KR101091138B1 - Glucansucrase Derived from Leuconostoc lactis and Method for Preparing the Same - Google Patents

Abstract

The present invention is a novel glucan sucrase derived from the Leuconostoc Lactis EG001 strain isolated from kimchi lactic acid bacteria, a gene encoding the glucan sucrase, a recombinant vector containing the gene, the gene or the recombinant The present invention relates to a recombinant microorganism having a glucan sucrase generating ability to which a vector is introduced. Glucan sucrase according to the present invention can be usefully used in the food, pharmaceutical industry by synthesizing polysaccharides using abundant carbohydrate resources such as sucrose, glucose.

Leukonostock lactis, glucan sucrase

Description

Glucansucrase Derived from Leuconostoc lactis and Method for Preparing the Same}

The present invention relates to a glucan sucrase (glucansucrase) that can be applied to synthesize a glucan polymer (gluan polymer) useful for industries such as food and pharmaceuticals, and more particularly, a leukonostock lock separated from kimchi The present invention relates to a novel glucan sucrase derived from Leuconostoc Lactis EG001 strain and a preparation method thereof.

Lactic acid bacteria have traditionally been used to make fermented foods. Lactic acid produced by lactic acid bacteria (lactic acid) has the effect of inhibiting the growth of microorganisms involved in decay. In addition, the exopolysaccharide (exopolysacharides) or polysaccharides (polysacharides) produced by the lactic acid bacteria serves as an antibiotic including a precursor of the compound, bacteriocins and the like to taste the unique flavor of fermented foods. The properties of these lactic acid bacteria are not harmful to the human body, and have traditionally been used in various fermented foods because of their advantages in food storage. In particular, polymers produced by lactic acid bacteria have recently been used as compounds useful in the health food and pharmaceutical industries (Maher Korakli and Rudi F. Vogel, App. Microbiol. Biotechnol., 71, 790-803, 2006).

Glucan sucrase (EC 2.4.1.5), on the other hand, is known as an enzyme that catalyzes the synthesis of D-glucose polymers, commonly called glucans, from sucrose in various lactic acid bacteria. In addition, enzymes mainly produced in Leuconostoc mesenteroides are dextransucrases, and enzymes commonly produced from Streptococcus species are also called glucosyltransferases (GTFs). The general structure of the enzyme consists of the N-terminal end, variable region, catalytic or sucrose binding domain, and C-terminal glucan binding domain with signal peptide. Glucan sucrose is produced mainly from lactic acid bacteria, such as Leuconostoc and Streptococcus , and then secreted extracellularly to protect cells from the external environment by synthesizing the surrounding sucrose substrate with polymer. These glucan sucrases are used in various fields, especially in the field of health applications, to synthesize oligosaccharides (FEMS Microbiology Reviews, 23, 131-151, 1999).

Glucan synthesized from sucrose has glucose units in the main chain of α- (1-2), α- (1-3), α- (1-4), and α- (1- 6) As homopolymers connected by various bonds, the type, ratio, and length of chain bonds vary depending on the type of glucan sucrase. Therefore, variously present glucan sucrase can synthesize various glucans according to the form of glucosidic linkages even though they have high similarity with each other. The glucans thus synthesized are classified into five groups as follows (S. Kralj et al ., Microbiology, 150, 3681-3690, 2004): (1) Synthesized mainly by α- (1-4) -glucosidic bonds Reuteran, (2) dextran, which has α- (1-6) -linked glucopyranosyl units as its backbone, (3) mutans with predominantly α- (1-3) linkages, (4) alteran alternating α- (1-6)-and α- (1-3) -linked glucopyranosyl units, (5) α- (1-2) linkages [α- ( 1-2, 6) glucan polymers mainly with branching points].

The lactic acid bacteria have traditionally been useful for the production of fermented foods, and glucan sucrose produced from lactic acid bacteria synthesizes compounds which are considered important not only in the food industry but also in the pharmaceutical industry, while having various metabolic potentials. The understanding of the mechanism of action of enzymes is not yet complete.

Accordingly, the present inventors have made efforts to understand the mechanism of action of glucan sucrase and to present various applicability. As a result, we cloned the glucan sucrase gene from leukonostock lactis EG001 isolated from kimchi lactic acid bacteria, and then cloned the gene. After expression in recombinant microorganisms, the activity of the expressed glucan sucrase was confirmed and the present invention was completed.

An object of the present invention is to provide a glucan sucrase derived from the leuconosstock lactis EG001, a gene encoding the glucan sucrase, and a recombinant vector containing the gene.

Another object of the present invention is to provide a recombinant microorganism into which the gene is introduced, and a method for producing glucan sucrase using the same.

In order to achieve the above object, the present invention is a glucan sucrase derived from leuconosstock lactis EG001 represented by the amino acid sequence of SEQ ID NO: 1, the gene encoding the glucan sucrase, recombinant containing the gene Provided are a vector and a recombinant microorganism into which the gene or the recombinant vector is introduced.

The present invention also provides a method for producing glucan sucrase, wherein the recombinant microorganism is cultured, followed by recovering glucan sucrase from the cultured microorganism.

Recombinant glucan sucrase according to the present invention can be utilized in the food, pharmaceutical industry, etc. by synthesizing polysaccharides using abundant carbohydrate resources such as sucrose, glucose. It is also expected to be used in other industries by applying to unknown reactions in the synthesis of glucan polymers.

