KR20020002588A - Method for production of isomaltooligosaccharides - Google Patents
Method for production of isomaltooligosaccharides Download PDFInfo
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Abstract
Description
본 발명은 이소말토올리고당을 생산하는 방법에 관한 것이다.The present invention relates to a method for producing isomaltooligosaccharide.
근래에 다양한 올리고당의 생리화학적 특성과 그로 인해 얻어지는 생리학적 효과에 대하여 보고되고 있다. 말토올리고당의 기능은 그것의 크기 및 결합 형태에 따라 다른데, 분지올리고당(branched maltooligosaccharides)은 주로 α-1,4-글라이코시딕 결합으로 연결되며 최소한 하나 이상의 α-1,6-글라이코시딕 결합을 가지는 α-D-글루코피라노에이즈(α-D-glucopyranoase)올리고머로, 이소말토오스, 이소말토트리오스, 패노즈 및 4-5개의 글루코오스 잔기로 이루어지는 올리고머를 의미한다.Recently, the physiological properties of various oligosaccharides and the physiological effects obtained thereby have been reported. The function of maltooligosaccharides depends on their size and binding form. Branched maltooligosaccharides are mainly linked by α-1,4-glycosidic bonds and at least one α-1,6-glycosidic Α-D-glucopyranoase oligomer having a bond means an oligomer consisting of isomaltose, isomaltotriose, panose and 4-5 glucose residues.
분지올리고당은 맛이 설탕보다 부드럽고 점도와 수분활성도를 상대적으로 낮출 수 있어 미생물 오염을 방지하는 효과가 크다. 또, 섭취하였을 때 설탕보다 대사에너지가 적고 장내 미생물군을 개선할 수 있으며 충치를 예방하는 효과가 있다.Branched oligosaccharides are softer than sugar and can lower the viscosity and water activity relatively, thus preventing microbial contamination. In addition, when ingested, less metabolic energy than sugar and can improve the intestinal microflora and has the effect of preventing tooth decay.
전분으로부터 이소말토올리고당을 제조하는 공정에는 아밀레이즈나 플루라네이즈와 같은 전분 분해성 효소와 당전이 효소들이 사용되고 있는데, 이와 같이 이소말토올리고당을 제조하는 방법은 시간과 노력이 많이 소요되는 공정이다.In the process for producing isomaltooligosaccharide from starch, starch degrading enzymes such as amylase and flulanease and sugar transfer enzymes are used. Thus, the method for preparing isomaltooligosaccharide is a time-consuming process.
아밀레이즈는 전분의 대부분의 α-(1,4)-글루코시딕 결합을 가수분해하여 글루코오스, 말토올리고당 및 α-한계 덱스트린을 생성하고, 잔존하는 α-(1,6)-글루코시딕 결합은 풀루라네이즈에 의해 가수분해된다. 따라서 아밀레이즈와 플루라네이즈를 사용한 전분 가수분해 공정에서는 덱스트린은 남아 있지 않다. 이러한 방법은 전분으로부터 원하는 최종 생성물을 보다 높은 수율로 얻을 수 있다.Amylase hydrolyzes most α- (1,4) -glucosidic bonds in starch to produce glucose, maltooligosaccharides, and α-limiting dextrins, and the remaining α- (1,6) -glucosidic bonds Is hydrolyzed by pullulanase. Therefore, dextrin does not remain in the starch hydrolysis process using amylase and flulanase. This method can obtain the desired final product in higher yield from starch.
다양한 올리고당들을 생산하기 위하여 아밀레이즈와 기타 관련 효소를 사용한 당전히 반응이 이용된다. 한 예로, α-아밀레이즈나 베타-아밀레이즈와 같은 전분 가수분해 효소로 액화시킨 액화전분에 말토게네이즈, 베타-아밀레이즈 및 풀루라네이즈를 처리하여 1차 당화시키고, 주로 글루코오스와 말토오스을 α-(1,6)ㅡ글루코시딜 결합으로 당전이시키는 트랜스글루코시데이즈를 처리하여 2차 당화시켜 이소올리고당을 생산하는 방법이 사용되고 있는데, 이 방법에서는 1차 당화에 약 72시간, 2차 당화에 약 48시간이 소요된다.Glucose reactions using amylase and other related enzymes are used to produce various oligosaccharides. For example, liquefied starch liquefied with starch hydrolase, such as α-amylase or beta-amylase, is treated with maltogenase, beta-amylase and pullulanase to primary glycosylation, and mainly glucose and maltose are α- ( 1,6)-Process of transglucosidase, which is glycosylated with glycosylated bonds, is used to produce isooligosaccharides by secondary glycosylation. In this method, about 72 hours for primary glycosylation and about 2 times glycosylation are used. It takes 48 hours.
한국특허출원 96-25135에는 말토오스생성 아밀레이즈를 사용하여 말토올리고당을 생산하는 방법이 기재되어 있다. 말토오스생성 아밀레이즈를 사용하는 경우 1차 당화와 2차 당화의 구분없이 약 12시간 만에 당화가 종료되어 말토올리고당을 얻을 수 있으나, 가수분해 활성 및 당전이 활성 모두를 가지는 말토오스생성 아밀레이즈의 특성으로 인하여 이소말토올리고당 함량을 일정 수치 이상으로 충분히 높일 수는 없다.Korean Patent Application 96-25135 describes a method for producing maltooligosaccharides using maltose-producing amylase. When maltose-producing amylase is used, the glycosylation is completed in about 12 hours without distinguishing between primary and secondary saccharification to obtain malto-oligosaccharide, but characteristics of maltose-producing amylase having both hydrolytic activity and sugar-transfer activity Due to this, the isomaltooligosaccharide content cannot be sufficiently increased above a certain level.
