KR101693476B1 - Method for production of alpha-cyclodextrin with CGTase mutant - Google Patents
Method for production of alpha-cyclodextrin with CGTase mutant Download PDFInfo
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Abstract
본 발명은 효소를 이용한 α-사이클로덱스트린(α-cyclodextrin, α-CD)의 수득방법에 관한 것으로, 본 발명의 사이클로덱스트린 글루카노트랜스퍼레이즈 변이효소를 이용할 경우, α-사이클로덱스트린,β-사이클로덱스트린, γ-사이클로덱스트린을 포함하는 혼합물 중 α-사이클로덱스트린만을 선택적으로 수득할 수 있다.The present invention relates to a method for obtaining an? -Cyclodextrin (? -CD) using an enzyme. When the cyclodextrin glucanotransferase mutant of the present invention is used,? -Cyclodextrin,? -Cyclodextrin , only? -cyclodextrin in the mixture containing? -cyclodextrin can be selectively obtained.
Description
본 발명은 효소를 이용한 α-사이클로덱스트린의 수득방법에 관한 것으로, 좀 더 구체적으로는 사이클로덱스트린 글루카노트랜스퍼레이즈(cyclodextrin glucanotransferase, CGTase) 변이효소를 이용해 α-사이클로덱스트린, β-사이클로덱스트린 및 γ-사이클로덱스트린을 포함하는 혼합물 중 α-사이클로덱스트린만을 선택적으로 수득하는 방법이다.
The present invention relates to a method for obtaining an? -Cyclodextrin using an enzyme, and more particularly, to a method for producing an? -Cyclodextrin using? -Cyclodextrin,? -Cyclodextrin and? -Cyclodextrin using a cyclodextrin glucanotransferase (CGTase) Cyclodextrin in the mixture containing cyclodextrin.
α-사이클로덱스트린(cyclodextrin)은 α-1,4 공유결합으로 연결된 6개의 글루코오스 단위로 구성되는 비 환원성 당질의 환상(環狀) 구조이다. α-사이클로덱스트린은 유리 지방산 용해에 매우 효과적이며, 식이섬유 대신 지방과 복합체를 형성하는 특성을 보인다. 대부분의 식이섬유가 지방과 1:1의 비율로 복합체를 형성하나, α-사이클로덱스트린은 지방과 1:9 (fiber:fat)의 비율로 복합체를 형성한다. Cyclodextrin is a non-reducing saccharide cyclic structure composed of six glucose units linked by? -1,4 covalent bonds. α-Cyclodextrin is very effective in dissolving free fatty acids and has the property of forming a complex with fat instead of dietary fiber. Most dietary fibers form a complex with fat at a ratio of 1: 1, while α-cyclodextrin forms a complex with fat at a ratio of 1: 9 (fiber: fat).
또한, α-사이클로덱스트린은 체내에서 생체 이용률을 감소시키고자 하는 식품 및 음료에 응용되고 있는데, 체중 조절, 지질 및 당 대사 개선 등의 목적으로 사용된다. 그외, α-사이클로덱스트린은 건조 혼합물, 구운 제품, 인스턴트 차, 커피와 같은 음식의 향, 색상, 감미료의 운반체로도 사용된다. In addition,? -Cyclodextrin is used in foods and beverages intended to reduce bioavailability in the body and is used for weight control, lipid and glucose metabolism improvement. In addition, α-cyclodextrin is used as a carrier of flavor, color and sweetener of food such as dry mixture, baked product, instant tea, coffee.
한편, α-사이클로덱스트린은 사이클로덱스트린 글루카노트랜스퍼레이즈(cyclodextrin glucanotransferase, CGTase)를 사용하여 전분으로부터 제조되는 것이 일반적인데, 이 반응에서는 α-사이클로덱스트린 외에 β-사이클로덱스트린, γ-사이클로덱스트린도 동시에 생산되는 문제가 있다. 따라서, 이들 β-사이클로덱스트린, γ-사이클로덱스트린로부터 α-사이클로덱스트린을 선택적으로 분리하는 공정이 필요하다.
On the other hand, α-cyclodextrin is generally prepared from starch using cyclodextrin glucanotransferase (CGTase). In this reaction, besides α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin are simultaneously produced There is a problem. Therefore, a process for selectively separating? -Cyclodextrin from? -Cyclodextrin and? -Cyclodextrin is needed.
본 발명에서는 α-사이클로덱스트린, β-사이클로덱스트린 및 γ-사이클로덱스트린을 포함하는 혼합물 중 α-사이클로덱스트린(α-cyclodextrin, α-CD)만을 효과적으로 수득하는 방법을 개발하여 제공하고자 하였다.
In the present invention, a method for effectively obtaining only? -Cyclodextrin (? -CD) in a mixture containing? -Cyclodextrin,? -Cyclodextrin and? -Cyclodextrin has been developed.
상기 목적을 달성하기 위하여 본 발명은, α-사이클로덱스트린(α-cyclodextrin, α-CD), β-사이클로덱스트린(β-cyclodextrin, β-CD) 및 γ-사이클로덱스트린(γ-cyclodextrin, γ-CD)을 포함하는 혼합물에, 서열번호 1에 기재된 아미노산 서열을 갖는 사이클로덱스트린 글루카노트랜스퍼레이즈(CGTase) 변이효소를 첨가하여 반응시키는 것을 특징으로 하는 α-사이클로덱스트린(α-cyclodextrin, α-CD)의 수득방법을 제공한다. In order to accomplish the above object, the present invention provides a process for producing a cyclodextrin-containing cyclodextrin (? -CD), comprising the steps of mixing? -Cyclodextrin,? -CD,? -Cyclodextrin, Cyclodextrin (? -CD), which is characterized in that a cyclodextrin glucanotransferase (CGTase) mutant having the amino acid sequence of SEQ ID NO: 1 is added to a mixture containing? -Cyclodextrin And a method for producing the same.
