KR101073990B1 - Method for Preparing Ascorbic Acid 2-Glucoside Using Glucansucrase - Google Patents
Method for Preparing Ascorbic Acid 2-Glucoside Using Glucansucrase Download PDFInfo
- Publication number
- KR101073990B1 KR101073990B1 KR1020090076848A KR20090076848A KR101073990B1 KR 101073990 B1 KR101073990 B1 KR 101073990B1 KR 1020090076848 A KR1020090076848 A KR 1020090076848A KR 20090076848 A KR20090076848 A KR 20090076848A KR 101073990 B1 KR101073990 B1 KR 101073990B1
- Authority
- KR
- South Korea
- Prior art keywords
- thr
- ala
- ascorbic acid
- asp
- gly
- Prior art date
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/01—Hexosyltransferases (2.4.1)
- C12Y204/01005—Dextransucrase (2.4.1.5)
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
본 발명은 아스코르브산 2-글루코시드의 생산방법에 관한 것으로, 보다 상세하게는 글루칸수크라제를 이용하여 L-아스코르브산의 글리코실화를 통하여 증가된 안정성을 가지는 아스코르브산 2-글루코시드를 생산하는 방법에 관한 것이다. 본 발명에 따른 방법은 다양한 산업분야에 사용되고 있는 L-아스코르브산 유도체인 아스코르브산 2-글루코시드를 수크로스와 같은 풍부한 탄수화물 자원을 이용하여 합성할 수 있어 경제적이며, 또한 우수한 효율로 아스코르브산 2-글루코시드를 생산하는바 유용하다.The present invention relates to a method for producing ascorbic acid 2-glucoside, and more particularly, to produce ascorbic acid 2-glucoside having increased stability through glycosylation of L-ascorbic acid using glucan sucralase. It is about a method. The method according to the present invention can synthesize ascorbic acid 2-glucoside, an L-ascorbic acid derivative used in various industrial fields, using abundant carbohydrate resources such as sucrose, which is economical and has high efficiency. It is useful for producing glucosides.
글루칸수크라제, 글리코실화, L-아스코르브산, 아스코르브산 2-글루코시드 Glucan sucrase, glycosylation, L-ascorbic acid, ascorbic acid 2-glucoside
Description
본 발명은 아스코르브산 2-글루코시드의 생산방법에 관한 것으로, 보다 상세하게는 글루칸수크라제를 이용하여 L-아스코르브산의 글리코실화를 통하여 증가된 안정성을 가지는 아스코르브산 2-글루코시드를 생산하는 방법에 관한 것이다. The present invention relates to a method for producing ascorbic acid 2-glucoside, and more particularly, to produce ascorbic acid 2-glucoside having increased stability through glycosylation of L-ascorbic acid using glucan sucralase. It is about a method.
L-아스코르브산(L-ascorbic acid, L-AsA)은 약학 제제, 사료, 식품 보존제 및 화장품을 포함한 다양한 산업적 응용에 사용되고 있다. 그러나, L-AsA가 산화 조건 및 열조건에서 불안정하기 때문에 L-AsA의 불안정성을 극복하기 위하여, 지금까지 L-아스코르브산 2-포스페이트(AA-2P), L-아스코르브산 2-설페이트(AA-2S) 및 L-아스코르브산 2-메틸 에테르(AA-2M)을 포함한 많은 안정한 유도체들이 연구되어져왔다.L-ascorbic acid (L-AsA) is used in a variety of industrial applications, including pharmaceutical formulations, feed, food preservatives and cosmetics. However, in order to overcome the instability of L-AsA because L-AsA is unstable in oxidizing conditions and thermal conditions, so far L-ascorbic acid 2-phosphate (AA-2P), L-ascorbic acid 2-sulfate (AA- Many stable derivatives have been studied including 2S) and L-ascorbic acid 2-methyl ether (AA-2M).
한편, 글리코실화 반응은 당 수용기의 수용성, 분자안정성 및 생물학적 활성을 향상시키며, 다양한 글리코실화된 산물은 항산화제, 항생제 및 항암제와 같은 생물학적 화합물로서 개발되고 있다. 이에 증가된 안정성을 가지는 글리코실 유도체인 L-아스코르브산 2-글루코사이드(L-ascorbic acid 2-glucoside, AA-2G)는 산업적으로 다양하게 사용되기 위하여, 글리코실화 반응에 의하여 효소학적으로 합성되고 있다. 즉, AA-2G는 L-AsA와 같은 수용 기질과, 말토오즈, 수크로즈 또는 다른 α-글루칸과 같은 공여 기질로부터 포유동물 및 벼 종자 α-글루코시다아제(I. Yamamoto et al., Chem . Pharm . Bull ., 38(11):3020, 1990), Bacillus stearothermophilus 싸이클로덱트린 글루카노트랜스퍼라제(CGTase)(M. Tanaka et al., Biochim. Biophys. Acta., 1078(2):127, 1991) 및 Bifidobacterium longum 슈크로스 포스포릴라아제(H.K. Kang et al., FEMS Microbiol. Lett., 292:33, 2009)에 의한 글리코실화 반응에 의하여 합성된다. 그러나, 여전히 산업적으로 유용한 AA-2G를 경제적으로 더욱 간이한 방법으로 효율적으로 합성할 수 있는 방법의 개발이 요구되어 왔다. On the other hand, glycosylation reactions improve the water solubility, molecular stability and biological activity of sugar receptors, and various glycosylated products are being developed as biological compounds such as antioxidants, antibiotics and anticancer agents. To this end, L-ascorbic acid 2-glucoside (AA-2G), a glycosyl derivative having increased stability, has been enzymatically synthesized by a glycosylation reaction for industrial use. . In other words, AA-2G is derived from mammalian and rice seed α-glucosidase (I. Yamamoto et al., Chem . A) from an accepting substrate such as L-AsA and a donor substrate such as maltose, sucrose or other α-glucan . Pharm . Bull ., 38 (11): 3020, 1990), Bacillus stearothermophilus cyclodextrin glucanotransferase (CGTase) (M. Tanaka et al., Biochim. Biophys. Acta., 1078 (2): 127, 1991 ) And Bifidobacterium longum sucrose phosphorylase (HK Kang et al., FEMS Microbiol. Lett., 292: 33, 2009). However, there is still a need for development of a method for efficiently synthesizing industrially useful AA-2G in an economically simpler manner.
한편, 글루칸수크라제(glucansucrases, EC 2.4.1.5)는 수크로스(sucrose)로부터 글루칸(glucan)의 합성하는 효소로서, 물이 수용체로서 사용될 때 글루코오스 모이어티를 전달함으로써 글루칸의 수용분해를 촉진할 수 있다. 이들은 또한 성장하는 글루칸 주쇄로 글루코오스 모이어티의 글루코실 전달에 의하여 글루칸을 합성할 수 있으며, 글루코어스 모이어티를 다른 사카라이드로 글리코실화하여 올리고사카라이드를 합성할 수 있다. 그러나, 상기와 같은 제한적인 용도만이 알려져 있을 뿐 아직까지 글루칸수크라제를 이용하여 L-AsA로부터 AA-2G를 합성한 예는 보고된 바 없다. Glucansucrases (EC 2.4.1.5), on the other hand, are enzymes that synthesize glucans from sucrose, which promotes water-soluble degradation of glucans by delivering glucose moieties when water is used as a receptor. Can be. They can also synthesize glucans by glucosyl delivery of glucose moieties to the growing glucan backbone, and glycosylate glucose moieties with other saccharides to synthesize oligosaccharides. However, only such limited uses are known, and no examples of synthesizing AA-2G from L-AsA using glucans sucrase have been reported.
이에 본 발명자들은 아스코르브산 2-글리코시드를 효율적이고 경제적으로 생산할 수 있는 방법을 개발하고자 예의 노력한 결과, 김치에서 분리한 L. latis EG001로부터 분리한 신규한 글루칸수크라제를 수크로스 및 L-아스코르브산과 함께 혼합하여 반응시키는 간이한 방법으로 아스코르브산 2-글리코시드를 효율적으로 생산할 수 있음을 확인하고, 본 발명을 완성하였다.Therefore, the present inventors have made diligent efforts to develop a method for efficiently and economically producing ascorbic acid 2-glycoside. As a result, the novel glucan sucrase isolated from L. latis EG001 isolated from kimchi is sucrose and L-ascorb. It was confirmed that ascorbic acid 2-glycoside could be efficiently produced by a simple method of mixing with an acid and reacting, thus completing the present invention.
본 발명의 목적은 산업적으로 유용한 아스코르브산 2-글리코시드를 효율적이고 경제적인 방법으로 합성할 수 있는 새로운 방법을 제공하는 데 있다. An object of the present invention is to provide a new method for synthesizing industrially useful ascorbic acid 2-glycoside in an efficient and economical manner.
상기 목적을 달성하기 위하여, 본 발명은 다음 단계를 포함하는, 글루칸수크라제(glucansucrase)를 이용한 아스코르브산 2-글루코시드(ascorbic acid 2-glucoside)의 생산방법을 제공한다:In order to achieve the above object, the present invention provides a method for producing ascorbic acid 2-glucoside using glucansucrase, comprising the following steps:
(a) L-아스코르브산 및 이당류를 글루칸수크라제와 함께 혼합하여 글리코실화 반응을 수행하는 단계; 및(a) mixing L-ascorbic acid and disaccharides with glucan sucrase to perform a glycosylation reaction; And
(b) 상기 글리코실화 반응으로 생성된 아스코르브산 2-글루코시드를 회수하는 단계.(b) recovering ascorbic acid 2-glucoside produced by the glycosylation reaction.
본 발명은 또한, 다음 단계를 포함하는, 글루칸수크라제(glucansucrase)를 이용한 아스코르브산 2-글루코시드(ascorbic acid 2-glucoside)의 생산방법을 제공한다:The present invention also provides a method for producing ascorbic acid 2-glucoside using glucansucrase, comprising the following steps:
(a) L-아스코르브산 및 수크로스를 서열번호 1의 아미노산 서열로 표시되는 글루칸수크라제와 함께 혼합하여 글리코실화 반응을 수행하는 단계; 및(a) mixing L-ascorbic acid and sucrose with glucan sucrase represented by the amino acid sequence of SEQ ID NO: 1 to perform a glycosylation reaction; And
(b) 상기 글리코실화 반응으로 생성된 아스코르브산 2-글루코시드를 회수하는 단계.(b) recovering ascorbic acid 2-glucoside produced by the glycosylation reaction.
본 발명은 글루칸수크라제를 이용하여 L-아스코르브산의 글리코실화를 통하여 아스코르브산 2-글루코시드를 생산하는 새로운 방법을 제공하는 효과가 있다. 본 발명에 따른 방법은 다양한 산업분야에 사용되고 있으며 증가된 안정성을 가지는 L-아스코르브산 유도체인 아스코르브산 2-글루코시드를 수크로스와 같은 풍부한 탄수화물 자원을 이용하여 합성할 수 있어 경제적이며, 또한 우수한 효율로 아스코르브산 2-글루코시드를 생산하는바 유용하다.The present invention has the effect of providing a new method for producing ascorbic acid 2-glucoside through glycosylation of L-ascorbic acid using glucan sucrase. The method according to the present invention is used in various industrial fields, and ascorbic acid 2-glucoside, an L-ascorbic acid derivative having increased stability, can be synthesized using abundant carbohydrate resources such as sucrose, which is economical and has excellent efficiency. It is useful to produce ascorbic acid 2-glucoside.
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 갖는다. 일반적으로, 본 명세서에서 사용된 명명법 은 본 기술분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.
본원에서, "L-아스코르브산"은 유리 L-아스코르브산과, 알칼리 금속염, 알칼리 토금속염 및 이들의 혼합물 등으로 된 L-아스코르브산염을 포함하는 개념이다. As used herein, "L-ascorbic acid" is a concept that includes free L-ascorbic acid and L-ascorbic acid salts of alkali metal salts, alkaline earth metal salts, mixtures thereof, and the like.
본원에서, "아미노산"은 자연적으로 발생하는 아미노산 및 합성아미노산과, 아미노산 유사체와 자연발생하는 아미노산과 기능이 유사한 아미노산의태체(amino acid minetics)를 의미한다. As used herein, "amino acid" refers to amino acid minetics of naturally occurring amino acids and synthetic amino acids, and amino acids analogs and amino acids similar in function to naturally occurring amino acids.
본 발명은 일 관점에서, 다음 단계를 포함하는 글루칸수크라제(glucansucrase)를 이용한 아스코르브산 2-글루코시드(ascorbic acid 2-glucoside)의 생산방법에 관한 것이다:In one aspect, the present invention relates to a method for producing ascorbic acid 2-glucoside using glucansucrase comprising the following steps:
(a) L-아스코르브산 및 이당류를 글루칸수크라제와 함께 혼합하여 글리코실화 반응을 수행하는 단계; 및(a) mixing L-ascorbic acid and disaccharides with glucan sucrase to perform a glycosylation reaction; And
(b) 상기 글리코실화 반응으로 생성된 아스코르브산 2-글루코시드를 회수하는 단계.(b) recovering ascorbic acid 2-glucoside produced by the glycosylation reaction.
