KR101693477B1 - Method for production of slowly digestible starch with alpha amylase - Google Patents
Method for production of slowly digestible starch with alpha amylase Download PDFInfo
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Abstract
본 발명은 전분에 서열번호 1의 아미노산 서열을 가지는 알파 아밀라아제를 처리하는 것을 특징으로 하는 지소화성 전분의 제조방법에 관한 것으로, 본 발명 서열번호 1의 아미노산 서열을 가지는 알파 아밀라아제는 상온에서 짧은 시간 안에 간편하게 지소화성 전분을 제조할 수 있다.The present invention relates to a method for producing a starch-digestible starch, which comprises treating an alpha amylase having an amino acid sequence of SEQ ID NO: 1 in starch, wherein the alpha amylase having an amino acid sequence of SEQ ID NO: It is possible to easily produce the starch-decomposable starch.
Description
본 발명은 지소화성 전분의 제조방법에 관한 것으로, 더욱 구체적으로는 서열번호 1의 아미노산 서열을 가지는 알파 아밀라아제를 이용한 지소화성 전분의 제조방법에 관한 것이다.
More particularly, the present invention relates to a method for producing a digestible starch using an alpha amylase having an amino acid sequence of SEQ ID NO: 1.
전분은 곡류, 서류 등의 식물체에 들어있는 저장 다당류로 인간의 주요 탄수화물 공급원이다. 전분은 영양학적으로 빠르게 소화되는 전분(rapidly digestible starch, RDS), 지소화성 전분(slowly digestible starch, SDS), 난소화성 전분(resistant starch, RS)으로 나누어진다. 지소화성 전분은 소장에서 완전히 소화되지만 소화 속도가 느린 전분이고, 난소화성 전분은 소장에서 소화되지 않고 대장에서 미생물에 의해 발효되는 전분이다.Starch is a storage polysaccharide contained in plants such as grains, papers, etc., and is a major source of human carbohydrates. Starch is divided into rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS). The hydrolyzable starch is a starch which is completely digested in the small intestine but has a slow digestion rate and the indigestible starch is a starch which is not digested in the small intestine but fermented by microorganisms in the large intestine.
일반적인 전분은 체내에서 포도당으로 전환되어 빠르게 흡수된 후, 에너지로 쓰이거나 저장된다. 이와 같은 빠른 소화는 충분한 포만감을 느끼지 못하게 하고, 체중을 쉽게 증가시키는 문제를 수반한다. Typical starches are converted to glucose in the body and are rapidly absorbed and then used or stored as energy. Such rapid digestion is accompanied by the problem of not feeling enough satiety and increasing the weight easily.
그러나, 지소화성 전분은 위와 소장을 거쳐 대장까지 도달하는 속도가 느리기 때문에 포만감의 지속시간이 길다. 또한, 혈당이 급격하게 상승하는 것을 막아 당뇨 관리에도 좋고, 콜레스테롤이 흡수되는 것도 막아 고지혈증과 같은 대사성 질환을 예방할 수도 있다.However, since the fatigueable starch has a slow rate of reaching the colon through the stomach and small intestine, the duration of satiety is long. In addition, it prevents blood sugar from rising sharply and is good for managing diabetes and prevents absorption of cholesterol, thereby preventing metabolic diseases such as hyperlipidemia.
또한, 혈당 수치를 빠르게 증가시키는 식품은 인슐린이 과량 분비되고 반복되면 췌장의 과부하로 인슐린이 분비되어도 그 역할을 제대로 하지 못하는 '인슐린 저항성'을 유발하는데, 혈당 수치를 천천히 증가시키는 지소화성 전분은 비만이나 당뇨병 환자들의 식이에 효과적으로 이용될 수 있다.In addition, foods that rapidly increase blood sugar levels cause excessive insulin secretion and repetition, resulting in 'insulin resistance' that does not play a role in insulin secretion due to overload of the pancreas. However, And diabetic patients.
한편, 지소화성 전분을 제조하기 위한 방법으로는, 통상적으로 물리적 처리, 화학적 처리 및 효소적 처리를 단독 또는 병행하는 방법이 사용된다. 이중 효소적 처리는 환경친화적이며, 소비자들에게 안전하고 건강지향적인 처리라 할 수 있다. 또한, 특이적인 반응만을 수행하기 때문에, 우리가 원하는 반응만을 유도할 수 있고, 수율도 높으며, 반응의 부산물이 적은 장점이 있다.
On the other hand, as a method for producing the hydrogenated starch, a physical treatment, a chemical treatment and an enzymatic treatment are usually used alone or in combination. Enzymatic treatment is eco-friendly and safe and health-oriented for consumers. In addition, since only a specific reaction is performed, it is possible to induce only the desired reaction, and the yield is high, and the byproduct of the reaction is small.
본 발명은 간편하고, 경제적인 방법으로 지소화성 전분을 제조하는 방법을 개발하여 제공하는 것을 목적으로 한다.
It is an object of the present invention to develop and provide a method for producing a digestible starch by a simple and economical method.
상기 목적을 달성하기 위하여, 본 발명은 전분에 서열번호 1의 아미노산 서열을 가지는 알파 아밀라아제를 처리하여 가수분해시키는 것을 특징으로 하는 지소화성 전분의 제조방법을 제공한다. In order to achieve the above object, the present invention provides a method for producing a digestible starch, which comprises hydrolyzing an alpha amylase having an amino acid sequence of SEQ ID NO: 1 into starch.
전분에 본 발명의 서열번호 1의 아미노산 서열을 갖는 효소를 처리하여 가수분해된 반응 생성물을 수득한 후, 이에 대한 알파 아밀라아제의 반응 정도를 측정한 결과, 본 발명의 서열번호 1의 아미노산 서열을 갖는 효소를 처리한 샘플이 그렇지 않은 대조군 샘플에 비해 반응 속도가 느려짐을 확인할 수 있었다. 즉, 본 발명의 효소 처리로 인하여, 소화가 쉬운 전분(RDS)이 지소화성 전분(SDS)으로 전환된 것을 확인할 수 있었던 것이다. The hydrolyzed reaction product was obtained by treating the starch with the enzyme having the amino acid sequence of SEQ ID NO: 1 of the present invention, and the degree of the reaction of the alpha amylase was measured. As a result, the amino acid sequence of SEQ ID NO: It was confirmed that the enzyme-treated sample had a slower reaction rate than the control sample. In other words, it was confirmed that starch (RDS), which is easy to digest, was converted into the digestible starch (SDS) due to the enzyme treatment of the present invention.
또한, 본 발명의 서열번호 1의 아미노산 서열을 갖는 효소는 반응 시간이 매우 빠르고, 상온 (20℃ 내외)에서 최적 반응 속도를 보이기 때문에, 짧은 시간에 경제적으로 지소화성 전분을 제조할 수 있는 장점이 있다. In addition, since the enzyme having the amino acid sequence of SEQ ID NO: 1 of the present invention has a very fast reaction time and exhibits an optimum reaction rate at room temperature (around 20 DEG C), it is advantageous to economically produce the digestible starch in a short time have.
한편, 본 발명에 있어서, 상기 알파 아밀라아제는, 바람직하게 비피도박테리움 롱검 서브스피시스 롱검 JCM 1217 (Bifidobacterium longum subsp. longgum JCM 1217) (KCTC 3127) 균주 유래인 것이 좋다.On the other hand, in the present invention, the alpha amylase is preferably Bifidobacterium ronggeom sub speaker system ronggeom JCM 1217 (Bifidobacterium longum subsp. longgum JCM 1217) (KCTC 3127).
또한, 본 발명에 있어서, 상기 알파 아밀라아제의 처리는, 바람직하게 10~25℃의 온도, 4.5~5.5의 pH 조건에서 수행하는 것이 좋다.