야생형 CGTase의 경우, 전분 또는 전분의 가수분해물을 기질로 하여 α-, β-, γ-사이클로덱스트린(cyclodextrin, CD)을 동시에 생산할 수 있다. 그런데, 이들로부터 α-사이클로덱스트린 만을 순수하게 분리하게 위해서는 β-, γ-사이클로덱스트린(cyclodextrin, CD)을 제거해야 한다. 하지만, 이들 β-, γ- 타입은 α-타입과 분자량이 동일하기 때문에 분리하기가 용이하지 않은 문제가 있다. In the case of the wild-type CGTase, α-, β-, γ-cyclodextrin (CD) can be simultaneously produced using hydrolyzate of starch or starch as a substrate. However, in order to purely separate only? -Cyclodextrin from these,? -,? -Cyclodextrin (CD) must be removed. However, these β- and γ-types are not easily separated because α-type and molecular weight are the same.
한편, 본 발명의 서열번호 1에 기재된 사이클로덱스트린 글루카노트랜스퍼레이즈(CGTase) 변이효소는 야생형 사이클로덱스트린 글루카노트랜스퍼레이즈(CGTase)의 아미노산 서열 중 233번이 His에서 Tyr로 변형된 것인데, α-타입을 개환시키지는 못하나, β-, γ- 타입은 개환시킬 수 있는 것으로 본 발명을 통해 확인되었다. 이는 곧 α-타입을 β-, γ- 타입으로부터 쉽게 분리할 수 있음을 의미한다. 즉, α-, β-, γ-사이클로덱스트린(cyclodextrin, CD)이 혼합되어 있는 혼합물에, 본 발명의 서열번호 1에 기재된 사이클로덱스트린 글루카노트랜스퍼레이즈(CGTase) 변이효소를 처리하면, β-, γ-타입은 개환되어, 선형 형태로 바뀌게 되고, 여기에 α-글루코시다제(α-glucosidase, AMG)를 처리하면, 이들 선형 형태의 β-, γ-타입은 단당류로 분해되기 때문에, 쉽게 제거할 수 있는 것이다. Meanwhile, the cyclodextrin glucanotransferase (CGTase) mutant according to SEQ ID NO: 1 of the present invention is obtained by modifying 233 amino acid residues of His-Tyr in the amino acid sequence of wild-type cyclodextrin glucanotransferase (CGTase) , But the β- and γ-types can be opened by the present invention. This means that α-type can be easily separated from β-, γ-type. That is, when the cyclodextrin glucanotransferase (CGTase) mutant enzyme described in SEQ ID NO: 1 of the present invention is treated with a mixture of α-, β-, γ-cyclodextrin (CD) When the? -type is opened and converted into a linear form and? -glucosidase (AMG) is treated with it, the?,? -type of these linear forms decomposes into monosaccharides, You can do it.
한편, 본 발명의 α-사이클로덱스트린의 수득방법에 있어서, 상기 반응은 바람직하게 α-사이클로덱스트린, β-사이클로덱스트린 및 γ-사이클로덱스트린을 포함하는 혼합물에 사이클로덱스트린 글루카노트랜스퍼레이즈(CGTase) 변이효소를 첨가하여 β-사이클로덱스트린 및 γ-사이클로덱스트린을 개환시키는 단계 (A); 개환된 β-사이클로덱스트린 및 γ-사이클로덱스트린을 가수분해시켜 글루코스(glucose)를 생성시키는 단계 (B); 및 상기 혼합물로부터 단계 (B)를 통해 생성된 글루코스를 제거함으로써 α-사이클로덱스트린을 수득하는 단계 (C);를 포함하는 과정으로 수행될 수 있다. On the other hand, in the process for obtaining an? -Cyclodextrin of the present invention, the reaction is preferably carried out by adding a cyclodextrin glucanotransferase (CGTase) mutant enzyme to a mixture containing? -Cyclodextrin,? -Cyclodextrin and? -Cyclodextrin (A) opening the? -Cyclodextrin and the? -Cyclodextrin by addition of? -Cyclodextrin and? -Cyclodextrin; Hydrolyzing the ring-opened? -Cyclodextrin and? -Cyclodextrin to produce glucose (B); And (C) obtaining α-cyclodextrin by removing glucose produced from step (B) from the mixture.
한편, 본 발명의 α-사이클로덱스트린 수득방법에 있어서, 상기 글루코스 생성을 위한 가수분해는 바람직하게 α-글루코시다제(α-glucosidase, AMG)를 사용하는 것이 좋다. On the other hand, in the method for obtaining an? -Cyclodextrin of the present invention, it is preferable to use? -Glucosidase (AMG) for hydrolysis for producing glucose.
한편, 본 발명의 α-사이클로덱스트린 수득방법에 있어서, 상기 단계 (B)의 글루코스(glucose)의 분리는 일 예로, 글루코스와 α-사이클로덱스트린의 수용화도의 차이에 따른 분리방법을 이용할 수 있고, 분자량의 차이를 이용하는 방법을 이용할 수도 있다. Meanwhile, in the method for obtaining an? -Cyclodextrin of the present invention, the separation of the glucose in the step (B) may be performed, for example, by a separation method depending on the degree of water-solubility of glucose and? -Cyclodextrin, A method using a difference in molecular weight may be used.