본 발명은 약학 제제, 사료, 식품 보존제 및 화장품 등 산업적으로 다양하게 이용될 수 있는 아스코르브산 2-글루코시드를 효소학적으로 합성하는 방법에 관한 것으로, 글루칸수크라제를 L-아스코르브산 및 수크로스와 함께 혼합하여 반응시키는 경우 글리코실화 반응을 수행하여 아스코르브산 2-글르코시드(AA-2G)를 생산함을 확인한 것에 기인한다. The present invention relates to a method for enzymatically synthesizing ascorbic acid 2-glucoside, which can be used in various industrial applications, such as pharmaceutical preparations, feed, food preservatives and cosmetics, wherein glucan sucrase is synthesized from L-ascorbic acid and sucrose. In the case of reacting with the mixture, the glycosylation reaction is performed to produce ascorbic acid 2-glycoside (AA-2G).
종래 아스코르브산 2-글루코시드(AA-2G)는 알파-글루코시다아제 및 싸이클로덱트린 글루카노트랜스퍼라제와 같은 몇몇 효소에 의한 글리코실화 반응에 의하여 합성되는 것이 보고된 바 있으나, 글루칸수크라제에 대한 AA-2G(안정한 L-AsA 글루코시드)의 형성은 보고된 적은 없었다. Ascorbic acid 2-glucoside (AA-2G) has been reported to be synthesized by glycosylation by several enzymes, such as alpha-glucosidase and cyclodextrin glucanotransferase. The formation of AA-2G (stable L-AsA glucoside) has not been reported.
본 발명에서는, 먼저 김치 유산균으로부터 페놀-황산법 (Phenol-sulfuric acid)을 이용하여 글루칸 함량이 높은 균주인 Leuconostoc l actis EG001를 선별 (Meulenbeld and Hartmans Biotechnol . and Bioeng., 70(4), 2000)한 후, 선별된 균주로부터 염색체 DNA를 분리한 다음, 이를 주형으로 PCR을 수행하여 글루칸수크 라제 관련 유전자를 증폭시키고, 또한 증폭된 글루칸수크라제 유전자를 함유하는 재조합 벡터를 대장균에 도입시켜 글루칸수크라제 유전자의 발현을 유도함으로써 글루칸수크라제를 대량으로 제조하였다. 이때, 제조된 글루칸수크라제는 약 165kDa의 분자량을 가지며 서열번호 1의 1500개의 아미노산 서열로 구성되는 효소로서, 이에 본 발명에 있어서, 상기 글루칸수크라제는 서열번호 1의 아미노산 서열로 표시되는 것임을 특징으로 한다. NCBI BlastP 검색 결과, 상기 L. l actis EG001로부터 유래된 글루칸수크라제는 Leuconostoc mesenteroides (AAN38835)의 dextransucrase (DsrR), Leuconostoc mesenteroides (CAB76565)의 dextransucrase (DsrB), Leuconostoc citreum KM20 (YP_001727410)의 glucosyltransferase에 각각 75%, 70%, 70%의 상동성을 갖는 것으로 나타났으며, 이에 신규한 글루칸수크라제인 것으로 확인되었다.In the present invention, first, Leuconostoc l actis , a strain having high glucan content, using phenol-sulfuric acid from kimchi lactic acid bacteria Screening for EG001 (Meulenbeld and Hartmans Biotechnol . And Bioeng ., 70 (4), 2000), and then chromosomal DNA is isolated from the selected strains, and then PCR is performed as a template to amplify the glucans sucrase related genes, and also contain the amplified glucans sucrase gene. Glucan sucrase was prepared in large quantities by introducing a recombinant vector into E. coli to induce the expression of the glucan sucrase gene. At this time, the prepared glucan sucrase is an enzyme having a molecular weight of about 165 kDa and composed of 1500 amino acid sequence of SEQ ID NO: 1, in the present invention, the glucan sucrase is represented by the amino acid sequence of SEQ ID NO: 1 It is characterized by. As a result of NCBI BlastP, glucan sucrase derived from the L. l actis EG001 is Leuconostoc dextransucrase of mesenteroides (AAN38835) ( DsrR ), Leuconostoc dextransucrase of mesenteroides (CAB76565) ( DsrB ), Leuconostoc The glucosyltransferase of citreum KM20 (YP_001727410) was shown to have 75%, 70%, and 70% homology, respectively, and thus was identified as a novel glucan sucrase.
또한, 상기 글루칸수크라제의 생산을 위하여 대장균에 도입되는 벡터의 글루칸수크라제 유전자는 TTG를 시작코돈으로 하고, TAA를 종결코돈으로 이루어지는 서열번호 2의 4503개의 염기로 구성되는 유전자로서, 이에 상기 글루칸수크라제는 서열번호 2의 염기서열로 표시되는 유전자에 의하여 코딩되는 것임을 특징으로 한다. In addition, the glucan sucrase gene of the vector introduced into Escherichia coli for the production of glucan sucrase is a gene composed of 4503 bases of SEQ ID NO: 2 consisting of TTG as a start codon and TAA as a stop codon. The glucan sucrase is characterized in that it is encoded by a gene represented by the nucleotide sequence of SEQ ID NO: 2.
본 발명에서는 또한, 제조한 글루칸수크라제를 수크로스 및 L-아스코르브산과 함께 혼합한 반응혼합물과 반응시킨 다음 그 반응산물에 대하여 TLC 분석을 수행한 결과, 수크로스 및 L-아스코르브산은 감소하고 표준 AA-2G와 동일한 Rf 값(Rf value, 0.35)을 가지는 산물이 생성됨을 확인하였다. 아울러, 상기 반응 산물에 대 하여 HPLC 분석을 추가적으로 수행한 결과, 표준 AA-2G와 동일한 retention time에서 피크(peak 2)를 보이는 것을 확인할 수 있었다. 이에 글루칸수크라제에 의한 글리코실화 반응에 의하여 L-아스코르브산 및 수크로스로부터 AA-2G (Ascorbic acid 2-glucoside)가 합성됨을 확인하였다. In the present invention, as a result of reacting the prepared glucan sucrase with a reaction mixture mixed with sucrose and L-ascorbic acid, and then performing TLC analysis on the reaction product, the sucrose and L-ascorbic acid are reduced and the standard is reduced. R f value equal to AA-2G (R f value, 0.35) was confirmed to produce the product. In addition, as a result of performing an HPLC analysis on the reaction product, it was confirmed that the peak (peak 2) at the same retention time as the standard AA-2G. As a result, it was confirmed that AA-2G (Ascorbic acid 2-glucoside) was synthesized from L-ascorbic acid and sucrose by glycosylation by glucan sucrase.
이때, 상기 글리코실화 반응을 위한 혼합배양물은 필수적으로 글루칸수크라제, 수크로스 및 L-아스코르브산을 포함하는데, 이외 통상적인 배양을 위한 물질을 포함할 수 있음은 본 발명이 속하는 기술분야에서 통상의 지식을 가지는 자에게 있어 자명할 것이다. 또한, 본 발명의 실시예에서는 이당류로서 수크로스를 사용하였으나, 이외 락토오스(lactose), 말토오스(Maltose), 트레할로스(trehalose), 셀로비오스(cellobiose), 코지비오스(kojibiose), 니게로스(nigerose), 이소말토스(isomaltose), β,β-트레할로스( β,β-trehalose), 소포로스(sophorose), 라미나리비오스(laminaribiose), 겐티오비오스(gentiobiose), 튜라노스(turanose), 말툴로스(maltulose), 팔라티노스(palatinose), 겐티오비울로스(gentiobiulose), 멜리비오스(melibiose), 루티노스(rutinose) 등 및 그 혼합물을 이용하여 글리코실화 반응을 수행할 수 있다. At this time, the mixed culture for the glycosylation reaction essentially includes glucan sucrase, sucrose and L-ascorbic acid, but may include a material for conventional culture in addition to that in the art It will be obvious to those of ordinary knowledge. In addition, in the embodiment of the present invention, sucrose was used as a disaccharide, but lactose, maltose, trehalose, cellobiose, kojibiose, nigerose, Isomaltose, β, β-trehalose, sophorose, laminaribiose, gentiobiose, turanose, maltulose ), Palatinose, gentiobiulose, melibiose, rutinose and the like and mixtures thereof may be used to perform glycosylation.
한편, Leuconostoc lactis EG001 균주 유래 글루칸수크라제의 최적 반응 조건을 알아보기 위하여, DNS 방법에 의해 반응온도의 영향을 조사한 결과, 효소의 최적 반응온도는 30℃이며, 25℃~35℃에서 상대적으로 높은 활성을 나타내는 것을 확인하였다. 따라서, 본 발명의 방법에 있어서, 상기 글리코실화 반응은 바람직하게는 25~35℃에서 수행하며, 더욱 바람직하게는 30℃에서 수행한다. Meanwhile, Leuconostoc lactis In order to determine the optimum reaction conditions of EG001 strain-derived glucans sucrase, the effect of reaction temperature was investigated by DNS method, and the optimum reaction temperature of enzyme was 30 ℃ and showed relatively high activity at 25 ℃ ~ 35 ℃. It was confirmed. Therefore, in the method of the present invention, the glycosylation reaction is preferably carried out at 25 ~ 35 ℃, more preferably at 30 ℃.
아울러, 상기 효소의 pH의 영향을 조사한 결과, pH 5에서 최대 활성을 나타내었으며, 산성 pH에서 비교적 안정하다는 것을 확인하였다. 따라서, 본 발명의 방법에 있어서, 상기 글리코실화 반응은 바람직하게는 pH 4.5~6에서 수행하며, 더욱 바람직하게는 pH 5에서 수행한다. In addition, as a result of examining the effect of the pH of the enzyme, it showed a maximum activity at
본 발명에 있어서 또한, 상기 합성된 아스코르브산 2-글루코시드의 회수는 반응결과물로부터 분리함으로써 이루어지는데, 예시적으로 분자량 및/또는 친화성 차이를 이용하는 정제방법, 즉, 멤브레인 븐리법, 겔 여과 크로마토그래피법, 컬럼 크로마토그래피법, 고속액체크로마토그래피법 및 이온 교환 크로마토그래피법 등을 이용하여 정제하여 수득할 수 있다. In the present invention, the recovery of the ascorbic acid 2-glucoside synthesized is achieved by separating from the reaction product. For example, a purification method using a molecular weight and / or affinity difference, that is, membrane avenue method, gel filtration chromatography It can be obtained by purification using a chromatography method, column chromatography method, high performance liquid chromatography method and ion exchange chromatography method.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.
실시예Example 1: One: LeuconostocLeuconostoc lactislactis EG001EG001 균주 유래 Strain derived 글루칸수크라제의Glucan sukrase 발현 및 분리 Expression and Separation
1-1: 균주의 분리 및 선별1-1: Isolation and Screening of Strains
한국전통발효음식인 김치로부터 유산균(lactic acid bacteria, LAB)의 혼합배양물을 분리하였다. 김치 유산균은 MRS 배지 (Proteose peptone No.3 10g/L, Beef Extract 10g/L, Yeast Extract 5g/L, Sucrose 20g/L, Polysorbate 80 1g/L, Ammonium citrate 2g/L, Sodium Acetate 5g/L, Magnesium Sulfate 0.1g/L, Manganese Sulfate 0.05g/L, Dipotassium phosphate 2g/L)에 접종하고 30℃에 배양하였다. 그 다음, 수집된 김치유산균에서 글루칸(glucan) 함량이 높은 균주를 분리하기 위하여 페놀-황산법(Phenol-sulfuric acid)을 실시하였다 (Meulenbeld and Hartmans, Biotechnol . and Bioeng ., 70(4), 2000). 즉, 김치 유산균을 배양한 후, 배양액 100㎕에 75% 에탄올 1㎖을 넣고 침전시켜 건조 후, 증류수 100㎕에 녹였다. 상기 준비된 100㎕의 시료와 5% 페놀 100㎕를 1.5㎖ 튜브에 넣고 실온에서 30초 동안 배양하였다. 튜브를 얼음에 넣고, 125㎕의 황산을 넣은 후 30초 동안 혼합하였다. 그리고 80℃ water bath에서 30분 동안 반응시키고, 튜브를 상온에서 식힌 후, Ultrospec 3100-Pro 스펙트로포토메터(Amersham Biosciences, USA)를 이용하여 흡광도 490nm에서 측정하였다. 이때 기준물질(standard)로 글루코스를 사용하여 글루칸 함량이 높은 균주를 선별하였다. A mixed culture of lactic acid bacteria (LAB) was isolated from kimchi, a Korean traditional fermented food. Kimchi Lactobacillus contains MRS medium (Proteose peptone No.3 10g / L, Beef Extract 10g / L, Yeast Extract 5g / L, Sucrose 20g / L, Polysorbate 80 1g / L, Ammonium citrate 2g / L, Sodium Acetate 5g / L, Magnesium Sulfate 0.1g / L, Manganese Sulfate 0.05g / L, Dipotassium phosphate 2g / L) and incubated at 30 ℃. Then, phenol-sulfuric acid was performed to isolate strains with high glucan content from the collected kimchi lactic acid bacteria (Meulenbeld and Hartmans, Biotechnol . And Bioeng ., 70 (4), 2000). That is, after lactic acid bacteria were cultured, 1 ml of 75% ethanol was added to 100 µl of the culture solution, precipitated, and dried, and then dissolved in 100 µl of distilled water. 100 μl of the prepared sample and 100 μl of 5% phenol were placed in a 1.5 ml tube and incubated at room temperature for 30 seconds. The tube was placed on ice, 125 μl of sulfuric acid was added and mixed for 30 seconds. After reacting for 30 minutes in an 80 ℃ water bath, the tube was cooled to room temperature, and the absorbance was measured at 490 nm using Ultrospec 3100-Pro spectrophotometer (Amersham Biosciences, USA). At this time, strains with high glucan content were selected using glucose as a standard.
