KR101073990B1 - Method for Preparing Ascorbic Acid 2-Glucoside Using Glucansucrase - Google Patents

Abstract

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.

Glucan sucrase, glycosylation, L-ascorbic acid, ascorbic acid 2-glucoside

Description

Method for preparing ascorbic acid 2-glucoside using glucansucrase

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-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).

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.

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.

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.

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.

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) mixing L-ascorbic acid and disaccharides with glucan sucrase to perform a glycosylation reaction; And

(b) recovering ascorbic acid 2-glucoside produced by the glycosylation reaction.

The present invention also provides a method for producing ascorbic acid 2-glucoside using glucansucrase, comprising the following steps:

(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) recovering ascorbic acid 2-glucoside produced by the glycosylation reaction.

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.

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.

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.

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.

In one aspect, the present invention relates to a method for producing ascorbic acid 2-glucoside using glucansucrase comprising the following steps:

(a) mixing L-ascorbic acid and disaccharides with glucan sucrase to perform a glycosylation reaction; And

(b) recovering ascorbic acid 2-glucoside produced by the glycosylation reaction.

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).

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.

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.

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.

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.

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.

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 ℃.

In addition, as a result of examining the effect of the pH of the enzyme, it showed a maximum activity at pH 5, it was confirmed that the relatively stable at acidic pH. Therefore, in the method of the present invention, the glycosylation reaction is preferably performed at pH 4.5-6, more preferably at pH 5.

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.

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.

Example  One: Leuconostoc lactis EG001  Strain derived Glucan sukrase  Expression and Separation

1-1: Isolation and Screening of Strains

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.

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: Glucans sucrase  Gene Isolation and Recombinant Vector Construction

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 '(SEQ ID NO: 3)

Reverse primer: 5'-ADRTCNCCRTARTANAVNYKNG-3 '(SEQ ID NO: 4)

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

That is, PCR reaction was performed on a mixture of 20 μl of the genomic DNA 100 ng, 10 × PCR buffer solution, 10 mM dNTP, each primer 5 μM, and 1 U EF- Taq polymerase (Solgent, Korea). The PCR reaction was denatured at 95 ° C. for 3 minutes, followed by 30 seconds of denaturation at 95 ° C., 1 minute annealing at 52 ° C., and 1 minute extension at 72 ° C .. And finally a 10 minute extension at 72 ° C. The obtained PCR product was cloned into pGEM T-easy vector (Promega) and nucleotide sequence was confirmed.

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.

One st PCR for primer

Forward primer: 5'-CACGATAGTGAAGTGCAAACG-3 '(SEQ ID NO: 5)

Reverse primer: 5'-ATCGTTGGCAGTAATCGAGC-3 '(SEQ ID NO: 6)

2 nd PCR for primer

Forward primer: 5'-ATAGCTTAGCACCAACAACAGAAC-3 '(SEQ ID NO: 7)

Reverse primer: 5'-GTTTCGCTGGCCTTGTTCA-3 '(SEQ ID NO: 8)

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.

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 '(SEQ ID NO: 9)

Primer: 5'-TGTGCGCGCATCTTGGGTA-3 '(SEQ ID NO: 10)

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.

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.

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.

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.

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 '(SEQ ID NO: 11)

Backward primer: 5'-AT GCGGCCGC CATATTGACGAGATCACCGTTG-3 '(SEQ ID NO: 12)

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: by recombinant E. coli culture Glucan sukrase  Expression and Purification

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.

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.

Example  2: L. lactis EG001  Strain derived Glucan sukrase  Analysis and Production of Ascorbic Acid 2-Glucoside Using the Same

2-1: L. lactis EG001  Strain derived Glucan sukrase  analysis

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 pH 5, it was confirmed that the relatively stable at acidic pH.

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 ℃.

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: Production of Ascorbic Acid 2-Glucoside

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.

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 (lane 4 and 5), Sucrose and L-ascorbic acid decreased and three new products (P1, P2 and P3) with different R _f values were observed (lane 1: sucrose; lane 2: L-ascorbic acid; lane 3: Ascorbic acid 2-Glucoside (AA-2G); lane 4: non-enzyme; lane 5: enzyme reaction (0h); lane 6: enzyme reaction (3h)). R _f value of P2 of this reaction product (R _f value, 0.35) is the R _f value of standard AA2G (R _f value, 0.35).

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 Gemini 5 μm C18 110A column (4.6 × 250 mm, Phenomenex, USA) chromatography). The peak of the reaction product was detected at 240 nm using a diode array detector (DAD).

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 (peak 1, L-ascorbic acid; peak 2, AA2G) . These results indicate that AA2G (Ascorbic acid 2-glucoside) was synthesized from L-ascorbic acid and sucrose by glycosylation reaction with glucan sucralase.

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.

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.

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  

Claims (9)

Method for producing ascorbic acid 2-glucoside using glucansucrase, comprising the following steps: (a) mixing L-ascorbic acid and a disaccharide with glucan sucrase represented by the amino acid sequence of SEQ ID NO: 1 to perform a glycosylation reaction; And (b) recovering ascorbic acid 2-glucoside produced by the glycosylation reaction. The method of claim 1, wherein the glycosylation reaction of step (a) is carried out at 25 ~ 35 ℃. The method for producing ascorbic acid 2-glucoside according to claim 1, wherein the glycosylation reaction of step (a) is performed at pH 4.5-6. delete The method of claim 1, wherein the glucan sucrase is encoded by a gene represented by the nucleotide sequence of SEQ ID NO: 2. A method for producing ascorbic acid 2-glucoside. The method of claim 1, wherein the glucan scrubber is derived from Leuconostoc lactis EG001. According to claim 1, wherein the disaccharide is sucrose (sucrose), lactose (lactose), maltose (Maltose), trehalose (trehalose), cellobiose (kojibiose), nigerose (nigerose), isomal Tomal (isomaltose), β, β-trehalose (β, β-trehalose), sophorose (sophorose), laminaribiose, gentiobiose, turanose, maltulose, Method for producing ascorbic acid 2-glucoside, characterized in that any one or more selected from the group consisting of palatinose, gentiobiulose, melibiose and rutinose. Method for producing ascorbic acid 2-glucoside using glucansucrase, comprising the following steps: (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) recovering ascorbic acid 2-glucoside produced by the glycosylation reaction. The method for producing ascorbic acid 2-glucoside according to claim 8, wherein the glycosylation reaction of step (a) is performed at a condition of pH 4.5-6 and 25-35 ° C.

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