In the present invention, the treatment of the alpha amylase is preferably performed at a temperature of 10 to 25 DEG C and a pH of 4.5 to 5.5.
본 발명에 의할 경우 전분에 서열번호 1의 아미노산 서열을 가지는 알파 아밀라아제를 처리하여 가수분해시킴으로써 지소화성 전분을 제조할 수 있다.According to the present invention, the starch can be hydrolyzed by treating with alpha amylase having the amino acid sequence of SEQ. ID.
또한, 본 발명은 상온에서 짧은 시간 안에 지소화성 전분을 제조할 수 있어 매우 경제적이다.
In addition, the present invention is capable of producing the digestible starch in a short time at room temperature, and is very economical.
도 1은 본 발명의 인서트(insert) DNA (bllj-0710)가 삽입된 재조합 벡터의 맵이다.
도 2는 정제된 본 발명 알파 아밀라아제 (BiLA) 효소의 SDS-PAGE 전기영동 사진이다.
도 3은 다양한 기질에, 본 발명 알파 아밀라아제 (BiLA) 효소를 처리한 TLC 결과이다. (A)는 {말토오스(maltose, G2), 말토트리오스(maltotriose, G3), 말토테트라오스(maltotetraose, G4), 말토헵타오스(maltoheptaose, G7), 알파-사이클로덱스트린(α-cyclodextrin, alpha-CD), 베타-사이클로덱스트린(β-cyclodextrin, beta-CD), 감마-사이클로덱스트린(γ-cyclodextrin, gamma-CD), 아밀로오스(amylose), 아밀로펙틴(amylopectin), 가용성 전분(soluble starch), 풀루란(pullulan)}에 대한 TLC 결과이고, (B)는 pNPG5에 대한 TLC 결과이다.
도 4는 본 발명 알파 아밀라아제 (BiLA) 효소의 반응 최적 온도 및 pH를 확인한 실험 결과이다. (A)는 BiLA 효소의 반응 최적 온도를 확인한 실험 결과이고, (B)는 BiLA 효소의 반응 최적 pH를 확인한 실험 결과이다.
도 5는 기질에 따른 본 발명 알파 아밀라아제 (BiLA) 효소의 반응성 (k cat/K m)을 확인한 결과이다. (A)는 가용성 전분(soluble starch), (B)는 아밀로펙틴(amylopectin), (C)는 아밀로오스(amylose), (D)는 옥수수 전분(corn starch), (E)는 감자 전분(potato starch)에 대한 결과이다.
도 6은 본 발명 알파 아밀라아제 (BiLA) 효소의 전분 분해능을 확인한 결과이다. 'control'은 BiLA 효소 무처리군 (대조군)이고, 'rxn'은 BiLA 효소 처리군이다.
도 7은 본 발명 알파 아밀라아제 (BiLA) 효소가 처리된 전분 용액의 HPAEC 결과이다.
도 8은 본 발명 알파 아밀라아제 (BiLA) 효소가 처리된 전분 용액을 에탄올 침전하여 수득한 반응 생성물에 대한 HPAEC 결과이다.
도 9는 본 발명 알파 아밀라아제 (BiLA) 효소가 처리된 전분 용액을 에탄올 침전하여 수득한 반응 생성물에 대한 알파 아밀라아제의 반응성 (k cat/K m)을 확인한 결과이다. (A)는 대조군 대한 결과이고, (B)는 본 발명 '알파 아밀라아제 (BiLA) 효소 처리 20분 샘플'에 대한 결과이고, (C)는 본 발명 '알파 아밀라아제 (BiLA) 효소 처리 12시간 샘플'에 대한 결과이다.1 is a map of a recombinant vector into which an insert DNA of the present invention ( bllj- 0710) is inserted.
FIG. 2 is an SDS-PAGE electrophoresis image of purified amorphous alpha amylase (BiLA) enzyme.
Figure 3 shows TLC results of various amylases treated with the invention alpha amylase (BiLA) enzyme. (A) is a mixture of maltose (G2), maltotriose (G3), maltotetraose (G4), maltoheptaose (G7), alpha- cyclodextrin CD, beta-cyclodextrin, beta-CD, gamma-cyclodextrin, amylose, amylopectin, soluble starch, (pullulan)}, and (B) is a TLC result for p NPG5.
FIG. 4 is a graph showing the results of an experiment for confirming the optimal reaction temperature and pH of the present invention alpha amylase (BiLA) enzyme. (A) is the result of the experiment confirming the optimal reaction temperature of the BiLA enzyme, and (B) is the experiment result of confirming the optimum pH of the reaction of the BiLA enzyme.
FIG. 5 shows the results of confirming the reactivity ( k cat / K m ) of the present invention alpha amylase (BiLA) enzyme according to the substrate. (A) is soluble starch, (B) is amylopectin, (C) is amylose, (D) is corn starch, (E) is potato starch, .
Figure 6 shows the results of confirming the starch resolution of the alpha amylase (BiLA) enzyme of the present invention. 'control' is the BiLA enzyme-free group (control group), and 'rxn' is the BiLA enzyme-treated group.
Figure 7 shows the HPAEC results of starch solutions treated with the invention alpha amylase (BiLA) enzyme.
Figure 8 is the HPAEC results for the reaction product obtained by ethanol precipitation of the starch solution treated with the present invention alpha amylase (BiLA) enzyme.
FIG. 9 is a result of confirming the reactivity ( k cat / K m ) of the alpha amylase to the reaction product obtained by ethanol precipitation of the starch solution treated with the alpha amylase (BiLA) enzyme of the present invention. (A) is the result for the control group, (B) is the result for the present invention '20 minute sample treated with Alpha Amylase (BiLA) enzyme', and (C) .
이하, 본 발명의 구성을 하기 실시예를 통해 구체적으로 설명하고자 한다. 다만, 본 발명의 권리범위가 하기 실시예에만 한정되는 것은 아니고, 그와 등가의 기술적 사상의 변형까지를 포함한다.
Hereinafter, the structure of the present invention will be described in detail with reference to the following examples. However, the scope of the present invention is not limited to the following embodiments, and includes modifications of equivalent technical ideas.
[[ 실시예Example 1: One: 비피도박테리움Bifidobacterium 롱검Long Sword 서브스피시스Subspecies 롱검Long Sword JCMJCM 1217 ( 1217 ( BifidobacteriumBifidobacterium longum longum subspsubsp . . longumlongum JCMJCM 1217) ( 1217) ( KCTCKCTC 3127) 균주 유래 알파 아밀라아제 유전자 3127) strain-derived alpha amylase gene 클로닝Cloning 및 정제] And tablets]
본 실시예에서는 비피도박테리움 롱검 서브스피시스 롱검 JCM 1217 (Bifidobacterium longum subsp . longum JCM 1217) (KCTC 3127) 균주로부터 알파 아밀라아제 유전자 (bllj-0710)를 클로닝하고, 정제하고자 하였다.
In this embodiment, Bifidobacterium ronggeom sub speaker system ronggeom JCM 1217 (Bifidobacterium longum subsp . longum Cloning alpha-amylase gene (bllj -0710) from JCM 1217) (KCTC 3127) strains, which was purified to.
(1) 중합효소연쇄반응(Polymerase chain reaction; PCR)(1) Polymerase chain reaction (PCR)
비피도박테리움 롱검 서브스피시스 롱검 JCM 1217 (Bifidobacterium longum subsp. longum JCM 1217) 균주로부터 본 발명의 알파 아밀라아제(alpha amylase)를 증폭하기 위하여 하기 표 1에 기재된 프라이머를 사용하였다.Bifidobacterium lematopsis subtilis JCM 1217 ( Bifidobacterium longum subsp. longum JCM 1217) were used to amplify the alpha amylase of the present invention.