선별된 균주는 16S rRNA 유전자서열을 NCBI 데이터베이스에서 검색한 결과, L. lactis의 16S rRNA와 99% 일치하는 것으로 나타났다. 이에 선별된 균주는 Leuconostoc lactis EG001로 명명하였다.The selected strains were found to be 99% identical to the 16S rRNA of L. lactis by searching the NCBI database for 16S rRNA gene sequences. The selected strain was named Leuconostoc lactis EG001.
1-2: 1-2: 글루칸수크라제Glucans sucrase 유전자 분리 및 재조합 벡터 제작 Gene Isolation and Recombinant Vector Construction
실시예 1-1에서 선별된 글루칸 함량이 높은 균주인 L. lactis EG001 균주를 LB broth (100㎍/㎖ ampicillin)에 배양한 후, 게노믹 DNA를 추출하였다. 샘플 균 주의 염기서열을 알기 위해, 추출한 게노믹 DNA를 template로 사용하고, 기존에 알려져 있는 Degenerate 프라이머(서열번호 3 및 4)를 사용하여 Degenerate PCR을 수행하였다(S. Kralj et al., Biocatalysis and Biotransformation, 21(4-5), 181-187, 2003). L. lactis EG001 strain, a strain of high glucan content selected in Example 1-1, was cultured in LB broth (100 µg / ml ampicillin), and then genomic DNA was extracted. In order to know the nucleotide sequence of the sample strain, extracted genomic DNA was used as a template, and Degenerate PCR was performed using known Degenerate primers (SEQ ID NOs: 3 and 4) (S. Kralj et al ., Biocatalysis and Biotransformation, 21 (4-5), 181-187, 2003).
Forward primer: 5'-GAYAAYWSNAAYCCNRYNGTNC-3' (서열번호 3)Forward primer: 5'-GAYAAYWSNAAYCCNRYNGTNC-3 '(SEQ ID NO: 3)
Reverse primer: 5'-ADRTCNCCRTARTANAVNYKNG-3' (서열번호 4)Reverse primer: 5'-ADRTCNCCRTARTANAVNYKNG-3 '(SEQ ID NO: 4)
상기에서, Y=C or T, R=A or G, W=A or T, S=G or C, D=A, T or G, V=A, G, or C, K=G or T, N=A,T,G or C임.In the above, Y = C or T, R = A or G, W = A or T, S = G or C, D = A, T or G, V = A, G, or C, K = G or T, N = A, T, G or C
즉, 상기 게노믹 DNA 100ng, 10×PCR 완충용액, 10mM dNTP, 각 프라이머 5μM, 1U EF-Taq polymerase(Solgent, Korea)를 포함하는 20㎕의 혼합물에 대하여 PCR 반응을 수행하였다. PCR 반응은 95℃에서 3분간 변성 (denaturation)을 수행한 후, 95℃에서 30초간 변성, 52℃에서 1분 풀림 (annealing), 72℃에서 1분 연장 (extension)을 30회 반복하였다. 그리고 마지막으로 72℃에서 10분 연장을 수행하였다. 확보된 PCR 산물은 pGEM T-easy 벡터(Promega)에 클로닝하고 염기서열을 확인하였다. That is, PCR reaction was performed on a mixture of 20 μl of the
그 다음, 전체 글루칸수크라제 단백질을 코딩하는 글루칸수크라제 유전자를 증폭하기 위하여 인버스 PCR(inverse PCR)을 수행하였다. 상기 L. lactis EG001 균주로부터 얻은 게노믹 DNA는 제한효소로 처리한 후, 셀프 라이게이션 (self-ligation)시켰다. 한편, 상기 Degenerate PCR의 결과로 얻은 염기서열로부터 하기 프라이머들을 제작하였다.Then, an inverse PCR was performed to amplify the glucan sucrase gene encoding the whole glucan sucrase protein. Genomic DNA obtained from the L. lactis EG001 strain was treated with restriction enzymes and then self-ligation. On the other hand, the following primers were prepared from the nucleotide sequence obtained as a result of the Degenerate PCR.
1One stst PCRPCR 용 for 프라이머primer
Forward primer: 5'-CACGATAGTGAAGTGCAAACG-3' (서열번호 5)Forward primer: 5'-CACGATAGTGAAGTGCAAACG-3 '(SEQ ID NO: 5)
Reverse primer: 5'-ATCGTTGGCAGTAATCGAGC-3' (서열번호 6)Reverse primer: 5'-ATCGTTGGCAGTAATCGAGC-3 '(SEQ ID NO: 6)
22 ndnd PCRPCR 용 for 프라이머primer
Forward primer: 5'-ATAGCTTAGCACCAACAACAGAAC-3' (서열번호 7)Forward primer: 5'-ATAGCTTAGCACCAACAACAGAAC-3 '(SEQ ID NO: 7)
Reverse primer: 5'-GTTTCGCTGGCCTTGTTCA-3' (서열번호 8)Reverse primer: 5'-GTTTCGCTGGCCTTGTTCA-3 '(SEQ ID NO: 8)
PCR 반응은 25ng의 원형 게노믹 DNA 단편, 10×PCR 완충용액, 10mM dNTP, 각 프라이머 2μM, 2U EF Taq polymerase(Solgent, Korea)를 포함하는 50㎕의 혼합물에 대하여 수행하였다. PCR 반응은 95℃에서 3분간 변성 (denaturation)을 수행한 후, 94℃에서 30초간 변성, 50℃에서 45초 어닐링 (annealing), 72℃에서 2분 연장 (extension)을 30회 반복하였다. 그리고 마지막으로 72℃에서 10분 연장을 수행하였다. 확보된 PCR 산물은 pGEM T-easy 벡터에 클로닝하여 시퀀싱하였다. PCR reactions were performed on a mixture of 25 μl of circular genomic DNA fragment, 10 × PCR buffer, 10 mM dNTP, 2 μM of each primer, and 2 U EF Taq polymerase (Solgent, Korea). The PCR reaction was denatured at 95 ° C. for 3 minutes, followed by denaturation at 94 ° C. for 30 seconds, annealing at 50 ° C. for 45 seconds, and extension at 72 ° C. for 30 minutes. And finally a 10 minute extension at 72 ° C. The obtained PCR product was sequenced by cloning into a pGEM T-easy vector.
그리고, Copycontrol Fosmid Library Production Kit을 이용하여 fosmid library를 제작하고 (fosmid library 제작은 Epicentre사의 manual에 따라 제작되었음), 제작된 library를 colony PCR 하여 insert가 제대로 삽입되어 있는 것을 확인 후 fosmid를 정제하였다. Colony PCR 수행전에 하기 서열번호 9 및 10의 프라이머를 제작하였다.Then, the fosmid library was prepared using the Copycontrol Fosmid Library Production Kit (fosmid library was manufactured according to Epicentre's manual), and the fosmid was purified after colony PCR. The primers of SEQ ID NOs: 9 and 10 were prepared before performing Colony PCR.
Primer: 5'-ACTGTGGCTTTCAACACCCA-3' (서열번호 9)Primer: 5'-ACTGTGGCTTTCAACACCCA-3 '(SEQ ID NO: 9)
Primer: 5'-TGTGCGCGCATCTTGGGTA-3' (서열번호 10)Primer: 5'-TGTGCGCGCATCTTGGGTA-3 '(SEQ ID NO: 10)
PCR 증폭을 위해 fosmid library를 D.W.에 희석해서 100ng을 사용하였다. PCR 증폭은 2U LA Taq polymerase(TaKaRa), 10배 polymerase buffer (MgCl2 포함) 5㎕, 10mM dNTP 1㎕, 각 프라이머 (5uM) 5㎕, 주형 DNA (염색체 DNA) 25ng을 함유하는 PCR 반응용액 (50㎕)을 준비한 후, 유전자 증폭기를 이용하여 수행하였다.For PCR amplification, 100ng of the fosmid library was diluted in DW. PCR amplification was performed using a PCR reaction solution containing 2U LA Taq polymerase (TaKaRa), 5 μl of 10-fold polymerase buffer (including MgCl 2 ), 1 μl of 10 mM dNTP, 5 μl of each primer (5 uM), and 25 ng of template DNA (chromosomal DNA). 50 μl) was prepared and performed using a gene amplifier.
PCR 반응은 94℃에서 1분간 변성 (denaturation)을 수행한 후, 94℃에서 30초간 변성, 68℃에서 1.5분 어닐링 (annealing)을 30회 반복하였다. 그리고 마지막으로 72℃에서 10분 연장을 수행하고, 4℃에서 유지시켰다. PCR 수행으로 fosmid library를 선택하여 Alkaline 방법으로 purification 한 뒤 염기서열을 확인하였다. The PCR reaction was denatured for 1 minute at 94 ° C., followed by 30 minutes of denaturation at 94 ° C. and 1.5 minutes annealing at 68 ° C. for 30 minutes. And finally a 10 min extension at 72 ° C. and maintained at 4 ° C. After PCR, fosmid library was selected and purified by Alkaline method.
상기 degenerate PCR, inverse PCR 결과 및 fosmid library의 서열분석 결과를 통하여 Leuconostoc lactis EG001 균주 유래의 글루칸수크라제 유전자 서열을 완결하였는데, 글루칸수크라제 유전자는 TTG를 시작코돈으로 하고, TAA를 종결코돈으로 이루어지는 4503개의 염기로 구성되는 서열번호 2의 핵산서열로 표시된다. Leuconostoc through the degenerate PCR, inverse PCR results and sequencing results of the fosmid library The glucan sucrase gene sequence derived from the lactis EG001 strain was completed, and the glucan sucrase gene is represented by the nucleic acid sequence of SEQ ID NO: 2 consisting of 4503 bases consisting of TTG as a start codon and TAA as a stop codon.
한편, Leuconostoc lactis EG001 균주 유래의 글루칸수크라제를 대장균 BL21(DE3)(Novagen)에서 대량 생산하기 위하여, 상기 확인한 글루칸수크라제 유전자를 발현 벡터인 pET-22b(+)의 NcoI-NotI 부위에 삽입하여 재조합 DNA를 제조하였다. Meanwhile, Leuconostoc In order to mass-produce glucan sucrase from lactis EG001 strain in E. coli BL21 (DE3) (Novagen), the glucan sucrase gene identified above was inserted into the Nco I- Not I site of pET-22b (+), an expression vector. Recombinant DNA was prepared.
먼저 글루칸수크라제의 PCR 증폭을 위해 Leuconostoc lactis EG001 균주의 게노믹 DNA를 분리하고, NcoI site을 삽입시킨 서열번호 11의 정방향 프라이머, NotI site를 삽입시킨 서열번호 12의 역방향 프라이머를 제작하였다. First Leuconostoc for PCR Amplification of Glucansucrase Isolate genomic DNA of lactis EG001 strain, NcoI A forward primer of SEQ ID NO: 11 having a site inserted therein and a reverse primer of SEQ ID NO: 12 having a Not I site inserted therein were prepared.
