였다. 혼합물을 MALDI 384 웰플레이트 상에 스폿(spot)하고 진공 건조시켰다. 생성물의 구성 당 분석은 UltraflexⅢTOF/TOF(Bruker Daltonics, bremen, Germany), 하기 표 6 및 표 7의 작동 조건에서 수행하였고 스펙트럼 획득 및 처리는 Flex Control ver. 3.0 소프트웨어를 사용하였다. Specifically, as in Example 3-1, after reacting the substrate with the glycosyl transfer enzyme (TP-DDase), 2 µl of the reaction-completed product was added to 2 µl of a matrix solution (α-Cyano-4-hydroxycinnamic acid in 0.1). % TFA/ACN (1:1, v/v) (SA matrix) and 2,5-Dihydroxybenzoic acid in 0.1% TFA/ACN (1:1, v/v) and 10 mM NaCl in a volume ratio of 7:3 It was mixed with a mixed DHB matrix). The mixture was spotted on a MALDI 384 well plate and vacuum dried. Analysis per composition of the product was performed under the operating conditions of UltraflexIIITOF/TOF (Bruker Daltonics, bremen, Germany), Tables 6 and 7 below, and spectrum acquisition and processing were performed by Flex Control ver. 3.0 software was used.
그 결과, 도 8에 나타난 바와 같이 이소말토오스를 기질로 사용한 경우 중합도가 2 내지 6인 이소말토올리고당이 생성되고, 이소말토트리오스 및 말토트리오스를 기질로 사용한 경우 중합도가 2 내지 8, 이소말토테트라오스 및 말토테트라오스를 기질로 사용한 경우 중합도가 2 내지 9, 이소말토펜토오스 및 말토펜토오스를 기질로 사용한 경우 중합도가 2 내지 10인 이소말토올리고당을 생성하는 것을 확인하였다. 또한, 각 피크 간 분자량 차이가 이소말토올리고당을 구성하는 단위 당(glucose) 1 개의 분자량인 162인 것으로 확인되어 α-1,6 결합을 가진 중합도가 8 이상인 고중합 이소말토올리고당 표준품을 생성할 수 있는 것으로 확인되었다.As a result, as shown in FIG. 8, when isomaltose is used as a substrate, isomaltooligosaccharide having a degree of polymerization of 2 to 6 is produced, and when isomalttotriose and maltotriose are used as a substrate, the degree of polymerization is 2 to 8, isomalto It was confirmed that when tetraose and maltotetraose were used as substrates, the degree of polymerization was 2 to 9, and when isomaltopentose and maltopentose were used as substrates, isomaltooligosaccharides having a polymerization degree of 2 to 10 were confirmed. In addition, the difference in molecular weight between each peak was found to be 162, which is the molecular weight of one unit sugar constituting the isomaltooligosaccharide, so that a high-polymerization isomaltooligosaccharide standard with a degree of polymerization of 8 or more with α-1,6 bonds can be produced. It was confirmed to be.
<110> REPUBLIC OF KOREA(MANAGEMENT : RURAL DEVELOPMENT ADMINISTRATION) <120> Glucosyltransferase producing high degree of polymerization of isomalto-oligosacharrides and manufacturing method of high degree of polymerization of isomalto-oligosacharrides using the same <130> 2019p-05-024 <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 4680 <212> DNA <213> Artificial Sequence <220> <223> TPCITase coding sequence <400> 1 atgttgtctc tttatagaag aaaactcttt ataacaattt taatagtaat tttcgtgttg 60 tctaactttt tcacattatt tacttatcct atctcaccag gagtttctgt tgcttatgcc 120 gcttcaacag gtaatttgat tcaacgggtt tatacagata aagctcgata taatccaggg 180 gatcttgtta ccattagtgc tgatttaatt aataaaactg gttcgacatg gtccggcact 240 ttaactcttc agattaataa actagaaagc caaatttaca ctgcaagtca gtcagttact 300 cttgccaatg gggattcaac tacaatcaca tttacatgga ctgctccacc taccgacttt 360 gttggttatt atgccggaat tgcggcagga agtacagatt tcaatggaac aggtatcgat 420 gtaagttcct ctccgcttcg gtttcctcgc tatggtttta tctcaaactt tcctgtttca 480 cagactgccc agcaatccac agatatagtt aaacagatgg tggaagacta tcatcttaat 540 atttttcaat tttacgactg gatgtggcgg catgagaagc taattaagcg aaccaatgga 600 gttatagatt ctacttgggt ggatcttttt gatcgcacac tttcttggca aactatacaa 660 aacaatgttg cagcagttca ttcctttaat gcttatgcca tggcttacgc tatgagttat 720 gcagctcgtg aaggatatga acaaatgtgg ggaatcagcc ctagctgggg tatctttcaa 780 gatacggcac atcaaagcca gtttaacgtg gattttcata atggcaagtt tctgtggctt 840 tttaatccag caaatgtgaa ttggcagagt tggatcatta gtgaatacaa agatgctatc 900 aatacggcag gttttgatgg catacagatc gatcaaatgg ggcagcgaga caatgtttac 960 gattatacta gcttttccgt tactttgcct agtacattcg ctcagtttct tcaacaagtt 1020 aagtcagaat tagaatctaa taatgcaaaa aaaaatgttg ttacttttaa tattgttgat 1080 ggtacagtga acggttgggg ggcgggagaa attgctcgtt atggtgccag cgactttgac 1140 ttcagcgaga tttggtggaa agctaacaca tataatgatc tccgaaatta catcgaatgg 1200 ctcaggcaaa acaatggtgg caagcctgtt gtacttgcag cttatatgaa ttacaacgag 1260 gaatacggtc caatatatga agctgaatcc gctattctat