멸균수 37.5 μL, 10X EX Taq 버퍼 5 μL, dNTP 4 μL, 정방향 프라이머(forward primer, 하기 표 1) 1 μL, 역방향 프라이머(reverse primer, 하기 표 1) 1 μL, DNA (KCTC 3127, Bifidobacterium longum subsp. longum JCM 1217) 1 μL, EX Taq 0.5 μL의 혼합물 (총 50 μL)을 사용하여 98℃에서 1분 동안 1회 실시한 후, 98℃에서 10초, 55℃에서 30초, 72℃에서 1분 30초의 과정을 총 30회 실시하는 PCR을 수행하였다. 5 μL of 10X EX Taq buffer, 4 μL of dNTP, 1 μL of a forward primer (see Table 1), 1 μL of a reverse primer (reverse primer, Table 1 below), DNA (KCTC 3127, Bifidobacterium longum subsp. longum JCM 1217) and 0.5 μL of EX Taq (total 50 μL) at 98 ° C. for 1 minute and then incubated at 98 ° C. for 10 seconds, at 55 ° C. for 30 seconds, at 72 ° C. for 1 minute and 30 seconds PCR was performed 30 times in total.
(forward primer)Forward primer
(forward primer)
(reverse primer)Reverse primer
(reverse primer)
(2) PCR 산물의 정제(2) Purification of PCR products
상기의 PCR 반응후, PCR 혼합물에 5배 부피만큼의 BNL 버퍼를 넣고 볼텍싱(voltexing)한 후, BNL 버퍼의 1.5배 부피만큼의 이소프로판올(isopropanol)을 넣고, 여러 번 피펫팅(pipetting)하며 섞어주었다. 그 후, 스핀컬럼(spin column)에 옮겨 담고, 11,000 rpm으로 1분간 원심분리하였다. 이후, 분리된 하층액을 버리고, 700 μL의 워싱버퍼(washing buffer)를 넣은 후, 11,000 rpm으로 1분간 원심분리하였다. 이후, 분리된 하층액을 제거하고, 아무것도 넣지 않은 상태로 다시 11,000 rpm으로 1분간 다시 원심분리하였다. 그 후, 스핀컬럼(spin column)의 윗부분을 마이크로센트리퓨즈 튜브(microcentrifuge tube)에 옮겨 담고, 멸균수 30 μL를 넣은 후, 1분간 방치한 뒤, 다시 11,000 rpm으로 1분간 원심분리하였다. 그 후, 나노드랍(nanodrop)으로 농도를 측정하였는데, 생산된 PCR 산물 (bllj-0710)의 농도가 157.1 ng/μL임을 확인하였다.
After the above PCR reaction, the PCR mixture was added with 5 times as much volume of BNL buffer, and then subjected to voltexing. 1.5 times as much isopropanol as that of the BNL buffer was added, and the mixture was pipetted several times. gave. Then, it was transferred to a spin column and centrifuged at 11,000 rpm for 1 minute. Then, the separated lower layer was discarded, 700 μL of washing buffer was added, and the mixture was centrifuged at 11,000 rpm for 1 minute. Thereafter, the separated submerged solution was removed and centrifuged again at 11,000 rpm for 1 minute in the absence of any solution. After that, the upper part of the spin column was transferred to a microcentrifuge tube, and 30 μL of sterilized water was added. The sample was allowed to stand for 1 minute and then centrifuged at 11,000 rpm for 1 minute. Thereafter, the concentration was measured by nanodrop, and it was confirmed that the concentration of produced PCR product ( bllj- 0710) was 157.1 ng / μL.
(3) 제한효소처리(3) Restriction enzyme treatment
상기에서 PCR 산물로 수득된 DNA (bllj-0710) 28 μL, 1.5×K 버퍼 7.5 μL, 멸균수 10.5 μL, Sal I 2 μL, Hind III 2 μL의 혼합물을 37℃에서 2시간 동안 반응시켰다. 벡터(vector)로는 p6xHTKNd를 사용하여 상기와 같은 방법으로 제한효소처리를 하였다. 이때, 벡터에만 다시 CIAP 1 μL를 넣고 37℃에서 1시간 동안 반응시켰다.
28 μL of DNA (bllj -0710) obtained above as a PCR product, 1.5 × K buffer, 7.5 μL, sterile water 10.5 μL, Sal I and 2 μL of Hind III was reacted at 37 ° C for 2 hours. As the vector, p6xHTKNd was used for restriction enzyme treatment in the same manner as described above. At this time, 1 μL of CIAP was added to the vector again and reacted at 37 ° C. for 1 hour.
(4) 겔 추출 (Gel extraction)(4) Gel extraction
상기에서 제한효소 처리를 한 인서트(insert) DNA (bllj-0710), 벡터 (p6xHTKNd) 각각에 로딩버퍼(loading buffer)를 5 μL씩 넣고, 1.2% 아가로스 겔(agarose gel)을 이용하여, 50 V로 전기영동하였다. 이후, 전기영동하여 나타난 밴드를 잘라서 멸균된 마이크로센트리퓨즈 튜브에 담고 무게를 측정하였다. 이후, 측정된 무게의 3배 양인 BNL 버퍼를 넣고 볼텍싱한 뒤, 55℃의 히팅 블럭(heating block)에서 10분 동안 반응시키며 녹여주었다. 여기에 겔(gel) 무게의 1배인 이소프로판올(isopropanol)을 넣고 여러 번 피펫팅 해 주었다. 그 후, 각각을 스핀컬럼에 옮겨 담고, 11,000 rpm으로 1분 원심분리하여 분리된 하층액은 제거하였다. 그 후, 워싱버퍼 700 μL를 넣고, 11,000 rpm으로 1분간 원심분리하여 분리된 하층액을 제거하고, 한번 더 원심분리하였다. 그 후, 스핀컬럼의 윗부분을 마이크로센트리퓨즈 튜브에 옮겨서 멸균수 30 μL를 넣고, 1분 동안 방치한 후, 다시 11,000 rpm으로 1분간 원심분리하였다. 농도를 측정한 결과, 삽입 DNA는 11.47 ng/μL, 벡터는 135.7 ng/μL으로 확인되었다.
5 μl of loading buffer was added to each of the insert DNA ( bllj- 0710) and vector (p6xHTKNd) treated with restriction enzymes and ligated to each other with 50% agarose gel using 1.2% agarose gel. V. Then, the band appeared by electrophoresis was cut and placed in a sterile microcentrifuge tube and weighed. Then, the BNL buffer, which is three times the measured weight, was vortexed and dissolved in a heating block at 55 ° C for 10 minutes. To this was added isopropanol, which is one time the weight of the gel, and pipetted several times. Then, each was transferred to a spin column and centrifuged at 11,000 rpm for 1 minute to remove the separated lower layer. Then, 700 μL of wash buffer was added, and centrifugation was performed at 11,000 rpm for 1 minute to remove the separated lower layer and centrifuged once more. Then, the upper portion of the spin column was transferred to a microcentrifuge tube, and 30 μL of sterilized water was added thereto. After allowing to stand for 1 minute, it was further centrifuged at 11,000 rpm for 1 minute. As a result of measurement of the concentration, it was confirmed that the inserted DNA was 11.47 ng / μL and the vector was 135.7 ng / μL.
(5) 라이게이션(Ligation) 및 형질전환(transformation)(5) Ligation and transformation
라이게이션(ligation, 실험군), 셀프 라이게이션(self-ligation, 대조군) 두 가지 샘플을 만들고자 하였다. 라이게이션 혼합물에서는 인서트 DNA와 벡터의 농도가 세 배 이상 차이 나야하는데, 인서트 DNA 8 μL (91.76 ng/μL), 5배 희석한 벡터 1 μL (27 ng/μL), 리가아제(ligase) 9 μL를 넣어주었다. Ligation (experimental), and self-ligation (self-ligation, control). In the ligation mixture, the concentration of insert DNA and vector should be at least three times higher than that of insert DNA (91.76 ng / μL), 1 μL of a 5-fold diluted vector (27 ng / μL), 9 μL of ligase .