Forward primer: 5'-AT CCATGG ATAGTAACCAAAACACAACTGGTT-3' (서열번호 11)Forward primer: 5'-AT CCATGG ATAGTAACCAAAACACAACTGGTT-3 '(SEQ ID NO: 11)
Backward primer: 5'-AT GCGGCCGC CATATTGACGAGATCACCGTTG-3' (서열번호 12)Backward primer: 5'-AT GCGGCCGC CATATTGACGAGATCACCGTTG-3 '(SEQ ID NO: 12)
PCR 반응은 분리한 게노믹 DNA 100ng, 10×PCR 완충용액, 10mM dNTP, 각 프라이머 0.2μM, 1U Taq polymerase(Solgent, Korea)를 포함하는 20㎕의 혼합물에 대하여 수행하였다. PCR 조건은 95℃에서 3분간 변성 (denaturation)을 수행한 후, 95℃에서 30초간 변성, 55℃에서 30초 풀림 (annealing), 72℃에서 1분 연장 (extension)을 25회 반복하였다. PCR 산물은 제한효소 NcoI 및 NotI로 자른 후, pET-22b(+) expression vector(Invitrogen, USA)의 NcoI-NotI 부위에 subcloning하여 재조합 벡터를 제조하였다.PCR reactions were performed on a mixture of 20 μl of genomic DNA isolated, 10 × PCR buffer, 10 mM dNTP, 0.2 μM of each primer, and 1 U Taq polymerase (Solgent, Korea). PCR conditions were denatured at 95 ° C. for 3 minutes, then denatured at 95 ° C. for 30 seconds, annealing at 55 ° C. for 30 seconds, and 1 minute extension at 72 ° C. for 25 times. PCR products were cut with restriction enzymes Nco I and Not I, and then recombinant vectors were prepared by subcloning the Nco I- Not I site of pET-22b (+) expression vector (Invitrogen, USA).
1-3: 재조합 대장균 배양에 의한 1-3: by recombinant E. coli culture 글루칸수크라제의Glucan sukrase 발현 및 정제 Expression and Purification
글루칸수크라제의 과잉 생산을 위하여, 실시예 1-2에서 제조된 재조합 벡터를 대장균 BL21(DE3) (Novagen)에 도입하였고, 얻어진 형질전환체를 100㎍/㎖ 앰피실린 (ampicillin)이 첨가된 LB 액체배지에서 37℃에서 배양하였다. 그 후, OD600이 0.5가 되었을 때, 20℃에서 12시간 동안 0.1mM의 IPTG를 처리함으로써 글루칸수크라제의 발현을 유도하였다. 배양체를 원심분리하여 침전된 세포(0.14g)를 회수하고, 회수한 세포를 100mM Tris-HCl 완충용액(pH7.4), 10mM imidazole, 1mM PMSF (Phenylmethylsulfonyl fluoride), 및 protease inhibitor cocktail (Roche, USA)을 포함한 5㎖ 용해 완충용액에 현탁하였다. 세포는 초음파로 파쇄한 후, 원심분 리(12000×g, 15min, 4℃)하여 상등액을 회수하였다. 상기 상등액은 2㎖ Ni-NTA agarose와 4℃에서 1시간 동안 혼합하여 크로마토그래피 컬럼에 로딩하였다. 컬럼은 20mM Tris-HCl (pH 8.0), 300mM NaCl, 및 200mM imidazole을 포함하는 완충용액으로 씻었으며, 250mM imidazole로 보충한 상기 완충용액을 이용하여 단백질을 컬럼에서 용출시켰다. 용출된 단백질은 Amicon Ultra-15 (Millipore, 50K NMWL device)로 농축한 후, 20% 글리세롤(glycerol), 100mM Tris-HCl 완충용액(pH 7.4)을 포함한 투석 완충용액에 넣어 4℃에서 투석 (dialysis)을 수행하였다. For overproduction of glucan sucrase, the recombinant vector prepared in Example 1-2 was introduced into Escherichia coli BL21 (DE3) (Novagen), and the resulting transformant was added with 100 µg / ml ampicillin. Incubated at 37 ° C. in LB liquid medium. Then, when OD 600 reached 0.5, expression of glucan sucrase was induced by treatment of 0.1 mM IPTG for 12 hours at 20 ° C. The cultured cells were centrifuged to recover the precipitated cells (0.14 g), and the recovered cells were treated with 100 mM Tris-HCl buffer (pH7.4), 10 mM imidazole, 1 mM PMSF (Phenylmethylsulfonyl fluoride), and protease inhibitor cocktail (Roche, USA In 5 ml lysis buffer). The cells were disrupted by ultrasonication and centrifuged (12000 x g, 15 min, 4 ° C) to recover the supernatant. The supernatant was mixed with 2 ml Ni-NTA agarose at 4 ° C. for 1 hour and loaded onto a chromatography column. The column was washed with a buffer containing 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 200 mM imidazole, and proteins were eluted from the column using the buffer supplemented with 250 mM imidazole. The eluted protein was concentrated in Amicon Ultra-15 (Millipore, 50K NMWL device) and then dialyzed at 4 ° C in dialysis buffer containing 20% glycerol and 100 mM Tris-HCl buffer (pH 7.4). ) Was performed.
정제된 효소는 약 165kDa의 분자량을 가지며, 서열번호 1로 표시되는 1500개의 아미노산 서열로 구성되는 것으로 확인되었다. The purified enzyme had a molecular weight of about 165 kDa and was confirmed to consist of 1500 amino acid sequences represented by SEQ ID NO: 1.
실시예Example 2: 2: L. L. lactislactis EG001EG001 균주 유래 Strain derived 글루칸수크라제의Glucan sukrase 분석 및 이를 이용한 아스코르브산 2-글루코시드의 생산 Analysis and Production of Ascorbic Acid 2-Glucoside Using the Same
2-1: 2-1: L. L. lactislactis EG001EG001 균주 유래 Strain derived 글루칸수크라제의Glucan sukrase 분석 analysis
실시예 1-3의 정제된 Leuconostoc lactis EG001 균주 유래 글루칸수크라제의 최적 반응 조건을 알아보기 위하여, DNS 방법에 의해 반응온도, pH의 영향을 조사하였다 (Gail Lorenz Miller, Analytical Chemistry, 31(3), 426-428). 즉, 50mM Sodium acetate (pH 5.2), 100mM sucrose, 1mM CaCl2, 적당히 희석된 정제된 효소를 포함하는 혼합물을 30℃에서 30분간 배양한 다음, 최종 과당(fructose)을 DNS법(DNS (3,5-dinitrosalicylic acid) 용액 처리 후, 흡광도 측정)으로 측정하였다. 효소의 유닛(U)은 30℃에서 수크로스가 분해되어 분당 생산되는 과당의 μmole 수를 의미한다. 최적 pH를 결정하기 위해서는, 표준 효소반응에서 각기 다른 완충용액을 사용하였다. 최적 pH 결정에 사용된 완충용액은 50mM sodium acetate buffer (pH 3-6), 50mM sodium phosphate buffer (pH 6-8), 그리고 50mM Tris-HCl buffer (pH 8-9)를 사용하였다. 각 pH buffer에서 상대적인 활성을 조사한 결과, 도 1(a)에 나타난 바와 같이, pH 5에서 최대 활성을 나타내었으며, 산성 pH에서 비교적 안정하다는 것을 확인하였다. Purified Leuconostoc of Examples 1-3 lactis In order to determine the optimal reaction conditions of EG001 strain-derived glucan sucrase, the effects of reaction temperature and pH were investigated by DNS method (Gail Lorenz Miller, Analytical Chemistry, 31 (3), 426-428). In other words, a mixture containing 50 mM Sodium acetate (pH 5.2), 100 mM sucrose, 1 mM CaCl 2 , and appropriately diluted purified enzyme was incubated at 30 ° C. for 30 minutes, and the final fructose was subjected to the DNS method (DNS (3, 5-dinitrosalicylic acid) solution treatment, and then measured by absorbance). The unit of enzyme (U) refers to the number of μmole of fructose produced per minute by the breakdown of sucrose at 30 ° C. To determine the optimal pH, different buffers were used in standard enzyme reactions. 50 mM sodium acetate buffer (pH 3-6), 50 mM sodium phosphate buffer (pH 6-8), and 50 mM Tris-HCl buffer (pH 8-9) were used for the determination of the optimal pH. As a result of examining the relative activity in each pH buffer, as shown in Figure 1 (a), it showed a maximum activity at
또한, 최적 반응온도를 알아보기 위해 정제된 효소를 사용하여 10~50℃ 범위에서 5℃ 간격으로 상대적인 활성을 측정하였다. 그 결과, 도 1(b)에 나타난 바와 같이, 효소의 최적 반응온도는 30℃이며, 25℃~35℃에서 상대적으로 높은 활성을 나타내는 것을 확인하였다. In addition, the relative activity was measured at intervals of 5 ° C. in the range of 10˜50 ° C. using purified enzyme to determine the optimum reaction temperature. As a result, as shown in Figure 1 (b), it was confirmed that the optimum reaction temperature of the enzyme is 30 ℃, showing a relatively high activity at 25 ℃ ~ 35 ℃.
또한, 열에 대한 안정성을 조사하기 위해 10~45℃ 범위에서 잔존 활성을 측정하였다. 각 지정된 온도에서 3시간 방치 후에 잔존 활성을 확인한 결과, 도 1(c)에 나타난 바와 같이, 30℃ 이하에서 잔존 활성이 높게 남아있는 것을 확인하였다.In addition, the residual activity was measured in the range of 10 ~ 45 ℃ to investigate the stability to heat. As a result of confirming the residual activity after standing for 3 hours at each specified temperature, as shown in Fig. 1 (c), it was confirmed that the residual activity remains high at 30 ℃ or less.
2-2: 아스코르브산 2-글루코시드의 생산2-2: Production of Ascorbic Acid 2-Glucoside
글리코실화 반응은 50mM sodium acetate (pH 5.2), 1 mM MgCl2, 0.1% L-아스코르브산(L-ascorbic acid), 100 mM sucrose, 및 적당히 희석된 정제된 효소를 혼합한 다음, 반응혼합물을 30℃에서 3시간 배양함으로써 수행되었다. 그 후 산물 은 TLC (thin layer chromatography)를 이용하여 분석하였다. 즉, 시료를 실리카 겔 60 F254 TLC 플레이트(Merck, Germany)에 스팟팅한 후, nitromethane/1-propanol/water (2:5:1.5, v/v/v)로 구성되는 용매 시스템으로 전개시켰다. 그리고 나서, 플레이트는 메탄올에서 10% H2SO4로 스프레이하여 비주얼화하였다. Glycosylation reaction was performed with 50 mM sodium acetate (pH 5.2), 1 mM MgCl 2 , 0.1% L-ascorbic acid, 100 mM sucrose, and appropriately diluted purified enzyme were mixed and then the reaction mixture was incubated at 30 ° C. for 3 hours. The product was then analyzed using TLC (thin layer chromatography). That is, the samples were spotted on silica gel 60 F254 TLC plates (Merck, Germany) and then developed into a solvent system consisting of nitromethane / 1-propanol / water (2: 5: 1.5, v / v / v). The plate was then visualized by spraying with 10% H 2 SO 4 in methanol.
그 결과, 도 2(a)에 나타난 바와 같이, 상기 효소 반응을 3시간 동안 수행한 결과(lane 6)를 효소를 포함하지 않거나 효소반응이 일어나기 전의 경우(lane 4 및 5)와 대비하여 보면, 수크로스 및 L-아스코르브산은 감소하고 각각 다른 Rf 값을 가지는 3개의 새로운 산물 (P1, P2 및 P3)이 나타난 것을 확인할 수 있었다 (lane 1: sucrose; lane 2: L-ascorbic acid; lane 3: AA-2G(Ascorbic acid 2-Glucoside); lane 4: non-enzyme; lane 5: enzyme reaction (0h); lane 6: enzyme reaction (3h)). 이러한 반응 산물 중 P2의 Rf 값(Rf value, 0.35)은 standard AA2G의 Rf 값(Rf value, 0.35)과 동일한 것으로 나타났다. As a result, as shown in Figure 2 (a), the result of performing the enzyme reaction for 3 hours (lane 6) compared to the case containing no enzyme or before the enzyme reaction (
한편, 추가적으로 상기 반응 산물에 대하여, HPLC 분석을 수행하였다. 즉, 이동상으로 0.1 M KH2PO4 buffer (pH 2.0)를 이용하고, 0.7 mL/min의 유속으로 하여 Gemini 5㎛ C18 110A column (4.6×250 mm, Phenomenex, USA)로 HPLC (high-performance liquid chromatography)를 수행하였다. 반응산물의 피크는 diode array detector (DAD)를 이용하여 240nm에서 검출되었다. On the other hand, for the reaction product, HPLC analysis was performed. That is, using a 0.1 M KH 2 PO 4 buffer (pH 2.0) as a mobile phase, at a flow rate of 0.7 mL / min, HPLC (high-performance liquid) on a
그 결과, 도 2(b)에 나타난 바와 같이, 상기 반응 산물이 표준 AA2G와 동일 한 retention time에서 피크(peak 2)를 보이는 것을 확인할 수 있었다 (peak 1, L-ascorbic acid; peak 2, AA2G). 이러한 결과는 글루칸수크라제에 의한 글리코실화 반응에 의하여 L-아스코르브산 및 수크로스로부터 AA2G (Ascorbic acid 2-glucoside)가 합성되었음을 의미한다. As a result, as shown in Figure 2 (b), it was confirmed that the reaction product shows a peak (peak 2) at the same retention time as the standard AA2G (
이상으로 본 발명의 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As described above in detail a specific part of the content of the present invention, for those skilled in the art, such a specific description is only a preferred embodiment, which is not limited by the scope of the present invention Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
도 1은 Leuconostoc lactis EG001 균주 유래 글루칸수크라제의 pH 변화에 따른 상대적인 활성(a), 온도변화에 따른 상대적인 활성(b) 및 온도변화에 따른 잔존활성의 측정결과(c)를 나타내는 그래프이다. 1 is Leuconostoc lactis It is a graph showing the measurement results (c) of the relative activity (a), relative activity (b) and residual activity according to the temperature change of the glucan sucrase derived from EG001 strain.