한편, 본 발명의 α-사이클로덱스트린 수득방법에 있어서, 상기 반응은 30~60℃에서 0.25~24시간 동안 반응을 수행하는 것이 좋다. 반응 후에는 바람직하게 가열함으로써 효소를 실활시켜 반응을 정지시키는 것이 좋다. 가열시 온도는 효소 실활 온도 이상으로 수행하면 된다.
Meanwhile, in the method for obtaining an? -Cyclodextrin of the present invention, the reaction is preferably carried out at 30 to 60 ° C for 0.25 to 24 hours. After the reaction, it is preferable to stop the reaction by inactivating the enzyme by heating preferably. The heating temperature may be higher than the enzyme deactivation temperature.
본 발명에서와 같이 사이클로덱스트린 글루카노트랜스퍼레이즈 변이효소(CGTase)를 이용할 경우, α-사이클로덱스트린, β-사이클로덱스트린 및 γ-사이클로덱스트린을 포함하는 혼합물 중 α-사이클로덱스트린(α-cyclodextrin, α-CD)만을 간편하게 특이적으로 수득할 수 있다.
When a cyclodextrin glucanotransferase mutant enzyme (CGTase) is used as in the present invention, a mixture of? -Cyclodextrin,? -Cyclodextrin,? -Cyclodextrin and? -Cyclodextrin in a mixture containing? -Cyclodextrin, CD) can be obtained simply and specifically.
도 1은 CGTase 변이효소가 삽입된 벡터 맵이다.
도 2는 시그날 펩타이드 포함 CGTase 변이효소의 아미노산 서열이다.
도 3은 정제된 CGTase 변이효소의 전기영동 사진이다.
도 4은 CGTase 변이효소 3.81 μL(0.0148 unit, 1.8 mg/mL)를 이용한 α-CD, β-CD, γ-CD의 가수분해 후 TLC 결과 사진이다.
도 5는 CGTase 변이효소 7.62 μL(0.0296 unit, 1.78 mg/mL)를 이용하고, 도 1에 비해 장시간 가수분해 후, α-CD, β-CD, γ-CD의 TLC 사진이다.
도 6은 CGTase 변이효소를 이용한 α-CD, β-CD, γ-CD 혼합물의 가수분해에 따른 TLC 사진이다.
도 7은 도 6에서 얻은 CGTase 변이효소 가수분해 용액에 α-글루코시다제(α-glucosidase, AMG)를 처리한 처리구의 TLC 사진이다.
도 8은 'Sep-Pak C18컬럼'을 이용하여 CD 혼합물로부터 글루코스(glucose)를 제거하면서, α-CD를 분리하는 과정을 보여주는 TLC 사진이다. Fig. 1 is a vector map in which a CGTase mutant enzyme is inserted.
Figure 2 is the amino acid sequence of the CGTase mutant enzyme including the signal peptide.
3 is an electrophoresis photograph of purified CGTase mutant enzyme.
FIG. 4 is a photograph of TLC after α-CD, β-CD and γ-CD hydrolysis using 3.81 μL of CGTase mutant enzyme (0.0148 unit, 1.8 mg / mL).
5 is a TLC photograph of? -CD,? -CD and? -CD after long-term hydrolysis using 7.62 μL of CGTase mutant enzyme (0.0296 unit, 1.78 mg / mL)
6 is a TLC photograph of the hydrolysis of a mixture of? -CD,? -CD and? -CD using a CGTase mutant enzyme.
FIG. 7 is a TLC photograph of the treatment of α-glucosidase (AMG) treated with the CGTase-mutated enzyme hydrolysis solution obtained in FIG.
8 is a TLC photograph showing the process of separating? -CD while removing glucose from a CD mixture using a 'Sep-Pak C18 column'.
이하, 본 발명의 내용을 하기 실시예 또는 실험예를 들어 더욱 상세히 설명하고자 한다. 다만, 본 발명의 권리범위가 하기 실시예에만 한정되는 것은 아니고, 그와 등가의 기술적 사상의 변형까지를 포함한다.
Hereinafter, the present invention will be described in more detail with reference to the following examples or experimental examples. However, the scope of the present invention is not limited to the following embodiments, and includes modifications of equivalent technical ideas.
[제조예 1: 사이클로덱스트린 글루카노트랜스퍼레이즈(CGTase) 변이효소의 제조][Preparation Example 1: Preparation of cyclodextrin glucanotransferase (CGTase) mutant enzyme]
본 제조예에서는 사이클로덱스트린 글루카노트랜스퍼레이즈(CGTase) 변이효소를 제조하고자 하였다. In this Example, a cyclodextrin glucanotransferase (CGTase) mutant enzyme was prepared.