본 발명은 효소 반응 시간이 짧은 이소말토올리고당을 생산하는 방법을 제공한다.The present invention provides a method for producing isomaltooligosaccharide with a short enzyme reaction time.
도 1은 본 발명에서 사용된 벡타 pGNX4F4M 제조에 사용된 F4와 MSI-344의 서열을 나타낸 것이다.Figure 1 shows the sequence of F4 and MSI-344 used in the preparation of the vector pGNX4F4M used in the present invention.
도 2은 BSMA의 농도에 따른 이소말토올리고당의 생산수율을 비교하여 나타낸 것이다.Figure 2 shows the comparison of the production yield of isomaltooligosaccharides according to the concentration of BSMA.
도 3는 ThMA의 농도에 따른 이소말토오리고당의 생산수율을 비교하여 나타낸 것이다.Figure 3 shows the comparison of the yield of isomaltoligosaccharides according to the concentration of ThMA.
도 4은 ThMA와 α-글루카노트랜스퍼레이즈의 동시 처리시의 이소말토올리고당의 생산수율의 변화를 나타낸 것이다.Figure 4 shows the change in the production yield of isomaltoligosaccharide upon simultaneous treatment of ThMA and α-glucanotransferase.
본 발명은 이소말토올리고당을 생산하는 방법에 관한 것이다.The present invention relates to a method for producing isomaltooligosaccharide.
본 발명의 방법은 액화전분에 말토오스생성 아밀레이즈와 α-글루카노트랜스퍼레이즈를 작용시켜 이소말토올리고당을 생산하는 것으로 이루어진다. 본 발명의 방법에서 말토오스생성 아밀레이즈와 α-글루카노트랜스퍼레이즈를 동시에 적용시키거나, 순차적으로 적용시킬 수 있다.The method of the present invention consists in producing isomaltooligosaccharide by acting maltose-producing amylase and α-glucanotransferase on liquefied starch. In the method of the present invention, maltose-producing amylase and α-glucanotransferase may be simultaneously applied or sequentially applied.
따라서 본 발명의 방법은 액화전분에 말토오스생성 아밀레이즈와 α-글루카노트랜스퍼레이즈를 동시에 적용시켜 반응시키는 것으로 이루어진다.Therefore, the method of the present invention consists in reacting liquefied starch by applying maltose-producing amylase and α-glucanotransferase simultaneously.
본 발명의 또 다른 방법은 액화전분에 말토오스생성 아밀레이즈를 적용하여 반응시킨 후, 여기에 α-글루카노트랜스퍼레이즈를 적용시켜 반응시키는 것으로 이루어진다.Another method of the present invention consists in reacting liquefied starch by applying maltose-producing amylase, followed by applying α-glucanotransferase.
본 발명의 또 다른 방법은 액화전분에 말토오스생성 아밀레이즈를 적용하여 반응시킨 후, 상기 말토오스생성 아밀레이즈를 불활성화시키고 여기에 α-글루카노트랜스퍼레이즈를 적용시켜 반응시키는 것으로 이루어진다.Another method of the present invention consists in reacting liquefied starch by applying maltose-producing amylase, then inactivating the maltose-producing amylase and applying it to α-glucanotransferase.
본 발명의 방법에서 전분에 α-글루카노트랜스퍼레이즈을 첨가할 때 필요한 경우 말토오스생성 아밀레이즈를 추가로 첨가할 수 있다.In the process of the invention, maltose amylase may be further added if necessary when the α-glucanotransferase is added to the starch.
본 발명에서 액화전분은 전분을 α-아밀레이즈와 같은 전분 가수분해효소로 액화시킨 것을 의미한다. 따라서 액화전분은 전분 슬러리에 전분 가수분해효소를 첨가하여 전분을 액화시켜 제조할 수 있으며, 시판되는 액화전분을 사용할 수도 있다.In the present invention, liquefied starch means that the starch is liquefied with starch hydrolase, such as α-amylase. Therefore, liquefied starch can be prepared by liquefying starch by adding starch hydrolase to the starch slurry, and commercially available liquefied starch can also be used.
본 발명에 따른 말토오스생성 아밀레이즈(maltogenic amylase)는 전분을 가수분해하여 주로 말토오스를 생산하고, 풀루란을 분해하여 주로 판노오스를 생성하며, 사이클로덱스트린을 강력하게 분해하는 특이한 효소적 특성을 가지는 아밀레이즈를 의미한다. 이러한 말토오스생성 아밀레이즈의 예로는 한국특허출원 96-25135에 기재된,Bacillus stearothermophilus(KFCC-10896)부터 생산되는 아밀레이즈(이하 BSMA라 함), 미국특허 5,583,039에 기재된Bacillus licheniformis(ATCC 27811), 한국특허출원 1998-63956에 기재된Thermussp. (IM6501 KCTC 0527BP)에서 분리된 아밀레이즈(이하 ThMA라 함), 또는 이들과 실질적으로 동일한 성질을 가지는 효소 및 이들과 실질적으로 동일한 성질을 가지며 유전자 재조합 기술에 생산되는 효소 등을 들 수 있다. 본 발명에서 사용된 발현벡타는 한국특허출원 1999-17920호에 기재된 방법을 참조하여 만들었으며, 상기 특허출원은 본 명세서의 내용에 포함된다.Maltogenic amylase according to the present invention is hydrolyzed starch to produce mainly maltose, and to break down pullulan to produce mainly pannose, amylose having a specific enzymatic properties that strongly degrade cyclodextrin It means raise. Examples of such maltose-producing amylase include amylase produced from Bacillus stearothermophilus (KFCC-10896) described in Korean Patent Application 96-25135 (hereinafter referred to as BSMA), Bacillus licheniformis (ATCC 27811) described in US Patent 5,583,039, and Korean Patent Thermus sp. Amylases (hereinafter referred to as ThMA) isolated from (IM6501 KCTC 0527BP), or enzymes having substantially the same properties as those, and enzymes having substantially the same properties as those produced in genetic recombination technology. Expression vector used in the present invention was made with reference to the method described in Korean Patent Application No. 1999-17920, the patent application is included in the content of the present specification.