한편, 대조군인 셀프 라이게이션 혼합물은 5배 희석한 벡터 1 μL, 인서트 DNA 대신 멸균수 8 μL, 리가아제 9 μL로 만들어주었다. On the other hand, the control self-ligation mixture was prepared by adding 1 μL of a 5-fold diluted vector, 8 μL of sterilized water and 9 μL of ligase instead of insert DNA.
상기에서 준비한 라이게이션 혼합물, 셀프 라이게이션 혼합물을 16℃에서 30분 동안 반응시켜 재조합 벡터로 각각 제조하였다 (도 1 참조). 도 1은 본 발명에서 제조한 인서트 DNA (bllj-0710)가 삽입된 재조합 벡터의 맵이다.The ligation mixture and the self-agitation mixture prepared above were reacted at 16 ° C for 30 minutes to prepare recombinant vectors, respectively (see FIG. 1). 1 is a map of a recombinant vector into which insert DNA ( bllj- 0710) prepared in the present invention is inserted.
한편, CaCl2법을 이용하여 대장균을 상기 재조합 벡터로 형질전환하였다. 각각의 대장균 컴피턴트 세포 (competent cell, MC1061) 200 μL에 라이게이션 혼합물, 셀프 라이게이션 혼합물을 각각 1 μL씩 넣고, 얼음 30분, 42℃ 2분, 얼음 2분 동안 반응시켰다. 이후, LB 배지를 800 μL 첨가하고, 37℃에서 1시간 동안 배양시킨 후, 5,000×g로 1분 동안 원심분리하였다. 그 후, 800 μL 버리고 나머지 200 μL를 취해 카나마이신(kanamycin) 배지에 도말한 다음, 37℃에서 24시간 동안 배양시켰다.
On the other hand, Escherichia coli was transformed with the above recombinant vector using the CaCl 2 method. 1 μL each of the ligation mixture and the self-ligation mixture was added to 200 μL of each competent cell (MC1061), and the mixture was reacted for 30 minutes on ice, 2 minutes on ice and 2 minutes on ice. Then, 800 μL of LB medium was added, and the mixture was incubated at 37 ° C. for 1 hour, followed by centrifugation at 5,000 × g for 1 minute. Then, 800 μL was discarded and the remaining 200 μL was plated on kanamycin medium and cultured at 37 ° C for 24 hours.
(6) 형질전환 대장균으로부터 본 발명 알파 아밀라아제 효소의 회수(6) Recovery of the present invention alpha amylase enzyme from transformed E. coli
라이게이션된 벡터 (실험군)가 삽입된 대장균을 1 L의 LBK 배지에 접종하여 37℃에서 200 rpm으로 20시간 동안 교반하여 키운 후, 원심분리기를 이용하여 세포를 회수하였다. 회수한 세포를 다시 pH 6.0의 50 mM NaOAC 완충용액에 재부유한 후, 초음파로 세포파쇄를 실시하였다. 세포파쇄로부터 수득된 파쇄액을 11,000 rpm으로 20분 동안 원심분리하여 분리된 상층액을 취한 후, 이를 Ni-NTA 컬럼에 흡착시켜 본 발명의 알파 아밀라아제 효소를 수득하였다. 그 후, 전기영동을 통하여 분리된 효소를 확인하였다 (도 2). 도 2는 분리된 본 발명 효소의 SDS-PAGE 전기영동 사진이다. Escherichia coli inserted with the lygated vector (experimental group) was inoculated into 1 L of LBK medium and cultured at 37 ° C for 20 hours at 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. The disruption obtained from the cell disruption was centrifuged at 11,000 rpm for 20 minutes, and the separated supernatant was taken and adsorbed on a Ni-NTA column to obtain the alpha amylase enzyme of the present invention. Thereafter, the separated enzyme was confirmed by electrophoresis (FIG. 2). 2 is an SDS-PAGE electrophoresis photograph of the separated enzyme of the present invention.
상기와 같이 분리된 본 발명의 효소는 서열번호 1의 아미노산 서열을 가지는 것으로 확인되었는데, 본 발명에서는 'BiLA 효소'라고 지칭하기로 한다.
The thus isolated enzyme of the present invention was found to have the amino acid sequence of SEQ ID NO: 1, which will be referred to as 'BiLA enzyme' in the present invention.
[[ 실시예Example 2: 본 발명 2: invention BiLABiLA 효소의 기질별 반응 시험] Reaction test of enzyme by substrate]
본 실시예에서는 본 발명 BiLA 효소의 기질별 반응성을 확인하고자 하였다.In this Example, the reactivity of the BiLA enzyme according to the present invention was examined.
pH 6.0의 50 mM NaOAC 완충용액에, 1% 기질 {(말토오스(maltose, G2), 말토트리오스(maltotriose, G3), 말토테트라오스(maltotetraose, G4), 말토헵타오스(maltoheptaose, G7), 알파-사이클로덱스트린(α-cyclodextrin, alpha-CD), 베타-사이클로덱스트린(β-cyclodextrin, beta-CD), 감마-사이클로덱스트린(γ-cyclodextrin, gamma-CD), 아밀로오스(amylose), 아밀로펙틴(amylopectin), 가용성 전분(soluble starch), 풀루란(pullulan)}과 BiLA 효소 0.5 unit/mg을 넣고 45℃에서 12시간 동안 반응시킨 후, TLC(thin layer chromatography)를 수행하였다. 그 결과는 도 3의 (A)에 나타내었다. (maltose, G2, maltotriose (G3), maltotetraose (G4), maltoheptaose (G7), alpha- alpha Cyclodextrin, alpha-CD, beta-cyclodextrin, gamma-cyclodextrin, gamma-CD, amylose, amylopectin, , Soluble starch, pullulan) and 0.5 unit / mg of BiLA enzyme were added to the reaction mixture at 45 ° C for 12 hours, and thin layer chromatography (TLC) was performed. A).
또한, pH 6.0의 50 mM NaOAC 완충용액에, 기질로 1% pNPG5(p-nitrophenol- α-D-maltopentaoside)와 BiLA 효소 0.5 unit/mg을 넣고, 45℃에서 0.5, 1, 3, 5, 24시간 동안 각각 반응시킨 후, TLC를 수행하였다. 그 결과는 도 3의 (B)에 나타내었다.In addition, 1% p NPG5 (p-nitrophenol- α-D-maltopentaoside) and 0.5 unit / mg of BiLA enzyme were added to a 50 mM NaOAC buffer solution of pH 6.0 at 45 ° C for 0.5, After each reaction for 24 hours, TLC was performed. The results are shown in Fig. 3 (B).
실험결과, 말토헵타오스, 아밀로펙틴, 가용성 전분을 기질로 할 경우, BiLA 효소에 반응함을 확인할 수 있었다 (도 3의 (A)). 도 3의 (A)는 본 발명 알파 아밀라아제 (BiLA) 효소를 처리한 기질 {(말토오스(maltose, G2), 말토트리오스(maltotriose, G3), 말토테트라오스(maltotetraose, G4), 말토헵타오스(maltoheptaose, G7), 알파-사이클로덱스트린(α-cyclodextrin, alpha-CD), 베타-사이클로덱스트린(β-cyclodextrin, beta-CD), 감마-사이클로덱스트린(γ-cyclodextrin, gamma-CD), 아밀로오스(amylose), 아밀로펙틴(amylopectin), 가용성 전분(soluble starch), 풀루란(pullulan)}의 TLC 결과이다.As a result of the experiment, it was confirmed that when maltoheptaose, amylopectin and soluble starch were used as a substrate, they reacted with BiLA enzyme (Fig. 3 (A)). FIG. 3 (A) is a graph showing the activity of the substrate of the present invention (FIG. 3) treated with an alpha amylase (BiLA) enzyme {maltose (G2), maltotriose (G3), maltotetraose (G4), maltoheptaose maltoheptaose, G7), alpha-cyclodextrin, alpha-CD, beta-cyclodextrin, beta-CD, gamma-cyclodextrin, gamma-CD, amylose ), Amylopectin, soluble starch, pullulan}.