호알칼리성 바실러스 균(Bacillus sp.)Ⅰ-5로부터 유래한 CGTase 유전자의 특정부위변이(site-directed mutagenesis)를 위하여 하기 표 1의 프라이머들을 사용하였다. 돌연변이를 주기 위하여 원본 DNA(pR2CGT Ⅰ-5)에 하기 표 1의 프라이머를 사용하여 95℃에서 2분 동안 1회 실시한 후, 95℃에서 30초, 53.5℃에서 45초, 72℃에서 7분을 30회 PCR을 실시하였다. The primers of Table 1 below were used for site-directed mutagenesis of the CGTase gene from Bacillus sp. I-5. In order to mutate, the original DNA (pR2CGT I-5) was subjected to PCR for 1 min at 95 ° C for 2 min using the primer set forth in Table 1, followed by 30 sec at 95 ° C, 45 sec at 53.5 ° C and 7 min at 72 °
상기 반응이 끝난 후 PCR 산물을 벡터 (도 1)에 클로닝하고, CaCl2법을 이용하여 대장균(E. coli) MC1016에 형질전환 하였다. 형질전환 유전자를 지니는 대장균 균주를 스크리닝(screening) 하기 위하여 앰피실린 (최종농도 100 ㎍/㎖)을 함유한 'LB agar' 배지에 평판 도말하여 내성을 지니는 균주를 선별하였다. 선별된 균주는 아미노산 서열 중 233번 아미노산이 His에서 Tyr로 변형된 상태인데, 서열번호 1과 같은 아미노산 서열을 갖는다 (도 2 참조). 도 1은 CGTase 변이효소가 삽입된 벡터 맵이고, 도 2는 시그날 펩타이드 포함 CGTase 변이효소의 아미노산 서열이다. After the reaction was completed, the PCR product was cloned into a vector (FIG. 1) and transformed into E. coli MC1016 using the CaCl 2 method. Strain-resistant strains were selected on 'LB agar' medium containing ampicillin (final concentration 100 μg / ml) in order to screen E. coli strains carrying the transgene. The selected strain has the amino acid sequence as shown in SEQ ID NO: 1 (see FIG. 2) in which amino acid 233 in the amino acid sequence is modified from His to Tyr. FIG. 1 is a vector map in which a CGTase mutant enzyme is inserted, and FIG. 2 is an amino acid sequence of a CGTase mutant enzyme including a signal peptide.
상기 선별된 균주를 1 L의 LB 배지에 접종하여 24시간 가량 37℃에서 교반(200 rpm)하여 키운 후, 원심분리기를 이용하여 세포를 회수하였다. 회수한 세포를 다시 pH 6.0의 50 mM NaOAC 완충용액에 재부유(resuspension)한 후, 초음파로 세포파쇄를 실시하였다. The selected strains were inoculated in 1 L of LB medium and cultured for 24 hours at 37 DEG C with stirring (200 rpm). Cells were recovered using a centrifuge. The recovered cells were resuspended in 50 mM NaOAc buffer solution at pH 6.0 and then subjected to cell disruption using ultrasound.
세포파쇄로부터 수득된 조효소액을 12,000 rpm으로 20분 동안 원심분리하여 상층액을 취하고, 이를 β-CD가 결합된 세파덱스 수지(sephadex resin)가 담긴 컬럼에 흡착시킨 후, 다시 1% β-CD를 포함하는 pH 6.0의 50 mM NaOAC 완충용액을 주입하는 'β-CD 친화 크로마토그래피법'으로 정제하여 CGTase 변이효소를 수득하였다 (도 3 참조). 도 3은 정제된 CGTase 변이효소의 전기영동 사진이다.
The crude enzyme solution obtained from the cell disruption was centrifuged at 12,000 rpm for 20 minutes to take up the supernatant. The supernatant was adsorbed on a column containing sephadex resin bound with β-CD, and then washed with 1% β-CD (Fig. 3). The < [beta] -CD affinity chromatography " 3 is an electrophoresis photograph of purified CGTase mutant enzyme.
[[
실시예Example
1: α- 1: α-
CDCD
, β-, β-
CDCD
, γ-, γ-
CD 의Of CD
개환 실험 1 - 0.0148 Opening experiment 1 - 0.0148
unitunit
효소
본 실험예에서는 상기에서 수득한 사이클로덱스트린 글루카노트랜스퍼레이즈(CGTase) 변이효소의 양과 반응 시간에 따라, β-CD 및 γ-CD가 반응(개환)하는지 여부를 확인하고자 하였다. In this experimental example, it was determined whether β-CD and γ-CD reacted (open) according to the amount of the cyclodextrin glucanotransferase (CGTase) mutant enzyme and the reaction time.
pH 6.0의 50 mM NaOAC 완충용액에, α-CD, β-CD, γ-CD (1%)과 상기 제조예 1에서 수득한 CGTase 변이효소 3.81 μL(0.0148 unit, 1.8 mg/mL)를 각각 넣고, 45℃에서 15분, 30분, 45분, 1시간 반응시킨 후, 5분간 끓여 반응을 정지하였다. 이후, TLC 분석을 실시하였다. 하기 표 2는 각 샘플별 처리 조건을 정리해서 보여준다. 본 실험예 및 하기 실험예에서 사용한 TLC의 전개용매 조건은 모두 'n-butanol:ethanol:water' (5:5:3, v/v/v)로 하였다.3.81 μL (0.0148 unit, 1.8 mg / mL) of α-CD, β-CD and γ-CD (1%) and the CGTase mutant enzyme obtained in Preparation Example 1 were added to a 50 mM NaOAc buffer solution of pH 6.0 , Followed by reaction at 45 ° C for 15 minutes, 30 minutes, 45 minutes, and 1 hour, followed by boiling for 5 minutes to stop the reaction. TLC analysis was then performed. Table 2 summarizes the processing conditions for each sample. The developing solvent conditions of the TLC used in this Experimental Example and the following Experimental Example were all 'n-butanol: ethanol: water' (5: 5: 3, v / v / v).