caggcgttag tgtcaataca 1320 aaccattctg gttatacggg cactggcttt gtcgatgggt ttgaaacggt gggtgattct 1380 atcacgtgga caattgactt ccctgagact ggtgattatt cgtttgtttt tcgttatgca 1440 aatgccactg gtgctactgc tactcgtaat gtttatgtag atggacgatt tctgggacaa 1500 gtctcatttg ctaatcaggt ggattgggat acttgggcga cggacgcgtg gattcaaata 1560 gagggactta ctgctggtac tcatagtgtg acattaaagt atgacagtga caatataggc 1620 gctataaacg tcgatcattt aactcttgga gagtttgagg aacattcagt tcgattggca 1680 gatgcaatga tgtttgctag cggtgctaca cacattgagc ttggtgatac taatcagatg 1740 ttagcgcatg aatattatcc gaaccgttcc aagtcaatgc gtaattctct caaggcagcg 1800 atgagagact actatagttt tgctactgct tatgaaaatt tactctttga tcctgatata 1860 gttccagctg accaaggcaa ccaatggatt gcactgacaa ccggacaacc tttaagcggc 1920 aacggaactt ctggtactat ttggcaaatg attaaacgca agtctgatta tgatatcata 1980 catttgatta atctaatggg caatgatgat cagtggcgta acccagctgt tcagcctact 2040 ttccaaagca acattggagt taaatactat ccaggaccta atgccgctgt cagtggcgta 2100 tatcttgcca gtccggatct cgatcacggt atgaccattc cactcactta tacgacgggt 2160 aatgatagcc gaggtaacta tatccagttt gttgtgccaa gcttaaagta ctgggatatg 2220 atttatgtaa agcgtacgat aaccacaccg ccagatggac agtatgaagc agaatatgct 2280 attaaaagtg gcactaacat caataccgat catacaggat atactggcag cggttttgta 2340 gacaactttg atgctagtga taaaggtgta tcgtttatta taaatgttcc aactagtgat 2400 acctacacgc ttcgattccg atatggaaac ggcggtacta ctattgccac gcgaacttta 2460 tttattgatg gtcagtatgc aggtactctc caattccgaa atctttataa ctgggatgtg 2520 tgggatacag tggaaaccac tgtgtggctt tcagcgggtg ttcatcaagt ggtactttgg 2580 tacagttccg aaaatgatgg tgctattaat cttgataacc tgattgttct acaacaaact 2640 actccaacaa gaacttcggc tcgctccttt tggatgaata actggtccaa cttgataggt 2700 attcacatgg ctagcaaatt aagcccgact gataatggaa attatggccc tcgcctggct 2760 gaacttcatt tcagaggcga ttggcctact aatcaaatcg tagatgcaac tgcttttttc 2820 cgagatgaaa cggatcttac tcccataaag tataccaatg ctcatagctt tgattctgaa 2880 gcatggtttg aaaacgacgg gactcttacc gtgagatatc tgacctataa tggctcagcc 2940 ttgccagtac agattaccaa acaatacgcc atggtgccta accaaaattt tcttgtgatc 3000 aaatatactt ttttaaacca gacaagtagc gcacgtactt taaactttct agaacaagtt 3060 catttgaaca acaggactag tagcgatccg aatccagggt ggcaacatgg ctggtgggac 3120 gtgagtcgaa atgcacttgg tactgacatg agccagactg gtcagttcta tattgaattg 3180 ggtgcttttc agactatgga tagttatcag gtgggtaatg acgccgattc caatcctaac 3240 agtcagacgt catcaccgtg gtatcagttc gacgccaacg gtgttctaaa taggtgcggt 3300 gatctttggt cacagaatct tagcatgggt tttcagaaac ttattaccgt gcctgctgga 3360 ggtagtgtaa cattggcttt ttattatgct atcggttcaa ctcaggagga agcggaagca 3420 gcagcagatt tagctcgatc tcagacagct gactactggt ttacccaaac tgctgccgag 3480 tataataatt ggcttaacag cggccagcgg gttaatacgt ctgatattgg tatcaatact 3540 gcctttgatc ggagcctcat cataaacaaa caggcacaac atccggagtt tggttcctgg 3600 cccgcggcta caaatccctc ttatcagtac aaggtgtggg ttcgtgactc agctgtaaca 3660 gctatgggaa tggatgctgc taatcatctt tctgaggcgg aaaagtattg gaactggatg 3720 gcctctgttc aaaatacaga tgggacatgg catacaaatt ataatgtatg gaaggcaaat 3780 gaatggatct cgtttgtgga gcctgaacac gatgccattg gactttttct catcggggtt 3840 tatcaacatt acagtttact caaatcccgc gattcctcgg cagcgacgac ttttctgaat 3900 aacatttgga cgcagataac ccgcgctggt gatttcatct acaaaaacat tggcgcttct 3960 gggtttggtc cggctgatgc ttctatctgg gaggagcagg tcgagtataa tatttttact 4020 caagttactt atgccgcagg attgaatgct ggacggttgt tagcccaaga aaaaggtgat 4080 attactcgtt caaataacta tttgtcgggt gctcaaacca tcaaggatgc gattctacga 4140 tcgtttttgt cttcaccacg aggtctctgg aataaatcta atcgctattt taatcgggcg 4200 atcaatacag acggtacagc gcgaacgacg gttgatgctt catcggactt gatttgggtt 4260 tttggtctcc tttcaccgac tgatactagg attcgagatc accgcattaa ggtactatca 4320 cggctgaccc atgataggta cggcattgct cgatatgaaa acgatgagtt ctattattct 4380 agtccatata gccccggtgg acagtatgag gctggcgcag ccgaaccagt ttggccacaa 4440 atgactatgt acgcgtctat gattgagcat tggaggggtg atgatgccac tgcactggct 4500 cggcttaagt ggtatgtcag ccgaactgct cgcggctatg tcacacccgg tgaagctgtg 4560 gactggacca atggtcaacc gcttatcagt acagcagttg aaccagtaac gggctcctgg 4620 tttcagatgg cggtccttac ttacttaaac cggtttgatc tacgattacc tgatttttaa 4680 4680 <210> 2 <211> 2463 <212> DNA <213> Artificial Sequence <220> <223> DDase coding sequence <400> 2 atgatttatg