본 발명에서 사용되는 α-글루코시딜트랜스퍼레이즈는 소당류를 α-(1,4)ㅡ글루코시딜 결합을 형성시키면서 말토올리고당을 전이하는 당전이효소를 의미한다. α-글루코시딜트랜스퍼레이즈의 종류는 특별히 한정되지 않으나, 공정상 내열성 α-글루코시딜 트랜스퍼레이즈가 바람직하다.The α-glucosidyltransferase used in the present invention means a glycotransferase that transfers maltooligosaccharides to small sugars while forming α- (1,4) -glucosidyl bonds. Although the kind of (alpha)-glucosidyl transferase is not specifically limited, A process heat resistant (alpha)-glucosidyl transferase is preferable.
본 발명에서 효소활성 측정방법 및 사용된 배지는 다음과 같다.Enzyme activity measurement method and the medium used in the present invention is as follows.
말토오스생성 아밀레이즈의 활성단위(CU)는 효소액 0.5ml에 pH 6.0의 50mM 소디움 사이트레이트 완충용액 0.4ml과 1%(w/v)의 β-사이클로덱스트린 용액 0.5ml을 넣은 후 55℃에서 30분간 반응시키고 DNS 용액을 넣어 반응을 중단시킨 뒤 5분간 끓여 발색시켜 575nm에서 흡광도를 측정하여 결정하였으며, 흡광도 차이가 1.0일 때 1단위로 정한다.The active unit (CU) of maltose-producing amylase was added to 0.5 ml of enzyme solution, 0.4 ml of 50 mM sodium citrate buffer solution at pH 6.0 and 0.5 ml of 1% (w / v) β-cyclodextrin solution, followed by 30 minutes at 55 ° C. After stopping the reaction by adding the DNS solution and boiled for 5 minutes to develop the color was measured by measuring the absorbance at 575nm, it is determined as 1 unit when the difference in absorbance is 1.0.
α-글루카노트랜스퍼레이즈의 활성단위(AU)는 효소액을 50mM Tris/HCl 완충용액 (pH 7.5)에 녹인 0.05% 아밀로오스, 0,05% 말토오스 용액과 60℃에서 혼합한다. 반응 0분, 15분 후에 각각 0.1ml을 취해 0.02% 아이오다인/포타슘아이오다인 용액 1ml과 혼합하여 620nm에서 흡광도를 측정한다. 측정한 흡광도 차이가 1이 될 때의 효소양을 1단위로 표시한다.The active unit (AU) of α-glucanotransferase is mixed with 0.05% amylose, 0,05% maltose solution dissolved in 50 mM Tris / HCl buffer (pH 7.5) at 60 ° C. After 0 min and 15 min of the reaction, 0.1 ml of each was mixed with 1 ml of a 0.02% iodide / potassium iodide solution and the absorbance was measured at 620 nm. The amount of enzyme when the measured absorbance difference becomes 1 is expressed by 1 unit.
LB 배지(박토-트립톤 10 g/l, 효모추출물 5 g/l, NaCl 10 g/l)LB medium (10 g / l bacto-tryptone, 5 g / l yeast extract, 10 g / l NaCl)
R 배지(Reisenberg 배지; KH2PO413.3 g/l, (NH4)2HPO44.0 g/l, 시트르산 0.17 g/l, MgSO4.7H2O 0.22 g/l, 글루코오스 20 g/l, 미량원소 용액 10ml/l)R medium (Reisenberg medium; KH 2 PO 4 13.3 g / l, (NH 4) 2 HPO 4 4.0 g / l, citric acid 0.17 g / l, MgSO 4 .7H 2 O 0.22 g / l, glucose 20 g / l, Trace element solution 10ml / l)
본 발명에서 이소말토올리고당 분석은 다음 조건에 따른 HPLC 방법으로 수행하였다.Isomaltooligosaccharide analysis in the present invention was carried out by HPLC method according to the following conditions.