변이효소CGTase
Mutant enzyme
3.81 μL
(0.0148 unit, 1.8 mg/mL)
3.81 μL
(0.0148 unit, 1.8 mg / mL)
1%
alpha -CD
One%
1%
β-CD
One%
1%γ-CD
One%
개환된 CD는 개환되지 않은 CD에 비해 TLC 상에서 높게 나타나는데, 도 4에서 보는 바와 같이, γ-CD는 CGTase 변이효소에 의해서 개환됨이 명확히 확인되었고, β-CD 오랜 반응시간(60분)에서 개환됨이 미미하나마 확인되었다. 하지만, α-CD는 시간이 지나도 개환되지 않았다 (도 4). As shown in FIG. 4, it was clearly confirmed that the γ-CD was converted by the CGTase mutant enzyme, and the β-CD was observed at the long reaction time (60 minutes) The reversion was confirmed to be minimal. However,? -CD did not open over time (Fig. 4).
한편, 하기 실험에서는 CGTase 변이효소의 첨가량과 반응 시간을 더 증가시켜 더욱 명확한 실험 결과를 도출하고자 하였다.
Meanwhile, in the following experiment, the amount of CGTase mutant and the reaction time were further increased to obtain more definite experimental results.
[[ 실시예Example 2: α- 2: α- CDCD , β-, β- CDCD , γ-, γ- CDCD 의 개환 실험 2 - 0.0296 Ring opening experiment 2 - 0.0296 unitunit 효소 enzyme 역가Potency 사용 0.5~2시간 반응] Use 0.5 ~ 2 hours reaction]
pH 6.0의 50 mM NaOAC 완충용액에, α-CD, β-CD, γ-CD(1%)과 상기 CGTase 변이효소 7.62 μL(0.0296 unit, 1.8 mg/mL)를 각각 넣고 45℃에서 0.5시간, 1시간, 2시간 반응시킨 후, 5분간 끓여 반응을 정지하여 TLC를 측정하였다. 하기 표 3은 각 샘플별 조건을 나타내었다.7.62 μL (0.0296 units, 1.8 mg / mL) of α-CD, β-CD and γ-CD (1%) and the CGTase mutant enzyme were added to a 50 mM NaOAc buffer solution at pH 6.0, After reacting for 1 hour and 2 hours, the reaction was stopped by boiling for 5 minutes and TLC was measured. Table 3 below shows the conditions for each sample.
변이효소CGTase
Mutant enzyme
7.62 μL
(0.0296 unit, 1.8 mg/mL)
7.62 μL
(0.0296 unit, 1.8 mg / mL)
1%alpha -CD
One%
1%β-CD
One%
1%
γ-CD
One%
실험결과, α-CD는 시간이 지나도 개환되지 않았고, β-CD는 초기에는 개환이 잘 되지 않았으나, 2시간 반응시 말토올리고당으로 개환됨을 명확히 확인할 수 있었다. 또한, γ-CD는 초기부터 개환이 잘 일어나는 것으로 확인되었다 (도 5). 이로부터, γ-CD>β-CD>α-CD 순으로 개환이 잘 되는 것을 확인할 수 있었다 .
The results showed that α-CD did not open over time, and β-CD was not re-opened at first, but it was clearly identified as maltooligosaccharide when reacted for 2 hours. In addition, it was confirmed that the γ-CD was changed from early on (FIG. 5). From this, it was confirmed that the rearrangement was successful in the order of γ-CD>β-CD> α-CD.
[[
실시예Example
3: α- 3: α-
CDCD
, β-, β-
CDCD
및 γ- And?
CD이 혼합된 CD mixed
혼합물에서의 개환 실험 - 0.08779 unit 효소 사용 2~24시간 반응]Opening experiment in mixture - 0.08779
pH 6.0의 50 mM NaOAC 완충용액에 α-CD, β-CD 및 γ-CD(1%) 혼합하여 혼합액(mixture)을 제조한 후, 상기 CGTase 변이효소 22.6 μL(0.0296 unit, 1.8 mg/mL)을 넣고, α-CD, β-CD 및 γ-CD(1%) 혼합액(mixture)을 45℃에서 2시간, 4시간, 16시간, 24시간 반응시킨 후, 5분간 끓여 반응을 정지하고 TLC 분석을 실시하였다. 실험결과, γ-CD는 2시간부터 분해되기 시작하여 24시간 때에는 완전히 분해된 것을 볼 수 있었으며, β-CD도 2시간부터 분해되기 시작하는 것을 볼 수 있었다 (도 6).The mixture was mixed with α-CD, β-CD and γ-CD (1%) in a 50 mM NaOAC buffer solution at pH 6.0. Then, 22.6 μL of the CGTase mutant enzyme (0.0296 units, 1.8 mg / mL) The reaction mixture was reacted at 45 ° C for 2 hours, 4 hours, 16 hours and 24 hours, and the mixture was boiled for 5 minutes to stop the reaction. TLC analysis Respectively. As a result of the experiment, γ-CD started to be decomposed from 2 hours and completely decomposed at 24 hours, and β-CD also started to decompose from 2 hours (FIG.
한편, 상기 'α-CD, β-CD 및 γ-CD(1%) 혼합액(mixture)에 CGTase 변이효소를 처리한 용액 300 μL'에 버퍼(50mM, NaOAC, PH 4.5) 290 μL, α-글루코시다제(α-glucosidase, AMG, 10 unit) 10 μL를 더 혼합하여 60℃에서 반응 후, TLC로 분석하였다. 실험결과, α-CD 외 나머지는 소당류로 잘린 것을 확인할 수 있었다 (도 7).Meanwhile, to 300 μL of the solution obtained by treating CGTase mutant enzyme in the mixture of α-CD, β-CD and γ-CD (1%), 290 μL of buffer (50 mM, NaOAC, pH 4.5) 10 μL of α-glucosidase (AMG, 10 unit) was further mixed and reacted at 60 ° C., and analyzed by TLC. As a result of the experiment, it was confirmed that the rest of the α-CD was truncated with the small saccharide (FIG. 7).