taaagcgtac gataaccaca ccgccagatg gacagtatga agcagaatat 60 gctattaaaa gtggcactaa catcaatacc gatcatacag gatatactgg cagcggtttt 120 gtagacaact ttgatgctag tgataaaggt gtatcgttta ttataaatgt tccaactagt 180 gatacctaca cgcttcgatt ccgatatgga aacggcggta ctactattgc cacgcgaact 240 ttatttattg atggtcagta tgcaggtact ctccaattcc gaaatcttta taactgggat 300 gtgtgggata cagtggaaac cactgtgtgg ctttcagcgg gtgttcatca agtggtactt 360 tggtacagtt ccgaaaatga tggtgctatt aatcttgata acctgattgt tctacaacaa 420 actactccaa caagaacttc ggctcgctcc ttttggatga ataactggtc caacttgata 480 ggtattcaca tggctagcaa attaagcccg actgataatg gaaattatgg ccctcgcctg 540 gctgaacttc atttcagagg cgattggcct actaatcaaa tcgtagatgc aactgctttt 600 ttccgagatg aaacggatct tactcccata aagtatacca atgctcatag ctttgattct 660 gaagcatggt ttgaaaacga cgggactctt accgtgagat atctgaccta taatggctca 720 gccttgccag tacagattac caaacaatac gccatggtgc ctaaccaaaa ttttcttgtg 780 atcaaatata cttttttaaa ccagacaagt agcgcacgta ctttaaactt tctagaacaa 840 gttcatttga acaacaggac tagtagcgat ccgaatccag ggtggcaaca tggctggtgg 900 gacgtgagtc gaaatgcact tggtactgac atgagccaga ctggtcagtt ctatattgaa 960 ttgggtgctt ttcagactat ggatagttat caggtgggta atgacgccga ttccaatcct 1020 aacagtcaga cgtcatcacc gtggtatcag ttcgacgcca acggtgttct aaataggtgc 1080 ggtgatcttt ggtcacagaa tcttagcatg ggttttcaga aacttattac cgtgcctgct 1140 ggaggtagtg taacattggc tttttattat gctatcggtt caactcagga ggaagcggaa 1200 gcagcagcag atttagctcg atctcagaca gctgactact ggtttaccca aactgctgcc 1260 gagtataata attggcttaa cagcggccag cgggttaata cgtctgatat tggtatcaat 1320 actgcctttg atcggagcct catcataaac aaacaggcac aacatccgga gtttggttcc 1380 tggcccgcgg ctacaaatcc ctcttatcag tacaaggtgt gggttcgtga ctcagctgta 1440 acagctatgg gaatggatgc tgctaatcat ctttctgagg cggaaaagta ttggaactgg 1500 atggcctctg ttcaaaatac agatgggaca tggcatacaa attataatgt atggaaggca 1560 aatgaatgga tctcgtttgt ggagcctgaa cacgatgcca ttggactttt tctcatcggg 1620 gtttatcaac attacagttt actcaaatcc cgcgattcct cggcagcgac gacttttctg 1680 aataacattt ggacgcagat aacccgcgct ggtgatttca tctacaaaaa cattggcgct 1740 tctgggtttg gtccggctga tgcttctatc tgggaggagc aggtcgagta taatattttt 1800 actcaagtta cttatgccgc aggattgaat gctggacggt tgttagccca agaaaaaggt 1860 gatattactc gttcaaataa ctatttgtcg ggtgctcaaa ccatcaagga tgcgattcta 1920 cgatcgtttt tgtcttcacc acgaggtctc tggaataaat ctaatcgcta ttttaatcgg 1980 gcgatcaata cagacggtac agcgcgaacg acggttgatg cttcatcgga cttgatttgg 2040 gtttttggtc tcctttcacc gactgatact aggattcgag atcaccgcat taaggtacta 2100 tcacggctga cccatgatag gtacggcatt gctcgatatg aaaacgatga gttctattat 2160 tctagtccat atagccccgg tggacagtat gaggctggcg cagccgaacc agtttggcca 2220 caaatgacta tgtacgcgtc tatgattgag cattggaggg gtgatgatgc cactgcactg 2280 gctcggctta agtggtatgt cagccgaact gctcgcggct atgtcacacc cggtgaagct 2340 gtggactgga ccaatggtca accgcttatc agtacagcag ttgaaccagt aacgggctcc 2400 tggtttcaga tggcggtcct tacttactta aaccggtttg atctacgatt acctgatttt 2460 taa 2463 <210> 3 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> primer1 forward <400> 3 ggatccccgg aattcatgat ttatgtaaag cgtacgataa ccac 44 <210> 4 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> primer2 reverse <400> 4 atgcggccgc tcgagttaaa aatcaggtaa tcgtagatca aacc 44 <210> 5 <211> 821 <212> PRT <213> Artificial Sequence <220> <223> TP-DDase <400> 5 Asp Met Ile Tyr Val Lys Arg Thr Ile Thr Thr Pro Pro Asp Gly Gln 1 5 10 15 Tyr Glu Ala Glu Tyr Ala Ile Lys Ser Gly Thr Asn Ile Asn Thr Asp 20 25 30 His Thr Gly Tyr Thr Gly Ser Gly Phe Val Asp Asn Phe Asp Ala Ser 35 40 45 Asp Lys Gly Val Ser Phe Ile Ile Asn Val Pro Thr Ser Asp Thr Tyr 50 55 60 Thr Leu Arg Phe Arg Tyr Gly Asn Gly Gly Thr Thr Ile Ala Thr Arg 65 70 75 80 Thr Leu Phe Ile Asp Gly Gln Tyr Ala Gly Thr Leu Gln Phe Arg Asn 85 90 95 Leu Tyr Asn Trp Asp Val Trp Asp Thr Val Glu Thr Thr Val Trp Leu 100 105 110 Ser Ala Gly Val His Gln Val Val Leu Trp Tyr Ser Ser Glu Asn Asp 115 120 125 Gly Ala Ile Asn Leu Asp Asn Leu Ile Val Leu Gln Gln Thr Thr Pro 130 135 140 Thr Arg Thr Ser Ala Arg Ser Phe Trp Met Asn Asn Trp Ser Asn Leu 145 150 155 160 Ile Gly Ile His Met Ala Ser Lys Leu Ser Pro Thr Asp Asn Gly Asn 165 170 175 Tyr Gly Pro Arg Leu Ala Glu