도 2는 Leuconostoc lactis EG001 균주 유래 글루칸수크라제에 의한 L-아스코르브산 및 수크로스의 글리코실화 반응산물의 TLC 분석결과(A) 및 HPLC 크로마토그래피 분석 결과(B)를 나타낸다. 2 is Leuconostoc lactis The results of TLC analysis (A) and HPLC chromatography analysis (B) of the glycosylation reaction products of L-ascorbic acid and sucrose with glucans sucrase derived from EG001 strain are shown.
<110> Korea Research Institute of Bioscience and Biotechnology <120> Method for Preparing Ascorbic Acid 2-Glucoside Using Glucansucrase <160> 12 <170> KopatentIn 1.71 <210> 1 <211> 1500 <212> PRT <213> amino acid sequence for glucansucrase derived from Leuconostoc lactis EG001 <400> 1 Leu Glu Asn Ala Thr Gln Val Arg Lys Lys Leu Tyr Lys Ala Gly Lys 1 5 10 15 Asn Trp Val Val Gly Gly Val Ile Thr Ala Gly Ala Ala Leu Ala Phe 20 25 30 Val Ala Gly Ala Thr Thr Ala Ala Ala Asp Ser Asn Gln Asn Thr Thr 35 40 45 Gly Ser Gln Val Thr Val Thr Ala Pro Glu Ala Ala Ala Ser Ala Thr 50 55 60 Thr Thr Thr Thr Thr Thr Thr Pro Thr Thr Ala Ala Ala Thr Thr Thr 65 70 75 80 Ala Ala Gln Ala Asp Thr Ser Ser Ala Thr Thr Pro Gln Pro Pro Val 85 90 95 Ala Pro Thr Thr Pro Val Ala Pro Thr Gln Ala Gln Pro Ala Thr Thr 100 105 110 Ala Ala Thr Thr Glu Gln Pro Gln Gln Pro Ala Ala Asp Thr Thr Pro 115 120 125 Ala Pro Gln Thr Gly Tyr Val Glu Lys Ala Gly Ala Trp Tyr Tyr Val 130 135 140 Asn Ala Asp Gln Ser Tyr Ala Lys Gly Leu Thr Thr Ile Ala Gly His 145 150 155 160 Leu Gln Tyr Phe Asp Thr Thr Gly Arg Gln Thr Lys Gly Ala Tyr Val 165 170 175 Thr Glu Asn Gly Lys Thr Tyr Tyr Phe Asp Ala Asn Thr Gly Asn Ala 180 185 190 Leu Thr Gly Leu Gln His Val Ala Gly Gln Thr Val Ala Phe Asn Thr 195 200 205 Gln Gly Glu Gln Ile Phe Ser Asp Phe Tyr Thr Ala Ala Asp Gly Gln 210 215 220 Thr Tyr Tyr Phe Gly Thr Asn Gly Gln Ala Ala Val Gly Val Thr Ser 225 230 235 240 Ile Ala Gly His Asn Tyr Tyr Phe Asp Ala Ala Gly Gln Leu Lys Lys 245 250 255 Gly Tyr Ala Gly Glu Ile Asp Gly Gln Met Arg Thr Phe Asp Ala Thr 260 265 270 Thr Gly Gln Glu Val Ser Ala Thr Thr Ser Gln Ile Thr Glu Gly Leu 275 280 285 Thr Ala Gln Asn Asp Asp Tyr Thr Ala His Asn Ala Val His Ser Thr 290 295 300 Ala Ser Ala Asp Phe Asp Asn Leu Asp Gly Tyr Leu Thr Ala Ser Ser 305 310 315 320 Trp Tyr Arg Pro Thr Asp Ile Leu Arg Asp Gly Asn Lys Trp Glu Ala 325 330 335 Ser Thr Ala Thr Asp Met Arg Pro Ile Leu Ser Val Trp Trp Pro Asp 340 345 350 Lys Gln Thr Gln Val Asp Tyr Leu Asn Tyr Met Ser Gln Leu Gly Leu 355 360 365 Val Glu Asn Pro Thr Pro Tyr Thr Leu Gln Asp Asp Gln Val Ala Leu 370 375 380 Asn Lys Ala Ser Glu Thr Leu Gln Gln Ala Ile Glu Thr Lys Ile Gly 385 390 395 400 Leu Thr Asn Ser Thr Asp Trp Leu Lys Thr Ala Met Ala Asn Phe Ile 405 410 415 Thr Thr Gln Pro Gln Trp Asn Gln Thr Ser Glu Asp Pro Asn Ser Asp 420 425 430 His Leu Gln Lys Gly Ala Leu Thr Phe Val Asn Ser Pro Leu Thr Pro 435 440 445 Asp Thr Asn Ser Ala Phe Arg Leu Leu Asn Arg Thr Pro Ala Asn Gln 450 455 460 Thr Asn Thr Gln Asn Tyr Thr Val Asp Asn Ser Lys Gly Gly Tyr Glu 465 470 475 480 Leu Leu Leu Ala Asn Asp Val Asp Asn Ser Asn Pro Val Val Gln Ala 485 490 495 Glu Gln Leu Asn Trp Leu His Tyr Leu Met Asn Phe Gly Ser Ile Thr 500 505 510 Ala Asn Asp Ala Asp Ala Asn Phe Asp Gly Ile Arg Val Asp Ala Val 515 520 525 Asp Asn Val Asp Ala Asp Leu Leu Gln Ile Ala Ala Asp Tyr Phe Lys 530 535 540 Ala Ala Tyr Gly Val Asp Lys Asn Asp Ala Thr Ala Asn Gln His Leu 545 550 555 560 Ser Ile Leu Glu Asp Trp Ser His Asn Asp Pro Leu Tyr Val Asn Asp 565 570 575 Phe Gly Asp Asn Gln Leu Thr Met Asp Asp Tyr Ala His Thr Gln Leu 580 585 590 Ile Trp Ser Leu Thr Lys Asn Ser Asp Ile Arg Gly Thr Met Gln Arg 595 600 605 Phe Met Asp Tyr Tyr Leu Val Asn Arg Ser Gln Asp Ser Thr Glu Asn 610 615 620 Thr Ala Thr Pro Asn Tyr Ser Phe Val Arg Ala His Asp Ser Glu Val 625 630 635 640 Gln Thr Val Ile Ala Gln Ile Val Ser Asp Leu His Pro Asp Val Glu 645 650 655 Asn Ser Leu Ala Pro Thr Thr Glu Gln Leu Leu Glu Ala Phe Lys Val 660 665 670 Tyr Asn Ala Asp Gln Lys Leu Ala Asp Lys Lys Tyr Thr Gln Tyr Asn 675 680 685 Met Pro Ser Ala Tyr Ala Met Leu Leu Thr Asn Lys Asp Thr Val Pro 690 695 700 Arg Val Tyr Tyr Gly Asp Leu Tyr Thr Asp Asp Gly Gln Tyr Met Ala 705 710 715 720 Thr Lys Ser Pro Tyr Phe Asn Ala Ile Asp Thr Leu Leu Lys Ala Arg 725 730 735 Ile Gln Tyr Val Ala Gly Gly Gln Ala Met Ala Val Asp Asn His Asp 740 745 750 Ile Leu Thr Ser Val Arg Tyr Gly Asn Gly Ala Met Thr Ala Thr Asp 755 760 765 Lys Gly Asp Ala Asp Thr Arg Thr Gln Gly Ile Gly Val Ile Ile Ser 770 775 780 Asn Asn Lys Asp Leu Ala Leu Gln Ala Gly Glu Thr Val Thr Leu His 785 790 795 800 Met Gly Ala Ala His Lys Lys Gln Ala Phe Arg Leu Leu Leu Gly Thr 805 810 815 Thr Gln Asp Gly Leu Asp Tyr Tyr Asn Thr Asp Asp Ala Pro Ile Arg 820 825 830 Tyr Thr Asp Asn Asn Gly Asp Leu Ile Phe Asn Ser Gln Asp Val Tyr 835 840 845 Gly Val Gln Asn Pro Gln Val Ser Gly Phe Leu Ala Val Trp Val Pro 850 855 860 Val Gly Ala Ser Ala Thr Gln Asp Ala Arg Thr Ala Ser Asp Thr Thr 865 870 875 880 Ser His Thr Asp Gly Lys Thr Phe His Ser Asn Ala Ala Leu Asp Ser 885 890 895 Gln Val Ile Tyr Glu Gly Phe Ser Asn Phe Gln Ala Phe Ala Thr Thr 900 905 910 Pro Asp Glu Tyr Thr Asn Ala Val Ile Ala Lys Asn Gly Ser Leu Phe 915 920 925 Lys Asp Trp Gly Val Thr Ser Phe Gln Leu Ala Pro Gln Tyr Arg Ser 930 935 940 Ser Thr Asp Thr Ser Phe Leu Asp Ser Ile Ile Gln Asn Gly Tyr Ala 945 950 955 960 Phe Thr Asp Arg Tyr Asp Leu Gly Phe Gly Thr Pro Thr Lys Tyr Gly 965 970 975 Thr Val Asp Gln Leu Arg Asp Ala Ile Lys Ala Leu His Ala Ser Gly 980 985 990 Ile Gln Ala Ile Ala Asp Trp Val Pro Asp Gln Ile Tyr Asn Leu Pro 995 1000 1005 Gly Gln Glu Leu Ala Thr Val Thr Arg Thr Asn Ser Tyr Gly Asp Lys 1010 1015 1020 Asp Pro Asn Ser Asp Ile Glu Asn Ser Leu Tyr Val Ile Gln Ser Arg 1025 1030 1035 1040 Gly Gly Gly Gln Tyr Gln Ala Gln Tyr Gly Gly Ala Phe Leu Ser Asp 1045 1050 1055 Leu Gln Ala Met Tyr Pro Ser Leu Phe Glu Thr Lys Gln Ile Ser Thr 1060 1065 1070 Asn Leu Pro Met Asp Pro Ser Thr Arg Ile Thr Glu Trp Ser Gly Lys 1075 1080 1085 Tyr Phe Asn Gly Ser Asn Ile Gln Gly Lys Gly Ala Gly Tyr Val Leu 1090 1095 1100 Lys Asp Ala Gly Ser Ser Thr Tyr Tyr His Val Val Ser Asn Asn Asn 1105 1110 1115 1120 Asp Ala Thr Tyr Leu Pro Lys Gln Leu Thr Asn Asp Leu Ser Glu Thr 1125 1130 1135 Gly Phe Thr His Asp Asn Gln Gly Ile Ile Tyr Tyr Thr Leu Ser Gly 1140 1145 1150 Thr Arg Ala Gln Asn Ser Phe Val Gln Asp Asn Ser Gly Asn Tyr Tyr 1155 1160 1165 Tyr Phe Asp Asn Thr Gly His Leu Val Thr Gly Ala Gln Thr Ile Asn 1170 1175 1180 Thr His His Tyr Phe Phe Leu Pro Asn Gly Ile Glu Leu Met Gln Ala 1185 1190 1195 1200 Phe Phe Gln Asn Ala Asp Gly Ser Thr Ile Tyr Phe Asp Lys Arg Gly 1205 1210 1215 Gln Gln Val Tyr Asn Gln Tyr Ile Val Asp Gln Thr Gly Ala Ala Tyr 1220 1225 1230 Tyr Phe Gly Thr Asp Gly Arg Met Ala Ile Asn Gly Phe Thr Asp Val 1235 1240 1245 Asp Gly His Arg Gln Tyr Phe Asp Gln Ser Gly His Gln Leu Lys Asp 1250 1255 1260 Gln Phe Met Thr Asp Thr Asn Gly His Val Tyr Tyr Phe Glu Ala Gly 1265 1270 1275 1280 Asn Gly Asn Met Ala Thr Tyr Arg Tyr Ala Gln Asn Ala Gln Gly Gln 1285 1290 1295 Trp Phe Tyr Leu Gly Gly Asp Gly Ile Ala Val Thr Gly Leu Gln Asn 1300 1305 1310 Ile Asn Gly Ala Asn Gln Tyr Phe Tyr Thr Asp Gly His Gln Ser Lys 1315 1320 1325 Gly Glu Phe Val Val Leu Thr Asp Lys Thr Ile Tyr Thr Asp Ala Thr 1330 1335 1340 Thr Gly Asn Leu Val Val Gly Val Gln Gln Leu Asp Gly Lys Thr Tyr 1345 1350 1355 1360 Val Phe Thr Thr Ala Gly Ala Met Leu Thr Asn Gln Tyr Tyr Glu Leu 1365 1370 1375 Ala Ala Asp Gln Trp Leu His Leu Ser Ala Gln Gly Gln Ala Asp Thr 1380 1385 1390 Gly Leu Thr Thr Ile Gly Asp Gln Leu Gln Tyr Phe Gly Thr Asp Gly 1395 1400 1405 Val Gln Val Lys Gly Ala Phe Val Thr Asp Pro Ala Thr Gln Ala Thr 1410 1415 1420 Tyr Tyr Phe Asn Ala Thr Thr Gly Asp Ala Val Ala Asn Gln Tyr Phe 1425 1430 1435 1440 His Ile Lys Gly Asp Trp Tyr Leu Thr Asp Asp Asn Ala Arg Leu Val 1445 1450 1455 Lys Gly Phe Lys Val Val Asn Asn Lys Val Gln His Phe Asp Glu Thr 1460 1465 1470 Thr Gly Val Gln Thr Lys Ser Thr His Leu Thr Lys Ala Gln Lys Gln 1475 1480 1485 Tyr Ile Phe Asp His Asn Gly Asp Leu Val Asn Met 1490 1495 1500 <210> 2 <211> 4503 <212> DNA <213> glucansucrase gene derived from Leuconostoc lactis EG001 <400> 2 ttggaaaacg caacacaagt tagaaaaaaa ctttataaag ccggtaaaaa ctgggtcgtc 60 ggtggtgtca ttacagccgg tgctgcccta gcctttgtcg cgggagcaac gactgctgca 120 gctgatagta accaaaacac aactggttca caggtaacag tgacagcgcc agaagccgca 180 gcgtcagcga cgactacaac gacaacgaca acgccgacca ctgctgcagc aacaacgaca 240 gccgctcagg cagataccag tagtgcaacg acaccacaac cccctgtcgc gcccactaca 300 cctgtagcac caacacaggc acaacctgcc acaaccgcag caacgactga gcaaccgcaa 360 caaccagcag ctgacactac ccctgcccca caaactggct acgtggaaaa agctggggct 420 tggtactatg