또한, pNPG5를 기질로 할 경우, 시간이 지남에 따라 pNPG5가 BiLA 효소에 의해 분해됨을 확인할 수 있었다 (도 3의 (B)). 도 3의 (B)는 본 발명 알파 아밀라아제 (BiLA) 효소를 처리한 기질 (pNPG5)의 TLC 결과이다.
In addition, when p NPG5 was used as a substrate, it was confirmed that p NPG5 was degraded by BiLA enzyme over time (Fig. 3 (B)). FIG. 3 (B) is a TLC result of the substrate ( p NPG5) treated with the present invention alpha amylase (BiLA) enzyme.
[[ 실시예Example 3: 본 발명 3: invention BiLABiLA 효소의 최적 반응 온도 및 Optimal reaction temperature of enzyme pHpH 확인 실험] Confirmation experiment]
본 실시예에서는 DNS(dinitrosalicylic acid)를 이용하여 본 발명 BiLA 효소의 반응 최적 온도 및 pH를 확인하고자 하였다.In this example, dinitrosalicylic acid (DNS) was used to determine the optimal reaction temperature and pH of the BiLA enzyme of the present invention.
(1) 최적 반응 온도 확인 실험(1) Experiments to determine optimum reaction temperature
버퍼 (50 mM Citric-NaOH, 50 mM NaOAC, 50 mM KH2PO4-NaOH)에, 기질 {0.5% 가용성 전분 - starch, soluble (Showa, Japan)}, BiLA 효소 0.5 unit/mg을 넣고, 매 분마다 샘플링(sampling)한 후, 샘플과 DNS의 비율이 1:3이 되게 혼합하였다. 이후, 이를 끓여 10~60℃가 되게 한 후, ELISA 리더 기기에 넣고 570 nm에서 흡광도를 측정하였다.Buffer (50 mM citric-NaOH, 50 mM NaOAC, 50 mM KH 2 PO 4 -NaOH) was added to the substrate {0.5% Soluble starch-starch, soluble (Showa, Japan)} and 0.5 unit / mg of BiLA enzyme were added, and the mixture was sampled every minute and then mixed with the sample to DNS ratio of 1: 3. After that, it was boiled to 10 ~ 60 ° C, and the absorbance was measured at 570 nm in an ELISA reader device.
실험결과, BiLA 효소는 20℃에서 역가가 가장 높음을 확인할 수 있었다 (도 4의 (A)). 도 4의 (A)는 본 발명 알파 아밀라아제 (BiLA) 효소의 반응 최적 온도를 확인한 실험 결과이다.
As a result of the experiment, it was confirmed that the BiLA enzyme had the highest titer at 20 ° C (FIG. 4 (A)). Fig. 4 (A) shows the results of experiments in which the optimum reaction temperature of the alpha amylase (BiLA) enzyme of the present invention was confirmed.
(2) 최적 pH 실험 (2) Optimal pH experiment
버퍼 (50 mM Citric-NaOH, 50 mM NaOAC, 50 mM KH2PO4-NaOH)에, 기질 {0.5% 가용성 전분 - starch, soluble (Showa, Japan)}, BiLA 효소 0.5 unit/mg을 넣고 분마다 샘플링한 후, 샘플과 DNS의 비율이 1:3이 되게 하였다. 이를 5분 동안 끓인 후, ELISA 리더 기기에 넣고, 570 nm에서 흡광도를 측정하였다. Buffer (50 mM citric-NaOH, 50 mM NaOAC, 50 mM KH 2 PO 4 -NaOH) was added to the substrate {0.5% Soluble Starch - starch, soluble (Showa, Japan)} and 0.5 unit / mg of BiLA enzyme were added to each sample every min and the ratio of sample to DNS was 1: 3. This was boiled for 5 minutes, then placed in an ELISA reader instrument and absorbance was measured at 570 nm.
실험결과, BiLA 효소는 pH 5에서 가장 역가가 높음을 확인할 수 있었다 (도 4의 B)). 도 4의 (B)는 본 발명 알파 아밀라아제 (BiLA) 효소의 반응 최적 pH를 확인한 실험결과이다.
As a result, it was confirmed that BiLA enzyme had the highest activity at pH 5 (FIG. 4B)). FIG. 4 (B) is a result of an experiment in which the optimum pH for the reaction of the present invention alpha amylase (BiLA) enzyme was confirmed.
[[ 실시예Example 4: 본 발명 4: invention BiLABiLA 효소의 Enzymatic 비역가Wrecker (( specificspecific activityactivity ) 측정]) Measure]
본 실시예에서는 본 발명 BiLA 효소의 비역가(specific activity)를 측정하고자 하였다.In this example, the specific activity of the present invention was measured.
pH 5.0의 50 mM NaOAC 완충용액에, 각 기질 {알파-사이클로덱스트린(α-cyclodextrin, alpha-CD), 베타-사이클로덱스트린(β-cyclodextrin, beta-CD), 감마-사이클로덱스트린(γ-cyclodextrin, gamma-CD), 아밀로오스(amylose), 아밀로펙틴(amylopectin), 가용성 전분(soluble starch), 풀루란(pullulan)}과 BiLA 효소 0.5 unit/mg을 넣고, 20℃에서 반응시키며 3분, 6분, 9분, 12분, 15분 마다 샘플링한 후, 샘플과 DNS의 비율이 1:3이 되게 하였다. 이후, 5분 동안 끓인 후, ELISA 리더 기기에 넣고, 570 nm에서 흡광도를 측정하였다. Cyclodextrin, beta-CD, gamma-cyclodextrin, and gamma-cyclodextrin were added to 50 mM NaOAc buffer, pH 5.0, Amylose, amylopectin, soluble starch, pullulan) and 0.5 unit / mg of BiLA enzyme were added to each well and reacted at 20 ° C for 3 minutes, 6 minutes, 9 Min, 12 min, and 15 min, and the ratio of sample to DNS was 1: 3. After boiling for 5 minutes, it was placed in an ELISA reader instrument and absorbance was measured at 570 nm.
또한, pH 5.0의 50 mM NaOAC 완충용액에, pNPG5(p-nitrophenol-α-D-maltopentaoside), BiLA 효소를 4:5:1 (완충용액:pNPG5:효소) 비율로 혼합하고, 20℃에서 반응시키며, 3분, 6분, 9분, 12분, 15분에서 샘플링하고, 405 nm에서 흡광도를 측정하였다. Further, p NPG5 (p-nitrophenol-α-D-maltopentaoside) and BiLA enzyme were mixed in a ratio of 4: 5: 1 (buffer solution: pNPG5: enzyme) to a 50 mM NaOAC buffer solution at pH 5.0, And samples were taken at 3 minutes, 6 minutes, 9 minutes, 12 minutes and 15 minutes, and the absorbance was measured at 405 nm.
그 결과는 하기 표 2에 나타내었다.The results are shown in Table 2 below.
실험결과, BiLA 효소는 가용성 전분(soluble starch)에서 18.62 U/mg으로 가장 우수한 비역가를 나타냄을 확인할 수 있었다. 다만, 알파-사이클로덱스트린(α-cyclodextrin, alpha-CD), 베타-사이클로덱스트린(β-cyclodextrin, beta-CD), 감마-사이클로덱스트린(γ-cyclodextrin, gamma-CD), 풀루란(pullulan)에서는 역가가 측정되지 않았다.