Leu His Phe Arg Gly Asp Trp Pro Thr 180 185 190 Asn Gln Ile Val Asp Ala Thr Ala Phe Phe Arg Asp Glu Thr Asp Leu 195 200 205 Thr Pro Ile Lys Tyr Thr Asn Ala His Ser Phe Asp Ser Glu Ala Trp 210 215 220 Phe Glu Asn Asp Gly Thr Leu Thr Val Arg Tyr Leu Thr Tyr Asn Gly 225 230 235 240 Ser Ala Leu Pro Val Gln Ile Thr Lys Gln Tyr Ala Met Val Pro Asn 245 250 255 Gln Asn Phe Leu Val Ile Lys Tyr Thr Phe Leu Asn Gln Thr Ser Ser 260 265 270 Ala Arg Thr Leu Asn Phe Leu Glu Gln Val His Leu Asn Asn Arg Thr 275 280 285 Ser Ser Asp Pro Asn Pro Gly Trp Gln His Gly Trp Trp Asp Val Ser 290 295 300 Arg Asn Ala Leu Gly Thr Asp Met Ser Gln Thr Gly Gln Phe Tyr Ile 305 310 315 320 Glu Leu Gly Ala Phe Gln Thr Met Asp Ser Tyr Gln Val Gly Asn Asp 325 330 335 Ala Asp Ser Asn Pro Asn Ser Gln Thr Ser Ser Pro Trp Tyr Gln Phe 340 345 350 Asp Ala Asn Gly Val Leu Asn Arg Cys Gly Asp Leu Trp Ser Gln Asn 355 360 365 Leu Ser Met Gly Phe Gln Lys Leu Ile Thr Val Pro Ala Gly Gly Ser 370 375 380 Val Thr Leu Ala Phe Tyr Tyr Ala Ile Gly Ser Thr Gln Glu Glu Ala 385 390 395 400 Glu Ala Ala Ala Asp Leu Ala Arg Ser Gln Thr Ala Asp Tyr Trp Phe 405 410 415 Thr Gln Thr Ala Ala Glu Tyr Asn Asn Trp Leu Asn Ser Gly Gln Arg 420 425 430 Val Asn Thr Ser Asp Ile Gly Ile Asn Thr Ala Phe Asp Arg Ser Leu 435 440 445 Ile Ile Asn Lys Gln Ala Gln His Pro Glu Phe Gly Ser Trp Pro Ala 450 455 460 Ala Thr Asn Pro Ser Tyr Gln Tyr Lys Val Trp Val Arg Asp Ser Ala 465 470 475 480 Val Thr Ala Met Gly Met Asp Ala Ala Asn His Leu Ser Glu Ala Glu 485 490 495 Lys Tyr Trp Asn Trp Met Ala Ser Val Gln Asn Thr Asp Gly Thr Trp 500 505 510 His Thr Asn Tyr Asn Val Trp Lys Ala Asn Glu Trp Ile Ser Phe Val 515 520 525 Glu Pro Glu His Asp Ala Ile Gly Leu Phe Leu Ile Gly Val Tyr Gln 530 535 540 His Tyr Ser Leu Leu Lys Ser Arg Asp Ser Ser Ala Ala Thr Thr Phe 545 550 555 560 Leu Asn Asn Ile Trp Thr Gln Ile Thr Arg Ala Gly Asp Phe Ile Tyr 565 570 575 Lys Asn Ile Gly Ala Ser Gly Phe Gly Pro Ala Asp Ala Ser Ile Trp 580 585 590 Glu Glu Gln Val Glu Tyr Asn Ile Phe Thr Gln Val Thr Tyr Ala Ala 595 600 605 Gly Leu Asn Ala Gly Arg Leu Leu Ala Gln Glu Lys Gly Asp Ile Thr 610 615 620 Arg Ser Asn Asn Tyr Leu Ser Gly Ala Gln Thr Ile Lys Asp Ala Ile 625 630 635 640 Leu Arg Ser Phe Leu Ser Ser Pro Arg Gly Leu Trp Asn Lys Ser Asn 645 650 655 Arg Tyr Phe Asn Arg Ala Ile Asn Thr Asp Gly Thr Ala Arg Thr Thr 660 665 670 Val Asp Ala Ser Ser Asp Leu Ile Trp Val Phe Gly Leu Leu Ser Pro 675 680 685 Thr Asp Thr Arg Ile Arg Asp His Arg Ile Lys Val Leu Ser Arg Leu 690 695 700 Thr His Asp Arg Tyr Gly Ile Ala Arg Tyr Glu Asn Asp Glu Phe Tyr 705 710 715 720 Tyr Ser Ser Pro Tyr Ser Pro Gly Gly Gln Tyr Glu Ala Gly Ala Ala 725 730 735 Glu Pro Val Trp Pro Gln Met Thr Met Tyr Ala Ser Met Ile Glu His 740 745 750 Trp Arg Gly Asp Asp Ala Thr Ala Leu Ala Arg Leu Lys Trp Tyr Val 755 760 765 Ser Arg Thr Ala Arg Gly Tyr Val Thr Pro Gly Glu Ala Val Asp Trp 770 775 780 Thr Asn Gly Gln Pro Leu Ile Ser Thr Ala Val Glu Pro Val Thr Gly 785 790 795 800 Ser Trp Phe Gln Met Ala Val Leu Thr Tyr Leu Asn Arg Phe Asp Leu 805 810 815 Arg Leu Pro Asp Phe 820 <110> REPUBLIC OF KOREA(MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Glucosyltransferase producing high degree of polymerization of isomalto-oligosacharrides and manufacturing method of high degree of polymerization of isomalto-oligosacharrides using the same <130> 2019p-05-024 <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 4680 <212> DNA <213> Artificial Sequence <220> <223> TPCITase coding sequence <400> 1 atgttgtctc tttatagaag aaaactcttt ataacaattt taatagtaat tttcgtgttg 60 tctaactttt tcacattatt tacttatcct atctcaccag gagtttctgt tgcttatgcc 120 gcttcaacag gtaatttgat tcaacgggtt tatacagata aagctcgata taatccaggg 180 gatcttgtta ccattagtgc tgatttaatt aataaaactg gttcgacatg gtccggcact 240 ttaactcttc agattaataa actagaaagc caaatttaca ctgcaagtca gtcagttact 300 cttgccaatg gggattcaac tacaatcaca tttacatgga ctgctccacc taccgacttt 360 gttggttatt atgccggaat tgcggcagga agtacagatt tcaatggaac aggtatcgat 420 gtaagttcct ctccgcttcg gtttcctcgc tatggtttta tctcaaactt tcctgtttca 480 cagactgccc agcaatccac agatatagtt aaacagatgg tggaagacta