DP(중합도) 분석 조건DP (polymerization) analysis conditions
기기: HPLC(waters)Instrument: HPLC (waters)
컬럼: Biorad Aminex-HPX42A 컬럼(Biorad; 7.8x300cm)Column: Biorad Aminex-HPX42A Column (Biorad; 7.8x300 cm)
검출기: RI 검출기Detector: RI Detector
컬럼온도: 80℃Column temperature: 80 ℃
이동상: 증류수Mobile phase: distilled water
유속: 0.4ml/분Flow rate: 0.4ml / min
BDP(분지중합도) 분석 조건Basis Polymerization Condition
기기: HPLC(waters)Instrument: HPLC (waters)
컬럼: Carbohydrate analysis 컬럼(waters; 길이 직경)Column: Carbohydrate analysis column (waters; length diameter)
검출기: RI 검출기Detector: RI Detector
컬럼온도: 35℃Column temperature: 35 ℃
이동상: 아세토나이트릴/물(65:35(w/w))Mobile phase: acetonitrile / water (65:35 (w / w))
유속: 2.0ml/분Flow rate: 2.0ml / min
이하 실시예를 통해 본 발명을 더욱 자세히 설명하고자 하나, 실시예에 의하여 본 발명의 범위가 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited by Examples.
실시예 1. 발현 벡타 제조Example 1 Expression Vector Preparation
한국특허출원 1999-17920에 기재된 방법을 참조하여 pGNX4F4M을 제조하였다.PGNX4F4M was prepared by referring to the method described in Korean Patent Application No. 1999-17920.
즉, 대한민국 대전광역시 유성구 어은동 52번지에 주소를 둔 생명공학연구소 부설 유전자은행(Korean Collection for Type Cultures)에 KCTC 0486BP로 기탁된 E.coli HM174(DE3)/pGNX2를 카사미노 산을 첨가한 R 배지에 접종하여 37℃에서 배양하고 Molecular cloning a laboratory manual 2nd(Sambrook 등, Cold Spring Habor laboratory Press, 1989)에 기재된 방법에 따라 발현 벡타 pGNX2를 분리하였다. 한국특허출원 1999-17920의 실시예 3과 도 4을 참고하여 pGNX2F4M을 제조하였다. 즉, F4(도 1 참조)의 3' 말단을SspI로 절단하고 MSI-344(도 1 참조)의 5' 말단을SmaI로 절단한 후 F4와 MSI-344를 연결하여 DNA 구조물 F4M 얻었다. F4M을NdeI과BamHI로 절단하여 얻은 단편을,NdeI과BamHI로 절단한 pGNX2에 삽입에 클로닝하여 플라즈미드 pGNX2F4M을 제조하였다.In other words, E.coli HM174 (DE3) / pGNX2 deposited as KCTC 0486BP in the Korean Collection for Type Cultures, located at 52, Eeun-dong, Yuseong-gu, Daejeon, Korea. The expression vector pGNX2 was isolated according to the method described in Molecular cloning a laboratory manual 2nd (Sambrook et al., Cold Spring Habor laboratory Press, 1989). PGNX2F4M was prepared by referring to Example 3 and FIG. 4 of Korean Patent Application No. 1999-17920. That is, the 3 'end of F4 (see Fig. 1) was cut with Ssp I, and the 5' end of MSI-344 (see Fig. 1) was cut with Sma I, and F4 and MSI-344 were connected to obtain a DNA construct F4M. A fragment obtained by cutting the F4M by Nde I and Bam HI, and cloning the insert into pGNX2 cut into Nde I and Bam HI to prepare a plasmid pGNX2F4M.
플라즈미드 pGNX3를 만들기 위하여, 먼저 pGNX2F4M을BspHI으로 부분절단하고, 하나의BspHI자리만 잘린 단편을 분리하여BamHI으로 절단하였다. T7 및 rrnBT1T2 터미네이터를 포함하는 단편을 만들기 위하여 pET11a(Novagen)로부터BamHI과EcoRV로 절단한 132 bp단편을 분리하고, 이를 ptrc99a(Pharmacia)를HincII와BspHI으로 절단하여 분리한 488 bp단편과 연결하였다. 연결된 단편들을BamHI과BspHI으로 절단하여 이를 앞에서 만든 벡터와 연결하여 플라즈미드 pGNX3F4M을 제조하였다.To make plasmid pGNX3, pGNX2F4M was first cleaved with Bsp HI, and fragments cut from only one Bsp HI locus were cut with Bam HI. To create fragments containing T7 and rrnBT1T2 terminators, 132 bp fragments cut with Bam HI and Eco RV were isolated from pET11a (Novagen), and 488 bp fragments isolated from ptrc99a (Pharmacia) with Hin cII and Bsp HI. Connected with. Plasmid pGNX3F4M was prepared by digesting the fragments with Bam HI and Bsp HI and linking them with the previously made vector.
플리즈미드 pGNX4F4M를 만들기 위하여, 플라즈미드 pETACc를XbaI과AlwN I으로 절단하여 3502 bp의 단편을 분리하고, 이를 pGNX3F4M을XbaI과AlwN I으로 절단하여 분리된 2405 bp의 단편과 연결하여 pGNX4F4M을 제조하였다. 플라즈미드 pETACc는 다음과 같이 제조하였다. 플라즈미드 pET21c(Novagen)을 XbaI으로 절단한 후 필링-인(filling-in) 하고 BglII로 절단하여 5375bp DNA 단편을 분리하고, 플라즈미드 pKK223-3(Pharmacia)를EcoRI으로 절단한 후 필링-인하고BamHI으로 절단하여 tac 프로모터가 포함된 259bp DNA 단편을 분리하였다. 이 두 DNA 단편을 연결하여 플라즈미드 pETACc를 얻었다.In order to make plasmid pGNX4F4M, plasmid pETACc was cleaved with Xba I and Alw N I to isolate 3502 bp fragment, which was then cleaved with pGNX3F4M with Xba I and Alw NI to connect 2405 bp fragment isolated to prepare pGNX4F4M. . Plasmid pETACc was prepared as follows. The plasmid pET21c (Novagen) was digested with XbaI, then peeled-in (filling-in) and digested with BglII to isolate 5375 bp DNA fragments.The plasmid pKK223-3 (Pharmacia) was digested with Eco RI, then peeled-in and Bam HI was isolated to 259bp DNA fragment containing the tac promoter was isolated. These two DNA fragments were linked to obtain plasmid pETACc.