ttaatgctga tcaatcctat gcaaaaggct taacgaccat tgcgggtcac 480 ttacaatatt ttgataccac tggccgccaa acaaagggcg cctacgtcac agaaaacggc 540 aagacttact attttgacgc taacacgggt aacgccttga ctggcttgca acacgttgcc 600 ggtcaaactg tggctttcaa cacccaaggt gaacaaattt tcagcgactt ttacacggcc 660 gctgacggcc aaacttatta ttttggtacc aacggtcaag ctgcagtcgg tgtcacgagt 720 atcgctggtc acaattatta tttcgacgct gcgggtcaac tgaagaaagg ctatgctggc 780 gaaattgacg gccaaatgcg gacatttgat gccacaactg gtcaagaagt gtcagccacc 840 acgtcacaaa tcacggaagg tttgactgcc caaaatgacg actacacggc ccacaacgcc 900 gtgcatagca ctgccagtgc agactttgat aaccttgatg gttatttgac agcatcatct 960 tggtatcgtc caaccgacat tttgcgcgat ggtaacaagt gggaagcttc aacagccacg 1020 gacatgcgcc caattttgtc ggtttggtgg cctgataagc aaacccaggt cgactacttg 1080 aactacatgt cacaacttgg ccttgtcgaa aacccaacgc cttataccct tcaagatgat 1140 caagtcgcct tgaacaaggc cagcgaaacg ttgcaacaag ccatcgaaac aaagattggg 1200 ttgacaaata gcacagactg gttgaagacg gcgatggcaa acttcatcac aacacaacca 1260 caatggaacc aaacgagtga agatccaaac agcgatcatt tgcaaaaggg tgccttgacg 1320 tttgtgaaca gccccctcac accagataca aattctgcat tccgcttact caaccgcacc 1380 ccagcgaacc aaaccaatac acaaaattat acagttgata attcaaaggg cggctacgaa 1440 ttgttgttgg ctaacgacgt ggataactca aaccccgtcg tgcaagccga acaattgaac 1500 tggttgcact acttgatgaa ctttggctcg attactgcca acgatgctga cgcgaacttt 1560 gacggtattc gtgtcgacgc tgtcgacaac gttgatgctg atttgttgca aattgccgcc 1620 gattacttca aagcggctta tggtgtcgac aaaaacgacg ccacagccaa ccaacacctc 1680 tcaattttgg aagattggag tcacaatgat cccctttacg tcaatgactt cggggacaac 1740 caactgacaa tggacgatta cgcccatacg caattgattt ggtcactaac caagaactca 1800 gatattcgtg gtaccatgca acgtttcatg gactactact tagttaaccg tagccaagat 1860 agcacggaaa acaccgccac accgaactat agtttcgtgc gcgcccacga tagtgaagtg 1920 caaacggtca ttgcccaaat cgtttctgat ttgcatcctg atgttgaaaa tagcttagca 1980 ccaacaacag aacaattgct tgaagccttc aaagtttata acgccgacca aaagctagcc 2040 gacaagaaat atacccaata caacatgcca agtgcctacg ccatgttgtt gactaataaa 2100 gacacagtcc cacgtgttta ctatggtgat ctttacacgg atgacggtca atatatggcc 2160 acaaagtccc cttatttcaa tgcgattgac acgttgctca aggcccgtat tcaatatgtt 2220 gccggtggtc aagccatggc tgtggacaac catgacattt taacttctgt ccgttacggt 2280 aatggtgcca tgactgccac tgacaagggc gatgctgaca cacggaccca agggattggg 2340 gtcatcatca gcaataacaa agatttggct ttgcaagctg gtgaaacggt gacgttgcac 2400 atgggggctg cccacaaaaa gcaagccttc cgtctgttgt taggcacaac acaagatggc 2460 ttggactatt acaacaccga tgatgcacct attcgttata ccgataacaa cggtgattta 2520 atctttaaca gccaagatgt ctacggcgtt caaaatccac aagtgtctgg cttcttagct 2580 gtttgggtac ccgttggggc ctcagctacc caagatgcgc gcacagcgtc agatacaact 2640 agccacactg atggtaagac tttccattcc aacgctgcat tggattcaca agtgatttac 2700 gaaggtttct caaacttcca agccttcgcc acaacccctg acgaatacac caacgccgtc 2760 attgccaaga atgggtcatt gtttaaggac tggggtgtca caagcttcca attggcccca 2820 caataccgtt caagtacaga tacgagtttc ttagattcaa tcattcaaaa cggctacgca 2880 tttaccgacc gttatgatct tggtttcggt acaccaacta agtacggcac agtagaccaa 2940 ttacgcgatg cgattaaagc cttgcatgcc agtggtattc aagcgattgc cgactgggtc 3000 cctgaccaaa tttacaacct acctggtcaa gaacttgcca cagtaacacg gacgaactca 3060 tatggtgaca aagatcctaa ttcagacatc gaaaattcac tttatgtgat tcaaagtcgt 3120 ggtggtgggc aatatcaagc ccaatacggt ggcgcattct tgagtgactt gcaagccatg 3180 tatccctcat tgtttgaaac aaagcaaatt tcaacgaact taccaatgga tccatcaact 3240 cgtatcacgg aatggtctgg taaatacttc aacggctcta acattcaagg taagggtgct 3300 ggctatgtgt tgaaagatgc tggttcaagt acttattacc atgtggtctc aaacaacaac 3360 gatgccactt acttacccaa gcaattgacc aatgacttgt cagaaactgg tttcacgcac 3420 gacaaccaag gtattattta ctacacatta agcggtacgc gtgcgcaaaa cagctttgtt 3480 caagacaatt cagggaacta ttactacttt gacaacacag gtcatttggt cactggtgcc 3540 caaaccatca acacgcatca ctacttcttc ttgccaaacg gcattgaatt gatgcaagcc 3600 ttcttccaaa atgctgacgg ctcaaccatt tacttcgata agcgcggcca acaagtctac 3660 aaccaataca tcgttgacca aactggggca gcttactact ttggcactga tggtcgtatg 3720 gcgatcaacg gcttcacgga tgtcgatggt caccgccaat actttgatca aagtggtcat 3780 caacttaagg atcagttcat gactgatacc aatggtcacg tgtactactt tgaagcgggt 3840 aacggtaaca tggccactta tcgctacgca caaaacgccc aaggtcaatg gttctacctt 3900 ggtggtgatg ggattgccgt gactggtttg caaaacatta acggcgctaa ccagtacttc 3960 tacaccgatg gccaccaaag taagggtgaa ttcgtcgtct taactgacaa gaccatttac 4020 acggatgcca caactggtaa tctagtcgtt ggcgtgcaac agcttgatgg caagacttac 4080 gtctttacca ctgctggtgc catgctcaca aaccaatact acgaacttgc tgctgaccaa 4140 tggttacacc taagtgcgca aggtcaagct gatactggtt tgacaaccat tggcgaccaa 4200 ttgcaatact ttgggactga cggtgtccaa gtgaagggtg cctttgtgac tgatccagcc 4260 actcaagcca cttactactt taacgccaca actggggatg cggttgccaa ccaatacttc 4320 cacatcaaag gcgattggta tttgacagat gataacgcac gtcttgtgaa gggctttaaa 4380 gtcgtgaaca acaaggtgca acattttgat gaaacaactg gtgtgcaaac caagtcaaca 4440 catttgacaa aagcacaaaa acaatatatt ttcgatcaca acggtgatct cgtcaatatg 4500 taa 4503 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 3 gayaaywsna ayccnryngt nc 22 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 4 adrtcnccrt artanavnyk ng 22 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 5 cacgatagtg aagtgcaaac g 21 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 6 atcgttggca gtaatcgagc 20 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 7 atagcttagc accaacaaca gaac 24 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 8 gtttcgctgg ccttgttca 19 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 actgtggctt tcaacaccca 20 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 tgtgcgcgca tcttgggta 19 <210> 11 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 11 atccatggat agtaaccaaa acacaactgg tt 32 <210> 12 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> backward primer <400> 12 atgcggccgc catattgacg agatcaccgt tg 32 <110> Korea Research Institute of Bioscience and Biotechnology <120> Method for Preparing Ascorbic Acid 2-Glucoside Using Glucansucrase <160> 12 <170> KopatentIn 1.71 <210> 1 <211> 1500 <212> PRT <213> amino acid sequence for glucansucrase derived from Leuconostoc lactis EG001 <400> 1 Leu Glu Asn Ala Thr Gln Val Arg Lys Lys Leu Tyr Lys Ala Gly Lys 1 5 10 15 Asn Trp Val Val Gly Gly Val Ile Thr Ala Gly Ala Ala Leu Ala Phe 20 25 30 Val Ala Gly Ala Thr Thr Ala Ala Ala Asp Ser Asn Gln Asn Thr Thr 35 40 45 Gly Ser Gln Val Thr Val Thr Ala Pro Glu Ala Ala Ala Ser Ala Thr 50 55 60 Thr Thr Thr Thr Thr Thr Thr Pro Thr Thr Ala Ala Ala Thr Thr Thr 65 70 75 80 Ala Ala Gln Ala Asp Thr Ser Ser Ala Thr Thr Pro Gln Pro Pro Val 85 90 95 Ala Pro Thr Thr Pro Val Ala Pro Thr Gln Ala Gln Pro Ala Thr Thr 100 105 110 Ala Ala Thr Thr Glu Gln Pro Gln Gln Pro Ala Ala Asp Thr Thr Pro 115 120 125 Ala Pro Gln Thr Gly Tyr Val Glu Lys Ala Gly Ala Trp Tyr Tyr Val 130 135 140 Asn Ala Asp Gln Ser Tyr Ala Lys Gly Leu Thr Thr Ile Ala Gly His 145 150 155 160 Leu Gln Tyr Phe Asp Thr Thr Gly Arg Gln Thr Lys Gly Ala Tyr Val 165 170 175 Thr Glu Asn Gly Lys Thr Tyr Tyr Phe Asp Ala Asn Thr Gly Asn Ala 180 185 190 Leu Thr Gly Leu Gln His Val Ala Gly Gln Thr Val Ala Phe Asn Thr 195 200 205 Gln Gly Glu Gln Ile Phe Ser Asp Phe Tyr Thr Ala Ala Asp Gly Gln 210 215 220 Thr Tyr Tyr Phe Gly Thr Asn Gly Gln Ala Ala Val Gly Val Thr Ser 225 230 235 240 Ile Ala Gly His Asn Tyr Tyr Phe Asp Ala Ala Gly Gln Leu Lys Lys 245 250 255 Gly Tyr Ala Gly Glu Ile Asp Gly Gln Met Arg Thr Phe Asp Ala Thr 260 265 270 Thr Gly Gln Glu Val Ser Ala Thr Thr Ser Gln Ile Thr Glu Gly Leu 275 280 285 Thr Ala Gln Asn Asp Asp Tyr Thr Ala His Asn Ala Val His Ser Thr 290 295 300 Ala Ser Ala Asp Phe Asp Asn Leu Asp Gly Tyr Leu Thr Ala Ser Ser 305 310 315 320 Trp Tyr Arg Pro Thr Asp Ile Leu Arg Asp Gly Asn Lys Trp Glu Ala 325 330 335 Ser Thr Ala Thr Asp Met Arg Pro Ile Leu Ser Val Trp Trp Pro Asp 340 345 350 Lys Gln Thr Gln Val Asp Tyr Leu Asn Tyr Met Ser Gln Leu Gly Leu 355 360 365 Val Glu Asn Pro Thr Pro Tyr Thr Leu Gln Asp Asp Gln Val Ala Leu 370 375 380 Asn Lys Ala Ser Glu Thr Leu Gln Gln Ala Ile Glu Thr Lys Ile Gly 385 390 395 400 Leu Thr Asn Ser Thr Asp Trp Leu Lys Thr Ala Met Ala Asn Phe Ile 405 410 415 Thr Thr Gln Pro Gln Trp Asn Gln Thr Ser Glu Asp Pro Asn Ser Asp 420 425 430 His Leu Gln Lys Gly Ala Leu Thr Phe Val Asn Ser Pro Leu Thr Pro 435 440 445 Asp Thr Asn Ser Ala Phe Arg Leu Leu Asn Arg Thr Pro Ala Asn Gln 450 455 460 Thr Asn Thr Gln Asn Tyr Thr Val Asp Asn Ser Lys Gly Gly Tyr Glu 465 470 475 480 Leu Leu Leu Ala Asn Asp Val Asp Asn Ser Asn Pro Val Val Gln Ala 485 490 495 Glu Gln Leu Asn Trp Leu His Tyr Leu Met Asn Phe Gly Ser Ile Thr 500 505 510 Ala Asn Asp Ala Asp Ala Asn Phe Asp Gly Ile Arg Val Asp Ala Val 515 520 525 Asp Asn