tcatcttaat 540 atttttcaat tttacgactg gatgtggcgg catgagaagc taattaagcg aaccaatgga 600 gttatagatt ctacttgggt ggatcttttt gatcgcacac tttcttggca aactatacaa 660 aacaatgttg cagcagttca ttcctttaat gcttatgcca tggcttacgc tatgagttat 720 gcagctcgtg aaggatatga acaaatgtgg ggaatcagcc ctagctgggg tatctttcaa 780 gatacggcac atcaaagcca gtttaacgtg gattttcata atggcaagtt tctgtggctt 840 tttaatccag caaatgtgaa ttggcagagt tggatcatta gtgaatacaa agatgctatc 900 aatacggcag gttttgatgg catacagatc gatcaaatgg ggcagcgaga caatgtttac 960 gattatacta gcttttccgt tactttgcct agtacattcg ctcagtttct tcaacaagtt 1020 aagtcagaat tagaatctaa taatgcaaaa aaaaatgttg ttacttttaa tattgttgat 1080 ggtacagtga acggttgggg ggcgggagaa attgctcgtt atggtgccag cgactttgac 1140 ttcagcgaga tttggtggaa agctaacaca tataatgatc tccgaaatta catcgaatgg 1200 ctcaggcaaa acaatggtgg caagcctgtt gtacttgcag cttatatgaa ttacaacgag 1260 gaatacggtc caatatatga agctgaatcc gctattctat caggcgttag tgtcaataca 1320 aaccattctg gttatacggg cactggcttt gtcgatgggt ttgaaacggt gggtgattct 1380 atcacgtgga caattgactt ccctgagact ggtgattatt cgtttgtttt tcgttatgca 1440 aatgccactg gtgctactgc tactcgtaat gtttatgtag atggacgatt tctgggacaa 1500 gtctcatttg ctaatcaggt ggattgggat acttgggcga cggacgcgtg gattcaaata 1560 gagggactta ctgctggtac tcatagtgtg acattaaagt atgacagtga caatataggc 1620 gctataaacg tcgatcattt aactcttgga gagtttgagg aacattcagt tcgattggca 1680 gatgcaatga tgtttgctag cggtgctaca cacattgagc ttggtgatac taatcagatg 1740 ttagcgcatg aatattatcc gaaccgttcc aagtcaatgc gtaattctct caaggcagcg 1800 atgagagact actatagttt tgctactgct tatgaaaatt tactctttga tcctgatata 1860 gttccagctg accaaggcaa ccaatggatt gcactgacaa ccggacaacc tttaagcggc 1920 aacggaactt ctggtactat ttggcaaatg attaaacgca agtctgatta tgatatcata 1980 catttgatta atctaatggg caatgatgat cagtggcgta acccagctgt tcagcctact 2040 ttccaaagca acattggagt taaatactat ccaggaccta atgccgctgt cagtggcgta 2100 tatcttgcca gtccggatct cgatcacggt atgaccattc cactcactta tacgacgggt 2160 aatgatagcc gaggtaacta tatccagttt gttgtgccaa gcttaaagta ctgggatatg 2220 atttatgtaa agcgtacgat aaccacaccg ccagatggac agtatgaagc agaatatgct 2280 attaaaagtg gcactaacat caataccgat catacaggat atactggcag cggttttgta 2340 gacaactttg atgctagtga taaaggtgta tcgtttatta taaatgttcc aactagtgat 2400 acctacacgc ttcgattccg atatggaaac ggcggtacta ctattgccac gcgaacttta 2460 tttattgatg gtcagtatgc aggtactctc caattccgaa atctttataa ctgggatgtg 2520 tgggatacag tggaaaccac tgtgtggctt tcagcgggtg ttcatcaagt ggtactttgg 2580 tacagttccg aaaatgatgg tgctattaat cttgataacc tgattgttct acaacaaact 2640 actccaacaa gaacttcggc tcgctccttt tggatgaata actggtccaa cttgataggt 2700 attcacatgg ctagcaaatt aagcccgact gataatggaa attatggccc tcgcctggct 2760 gaacttcatt tcagaggcga ttggcctact aatcaaatcg tagatgcaac tgcttttttc 2820 cgagatgaaa cggatcttac tcccataaag tataccaatg ctcatagctt tgattctgaa 2880 gcatggtttg aaaacgacgg gactcttacc gtgagatatc tgacctataa tggctcagcc 2940 ttgccagtac agattaccaa acaatacgcc atggtgccta accaaaattt tcttgtgatc 3000 aaatatactt ttttaaacca gacaagtagc gcacgtactt taaactttct agaacaagtt 3060 catttgaaca acaggactag tagcgatccg aatccagggt ggcaacatgg ctggtgggac 3120 gtgagtcgaa atgcacttgg tactgacatg agccagactg gtcagttcta tattgaattg 3180 ggtgcttttc agactatgga tagttatcag gtgggtaatg acgccgattc caatcctaac 3240 agtcagacgt catcaccgtg gtatcagttc gacgccaacg gtgttctaaa taggtgcggt 3300 gatctttggt cacagaatct tagcatgggt tttcagaaac ttattaccgt gcctgctgga 3360 ggtagtgtaa cattggcttt ttattatgct atcggttcaa ctcaggagga agcggaagca 3420 gcagcagatt tagctcgatc tcagacagct gactactggt ttacccaaac tgctgccgag 3480 tataataatt ggcttaacag cggccagcgg gttaatacgt ctgatattgg tatcaatact 3540 gcctttgatc ggagcctcat cataaacaaa caggcacaac atccggagtt tggttcctgg 3600 cccgcggcta caaatccctc ttatcagtac aaggtgtggg ttcgtgactc agctgtaaca 3660 gctatgggaa tggatgctgc taatcatctt tctgaggcgg aaaagtattg gaactggatg 3720 gcctctgttc aaaatacaga tgggacatgg catacaaatt ataatgtatg gaaggcaaat 3780 gaatggatct cgtttgtgga gcctgaacac gatgccattg gactttttct catcggggtt 3840 tatcaacatt acagtttact caaatcccgc gattcctcgg cagcgacgac ttttctgaat 3900 aacatttgga cgcagataac ccgcgctggt gatttcatct acaaaaacat tggcgcttct 3960 gggtttggtc cggctgatgc ttctatctgg gaggagcagg tcgagtataa