실시예 2. 효소 생산Example 2. Enzyme Production
1) BSMA 제조1) BSMA manufacture
한국특허출원 1996-25135에 기재된, 기탁번호 KFCC-10896로 E.coli/pSG18 를 LB-엠피실린 배지에서 37℃에서 배양하여, 위에 기재된 Sambrook 등의 참고문헌에 기재된 방법에 따라 플라즈미드 pSG18을 분리하고 이로부터 BSMA 유전자(NCI Accession No.: U50744)를 분리하였다. 분리한 BSMA 유전와 아래의 프라이머를 이용하여 중합효소연쇄반응(PCR) 반응에 의해 5' 말단에 제한효소NdeI 절단자리를 가지고 3' 말단에 제한효소BamHI 절단자리를 가지는 BSMA 유전자를 만들었다. 이 유전자를 제한효소NdeI로 부분절단하고BamHI으로 완전 절단한 것을, 제한효소NdeI와BamHI으로 절단한 플라즈미드 pGNX4F4M에 클로닝시켜 pGNX4-BSMA를 얻었다.E. coli / pSG18 was incubated at 37 ° C. in LB-Epicillin medium with Accession No. KFCC-10896, described in Korean Patent Application 1996-25135, to separate plasmid pSG18 according to the method described in the reference of Sambrook et al. From this, the BSMA gene (NCI Accession No .: U50744) was isolated. Using the isolated BSMA gene and the primers below, a BSMA gene was produced having a restriction enzyme Nde I cleavage site at 5 'end and a restriction enzyme Bam HI cleavage site at 3' end by polymerase chain reaction (PCR) reaction. Parts cut the gene with restriction enzymes Nde I and that a complete cut with Bam HI, was cloned into a plasmid pGNX4F4M cut with restriction enzymes Nde I and Bam HI to obtain a pGNX4-BSMA.
프라이머primer
BMNdeI 5'-CCCCATATGTTCAAAGAAGCC-3'BMNdeI 5'-CCC CATATG TTCAAAGAAGCC-3 '
BMBmI 5'-CCCGGATCCTTATAACCAATCTTCG-3' BMBmI 5'-CCC GGATCC TTATAACCAATCTTCG-3 '
pGNX4-BSMA를E. coliTG1에 형질전환시켜 얻은 형질전환체를 카사미노 산을 첨가한 R 배지 1.5L를 포함하는 5L 발효조에서, 30℃에서 배양하였다. 배양액이 OD66080일 때 1mM IPTG를 첨가하여 효소 발현을 유도한 후 5시간 더 배양하였다. 균체를 회수하고, 회수한 균체를 pH 7.5의 50 mM Tris-HCl 완충용액에 현탁시킨 다음 균체를 호모게나이저로 파괴하였다. 핵산을 제거하기 위하여 폴리에틸렌이민 0.5%(w/v)을 처리하였다. 12,000g에서 30분간 원심분리하여 상등액을 취하고 pH 7.5의 50 mM Tris-HCl 완충용액으로 투석하고, 컷오프 10,000인 펠리콘 울트라필트레이션 키트(Millipore, 미국)로 농축하였다. 농축액을 20%(w/v) 황산암모니움으로 분획하여 4 ℃에서 2시간 방치한 후 원심분리하고, 얻어진 상등액을 60%(w/v) 황산암모니움으로 분획하여 4 ℃에서 2시간 방치한 후 원심분리하여 상등액을 제거하였다. 원심분리하여 얻어진 팰렛을 pH 7.5의 50 mM Tris-HCl 완충용액에 현탁시킨 후 투석하고, 펠리콘 울트라필트레이션 키트(Millipore, 미국)로 농축하여 부분 정제된 BSMA를 얻었다.Transformants obtained by transforming pGNX4-BSMA into E. coli TG1 were incubated at 30 ° C. in a 5 L fermenter containing 1.5 L of R medium to which cassanoic acid was added. When the culture medium was OD 660 80, 1mM IPTG was added to induce enzyme expression, followed by further incubation for 5 hours. The cells were recovered, and the recovered cells were suspended in 50 mM Tris-HCl buffer at pH 7.5 and the cells were disrupted by a homogenizer. Polyethyleneimine 0.5% (w / v) was treated to remove the nucleic acid. The supernatant was collected by centrifugation at 12,000 g for 30 minutes, dialyzed with 50 mM Tris-HCl buffer at pH 7.5, and concentrated with a Pelicon Ultrafiltration Kit (Millipore, USA) with a cutoff of 10,000. The concentrate was partitioned with 20% (w / v) ammonium sulfate and left at 4 ° C. for 2 hours, followed by centrifugation. The resulting supernatant was partitioned with 60% (w / v) ammonium sulfate and left for 4 hours at 4 ° After centrifugation, the supernatant was removed. The pellet obtained by centrifugation was suspended in 50 mM Tris-HCl buffer at pH 7.5, then dialyzed, and concentrated with a Pelicon Ultrafiltration Kit (Millipore, USA) to obtain partially purified BSMA.