As a result of the experiment, it was confirmed that BiLA enzyme showed the most excellent immunoglobulin at 18.62 U / mg in soluble starch. However, in the case of alpha-cyclodextrin, alpha-CD, beta-cyclodextrin, gamma-cyclodextrin, and pullulan, No potency was measured.
[[ 실시예Example 5: 본 발명 5: invention BiLABiLA 효소의 반응성 ( Reactivity of Enzymes ( kk catcat // KK mm ) 측정]) Measure]
본 실시예에서는 본 발명 BiLA 효소의 반응성 (k cat/K m) 측정하고자 하였다.In this example, the reactivity ( k cat / K m ) of the present BiLA enzyme was measured.
pH 5.0인 50 mM NaOAC 완충용액에, 1% 기질, BiLA 효소 0.5 unit/mg을 넣고 20℃에서 반응시키며 0분, 2분, 4분, 6분, 8분, 10분에 샘플링한 후 샘플과 DNS의 비율이 1:3이 되게 하였다. 이를 5분 동안 끓인 후 ELISA 리더 기기에 넣고 570 nm에서 흡광도를 측정하였다. 이때, 기질은 가용성 전분(soluble starch), 아밀로펙틴(amylopectin), 아밀로오스(amylose), 옥수수 전분(corn starch), 감자 전분(potato starch)을 사용하여 농도별로 실험하였다. 그 결과는 하기 표 3 및 도 5에 나타내었다.The samples were incubated at 0, 2, 4, 6, 8, and 10 min with 1% substrate and 0.5 unit / mg BiLA enzyme in a 50 mM NaOAC buffer solution at pH 5.0. DNS ratio is 1: 3. This was boiled for 5 minutes and then placed in an ELISA reader instrument and absorbance was measured at 570 nm. At this time, the substrate was tested by concentration using soluble starch, amylopectin, amylose, corn starch and potato starch. The results are shown in Table 3 and FIG.
실험결과, 가용성 전분에서의 k cat/K m는 1.87441 ㎖/mg·sec, 아밀로펙틴에서는 0.373 ㎖/mg·sec, 아밀로오스에서는 3.4191 ㎖/mg·sec, 옥수수 전분에서는 0.4865 ㎖/mg·sec, 감자 전분에서는 0.5774 ㎖/mg·sec를 나타내었다. As a result, k cat / K m in soluble starch was 1.87441 ml / mg · sec, amylopectin 0.373 ml / mg · sec, amylose 3.4191 ml / mg · sec, corn starch 0.4865 ml / mg · sec, Lt; RTI ID = 0.0 > mg / sec. ≪ / RTI >
아밀로오스에서의 반응속도가 가장 빠르며, 그 다음으로 가용성 전분에서 반응속도가 빠름을 확인할 수 있었다 (도 5). 도 5는 기질에 따른 본 발명 알파 아밀라아제 (BiLA) 효소의 반응성(k cat/K m)을 확인한 결과이다. (A)는 가용성 전분(soluble starch), (B)는 아밀로펙틴(amylopectin), (C)는 아밀로오스(amylose), (D)는 옥수수 전분(corn starch), (E)는 감자 전분(potato starch)에 대한 결과이다.
It was confirmed that the reaction rate in amylose was the fastest, followed by the faster reaction rate in the soluble starch (FIG. 5). FIG. 5 shows the results of confirming the reactivity ( k cat / K m ) of the present invention alpha amylase (BiLA) enzyme according to the substrate. (A) is soluble starch, (B) is amylopectin, (C) is amylose, (D) is corn starch, (E) is potato starch, .
[[ 실시예Example 6: 본 발명 6: invention BiLABiLA 효소의 전분 분해능 확인] Determination of starch resolution of enzyme]
본 실시예에서는 본 발명 BiLA 효소의 전분 분해능을 확인하고자 하였다.In this example, the starch resolution of the present BiLA enzyme was examined.
pH 5.0인 50 mM NaOAC 완충용액에, 옥수수 전분과 BiLA 효소 0.5 unit/mg을 넣어 제조된 반응액 (최종 농도가 4, 8, 16%가 되도록 옥수수 전분 첨가) 500 μL를 20℃에서 1시간, 2시간, 3시간, 4시간, 5시간, 6시간, 7시간, 24시간 반응시킨 후, 각각의 상태를 육안으로 확인하였다. 대조군(control)은 BiLA 효소 무처리군이 사용되었다.500 μL of the reaction mixture (corn starch added to final concentrations of 4, 8, and 16%) prepared by adding corn starch and 0.5 unit / mg of BiLA enzyme to a 50 mM NaOAC buffer solution at pH 5.0 was added at 20 ° C. for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, and 24 hours, respectively. As a control, BiLA enzyme-free treatment group was used.
실험결과, 시간이 지남에 따라 BiLA 효소 처리 용액은 대조군 대비 혼탁도가 낮아진 것 (즉, 맑아진 것)을 확인할 수 있었다. 특히, 4% 전분 반응액이 대조군에 비해서 가장 많이 분해되어 가장 맑아진 것으로 확인할 수 있었다. 또한, 전분의 농도가 높아질수록 전분 분해 정도가 낮아지며, 2시간 반응부터 가시적인 차이가 나타남을 확인할 수 있었다 (도 6). 도 6은 본 발명 알파 아밀라아제 (BiLA) 효소의 전분 분해능을 확인한 결과이다. 'control'은 BiLA 효소 무처리군 (대조군)이고, 'rxn'은 BiLA 효소 처리군이다.
As a result of the experiment, it was confirmed that the BiLA enzyme-treated solution had a lower turbidity (that is, clarified) as compared with the control over time. In particular, it was confirmed that the 4% starch reaction solution was the most decomposed and clearest as compared with the control group. In addition, the higher the starch concentration, the lower the degree of starch decomposition, and the visible difference was observed from the 2 hour reaction (Fig. 6). Figure 6 shows the results of confirming the starch resolution of the alpha amylase (BiLA) enzyme of the present invention. 'control' is the BiLA enzyme-free group (control group), and 'rxn' is the BiLA enzyme-treated group.
[[ 실시예Example 7: 본 발명 7: invention BiLABiLA 효소 처리 전분 Enzyme treated starch 용액의 Solution 지소화성Geotoxicity 확인] Confirm]
본 실시예에서는 본 발명 BiLA 효소가 처리된 전분 용액의 지소화성을 확인하고자 하였다.
In this Example, the biodegradability of the starch solution treated with the BiLA enzyme of the present invention was confirmed.
(1) 한계 덱스트린(limit dextrin) 제조(1) Manufacture of limit dextrin
pH 5.0의 50 mM NaOAC 완충액에, 1% 전분 기질 {가용성 전분 - starch, soluble (Showa, Japan)}과 BiLA 효소 0.5 unit/mg을 넣고 20℃에서 반응시켜 0분, 20분, 12시간 단위로 샘플링한 후, 5분 동안 끓이고 식혔다. 그 후, HPAEC(High Pressured Anion Exchange Chrpmatography)를 수행하였다.To the 50 mM NaOAC buffer solution of pH 5.0, 1% starch substrate (soluble starch-starch, soluble (Showa, Japan)) and 0.5 unit / mg of BiLA enzyme were added and reacted at 20 ° C in 0, 20, After sampling, it was boiled and cooled for 5 minutes. Then, HPAEC (High Pressured Anion Exchange Chrpmatography) was performed.