tatttttact 4020 caagttactt atgccgcagg attgaatgct ggacggttgt tagcccaaga aaaaggtgat 4080 attactcgtt caaataacta tttgtcgggt gctcaaacca tcaaggatgc gattctacga 4140 tcgtttttgt cttcaccacg aggtctctgg aataaatcta atcgctattt taatcgggcg 4200 atcaatacag acggtacagc gcgaacgacg gttgatgctt catcggactt gatttgggtt 4260 tttggtctcc tttcaccgac tgatactagg attcgagatc accgcattaa ggtactatca 4320 cggctgaccc atgataggta cggcattgct cgatatgaaa acgatgagtt ctattattct 4380 agtccatata gccccggtgg acagtatgag gctggcgcag ccgaaccagt ttggccacaa 4440 atgactatgt acgcgtctat gattgagcat tggaggggtg atgatgccac tgcactggct 4500 cggcttaagt ggtatgtcag ccgaactgct cgcggctatg tcacacccgg tgaagctgtg 4560 gactggacca atggtcaacc gcttatcagt acagcagttg aaccagtaac gggctcctgg 4620 tttcagatgg cggtccttac ttacttaaac cggtttgatc tacgattacc tgatttttaa 4680 4680 <210> 2 <211> 2463 <212> DNA <213> Artificial Sequence <220> <223> DDase coding sequence <400> 2 atgatttatg taaagcgtac gataaccaca ccgccagatg gacagtatga agcagaatat 60 gctattaaaa gtggcactaa catcaatacc gatcatacag gatatactgg cagcggtttt 120 gtagacaact ttgatgctag tgataaaggt gtatcgttta ttataaatgt tccaactagt 180 gatacctaca cgcttcgatt ccgatatgga aacggcggta ctactattgc cacgcgaact 240 ttatttattg atggtcagta tgcaggtact ctccaattcc gaaatcttta taactgggat 300 gtgtgggata cagtggaaac cactgtgtgg ctttcagcgg gtgttcatca agtggtactt 360 tggtacagtt ccgaaaatga tggtgctatt aatcttgata acctgattgt tctacaacaa 420 actactccaa caagaacttc ggctcgctcc ttttggatga ataactggtc caacttgata 480 ggtattcaca tggctagcaa attaagcccg actgataatg gaaattatgg ccctcgcctg 540 gctgaacttc atttcagagg cgattggcct actaatcaaa tcgtagatgc aactgctttt 600 ttccgagatg aaacggatct tactcccata aagtatacca atgctcatag ctttgattct 660 gaagcatggt ttgaaaacga cgggactctt accgtgagat atctgaccta taatggctca 720 gccttgccag tacagattac caaacaatac gccatggtgc ctaaccaaaa ttttcttgtg 780 atcaaatata cttttttaaa ccagacaagt agcgcacgta ctttaaactt tctagaacaa 840 gttcatttga acaacaggac tagtagcgat ccgaatccag ggtggcaaca tggctggtgg 900 gacgtgagtc gaaatgcact tggtactgac atgagccaga ctggtcagtt ctatattgaa 960 ttgggtgctt ttcagactat ggatagttat caggtgggta atgacgccga ttccaatcct 1020 aacagtcaga cgtcatcacc gtggtatcag ttcgacgcca acggtgttct aaataggtgc 1080 ggtgatcttt ggtcacagaa tcttagcatg ggttttcaga aacttattac cgtgcctgct 1140 ggaggtagtg taacattggc tttttattat gctatcggtt caactcagga ggaagcggaa 1200 gcagcagcag atttagctcg atctcagaca gctgactact ggtttaccca aactgctgcc 1260 gagtataata attggcttaa cagcggccag cgggttaata cgtctgatat tggtatcaat 1320 actgcctttg atcggagcct catcataaac aaacaggcac aacatccgga gtttggttcc 1380 tggcccgcgg ctacaaatcc ctcttatcag tacaaggtgt gggttcgtga ctcagctgta 1440 acagctatgg gaatggatgc tgctaatcat ctttctgagg cggaaaagta ttggaactgg 1500 atggcctctg ttcaaaatac agatgggaca tggcatacaa attataatgt atggaaggca 1560 aatgaatgga tctcgtttgt ggagcctgaa cacgatgcca ttggactttt tctcatcggg 1620 gtttatcaac attacagttt actcaaatcc cgcgattcct cggcagcgac gacttttctg 1680 aataacattt ggacgcagat aacccgcgct ggtgatttca tctacaaaaa cattggcgct 1740 tctgggtttg gtccggctga tgcttctatc tgggaggagc aggtcgagta taatattttt 1800 actcaagtta cttatgccgc aggattgaat gctggacggt tgttagccca agaaaaaggt 1860 gatattactc gttcaaataa ctatttgtcg ggtgctcaaa ccatcaagga tgcgattcta 1920 cgatcgtttt tgtcttcacc acgaggtctc tggaataaat ctaatcgcta ttttaatcgg 1980 gcgatcaata cagacggtac agcgcgaacg acggttgatg cttcatcgga cttgatttgg 2040 gtttttggtc tcctttcacc gactgatact aggattcgag atcaccgcat taaggtacta 2100 tcacggctga cccatgatag gtacggcatt gctcgatatg aaaacgatga gttctattat 2160 tctagtccat atagccccgg tggacagtat gaggctggcg cagccgaacc agtttggcca 2220 caaatgacta tgtacgcgtc tatgattgag cattggaggg gtgatgatgc cactgcactg 2280 gctcggctta agtggtatgt cagccgaact gctcgcggct atgtcacacc cggtgaagct 2340 gtggactgga ccaatggtca accgcttatc agtacagcag ttgaaccagt aacgggctcc 2400 tggtttcaga tggcggtcct tacttactta aaccggtttg atctacgatt acctgatttt 2460 taa 2463 <210> 3 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> primer1 forward <400> 3 ggatccccgg aattcatgat ttatgtaaag cgtacgataa ccac 44 <210> 4 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> primer2 reverse <400> 4 atgcggccgc tcgagttaaa aatcaggtaa tcgtagatca aacc 44 <210> 5 <211> 821 <212> PRT <213> Artificial