2) ThMA 제조2) ThMA manufacture
한국특허출원 1998-63956에 기재된, 기탁번호 KCTC-0528BP로 기탁된 E.coli/pThMA119 를 LB-엠피실린 배지에서 37℃에서 배양하여, 위에 기재된 Sambrook 등의 참고문헌에 기재된 방법에 따라 플라즈미드 pThMA119를 분리하였다. 보고된 ThMA 코딩 DNA 서열(NCI Accession No.: AF060204)과 다음에 기재된 프라이머를 사용하여 중합효소연쇄반응으로 ThMA 유전자를 얻은 후NdeI와HindIII로 절단하여 서머스 말토오스생성 아밀레이즈(ThMA)의 유전자를 분리하였다.E. coli / pThMA119 deposited with accession number KCTC-0528BP described in Korean Patent Application 1998-63956 was incubated at 37 ° C. in LB-empicillin medium, and the plasmid pThMA119 was prepared according to the method described in the reference of Sambrook et al. Separated. Using the reported ThMA coding DNA sequence (NCI Accession No .: AF060204) and the primers described below, the ThMA gene was obtained by polymerase chain reaction, and then cleaved with Nde I and Hin dIII to generate a Somers maltose-producing amylase (ThMA) gene. Was separated.
프라이머primer
TMNdeITMNdeI
5'-GGAGGAAAAAGCATATGAGGAAAGAAGCC-3'5'-GGAGGAAAAAG CATATG AGGAAAGAAGCC-3 '
TMHdIIITMHdIII
5'-AAAGGCAAAGCTTTGGTTGGCGGAGACG-3'5'-AAAGGCA AAGCTT TGGTTGGCGGAGACG-3 '
얻어진 ThMA 유전자를,NdeI와HindIII로 자른 pGNX4F4M에 클로닝하여 플라즈미드 pGNX4-ThMA를 얻었다. pGNX4-ThMA를E. coliTG1에 형질전환하여 얻은 형질전환체를 BSMA 형질전환체와 동일한 방법으로 배양하여 회수한 균체를 pH 7.5의 50 mM Tris-HCl 완충용액에 현탁시킨 다음 균체를 호모게나이저로 파괴하였다. 60℃ 수조에서 20분간 열처리하고, 핵산을 제거하기 위하여 폴리에틸렌이민 0.5%(w/v)을 처리하고 12,000g에서 30분간 원심분리하여 펠렛을 제거하였다. 상등액을 pH 7.5의 50 mM Tris-HCl 완충용액으로 투석하고, 컷오프 10,000인 펠리콘 울트라필트레이션 키트(Millipore, 미국)로 농축하여 부분 정제된 ThMA를 얻었다.The obtained ThMA gene was cloned into pGNX4F4M cut with Nde I and Hin dIII to obtain plasmid pGNX4-ThMA. The transformed cells obtained by transforming pGNX4-ThMA to E. coli TG1 were cultured in the same manner as the BSMA transformants, and the recovered cells were suspended in 50 mM Tris-HCl buffer at pH 7.5, and the cells were homogenized. Destroyed. Heat treated for 20 minutes in a 60 ℃ water bath, and treated with polyethyleneimine 0.5% (w / v) to remove the nucleic acid and centrifuged for 30 minutes at 12,000g to remove the pellets. The supernatant was dialyzed with 50 mM Tris-HCl buffer at pH 7.5 and concentrated with a Pelicon Ultrafiltration Kit (Millipore, USA) with a cutoff of 10,000 to obtain partially purified ThMA.
3) α-글루카노트랜스퍼레이즈 제조3) α-glucanotransferase production
기탁된 균주인 Thermotoga maritima MSB8 JCM10099 (Japan Collection of Microorganisms)의 염색체를 템플레이트로 사용하고, 보고된 α-글루카노트랜스퍼레이즈 유전자 서열(NCI Accession No.: Z50813)을 기초로 한 다음의 프라이머를 사용하여 중합효소연쇄반응(PCR)으로 α-글루카노트랜스퍼레이즈 유전자를 포함하는 DNA 단편을 얻었다.Using the chromosome of the deposited strain Thermotoga maritima MSB8 JCM10099 (Japan Collection of Microorganisms) as a template, and using the following primers based on the reported α-glucanotransferase gene sequence (NCI Accession No .: Z50813) Polymerase chain reaction (PCR) yielded a DNA fragment containing the α-glucanotransferase gene.
프라이머primer
TmAGT5 : 5'-GACGGCTCAGAAGTTCTACAATACC-3'TmAGT5: 5'-GACGGCTCAGAAGTTCTACAATACC-3 '
TmAGT3 : 5'-CCCGCGAAGTCTTCAAGAGGAACG-3'TmAGT3: 5'-CCCGCGAAGTCTTCAAGAGGAACG-3 '
얻어진 α-글루카노트랜스퍼레이즈 유전자를 포함하는 DNA 단편을HincII로 절단한 pUC119에 클로닝하여 pα-GT119를 얻었다.The DNA fragment containing the obtained α-glucanotransferase gene was cloned into pUC119 digested with Hin cII to obtain pα-GT119.
pα-GT119를 기초로 다음의 프라이머를 사용하여 PCR한 후NdeI으로 완전 절단하고HindIII로 부분절단하여 얻어진 α-글루카노트랜스퍼레이즈 유전자 포함 DNA 단편을NdeI와HindIII로 자른 pGNX4F4M에 클로닝하여 플라즈미드 pGNX4-αGT를 얻었다.After PCR using the following primers based on the pα-GT119 completely cut with Nde I and cut the α- glue transfer Kano raised gene comprising a DNA fragment obtained by partial cleavage with Hin dIII to Nde I and Hin dIII was cloned in pGNX4F4M The plasmid pGNX4-αGT was obtained.