Val Asp Ala Asp Leu Leu Gln Ile Ala Ala Asp Tyr Phe Lys 530 535 540 Ala Ala Tyr Gly Val Asp Lys Asn Asp Ala Thr Ala Asn Gln His Leu 545 550 555 560 Ser Ile Leu Glu Asp Trp Ser His Asn Asp Pro Leu Tyr Val Asn Asp 565 570 575 Phe Gly Asp Asn Gln Leu Thr Met Asp Asp Tyr Ala His Thr Gln Leu 580 585 590 Ile Trp Ser Leu Thr Lys Asn Ser Asp Ile Arg Gly Thr Met Gln Arg 595 600 605 Phe Met Asp Tyr Tyr Leu Val Asn Arg Ser Gln Asp Ser Thr Glu Asn 610 615 620 Thr Ala Thr Pro Asn Tyr Ser Phe Val Arg Ala His Asp Ser Glu Val 625 630 635 640 Gln Thr Val Ile Ala Gln Ile Val Ser Asp Leu His Pro Asp Val Glu 645 650 655 Asn Ser Leu Ala Pro Thr Thr Glu Gln Leu Leu Glu Ala Phe Lys Val 660 665 670 Tyr Asn Ala Asp Gln Lys Leu Ala Asp Lys Lys Tyr Thr Gln Tyr Asn 675 680 685 Met Pro Ser Ala Tyr Ala Met Leu Leu Thr Asn Lys Asp Thr Val Pro 690 695 700 Arg Val Tyr Tyr Gly Asp Leu Tyr Thr Asp Asp Gly Gln Tyr Met Ala 705 710 715 720 Thr Lys Ser Pro Tyr Phe Asn Ala Ile Asp Thr Leu Leu Lys Ala Arg 725 730 735 Ile Gln Tyr Val Ala Gly Gly Gln Ala Met Ala Val Asp Asn His Asp 740 745 750 Ile Leu Thr Ser Val Arg Tyr Gly Asn Gly Ala Met Thr Ala Thr Asp 755 760 765 Lys Gly Asp Ala Asp Thr Arg Thr Gln Gly Ile Gly Val Ile Ile Ser 770 775 780 Asn Asn Lys Asp Leu Ala Leu Gln Ala Gly Glu Thr Val Thr Leu His 785 790 795 800 Met Gly Ala Ala His Lys Lys Gln Ala Phe Arg Leu Leu Leu Gly Thr 805 810 815 Thr Gln Asp Gly Leu Asp Tyr Tyr Asn Thr Asp Asp Ala Pro Ile Arg 820 825 830 Tyr Thr Asp Asn Asn Gly Asp Leu Ile Phe Asn Ser Gln Asp Val Tyr 835 840 845 Gly Val Gln Asn Pro Gln Val Ser Gly Phe Leu Ala Val Trp Val Pro 850 855 860 Val Gly Ala Ser Ala Thr Gln Asp Ala Arg Thr Ala Ser Asp Thr Thr 865 870 875 880 Ser His Thr Asp Gly Lys Thr Phe His Ser Asn Ala Ala Leu Asp Ser 885 890 895 Gln Val Ile Tyr Glu Gly Phe Ser Asn Phe Gln Ala Phe Ala Thr Thr 900 905 910 Pro Asp Glu Tyr Thr Asn Ala Val Ile Ala Lys Asn Gly Ser Leu Phe 915 920 925 Lys Asp Trp Gly Val Thr Ser Phe Gln Leu Ala Pro Gln Tyr Arg Ser 930 935 940 Ser Thr Asp Thr Ser Phe Leu Asp Ser Ile Ile Gln Asn Gly Tyr Ala 945 950 955 960 Phe Thr Asp Arg Tyr Asp Leu Gly Phe Gly Thr Pro Thr Lys Tyr Gly 965 970 975 Thr Val Asp Gln Leu Arg Asp Ala Ile Lys Ala Leu His Ala Ser Gly 980 985 990 Ile Gln Ala Ile Ala Asp Trp Val Pro Asp Gln Ile Tyr Asn Leu Pro 995 1000 1005 Gly Gln Glu Leu Ala Thr Val Thr Arg Thr Asn Ser Tyr Gly Asp Lys 1010 1015 1020 Asp Pro Asn Ser Asp Ile Glu Asn Ser Leu Tyr Val Ile Gln Ser Arg 1025 1030 1035 1040 Gly Gly Gly Gln Tyr Gln Ala Gln Tyr Gly Gly Ala Phe Leu Ser Asp 1045 1050 1055 Leu Gln Ala Met Tyr Pro Ser Leu Phe Glu Thr Lys Gln Ile Ser Thr 1060 1065 1070 Asn Leu Pro Met Asp Pro Ser Thr Arg Ile Thr Glu Trp Ser Gly Lys 1075 1080 1085 Tyr Phe Asn Gly Ser Asn Ile Gln Gly Lys Gly Ala Gly Tyr Val Leu 1090 1095 1100 Lys Asp Ala Gly Ser Ser Thr Tyr Tyr His Val Val Ser Asn Asn Asn 1105 1110 1115 1120 Asp Ala Thr Tyr Leu Pro Lys Gln Leu Thr Asn Asp Leu Ser Glu Thr 1125 1130 1135 Gly Phe Thr His Asp Asn Gln Gly Ile Ile Tyr Tyr Thr Leu Ser Gly 1140 1145 1150 Thr Arg Ala Gln Asn Ser Phe Val Gln Asp Asn Ser Gly Asn Tyr Tyr 1155 1160 1165 Tyr Phe Asp Asn Thr Gly His Leu Val Thr Gly Ala Gln Thr Ile Asn 1170 1175 1180 Thr His His Tyr Phe Phe Leu Pro Asn Gly Ile Glu Leu Met Gln Ala 1185 1190 1195 1200 Phe Phe Gln Asn Ala Asp Gly Ser Thr Ile Tyr Phe Asp Lys Arg Gly 1205 1210 1215 Gln Gln Val Tyr Asn Gln Tyr Ile Val Asp Gln Thr Gly Ala Ala Tyr 1220 1225 1230 Tyr Phe Gly Thr Asp Gly Arg Met Ala Ile Asn Gly Phe Thr Asp Val 1235 1240 1245 Asp Gly His Arg Gln Tyr Phe Asp Gln Ser Gly His Gln Leu Lys Asp 1250 1255 1260 Gln Phe Met Thr Asp Thr Asn Gly His Val Tyr Tyr Phe Glu Ala Gly 1265 1270 1275 1280 Asn Gly Asn Met Ala Thr Tyr Arg Tyr Ala Gln Asn Ala Gln Gly Gln 1285 1290 1295 Trp Phe Tyr Leu Gly Gly Asp Gly Ile Ala Val Thr Gly Leu Gln Asn 1300 1305 1310 Ile Asn Gly Ala Asn Gln Tyr Phe Tyr Thr Asp Gly His Gln Ser Lys 1315 1320 1325 Gly Glu Phe Val Val Leu Thr Asp Lys Thr Ile Tyr Thr Asp Ala Thr 1330 1335 1340 Thr Gly Asn Leu Val Val Gly Val Gln Gln Leu Asp Gly Lys Thr Tyr 1345 1350 1355 1360 Val Phe Thr Thr Ala Gly Ala Met Leu Thr Asn Gln Tyr Tyr Glu Leu 1365 1370 1375 Ala Ala Asp Gln Trp Leu His Leu Ser Ala Gln Gly Gln Ala Asp Thr 1380 1385 1390 Gly Leu Thr Thr Ile Gly Asp Gln Leu Gln Tyr Phe Gly Thr Asp Gly 1395 1400 1405 Val Gln Val Lys Gly Ala Phe Val Thr Asp Pro Ala Thr Gln Ala Thr 1410 1415 1420 Tyr Tyr Phe Asn Ala Thr Thr Gly Asp Ala Val Ala Asn Gln Tyr Phe 1425 1430 1435 1440 His Ile Lys Gly Asp Trp Tyr Leu Thr Asp Asp Asn Ala Arg Leu Val 1445 1450 1455 Lys Gly Phe Lys Val Val Asn Asn Lys Val Gln His Phe Asp Glu Thr 1460 1465 1470 Thr Gly Val Gln Thr Lys Ser Thr His Leu Thr Lys Ala Gln Lys Gln 1475 1480 1485 Tyr Ile Phe Asp His Asn Gly Asp Leu Val Asn Met 1490 1495 1500 <210> 2 <211> 4503 <212> DNA <213> Glucansucrase gene derived from Leuconostoc lactis EG001 <400> 2 ttggaaaacg caacacaagt tagaaaaaaa ctttataaag ccggtaaaaa ctgggtcgtc 60 ggtggtgtca ttacagccgg tgctgcccta gcctttgtcg cgggagcaac gactgctgca 120 gctgatagta accaaaacac aactggttca caggtaacag tgacagcgcc agaagccgca 180 gcgtcagcga cgactacaac gacaacgaca acgccgacca ctgctgcagc aacaacgaca 240 gccgctcagg cagataccag tagtgcaacg acaccacaac cccctgtcgc gcccactaca 300 cctgtagcac caacacaggc acaacctgcc acaaccgcag caacgactga gcaaccgcaa 360 caaccagcag ctgacactac ccctgcccca caaactggct acgtggaaaa agctggggct 420 tggtactatg ttaatgctga tcaatcctat gcaaaaggct taacgaccat tgcgggtcac 480 ttacaatatt ttgataccac tggccgccaa acaaagggcg cctacgtcac agaaaacggc 540 aagacttact attttgacgc taacacgggt aacgccttga ctggcttgca acacgttgcc 600 ggtcaaactg tggctttcaa cacccaaggt gaacaaattt tcagcgactt ttacacggcc 660 gctgacggcc aaacttatta ttttggtacc aacggtcaag ctgcagtcgg tgtcacgagt 720 atcgctggtc acaattatta tttcgacgct gcgggtcaac tgaagaaagg ctatgctggc 780 gaaattgacg gccaaatgcg gacatttgat gccacaactg gtcaagaagt gtcagccacc 840 acgtcacaaa tcacggaagg tttgactgcc caaaatgacg actacacggc ccacaacgcc 900 gtgcatagca ctgccagtgc agactttgat aaccttgatg gttatttgac agcatcatct 960 tggtatcgtc caaccgacat tttgcgcgat ggtaacaagt gggaagcttc aacagccacg 1020 gacatgcgcc caattttgtc ggtttggtgg cctgataagc aaacccaggt cgactacttg 1080 aactacatgt cacaacttgg ccttgtcgaa aacccaacgc cttataccct tcaagatgat 1140 caagtcgcct tgaacaaggc cagcgaaacg ttgcaacaag ccatcgaaac aaagattggg 1200 ttgacaaata gcacagactg gttgaagacg gcgatggcaa acttcatcac aacacaacca 1260 caatggaacc aaacgagtga agatccaaac agcgatcatt tgcaaaaggg tgccttgacg 1320 tttgtgaaca gccccctcac accagataca aattctgcat tccgcttact caaccgcacc 1380 ccagcgaacc aaaccaatac acaaaattat acagttgata attcaaaggg cggctacgaa 1440 ttgttgttgg ctaacgacgt ggataactca aaccccgtcg tgcaagccga acaattgaac 1500 tggttgcact acttgatgaa ctttggctcg attactgcca acgatgctga cgcgaacttt 1560 gacggtattc gtgtcgacgc tgtcgacaac gttgatgctg atttgttgca aattgccgcc 1620 gattacttca aagcggctta tggtgtcgac aaaaacgacg ccacagccaa ccaacacctc 1680 tcaattttgg aagattggag tcacaatgat cccctttacg tcaatgactt cggggacaac 1740 caactgacaa tggacgatta cgcccatacg caattgattt ggtcactaac caagaactca 1800 gatattcgtg gtaccatgca acgtttcatg gactactact tagttaaccg tagccaagat 1860 agcacggaaa acaccgccac accgaactat agtttcgtgc gcgcccacga tagtgaagtg 1920 caaacggtca ttgcccaaat cgtttctgat ttgcatcctg atgttgaaaa tagcttagca 1980 ccaacaacag aacaattgct tgaagccttc aaagtttata acgccgacca aaagctagcc 2040 gacaagaaat atacccaata caacatgcca agtgcctacg ccatgttgtt gactaataaa 2100 gacacagtcc cacgtgttta ctatggtgat ctttacacgg atgacggtca atatatggcc 2160 acaaagtccc cttatttcaa tgcgattgac acgttgctca aggcccgtat tcaatatgtt 2220 gccggtggtc aagccatggc tgtggacaac catgacattt taacttctgt ccgttacggt 2280 aatggtgcca tgactgccac tgacaagggc gatgctgaca cacggaccca agggattggg 2340 gtcatcatca gcaataacaa agatttggct ttgcaagctg gtgaaacggt gacgttgcac 2400 atgggggctg cccacaaaaa gcaagccttc cgtctgttgt taggcacaac acaagatggc 2460 ttggactatt acaacaccga tgatgcacct attcgttata ccgataacaa cggtgattta 2520 atctttaaca gccaagatgt ctacggcgtt caaaatccac aagtgtctgg cttcttagct 2580 gtttgggtac ccgttggggc ctcagctacc caagatgcgc gcacagcgtc agatacaact 2640 agccacactg atggtaagac tttccattcc aacgctgcat tggattcaca agtgatttac 2700 gaaggtttct caaacttcca agccttcgcc acaacccctg acgaatacac caacgccgtc 2760 attgccaaga atgggtcatt gtttaaggac tggggtgtca caagcttcca attggcccca 2820 caataccgtt caagtacaga tacgagtttc ttagattcaa tcattcaaaa cggctacgca 2880 tttaccgacc gttatgatct tggtttcggt acaccaacta agtacggcac agtagaccaa 2940 ttacgcgatg cgattaaagc cttgcatgcc agtggtattc aagcgattgc cgactgggtc 3000 cctgaccaaa tttacaacct acctggtcaa gaacttgcca cagtaacacg gacgaactca 3060 tatggtgaca aagatcctaa ttcagacatc gaaaattcac