Sequence <220> <223> TP-DDase <400> 5 Asp Met Ile Tyr Val Lys Arg Thr Ile Thr Thr Pro Pro Asp Gly Gln 1 5 10 15 Tyr Glu Ala Glu Tyr Ala Ile Lys Ser Gly Thr Asn Ile Asn Thr Asp 20 25 30 His Thr Gly Tyr Thr Gly Ser Gly Phe Val Asp Asn Phe Asp Ala Ser 35 40 45 Asp Lys Gly Val Ser Phe Ile Ile Asn Val Pro Thr Ser Asp Thr Tyr 50 55 60 Thr Leu Arg Phe Arg Tyr Gly Asn Gly Gly Thr Thr Ile Ala Thr Arg 65 70 75 80 Thr Leu Phe Ile Asp Gly Gln Tyr Ala Gly Thr Leu Gln Phe Arg Asn 85 90 95 Leu Tyr Asn Trp Asp Val Trp Asp Thr Val Glu Thr Thr Val Trp Leu 100 105 110 Ser Ala Gly Val His Gln Val Val Leu Trp Tyr Ser Ser Glu Asn Asp 115 120 125 Gly Ala Ile Asn Leu Asp Asn Leu Ile Val Leu Gln Gln Thr Thr Pro 130 135 140 Thr Arg Thr Ser Ala Arg Ser Phe Trp Met Asn Asn Trp Ser Asn Leu 145 150 155 160 Ile Gly Ile His Met Ala Ser Lys Leu Ser Pro Thr Asp Asn Gly Asn 165 170 175 Tyr Gly Pro Arg Leu Ala Glu Leu His Phe Arg Gly Asp Trp Pro Thr 180 185 190 Asn Gln Ile Val Asp Ala Thr Ala Phe Phe Arg Asp Glu Thr Asp Leu 195 200 205 Thr Pro Ile Lys Tyr Thr Asn Ala His Ser Phe Asp Ser Glu Ala Trp 210 215 220 Phe Glu Asn Asp Gly Thr Leu Thr Val Arg Tyr Leu Thr Tyr Asn Gly 225 230 235 240 Ser Ala Leu Pro Val Gln Ile Thr Lys Gln Tyr Ala Met Val Pro Asn 245 250 255 Gln Asn Phe Leu Val Ile Lys Tyr Thr Phe Leu Asn Gln Thr Ser Ser 260 265 270 Ala Arg Thr Leu Asn Phe Leu Glu Gln Val His Leu Asn Asn Arg Thr 275 280 285 Ser Ser Asp Pro Asn Pro Gly Trp Gln His Gly Trp Trp Asp Val Ser 290 295 300 Arg Asn Ala Leu Gly Thr Asp Met Ser Gln Thr Gly Gln Phe Tyr Ile 305 310 315 320 Glu Leu Gly Ala Phe Gln Thr Met Asp Ser Tyr Gln Val Gly Asn Asp 325 330 335 Ala Asp Ser Asn Pro Asn Ser Gln Thr Ser Ser Pro Trp Tyr Gln Phe 340 345 350 Asp Ala Asn Gly Val Leu Asn Arg Cys Gly Asp Leu Trp Ser Gln Asn 355 360 365 Leu Ser Met Gly Phe Gln Lys Leu Ile Thr Val Pro Ala Gly Gly Ser 370 375 380 Val Thr Leu Ala Phe Tyr Tyr Ala Ile Gly Ser Thr Gln Glu Glu Ala 385 390 395 400 Glu Ala Ala Ala Asp Leu Ala Arg Ser Gln Thr Ala Asp Tyr Trp Phe 405 410 415 Thr Gln Thr Ala Ala Glu Tyr Asn Asn Trp Leu Asn Ser Gly Gln Arg 420 425 430 Val Asn Thr Ser Asp Ile Gly Ile Asn Thr Ala Phe Asp Arg Ser Leu 435 440 445 Ile Ile Asn Lys Gln Ala Gln His Pro Glu Phe Gly Ser Trp Pro Ala 450 455 460 Ala Thr Asn Pro Ser Tyr Gln Tyr Lys Val Trp Val Arg Asp Ser Ala 465 470 475 480 Val Thr Ala Met Gly Met Asp Ala Ala Asn His Leu Ser Glu Ala Glu 485 490 495 Lys Tyr Trp Asn Trp Met Ala Ser Val Gln Asn Thr Asp Gly Thr Trp 500 505 510 His Thr Asn Tyr Asn Val Trp Lys Ala Asn Glu Trp Ile Ser Phe Val 515 520 525 Glu Pro Glu His Asp Ala Ile Gly Leu Phe Leu Ile Gly Val Tyr Gln 530 535 540 His Tyr Ser Leu Leu Lys Ser Arg Asp Ser Ser Ala Ala Thr Thr Phe 545 550 555 560 Leu Asn Asn Ile Trp Thr Gln Ile Thr Arg Ala Gly Asp Phe Ile Tyr 565 570 575 Lys Asn Ile Gly Ala Ser Gly Phe Gly Pro Ala Asp Ala Ser Ile Trp 580 585 590 Glu Glu Gln Val Glu Tyr Asn Ile Phe Thr Gln Val Thr Tyr Ala Ala 595 600 605 Gly Leu Asn Ala Gly Arg Leu Leu Ala Gln Glu Lys Gly Asp Ile Thr 610 615 620 Arg Ser Asn Asn Tyr Leu Ser Gly Ala Gln Thr Ile Lys Asp Ala Ile 625 630 635 640 Leu Arg Ser Phe Leu Ser Ser Pro Arg Gly Leu Trp Asn Lys Ser Asn 645 650 655 Arg Tyr Phe Asn Arg Ala Ile Asn Thr Asp Gly Thr Ala Arg Thr Thr 660 665 670 Val Asp Ala Ser Ser Asp Leu Ile Trp Val Phe Gly Leu Leu Ser Pro 675 680 685 Thr Asp Thr Arg Ile Arg Asp His Arg Ile Lys Val Leu Ser Arg Leu 690 695 700 Thr His Asp Arg Tyr Gly Ile Ala Arg Tyr Glu Asn Asp Glu Phe Tyr 705 710 715 720 Tyr Ser Ser Pro Tyr Ser Pro Gly Gly Gln Tyr Glu Ala Gly Ala Ala 725 730 735 Glu Pro Val Trp Pro Gln Met Thr Met Tyr Ala Ser Met Ile Glu His 740 745 750 Trp Arg Gly Asp Asp Ala Thr Ala Leu Ala Arg Leu Lys Trp Tyr Val 755 760 765 Ser Arg Thr Ala Arg Gly Tyr Val Thr Pro Gly Glu Ala Val Asp Trp 770 775 780 Thr Asn Gly Gln Pro Leu Ile Ser Thr Ala Val Glu Pro Val Thr Gly 785 790 795 800 Ser Trp Phe Gln Met Ala Val Leu Thr Tyr Leu Asn Arg Phe Asp Leu 805 810 815 Arg Leu Pro Asp Phe 820
Claims (12)
Glucose transferase for preparing highly polymerized isomaltooligosaccharide consisting of the amino acid sequence of SEQ ID NO: 5.