프라이머primer
TGTNd : 5'-CAAGGAGGATAGACATATGATAGGCTATCA G -3'TGTNd: 5'-CAAGGAGGATAGA CATATG ATAGGCTATCA G -3 '
Reverse primer (sequencing) : 5'-AGCGGATAACAATTTCACACAGGA-3'Reverse primer (sequencing): 5'-AGCGGATAACAATTTCACACAGGA-3 '
pGNX4-αGT를E. coliTG1에 형질전환하여 얻은 형질전환체를 BSMA 형질전환체와 동일한 방법으로 배양하여 회수한 균체를 pH 7.5의 50 mM Tris-HCl 완충용액에 현탁시킨 다음 균체를 호모게나이저로 파괴하였다. 80℃ 수조에서 20분간 열처리하고, 핵산을 제거하기 위하여 폴리에틸렌이민 0.5%(w/v)을 처리하였다. 12,000g에서 30분간 원심분리하여 상등액을 제거하고 1회 세척한 후, pH 7.5의 50 mM Tris-HCl 완충용액으로 투석하고, 컷오프 10,000인 펠리콘 울트라필트레이션 키트(Millipore, 미국)로 농축하여 부분 정제된 α-클루카노트랜스퍼레이즈를 얻었다.The transformed cells obtained by transforming pGNX4-αGT into E. coli TG1 were cultured in the same manner as the BSMA transformants, and the recovered cells were suspended in 50 mM Tris-HCl buffer at pH 7.5 and the cells were homogenized. Destroyed. Heat treated for 20 minutes in an 80 ℃ water bath, and treated with 0.5% polyethylenimine (w / v) to remove the nucleic acid. The supernatant was removed by centrifugation at 12,000g for 30 minutes, washed once, dialyzed with 50 mM Tris-HCl buffer at pH 7.5, concentrated with a Pelicon Ultrafiltration Kit (Millipore, USA) with a cutoff of 10,000. Purified α-glucanotransferase was obtained.
비교예. 말토오스생산 아밀레이즈를 이용한 분지올리고당 생산Comparative example. Branch oligosaccharide production using maltose-produced amylase
액화 옥수수 시럽(Brix33, DE22)를 사용하여 분지올리고당 생산 반응을 수행하였다.Branched oligosaccharide production reactions were performed using liquefied corn syrup (Brix33, DE22).
액화 옥수수 시럽에 BSMA를 각각 100, 200, 400 CU/g기질 첨가하고 50℃에서 65시간동안 당화반응시키면서 시간에 따른 분지올리고당 생산량을 측정하였다. 그 결과를 도 2에 나타낸다.BSMA was added to liquefied corn syrup 100, 200, and 400 CU / g substrate, respectively, and the yield of branched oligosaccharides over time was measured while saccharifying at 50 ° C. for 65 hours. The result is shown in FIG.
BSMA 대신 ThMA를 사용하여, 당화반응 온도를 55℃로 하는 것을 제외하고는 동일한 조건으로 실험하였으며, 그 결과를 도 3에 나타낸다.Using ThMA instead of BSMA, the experiment was conducted under the same conditions except that the saccharification temperature was 55 ° C., and the results are shown in FIG. 3.
실시예 3. BSMA 및 α-글루카노트랜스퍼레이즈를 이용한 분지올리고당 생산Example 3. Branch Oligosaccharide Production Using BSMA and α-Glucanotransferase
액화 옥수수 시럽(Brix33, DE22)에 실시예 2에서 생산한 BSMA 200 CU/g기질과, α-글루카노트랜스퍼레이즈를 3000, 6,000, 12,000 AU/g기질을 각각 첨가하고 55℃에서 0-50 시간 반응시켰다. 시간별로 생산된 말토올리고당 및 이소말토올리고당을 분석하여 그 결과를 표 1에 나타낸다.BSMA 200 CU / g substrate produced in Example 2 and α-glucanotransferase were added to liquefied corn syrup (Brix33, DE22) 3000, 6,000, and 12,000 AU / g substrate, respectively, and 0-50 hours at 55 ° C. Reacted. Malto oligosaccharides and isomaltooligosaccharides produced over time are analyzed and the results are shown in Table 1.