tttatgtgat tcaaagtcgt 3120 ggtggtgggc aatatcaagc ccaatacggt ggcgcattct tgagtgactt gcaagccatg 3180 tatccctcat tgtttgaaac aaagcaaatt tcaacgaact taccaatgga tccatcaact 3240 cgtatcacgg aatggtctgg taaatacttc aacggctcta acattcaagg taagggtgct 3300 ggctatgtgt tgaaagatgc tggttcaagt acttattacc atgtggtctc aaacaacaac 3360 gatgccactt acttacccaa gcaattgacc aatgacttgt cagaaactgg tttcacgcac 3420 gacaaccaag gtattattta ctacacatta agcggtacgc gtgcgcaaaa cagctttgtt 3480 caagacaatt cagggaacta ttactacttt gacaacacag gtcatttggt cactggtgcc 3540 caaaccatca acacgcatca ctacttcttc ttgccaaacg gcattgaatt gatgcaagcc 3600 ttcttccaaa atgctgacgg ctcaaccatt tacttcgata agcgcggcca acaagtctac 3660 aaccaataca tcgttgacca aactggggca gcttactact ttggcactga tggtcgtatg 3720 gcgatcaacg gcttcacgga tgtcgatggt caccgccaat actttgatca aagtggtcat 3780 caacttaagg atcagttcat gactgatacc aatggtcacg tgtactactt tgaagcgggt 3840 aacggtaaca tggccactta tcgctacgca caaaacgccc aaggtcaatg gttctacctt 3900 ggtggtgatg ggattgccgt gactggtttg caaaacatta acggcgctaa ccagtacttc 3960 tacaccgatg gccaccaaag taagggtgaa ttcgtcgtct taactgacaa gaccatttac 4020 acggatgcca caactggtaa tctagtcgtt ggcgtgcaac agcttgatgg caagacttac 4080 gtctttacca ctgctggtgc catgctcaca aaccaatact acgaacttgc tgctgaccaa 4140 tggttacacc taagtgcgca aggtcaagct gatactggtt tgacaaccat tggcgaccaa 4200 ttgcaatact ttgggactga cggtgtccaa gtgaagggtg cctttgtgac tgatccagcc 4260 actcaagcca cttactactt taacgccaca actggggatg cggttgccaa ccaatacttc 4320 cacatcaaag gcgattggta tttgacagat gataacgcac gtcttgtgaa gggctttaaa 4380 gtcgtgaaca acaaggtgca acattttgat gaaacaactg gtgtgcaaac caagtcaaca 4440 catttgacaa aagcacaaaa acaatatatt ttcgatcaca acggtgatct cgtcaatatg 4500 taa 4503 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 3 gayaaywsna ayccnryngt nc 22 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 4 adrtcnccrt artanavnyk ng 22 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 5 cacgatagtg aagtgcaaac g 21 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 6 atcgttggca gtaatcgagc 20 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 7 atagcttagc accaacaaca gaac 24 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 8 gtttcgctgg ccttgttca 19 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 actgtggctt tcaacaccca 20 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 tgtgcgcgca tcttgggta 19 <210> 11 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 11 atccatggat agtaaccaaa acacaactgg tt 32 <210> 12 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> backward primer <400> 12 atgcggccgc catattgacg agatcaccgt tg 32
Claims (9)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1020090076848A KR101073990B1 (en) | 2009-08-19 | 2009-08-19 | Method for Preparing Ascorbic Acid 2-Glucoside Using Glucansucrase |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1020090076848A KR101073990B1 (en) | 2009-08-19 | 2009-08-19 | Method for Preparing Ascorbic Acid 2-Glucoside Using Glucansucrase |
Publications (2)
Publication Number | Publication Date |
---|---|
KR20110019226A KR20110019226A (en) | 2011-02-25 |
KR101073990B1 true KR101073990B1 (en) | 2011-10-17 |
Family
ID=43776622
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
KR1020090076848A KR101073990B1 (en) | 2009-08-19 | 2009-08-19 | Method for Preparing Ascorbic Acid 2-Glucoside Using Glucansucrase |
Country Status (1)
Country | Link |
---|---|
KR (1) | KR101073990B1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20150101076A (en) * | 2014-02-26 | 2015-09-03 | 대한민국(관리부서 : 농림축산식품부 농림축산검역본부) | Method for producing of hot water molding filter for separating nematode and hot water molding filter produced by the same |
KR20200081038A (en) | 2018-12-27 | 2020-07-07 | 건양대학교산학협력단 | The method for producing ascorbic acid-2-glucoside(AA-2G) using the immobilization of recombinant cyclodextrin glucanotransferase (CGTase) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20130014227A (en) * | 2011-07-29 | 2013-02-07 | 한국생명공학연구원 | NOVEL α-GLUCOSYL STEVIOSIDES AND PROCESS FOR PRODUCING THE SAME |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20060216792A1 (en) | 2002-08-06 | 2006-09-28 | Kazuhisa Mukai | Process for producing 2-0-alpha-d-glucopyranosyl-l-ascorbic acid |
-
2009
- 2009-08-19 KR KR1020090076848A patent/KR101073990B1/en active IP Right Grant
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20060216792A1 (en) | 2002-08-06 | 2006-09-28 | Kazuhisa Mukai | Process for producing 2-0-alpha-d-glucopyranosyl-l-ascorbic acid |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20150101076A (en) * | 2014-02-26 | 2015-09-03 | 대한민국(관리부서 : 농림축산식품부 농림축산검역본부) | Method for producing of hot water molding filter for separating nematode and hot water molding filter produced by the same |
KR20200081038A (en) | 2018-12-27 | 2020-07-07 | 건양대학교산학협력단 | The method for producing ascorbic acid-2-glucoside(AA-2G) using the immobilization of recombinant cyclodextrin glucanotransferase (CGTase) |
Also Published As
Publication number | Publication date |
---|---|
KR20110019226A (en) | 2011-02-25 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Oliveira et al. | Recombinant microbial systems for improved β-galactosidase production and biotechnological applications | |
US20020086358A1 (en) | Enzyme isolated from a Bifidobacterium | |
Kim et al. | Purification and characterization of a novel glucansucrase from Leuconostoc lactis EG001 | |
AU2001262068A1 (en) | A new enzyme isolated from a bifidobacterium | |
JP5224572B2 (en) | Dextran producing enzyme gene, dextran producing enzyme and method for producing the same, and method for producing dextran | |
JP2009034028A (en) | beta-GLUCOSIDASE, METHOD FOR PRODUCING THE SAME, AND METHOD FOR PRODUCING beta-TYPE GLYCOSIDE USING THE SAME | |
JPWO2018016483A1 (en) | Method of producing mogrol or mogrol glycoside | |
KR101091138B1 (en) | Glucansucrase Derived from Leuconostoc lactis and Method for Preparing the Same | |
KR101073990B1 (en) | Method for Preparing Ascorbic Acid 2-Glucoside Using Glucansucrase | |
Zhao et al. | β-Galactosidase BMG without galactose and glucose inhibition: Secretory expression in Bacillus subtilis and for synthesis of oligosaccharide | |
WO2005105991A1 (en) | Amp deaminase originating in streptomyces and utilization thereof | |
US11884942B2 (en) | Compositions and methods comprising the use of Exiguobacterium acetylicum and Bacillus coagluans α-glucanotransferase enzymes | |
Dion et al. | Modulation of the regioselectivity of a Bacillus α-galactosidase by directed evolution | |
KR101392987B1 (en) | Production of the rare ginsenosides compound Mc, compound Y, aglycon protopanaxadiol by a thermostable beta-glucosidase | |
AU784466B2 (en) | Cyclic depsipeptide synthases, genes thereof and mass production system of cyclic depsipeptide | |
US10745675B2 (en) | Modified enzymes for producing increased isomelezitose | |
CN116057179A (en) | Enzyme for catalyzing epimerization reaction of sugar, process for producing epimerization reaction product, and epimerization reaction product | |
KR20230006803A (en) | Production of NMN and its derivatives through microbial processes | |
CN115449514A (en) | Beta-1, 2-glycosyltransferase and application thereof | |
CN106367458B (en) | Recombinant sucrose phosphorylase is preparing the application in functional oligose | |
US20240124909A1 (en) | Composition and method for production of fructose | |
JPWO2018056388A1 (en) | Method of producing sesaminol or sesaminol glycoside | |
US6916641B2 (en) | (R)-2-hydroxy-3-phenylpropionate (d-phenyllactate) dehydrogenase and gene encoding the same | |
JPH09252789A (en) | Production of acarbiosyltransferase and its use in conversion of acarbose homologue to acarbose and for preparing acarbose homologue | |
JPS61135581A (en) | Polypeptide having cyclomaltodextrin glucanotransferase activity |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A201 | Request for examination | ||
E902 | Notification of reason for refusal | ||
E701 | Decision to grant or registration of patent right | ||
GRNT | Written decision to grant | ||
FPAY | Annual fee payment |
Payment date: 20140903 Year of fee payment: 4 |
|
FPAY | Annual fee payment |
Payment date: 20150922 Year of fee payment: 5 |
|
FPAY | Annual fee payment |
Payment date: 20161010 Year of fee payment: 6 |
|
FPAY | Annual fee payment |
Payment date: 20171026 Year of fee payment: 7 |
|
FPAY | Annual fee payment |
Payment date: 20181205 Year of fee payment: 8 |
|
FPAY | Annual fee payment |
Payment date: 20181226 Year of fee payment: 18 |