한편, 상기 「'α-CD, β-CD 및 γ-CD(1%) 혼합액(mixture)에 CGTase 변이효소를 처리한 용액 300 μL'에 버퍼(50mM, NaOAC, PH 4.5) 290 μL, α-글루코시다제(α-glucosidase, AMG, 10 unit) 10 μL를 더 혼합하여 60℃에서 반응시킨 반응물」을 농축하여 'Sep-Pak C18 컬럼'에 주입하고, 증류수와 메탄올 순으로 세척하였다. 세척 후, 용출된 물질을 'Speed-Vac'으로 감압농축하고, TLC 분석을 수행하였다. 실험결과, 증류수 세척을 통해, 수용성이 높은 글루코스(glucose)를 씻겨 내고, 메탄올로 용출하여 α-CD를 선택적으로 수득할 수 있었다 (도 8).To 300 μL of the solution obtained by treating the CGTase mutant enzyme with the α-CD, β-CD and γ-CD (1%) mixture, 290 μL of buffer (50 mM NaOAC, pH 4.5) 10 μL of α-glucosidase (AMG, 10 units) was further mixed and reacted at 60 ° C. The reaction mixture was concentrated, poured into a Sep-Pak C18 column, and washed with distilled water and methanol. After washing, the eluted material was concentrated under reduced pressure to 'Speed-Vac', and TLC analysis was performed. As a result of the experiment, glucose with high water-solubility was washed out by distilled water washing and eluted with methanol to selectively obtain? -CD (FIG. 8).
<110> Industry Academic Cooperation Foundation, Hallym University <120> Method for production of alpha-cyclodextrin with CGTase mutant <130> AP-2015-0052 <160> 1 <170> KopatentIn 2.0 <210> 1 <211> 685 <212> PRT <213> Bacillus sp. 1-5 <400> 1 Ala Pro Asp Thr Ser Val Ser Asn Lys Gln Asn Phe Ser Thr Asp Val 1 5 10 15 Ile Tyr Gln Ile Phe Thr Asp Arg Phe Ser Asp Gly Asn Pro Ala Asn 20 25 30 Asn Pro Thr Gly Ala Ala Phe Asp Gly Ser Cys Thr Asn Leu Arg Leu 35 40 45 Tyr Cys Gly Gly Asp Trp Gln Gly Ile Ile Asn Lys Ile Asn Asp Gly 50 55 60 Tyr Leu Thr Gly Met Gly Ile Thr Ala Ile Trp Ile Ser Gln Pro Val 65 70 75 80 Glu Asn Ile Tyr Ser Val Ile Asn Tyr Ser Gly Val His Asn Thr Ala 85 90 95 Tyr His Gly Tyr Trp Ala Arg Asp Phe Lys Lys Thr Asn Pro Ala Tyr 100 105 110 Gly Thr Met Gln Asp Phe Lys Asn Leu Ile Asp Thr Ala His Ala His 115 120 125 Asn Ile Lys Val Ile Ile Asp Phe Ala Pro Asn His Thr Ser Pro Ala 130 135 140 Ser Ser Asp Asp Pro Ser Phe Ala Glu Asn Gly Arg Leu Tyr Asp Asn 145 150 155 160 Gly Asn Leu Leu Gly Gly Tyr Thr Asn Asp Thr Gln Asn Leu Phe His 165 170 175 His Tyr Gly Gly Thr Asp Phe Ser Thr Ile Glu Asn Gly Ile Tyr Lys 180 185 190 Asn Leu Tyr Asp Leu Ala Asp Leu Asn His Asn Asn Ser Ser Val Asp 195 200 205 Val Tyr Leu Lys Asp Ala Ile Lys Met Trp Leu Asp Leu Gly Val Asp 210 215 220 Gly Ile Arg Val Asp Ala Val Lys Tyr Met Pro Phe Gly Trp Gln Lys 225 230 235 240 Ser Phe Met Ser Thr Ile Asn Asn Tyr Lys Pro Val Phe Thr Phe Gly 245 250 255 Glu Trp Phe Leu Gly Val Asn Glu Ile Ser Pro Glu Tyr His Gln Phe 260 265 270 Ala Asn Glu Ser Gly Met Ser Leu Leu Asp Phe Arg Phe Ala Gln Lys 275 280 285 Ala Arg Gln Val Phe Arg Asp Asn Thr Asp Asn Met Tyr Gly Leu Lys 290 295 300 Ala Met Leu Glu Gly Ser Glu Val Asp Tyr Ala Gln Val Asn Asp Gln 305 310 315 320 Val Thr Phe Ile Asp Asn His Asp Met Glu Arg Phe His Thr Ser Asn 325 330 335 Gly Asp Arg Arg Lys Leu Glu Gln Ala Leu Ala Phe Thr Leu Thr Ser 340 345 350 Arg Gly Val Pro Ala Ile Tyr Tyr Gly Ser Glu Gln Tyr Met Ser Gly 355 360 365 Gly Asn Asp Pro Asp Asn Arg Ala Arg Ile Pro Ser Phe Ser Thr Thr 370 375 380 Thr Thr Ala Tyr Gln Val Ile Gln Lys Leu Ala Pro Leu Arg Lys Ser 385 390 395 400 Asn Pro Ala Ile Ala Tyr Gly Ser Thr Gln Glu Arg Trp Ile Asn Asn 405 410 415 Asp Val Ile Ile Tyr Glu Arg Lys Phe Gly Asn Asn Val Ala Val Val 420 425 430 Ala