According to claim 1, wherein the sugar transfer enzyme is dextran dextrinase (Dextran dextrinase) is a sugar transfer enzyme for the production of high polymerization isomaltooligosaccharide.
The sugar transfer enzyme according to claim 1, wherein the highly polymerized isomaltooligosaccharide has a degree of polymerization of 7 to 10.
According to claim 1, wherein the glycosyl transfer enzyme (TP-DDase) is a thermoan aerobacter thermocopriae (Thermoanaerobacter thermocopriae) strain derived from a high-polymerization isomaltooligosaccharide sugar transfer enzyme for production.
Recombinant vector comprising a glycotransferase (TP-DDase) gene consisting of a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 5.
The recombinant vector of claim 5, wherein the vector is one vector selected from the group consisting of pET28a, pET, pGEX, pQE, pDEST and pCOLD.
A transformant transformed with the recombinant vector of claim 5.
The method of claim 7, wherein the transformant is the recombinant vector of claim 5, BL21 (DE3), Rosetta (DE3), Rosetta2 (DE3), ArcticExpress (DE3), STAR (DE3), C41 (DE3) and C43 (DE3 ) Transformed by introducing into one E. coli selected from the group consisting of.
2) 상기 배양물로부터 당전이효소(TP-DDase)를 수득하는 단계;를 포함하는 당전이효소(TP-DDase)의 제조방법.
1) preparing a culture obtained by culturing the transformant of claim 7; And
2) obtaining a sugar transfer enzyme (TP-DDase) from the culture; a method for producing a sugar transfer enzyme (TP-DDase) comprising.
2) 상기 배양물로부터 당전이효소(TP-DDase)를 수득하는 단계; 및
3) 상기 단계 2)에서 수득한 당전이효소(TP-DDase)를 기질에 첨가하여 반응시키는 단계; 를 포함하는 고중합 이소말토올리고당을 제조하는 방법.
1) preparing a culture obtained by culturing the transformant of claim 7;
2) obtaining a glycosyltransferase (TP-DDase) from the culture; And
3) reacting by adding the glycotransferase (TP-DDase) obtained in step 2) to the substrate; Method for producing a highly polymerized isomaltooligosaccharide comprising a.
11. The method of claim 10, wherein the highly polymerized isomaltooligosaccharide has a degree of polymerization of 7 to 10.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20210050631A (en) * | 2019-10-28 | 2021-05-10 | 대한민국(농촌진흥청장) | Method for producing oligosaccharides derived from isomaltulose using sugar transferase for preparing highly polymerized isomaltooligosaccharide |
| KR20220060001A (en) * | 2020-11-02 | 2022-05-11 | 대한민국(농촌진흥청장) | Method for preparing dextrin-derived highly polymerized isomaltooligosaccharide by using a novel sugar transferase and the dextrin-derived highly polymerized isomaltooligosaccharide with the effect of improving bowel movements by the method |
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| KR20020002588A (en) * | 2000-06-30 | 2002-01-10 | 김일웅 | Method for production of isomaltooligosaccharides |
| KR20120103545A (en) * | 2009-07-01 | 2012-09-19 | 아마노 엔자임 가부시키가이샤 | Maltotriocil transferase, process for production thereof, and use thereof |
| KR101987262B1 (en) * | 2018-03-05 | 2019-09-27 | 전남대학교산학협력단 | Manufacturing Method of Cyclodextran Using Thermostable Cyclodextran Glucanotransferase |
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| KR20020002588A (en) * | 2000-06-30 | 2002-01-10 | 김일웅 | Method for production of isomaltooligosaccharides |
| KR20120103545A (en) * | 2009-07-01 | 2012-09-19 | 아마노 엔자임 가부시키가이샤 | Maltotriocil transferase, process for production thereof, and use thereof |
| KR101987262B1 (en) * | 2018-03-05 | 2019-09-27 | 전남대학교산학협력단 | Manufacturing Method of Cyclodextran Using Thermostable Cyclodextran Glucanotransferase |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20210050631A (en) * | 2019-10-28 | 2021-05-10 | 대한민국(농촌진흥청장) | Method for producing oligosaccharides derived from isomaltulose using sugar transferase for preparing highly polymerized isomaltooligosaccharide |
| KR102320952B1 (en) | 2019-10-28 | 2021-11-04 | 대한민국(농촌진흥청장) | Method for producing oligosaccharides derived from isomaltulose using sugar transferase for preparing highly polymerized isomaltooligosaccharide |
| KR20220060001A (en) * | 2020-11-02 | 2022-05-11 | 대한민국(농촌진흥청장) | Method for preparing dextrin-derived highly polymerized isomaltooligosaccharide by using a novel sugar transferase and the dextrin-derived highly polymerized isomaltooligosaccharide with the effect of improving bowel movements by the method |
| KR102599134B1 (en) * | 2020-11-02 | 2023-11-09 | 대한민국 | Method for preparing dextrin-derived highly polymerized isomaltooligosaccharide by using a novel sugar transferase and the dextrin-derived highly polymerized isomaltooligosaccharide with the effect of improving bowel movements by the method |
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