실험결과, 대조군 (BiLA 효소 미처리군)은 중합도가 12, 30인 말토덱스트린이 확인되었으나, BiLA 효소 처리 전분 용액은 대조군에 비하여 중합도가 낮은 말토덱스트린 조성을 보임을 확인할 수 있었다 (도 7). 도 7은 본 발명 알파 아밀라아제 (BiLA) 효소가 처리된 전분 용액의 HPAEC 결과이다.
As a result, maltodextrin having a degree of polymerization of 12 and 30 was found in the control group (untreated BiLA enzyme group), but it was confirmed that the biodegradable starch solution showed maltodextrin composition having a lower degree of polymerization than the control group (Fig. 7). Figure 7 shows the HPAEC results of starch solutions treated with the invention alpha amylase (BiLA) enzyme.
(2) 에탄올 침전반응을 통한 반응 생성물의 확인 (2) Identification of reaction products through ethanol precipitation reaction
상기 BiLA 효소 처리된 전분 용액 (반응 생성물)에 2배 부피의 에탄올을 넣고 5시간 냉동하였다. 그 후, 7,9000 rpm으로 원심분리하고, 37℃ 인큐베이터(incubator)에서 건조시켜 효소 반응된 전분 (반응 생성물)을 수득하였다. 여기에 pH 5.0의 50 mM 소디움 아세테이트(sodium acetate)를 480 μL 넣고, 2 U의 이소아밀라아제(isoamylase)를 넣은 후, 40℃에서 4시간 동안 반응시켰다. 그 후, HPAEC를 수행하였다. Two times volume of ethanol was added to the BiLA enzyme-treated starch solution (reaction product) and frozen for 5 hours. Thereafter, the mixture was centrifuged at 7,900 rpm and dried in an incubator at 37 ° C to obtain an enzyme-reacted starch (reaction product). 480 μL of 50 mM sodium acetate at pH 5.0 was added thereto, and 2 U of isoamylase was added thereto, followed by reaction at 40 ° C. for 4 hours. HPAEC was then performed.
실험결과, 대조군 (BiLA 효소 미처리군)에서는 말토덱스트린이 발견되지 않음에 반해, BiLA 효소 처리 전분 용액에서는 중합도가 3, 4, 13인 말토덱스트린이 발견되었다 (도 8). 도 8은 BiLA 효소가 처리된 전분 용액을 에탄올 침전하여 수득한 반응 생성물에 대한 HPAEC 결과이다.
Experimental results showed that maltodextrin was not found in the control (untreated BiLA enzyme group), but maltodextrin having a degree of polymerization of 3, 4, 13 was found in the BiLA enzyme treated starch solution (Fig. 8). Figure 8 shows the HPAEC results for the reaction product obtained by ethanol precipitation of the BiLA enzyme treated starch solution.
(3) 알파 아밀라아제에 의한 반응성 (k cat/K m) 측정(3) Measurement of reactivity by alpha amylase ( k cat / K m )
상기 에탄올 침전(EtOH precipitation)으로 수득한 반응 생성물 (대조군, BiLA 효소로 20분간 반응된 전분 용액, BiLA 효소로 12시간 반응된 전분 용액)에 알파 아밀라아제 {시판 중인 일반 효소 - alpha amylase from porcine pancreas (Sigma-Aldrich Co.)}를 처리하여 반응속도를 측정하였다.Alpha amylase from porcine pancreas (commercially available alpha-amylase from porcine pancreas (commercially available enzyme-labeled amylase) was added to the reaction product obtained by the above ethanol precipitation (control group, starch solution reacted with BiLA enzyme for 20 minutes, starch solution reacted with BiLA enzyme for 12 hours) Sigma-Aldrich Co.) to measure the reaction rate.
pH 7.0의 50 mM KHPO 완충액에, 각각의 기질 (하기 표 4)과 알파 아밀라아제를 넣고, 알파 아밀라아제의 최적온도인 20℃에서 반응시켰다. 반응 후, 0분, 2분, 4분, 6분, 8분, 10분 단위로 샘플링한 후, 샘플과 DNS가 10:1 비율이 되게 DNS를 첨가하였다. 이후, 5분 동안 끓인 후, ELISA 리더 기기에 넣고 570 nm에서 흡광도를 측정하였다. 측정된 수치로부터 계산한 k cat/K m 값은 하기 표 4 및 도 9에 나타내었다.Each substrate (Table 4 below) and alpha amylase were added to a 50 mM KHPO buffer solution at pH 7.0 and reacted at 20 ° C, which is the optimum temperature of alpha amylase. After the reaction, the samples were sampled at 0 minute, 2 minutes, 4 minutes, 6 minutes, 8 minutes, and 10 minutes, and DNS was added so that the samples and the DNS were in a ratio of 10: 1. After boiling for 5 minutes, the absorbance was measured at 570 nm in an ELISA reader instrument. The values of k cat / K m calculated from the measured values are shown in Table 4 and FIG.
실험결과, 12시간 동안 반응한 BiLA 효소 처리 전분의 알파 아밀라아제의 k cat/K m은 0.23 ㎖/mg·sec, 20분 동안 반응한 BiLA 효소 처리 전분은 0.44 ㎖/mg·sec, 대조군은 0.66 ㎖/mg·sec을 나타내었다 (도 9). 도 9는 본 발명 알파 아밀라아제 (BiLA) 효소가 처리된 전분 용액을 에탄올 침전하여 수득한 반응 생성물에 대한 알파 아밀라아제의 반응성 (k cat/K m)을 확인한 결과이다. (A)는 대조군에 대한 결과이고, (B)는 BiLA 효소 처리 전분 용액 (20분)에 대한 결과이고, (C)는 BiLA 효소 처리 전분 용액 (12시간)에 대한 결과이다.Experimental results, k cat / K m of the alpha-amylase of BiLA enzyme-treated starch to react for 12 hours BiLA enzyme treatment starches reacted for 0.23 ㎖ / mg · sec, 20 minutes, 0.44 ㎖ / mg · sec, the control group, 0.66 ㎖ / mg · sec (Fig. 9). FIG. 9 is a result of confirming the reactivity ( k cat / K m ) of the alpha amylase to the reaction product obtained by ethanol precipitation of the starch solution treated with the alpha amylase (BiLA) enzyme of the present invention. (A) is the result for the control group, (B) is for the BiLA enzyme treated starch solution (20 min), and (C) is the result for the BiLA enzyme treated starch solution (12 hr).
이와 같은 결과는, BiLA 효소를 처리한 샘플이 그렇지 않은 대조군에 비해, 알파 아밀라아제에 의한 분해능이 낮은 것을 의미하는데, 이는 BiLA 효소를 처리한 샘플이 그렇지 않은 경우에 비해 느린 소화도, 즉 지소화도를 갖는 것을 의미한다. 또한, BiLA 효소 처리 샘플 간에는 BiLA 효소를 더 오랫동안 처리한 샘플이 더 높은 지소화도를 갖는 것으로 나타났다. These results indicate that the samples treated with BiLA enzyme had lower resolution by alpha amylase than the control samples without BiLA enzyme, indicating that samples treated with BiLA enzyme had a slower digestibility, . In addition, among samples treated with BiLA enzymes, longer samples of BiLA enzyme were found to have a higher degree of lipidation.