실시예 4. BSMA 및 α-글루카노트랜스퍼레이즈를 이용한 분지올리고당 생산Example 4 Branch Oligosaccharide Production Using BSMA and α-Glucanotransferase
액화 옥수수 시럽(Brix33, DE22)에 BSMA 100 CU/g기질을 첨가하여 50℃에서 20시간 반응시켰다. BSMA를 불활성화시키고, 시료를 4개의 플라스크에 나누어 담았다. 플라스크 1개에는 BSMA 50 CU/g기질만 추가하고, 나머지 3개의 플라스크에는 각각 BSMA 50 CU/g기질 + α-글루카노트랜스퍼레이즈 3000 AU/g기질, BSMA 50 CU/g기질 + α-글루카노트랜스퍼레이즈 6000 AU/g기질, BSMA 50 CU/g기질 + α-글루카노트랜스퍼레이즈 12000 AU/g기질을 추가하여 50℃에서 반응시켰다. 생산된 말토올리고당 및 이소말토올리고당을 분석하여 그 결과를 표 2에 나타낸다.BSMA 100 CU / g substrate was added to the liquefied corn syrup (Brix33, DE22) and reacted at 50 ° C. for 20 hours. BSMA was inactivated and the sample was divided into four flasks. Add only BSMA 50 CU / g substrate to one flask, BSMA 50 CU / g substrate + α-Glucanotransferase 3000 AU / g substrate, BSMA 50 CU / g substrate + α-glucano A transferase 6000 AU / g substrate, BSMA 50 CU / g substrate + α-glucanotransferase 12000 AU / g substrate were added and reacted at 50 ° C. The produced maltooligosaccharides and isomaltooligosaccharides are analyzed and the results are shown in Table 2.
실시예 5. BSMA 및 α-글루카노트랜스퍼레이즈를 이용한 분지올리고당 생산Example 5 Branched Oligosaccharide Production Using BSMA and α-Glucanotransferase
액화 옥수수 시럽(Brix33, DE22)에 BSMA 100 CU/g기질을 첨가하여 50℃에서 20시간 반응시켰다. 시료를 4개의 플라스크에 나누어 1개의 플라스크는 α-글루카노트랜스퍼레이즈 첨가를 하지 않고 나머지 플라스크 3개는 BSMA를 불활성화시키고 각각 α-글루카노트랜스퍼레이즈 3,000, 6,000, 12,000 AU/g기질을 추가한 후 50℃에서 계속 반응시켰다. 생산된 말토올리고당 및 이소말토올리고당을 분석하여 그 결과를 표 3에 나타낸다. BSMA 100 CU / g substrate was added to the liquefied corn syrup (Brix33, DE22) and reacted at 50 ° C. for 20 hours. Samples were divided into four flasks, one flask without the addition of α-glucanotransferase, and the remaining three flasks inactivated BSMA and added the α-glucanotransferase 3,000, 6,000 and 12,000 AU / g substrate, respectively. After the reaction was continued at 50 ℃. The produced maltooligosaccharides and isomaltooligosaccharides are analyzed and the results are shown in Table 3.
실시예 6. ThMA 및 α-글루카노트랜스퍼레이즈를 이용한 분지올리고당 생산Example 6 Branched Oligosaccharide Production Using ThMA and α-Glucanotransferase
두 개의 플라스크에 액화 옥수수 시럽(Brix33, DE22)에 각각 ThMA 200 CU/g기질, ThMA 200CU/g기질 + α-글루카노트랜스퍼레이즈 6000AU/g기질을 첨가하고 55℃에서 0-50시간 반응시켰다. 두 개의 다른 플라스크에는 액화 옥수수 시럽(Brix33, DE22)에 ThMA 200CU/g기질을 각각 첨가하여 20시간까지 반응시킨 후 하나에는 α-글루카노트랜스퍼레이즈를 6000AU/g기질을 추가하고 다른 하나에는 ThMA를 불활성화 시킨 후 α-글루카노트랜스퍼레이즈 6000AU/g기질을 추가하여 55℃에서 50시간 까지 반응시켰다. 생산된 말토올리고당 및 이소말토올리고당을 분석하여 그 결과를 도 4에 나타낸다.To two flasks, ThMA 200 CU / g substrate, ThMA 200CU / g substrate + α-glucanotransferase 6000AU / g substrate were added to liquefied corn syrup (Brix33, DE22), respectively, and reacted at 55 ° C. for 0-50 hours. Two different flasks were reacted with liquefied corn syrup (Brix33, DE22) with ThMA 200 CU / g substrate for up to 20 hours, and one α-glucanotransferase with 6000AU / g substrate and the other with ThMA. After inactivation, the α-glucanotransferase 6000AU / g substrate was added and reacted at 55 ° C. for 50 hours. The produced maltooligosaccharides and isomaltooligosaccharides are analyzed and the results are shown in FIG. 4.
본 발명의 제조방법에 의해, 이소말토올리고당 제조 시간을 단축할 수 있으며, 이소말토올리고당 수율을 향상시킬 수 있다.By the production method of the present invention, it is possible to shorten the production time of isomaltooligosaccharide and to improve the yield of isomaltooligosaccharide.
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KR102170296B1 (en) * | 2019-06-17 | 2020-10-27 | 대한민국(농촌진흥청장) | Glycosyltransferase producing high degree of polymerization of isomalto-oligosacharrides and manufacturing method of high degree of polymerization of isomalto-oligosacharrides using the same |
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 |
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KR102411709B1 (en) | 2020-01-31 | 2022-06-21 | 부경대학교 산학협력단 | Manufacturing method of isomalto-oligosaccharide using rice powder |
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EP1601699A4 (en) * | 2003-03-10 | 2011-03-16 | Genencor Int | Grain compositions containing pre-biotic isomalto-oligosaccharides and methods of making and using same |
KR102170296B1 (en) * | 2019-06-17 | 2020-10-27 | 대한민국(농촌진흥청장) | Glycosyltransferase producing high degree of polymerization of isomalto-oligosacharrides and manufacturing method of high degree of polymerization of isomalto-oligosacharrides using the same |
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 |
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