Ile Asn Arg Asn Met Asn Thr Pro Ala Ser Ile Thr Gly Leu Val 435 440 445 Thr Ser Leu Pro Gln Gly Ser Tyr Asn Asp Val Leu Gly Gly Ile Leu 450 455 460 Asn Gly Asn Thr Leu Thr Val Gly Ala Gly Gly Ala Ala Ser Asn Phe 465 470 475 480 Thr Leu Ala Pro Gly Gly Thr Ala Val Trp Gln Tyr Thr Thr Asp Ala 485 490 495 Thr Ala Pro Ile Ile Gly Asn Val Gly Pro Met Met Ala Lys Pro Gly 500 505 510 Val Thr Ile Thr Ile Asp Gly Arg Ala Ser Ala Arg Gln Gly Thr Val 515 520 525 Tyr Phe Gly Thr Thr Ala Val Thr Gly Ala Asp Ile Val Ala Trp Glu 530 535 540 Asp Thr Gln Ile Gln Val Lys Ile Leu Arg Val Pro Gly Gly Ile Tyr 545 550 555 560 Asp Ile Arg Val Ala Asn Ala Ala Gly Ala Ala Ser Asn Ile Tyr Asp 565 570 575 Asn Phe Glu Val Leu Thr Gly Asp Gln Val Thr Val Arg Phe Val Ile 580 585 590 Asn Asn Ala Thr Thr Ala Leu Gly Gln Asn Val Phe Leu Thr Gly Asn 595 600 605 Val Ser Glu Leu Gly Asn Trp Asp Pro Asn Asn Ala Ile Gly Pro Met 610 615 620 Tyr Asn Gln Val Val Tyr Gln Tyr Pro Thr Trp Tyr Tyr Asp Val Ser 625 630 635 640 Val Pro Ala Gly Gln Thr Ile Glu Phe Lys Phe Leu Lys Lys Gln Gly 645 650 655 Ser Thr Val Thr Trp Glu Gly Gly Ala Asn Arg Thr Phe Thr Thr Pro 660 665 670 Thr Ser Gly Thr Ala Thr Val Asn Val Asn Trp Gln Pro 675 680 685 <110> Industry Academic Cooperation Foundation, Hallym University <120> Method for production of alpha-cyclodextrin with CGTase mutant <130> AP-2015-0052 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 685 <212> PRT <213> Bacillus sp. 1-5 <400> 1 Ala Pro Asp Thr Ser Val Ser Asn Lys Gln Asn Phe Ser Thr Asp Val 1 5 10 15 Ile Tyr Gln Ile Phe Thr Asp Arg Phe Ser Asp Gly Asn Pro Ala Asn 20 25 30 Asn Pro Thr Gly Ala Ala Phe Asp Gly Ser Cys Thr Asn Leu Arg Leu 35 40 45 Tyr Cys Gly Gly Asp Trp Gln Gly Ile Ile Asn Lys Ile Asn Asp Gly 50 55 60 Tyr Leu Thr Gly Met Gly Ile Thr Ala Ile Trp Ile Ser Gln Pro Val 65 70 75 80 Glu Asn Ile Tyr Ser Val Ile Asn Tyr Ser Gly Val His Asn Thr Ala 85 90 95 Tyr His Gly Tyr Trp Ala Arg Asp Phe Lys Lys Thr Asn Pro Ala Tyr 100 105 110 Gly Thr Met Gln Asp Phe Lys Asn Leu Ile Asp Thr Ala His Ala His 115 120 125 Asn Ile Lys Val Ile Ile Asp Phe Ala Pro Asn His Thr Ser Pro Ala 130 135 140 Ser Ser Asp Asp Ser Ser Phe Ala Glu Asn Gly Arg Leu Tyr Asp Asn 145 150 155 160 Gly Asn Leu Leu Gly Gly Tyr Thr Asn Asp Thr Gln Asn Leu Phe His 165 170 175 His Tyr Gly Gly Thr Asp Phe Ser Thr Ile Glu Asn Gly Ile Tyr Lys 180 185 190 Asn Leu Tyr Asp Leu Ala Asp Leu Asn His Asn Asn Ser Ser Val Asp 195 200 205 Val Tyr Leu Lys Asp Ala Ile Lys Met Trp Leu Asp Leu Gly Val Asp 210 215 220 Gly Ile Arg Val Asp Ala Val Lys Tyr Met Pro Phe Gly Trp Gln Lys 225 230 235 240 Ser Phe Met Ser Thr Ile Asn Asn Tyr Lys Pro Val Phe Thr Phe Gly 245 250 255 Glu Trp Phe Leu Gly Val Asn Glu Ile Ser Pro Glu Tyr His Gln Phe 260 265 270 Ala Asn Glu Ser Gly Met Ser Leu Leu Asp Phe Arg Phe Ala Gln Lys 275 280 285 Ala Arg Gln Val Phe Arg Asp Asn Thr Asp Asn Met Tyr Gly Leu Lys 290 295 300 Ala Met Leu Glu Gly Ser Glu Val Asp Tyr Ala Gln Val Asn Asp Gln 305 310 315 320 Val Thr Phe Ile Asp Asn His Asp Met Glu Arg Phe His Thr Ser Asn 325 330 335 Gly Asp Arg Arg Lys Leu Glu Gln Ala Leu Ala Phe Thr Leu Thr Ser 340 345 350 Arg Gly Val Pro Ala Ile Tyr Tyr Gly Ser Glu Gln Tyr Met Ser Gly 355 360 365 Gly Asn Asp Pro Asp Asn Arg Ala Arg Ile Pro Ser Phe Ser Thr Thr 370 375 380 Thr Thr Ala Tyr Gln Val