<110> Industry Academic Cooperation Foundation, Hallym University <120> Method for production of slowly digestible starch with alpha amylase <130> AP-2015-0053 <160> 1 <170> KopatentIn 2.0 <210> 1 <211> 431 <212> PRT <213> Bifidobacterium longum subsp. longum JCM 1217 (KCTC 3127) <400> 1 Val Val Val Ser Met Asp Ser Ser Arg Thr Met Pro Asp Trp Val Lys 1 5 10 15 Tyr Gly Val Phe Trp His Val Tyr Pro Leu Gly Phe Cys Gly Ala Asp 20 25 30 Ile Arg Pro Ala Gly Pro Arg Gln Phe His Gly Arg Gly Leu Asp Ala 35 40 45 Ile Ile Pro Trp Leu Glu Tyr Val Arg Asp Leu Gly Ala Ser Gly Leu 50 55 60 Leu Leu Gly Pro Val Phe Glu Ser Ala Thr His Gly Tyr Asp Thr Leu 65 70 75 80 Asp His Met His Ile Asp Val Arg Leu Gly Gly Asp Ala Ala Phe Asp 85 90 95 Gln Leu Val Ala Ala Cys Arg Asp Met Gly Leu Gln Val Met Leu Asp 100 105 110 Gly Val Phe Asn His Val Ser Arg Asn His Pro Ala Val Gln Thr Ala 115 120 125 Leu Asp Glu Thr Ala Gly Arg Leu Asn Pro Ala Asp Asn Pro Trp His 130 135 140 Gly Leu Val Arg Ala His Val Gly Asp Asn Gly Glu Pro Ala Leu Asp 145 150 155 160 Val Phe Glu Gly His Gly Asp Leu Val Ala Leu Asp His Ser Ala Asp 165 170 175 Glu Thr Val Asp Tyr Val Ala His Val Met Ser His Trp Leu Asp Arg 180 185 190 Gly Ala Ala Gly Trp Arg Leu Asp Ala Ala Tyr Ala Val Pro Ser Pro 195 200 205 Phe Trp Ala Arg Val Leu Pro Gln Val Lys Ala Ala His Pro Tyr Ser 210 215 220 Trp Ile Phe Gly Glu Val Ile His Gly Asp Tyr Pro Arg Ile Ile Glu 225 230 235 240 Glu Ser Thr Met Asp Ser Ile Thr Gln Tyr Glu Leu Trp Lys Ser Ile 245 250 255 Gln His Ala Leu Glu Thr Glu Asn Phe Phe Glu Leu Asp Trp Asn Leu 260 265 270 Lys Arg His Asn Ala Phe Leu Asp Ser Phe Val Pro Gln Thr Phe Ile 275 280 285 Gly Asn His Asp Val Thr Arg Ile Ala Ser Gln Ile Gly Pro Ala Lys 290 295 300 Ala Ala Leu Ala Leu Ala Val Leu Met Thr Val Gly Gly Val Pro Ser 305 310 315 320 Ile Tyr Tyr Gly Asp Glu Gln Gly Tyr Val Gly Val Lys Gln Glu Arg 325 330 335 Phe Gly Gly Asp Asp Asp Val Arg Pro Lys Phe Pro Asp Ser Pro Ala 340 345 350 Glu Leu Ser Thr Leu Gly Glu Pro Thr Tyr Arg Leu His Gln Ala Leu 355 360 365 Ile Ala Leu Arg Arg Arg Asn Pro Trp Leu Leu Asp Ala Arg Thr Glu 370 375 380 Ala Val Lys Leu Glu Asn Lys His Phe Val Tyr Arg Ser Thr Ser Ala 385 390 395 400 Asp Ala Gln His Ser Leu Thr Val Asp Leu Asn Ile Glu Gln Ser Pro 405 410 415 Thr Phe Thr Ile Arg Asn Ala Asp Gly Ser Thr Ala Tyr Gln Tyr 420 425 430 <110> Industry Academic Cooperation Foundation, Hallym University <120> Method for production of slowly digestible starch with alpha amylase <130> AP-2015-0053 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 431 <212> PRT <213> Bifidobacterium longum subsp. longum JCM 1217 (KCTC 3127) <400> 1 Val Val Val Ser Met Asp Ser Ser Arg Thr Met Pro Asp Trp Val Lys 1 5 10 15 Tyr Gly Val Phe Trp His Val Tyr Pro Leu Gly Phe Cys Gly Ala Asp 20 25 30 Ile Arg Pro Ala Gly Pro Arg Gln Phe His Gly Arg Gly Leu Asp Ala 35 40 45 Ile Ile Pro Trp Leu Glu Tyr Val Arg Asp Leu Gly Ala Ser Gly Leu 50 55 60 Leu Leu Gly Pro Val Phe Glu Ser Ala Thr His Gly Tyr Asp Thr Leu 65 70 75 80 Asp His Met His Ile Asp Val Arg Leu Gly Gly Asp Ala Ala Phe Asp 85 90 95 Gln Leu Val Ala Ala Cys Arg Asp Met Gly Leu Gln Val Met Leu Asp 100 105 110 Gly Val Phe Asn His Val Ser Ser Asn His Pro Ala Val Gln Thr Ala 115 120 125 Leu Asp Glu Thr Ala Gly Arg Leu Asn Pro Ala Asp Asn Pro Trp His 130 135 140 Gly Leu Val Arg Ala His Val Gly Asp Asn Gly Glu Pro Ala Leu Asp 145 150 155 160 Val Phe Glu Gly His Gly Asp Leu Val Ala Leu Asp His Ser Ala Asp 165 170 175 Glu Thr Val Asp Tyr Val Ala His Val Met Ser His Trp Leu Asp Arg 180 185 190 Gly Ala Gly Trp Arg Leu Asp Ala Ala Tyr Ala Val Pro Ser Pro 195 200 205 Phe Trp Ala Arg Val Leu Pro Gln Val Lys Ala Ala His Pro Tyr Ser 210 215 220 Trp Ile Phe Gly Glu Val Ile His Gly Asp Tyr Pro Arg Ile Ile Glu 225 230 235 240 Glu Ser Thr Met Asp Ser Ile Thr Gln Tyr Glu Leu Trp Lys Ser Ile 245 250 255 Gln His Ala Leu Glu Thr Glu Asn Phe Phe Glu Leu Asp Trp Asn Leu 260 265 270 Lys Arg His Asn Ala Phe Leu Asp Ser Phe Val Pro Gln Thr Phe Ile 275 280 285 Gly Asn His Asp Val Thr Arg Ile Ala Ser Gln Ile Gly Pro Ala Lys 290 295 300 Ala Ala Leu Ala Leu Ala Val Leu Met Thr Val Gly Gly Val Ser Ser 305 310 315 320 Ile Tyr Tyr Gly Asp Glu Gln Gly Tyr Val Gly Val Lys Gln Glu Arg 325 330 335 Phe Gly Gly Asp Asp Asp Val Arg Pro Lys Phe Pro Asp Ser Pro Ala 340 345 350 Glu Leu Ser Thr Leu Gly Glu Pro Thr Tyr Arg Leu His Gln Ala Leu 355 360 365 Ile Ala Leu Arg Arg Arg Asn Pro Trp Leu Leu Asp Ala Arg Thr Glu 370 375 380 Ala Val Lys Leu Glu Asn Lys His Phe Val Tyr Arg Ser Thr Ser Ala 385 390 395 400 Asp Ala Gln His Ser Leu Thr Val Asp Leu Asn Ile Glu Gln Ser Pro 405 410 415 Thr Phe Thr Ile Arg Asn Ala Asp Gly Ser Thr Ala Tyr Gln Tyr 420 425 430
Claims (3)
Characterized in that the starch has an amino acid sequence of SEQ ID NO: 1 and is hydrolyzed by treating an alpha amylase derived from a strain of Bifidobacterium longum subsp. Longum JCM 1217 (KCTC 3127), Bacillus subtilis JCM 1217 (Method for producing starch of chemical conversion).
상기 알파 아밀라아제의 처리는,
10~25℃의 온도, 4.5~5.5의 pH 조건에서 수행하는 것을 특징으로 하는 지소화성 전분의 제조방법.
The method according to claim 1,
The treatment of the alpha amylase,
Wherein the hydrolysis is carried out at a temperature of 10 to 25 DEG C and a pH of 4.5 to 5.5.
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