Ile Gln Lys Leu Ala Pro Leu Arg Lys Ser 385 390 395 400 Asn Pro Ala Ile Ala Tyr Gly Ser Thr Gln Glu Arg Trp Ile Asn Asn 405 410 415 Asp Val Ile Ile Tyr Glu Arg Lys Phe Gly Asn Asn Val Ala Val Val 420 425 430 Ala Ile Asn Arg Asn Met Asn Thr Pro Ala Ser Ile Thr Gly Leu Val 435 440 445 Thr Ser Leu Pro Gln Gly Ser Tyr Asn Asp Val Leu Gly Gly Ile Leu 450 455 460 Asn Gly Asn Thr Leu Thr Val Gly Ala Gly Gly Ala Ala Ser Asn Phe 465 470 475 480 Thr Leu Ala Pro Gly Gly Thr Ala Val Trp Gln Tyr Thr Thr Asp Ala 485 490 495 Thr Ala Pro Ile Gly Asn Val Gly Pro Met Met Ala Lys Pro Gly 500 505 510 Val Thr Ile Thr Ile Asp Gly Arg Ala Ser Ala Arg Gln Gly Thr Val 515 520 525 Tyr Phe Gly Thr Thr Ala Val Thr Gly Ala Asp Ile Val Ala Trp Glu 530 535 540 Asp Thr Gln Ile Gln Val Lys Ile Leu Arg Val Pro Gly Gly Ile Tyr 545 550 555 560 Asp Ile Arg Val Ala Asn Ala Ala Gly Ala Ala Ser Asn Ile Tyr Asp 565 570 575 Asn Phe Glu Val Leu Thr Gly Asp Gln Val Thr Val Arg Phe Val Ile 580 585 590 Asn Asn Ala Thr Thr Ala Leu Gly Gln Asn Val Phe Leu Thr Gly Asn 595 600 605 Val Ser Glu Leu Gly Asn Trp Asp Pro Asn Asn Ale Gly Pro Met 610 615 620 Tyr Asn Gln Val Val Tyr Gln Tyr Pro Thr Trp Tyr Tyr Asp Val Ser 625 630 635 640 Val Pro Ala Gly Gln Thr Ile Glu Phe Lys Phe Leu Lys Lys Gln Gly 645 650 655 Ser Thr Val Thr Trp Glu Gly Gly Ala Asn Arg Thr Phe Thr Thr Pro 660 665 670 Thr Ser Gly Thr Ala Thr Val Asn Val Asn Trp Gln Pro 675 680 685
Claims (5)
To a mixture comprising? -cyclodextrin,? -cyclodextrin and? -cyclodextrin, A method for obtaining? -Cyclodextrin, which comprises reacting a cyclodextrin glucanotransferase (CGTase) mutant having the amino acid sequence set forth in SEQ ID NO:
상기 반응은,
α-사이클로덱스트린, β-사이클로덱스트린 및 γ-사이클로덱스트린을 포함하는 혼합물에 사이클로덱스트린 글루카노트랜스퍼레이즈 변이효소를 첨가하여 β-사이클로덱스트린 및 γ-사이클로덱스트린을 개환시키는 단계 (A);
개환된 β-사이클로덱스트린 및 γ-사이클로덱스트린을 가수분해시켜 글루코스(glucose)를 생성시키는 단계 (B); 및
상기 혼합물로부터 단계 (B)를 통해 생성된 글루코스를 제거함으로써 α-사이클로덱스트린을 수득하는 단계 (C);를 포함하는 것을 특징으로 하는 α-사이클로덱스트린의 수득방법.
The method according to claim 1,
The above-
(A) opening the? -cyclodextrin and? -cyclodextrin by adding a cyclodextrin glucanotransferase mutant to a mixture comprising? -cyclodextrin,? -cyclodextrin and? -cyclodextrin;
Hydrolyzing the ring-opened? -Cyclodextrin and? -Cyclodextrin to produce glucose (B); And
(C) obtaining alpha -cyclodextrin by removing glucose produced from step (B) from said mixture.
상기 글루코스 생성을 위한 가수분해는,
α-글루코시다제를 사용하는 것을 특징으로 하는 α-사이클로덱스트린의 수득방법.
3. The method of claim 2,
The hydrolysis for the production of glucose,
A method for obtaining an? -cyclodextrin using? -glucosidase.
상기 사이클로덱스트린 글루카노트랜스퍼레이즈 변이효소는,
야생형 사이클로덱스트린 글루카노트랜스퍼레이즈의 아미노산 서열 중 233번이 His에서 Tyr로 변형된 것을 특징으로 하는 α-사이클로덱스트린의 수득방법.
The method according to claim 1,
The above-mentioned cyclodextrin glucanotransferase mutant enzyme can be produced,
Wherein the amino acid sequence of wild-type cyclodextrin glucanotransferase is modified from His to Tyr 233.
상기 반응은,
30~60℃에서 0.25~24시간 동안 수행하는 것을 특징으로 하는 α-사이클로덱스트린의 수득방법.The method according to claim 1,
The above-
At 30 to 60 DEG C for 0.25 to 24 hours.
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Appl. Microbiol. Biotechnol., Vol. 84, pp. 119-133 (2009.) |
Biochemistry, Vol. 32, pp. 6624-6631 (1993.) |
Starch, Vol. 53, pp. 75-83 (2001.) |
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