In the present invention, the chromosomal DNA encoding the glucan sucrase from the lactic acid bacteria was cloned, and its nucleotide sequence and amino acid sequence of glucan sucrase were confirmed, and recombinant vectors and recombinant microorganisms were prepared based on the nucleotide sequences of the identified genes. After that, it was used to produce glucan sucrase useful in the food and pharmaceutical industries.

That is, in the present invention, strains with high glucan content were selected from the kimchi lactic acid bacteria (Phenol-sulfuric acid) (Meulenbeld and Hartmans Biotechnol. And Bioeng ., 70 (4), 2000) After chromosomal DNA was isolated from the strain, PCR was carried out with the template to amplify the glucans sucrase-related gene, confirm the nucleotide sequence of the gene, and prepare a recombinant vector containing the amplified gene. It was confirmed that glucan sucrase can be produced in large quantities by introducing into and inducing the expression of the glucan sucrase gene.

Therefore, in one aspect, the present invention provides a glucansucrase represented by the amino acid sequence of SEQ ID NO: 1, a gene encoding the glucan sucrase, a recombinant vector containing the gene, the gene or the recombinant The present invention relates to a recombinant microorganism having a glucan sucrase generating ability to which a vector is introduced.

In the present invention, the gene is represented by the nucleotide sequence of SEQ ID NO: 2, characterized in that derived from Leuconostoc lactis (EG001).

In the present invention, in order to find a novel glucan sucrase derived from lactic acid bacteria, kimchi lactic acid bacteria were collected, and among the above strains, strains with high glucan content were selected using the phenol-sulfuric acid method. The 16s rRNA sequences of the selected strains were confirmed, and the selected strains were named Leuconostoc Lactis EG001.

In the present invention, in order to secure the glucan sucrase gene from the strain, a degenerate primer was prepared to PCR amplify some sequences of the gene encoding the enzyme, and then determine the sequence. In addition, PCR was performed by preparing an inverse PCR primer from a partially found base sequence to know the entire sequence of the gene. As a result of performing the degenerate PCR and the inverse PCR, the base sequence of about 2.4 kb was determined from the expected length of the base sequence of about 4.5 kb. In order to determine the remaining sequence, the chromosomal DNA of Leuconostoc Lactis EG001 was cut randomly, cloned into the fosmid vector, and a library was prepared. The prepared library was transformed into EPI300-T1 E. coli strain (EPICENTRE), and colony PCR was selected to determine the presence of the gene by colony PCR. And DNA was obtained from the selected colonies to determine the nucleotide sequence. This was able to determine the glucan sucrase gene sequence expected to be about 4.5kb. As a result, it was found that glucan sucrase was composed of 4503 bases consisting of TTG as a start codon and TAA as a stop codon. The enzyme encoded by the gene was found to have a molecular weight of about 165 kDa and consist of 1500 amino acid sequences. In addition, the amino acid sequence of the enzyme was detected by NCBI BlastP, dextransucrase (DsrR) of Leuconostoc mesenteroides (AAN38835), dextransucrase ( DsrB ) of Leuconostoc mesenteroides ( CAB76565 ), and Leuconostoc citreum KM20 (YP_001727410, 70% of glucos 75%). , A novel glucans sucrase with a homology of 70%.

The present inventors prepared a primer from the gene sequence of the enzyme identified above to clone the glucan sucrase, PCR amplification of the glucan sucrase gene using the chromosomal DNA of Leuconostoc lactis EG001 as a template. The amplified gene was cloned between Nco I and Not I of the pET22b (+) expression vector.

In the present invention, the recombinant vector containing the gene may exemplify a protein expression vector such as pET22b (+), pET family vector, pHCE, pGEX family, pTRP, pMAL family, but is not limited thereto.

In the present invention, "vector" refers to a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA in a suitable host. Vectors can be plasmids, phage particles or simply potential genomic inserts. Once transformed into the appropriate host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself. Since plasmids are the most commonly used form of current vectors, "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention. For the purposes of the present invention, it is preferred to use plasmid vectors. Typical plasmid vectors that can be used for this purpose include (a) a replication initiation point that allows for efficient replication to include hundreds of plasmid vectors per host cell, and (b) host cells transformed with the plasmid vector. It has a structure comprising an antibiotic resistance gene and (c) a restriction enzyme cleavage site into which foreign DNA fragments can be inserted. Although no appropriate restriction enzyme cleavage site is present, the use of synthetic oligonucleotide adapters or linkers according to conventional methods facilitates ligation of the vector and foreign DNA.

After ligation, the vector should be transformed into the appropriate host cell. Transformation is described by Sambrook, et. al., easily achieved using the calcium chloride method described in section 1.82 of supra . Optionally, electroporation [Neumann, et. al., EMBO J. , 1: 841, 1982] may also be used for transformation of such cells.

Nucleic acids are "operably linked" when placed in a functional relationship with other nucleic acid sequences. This may be genes and regulatory sequence (s) linked in such a way as to enable gene expression when appropriate molecules (eg, transcriptional activating proteins) are bound to regulatory sequence (s). For example, DNA for a pre-sequence or secretion leader is operably linked to DNA for a polypeptide when expressed as a shear protein that participates in the secretion of the polypeptide; A promoter or enhancer is operably linked to a coding sequence when it affects the transcription of the sequence; Or the ribosomal binding site is operably linked to a coding sequence when it affects the transcription of the sequence; Or the ribosomal binding site is operably linked to a coding sequence when positioned to facilitate translation. In general, "operably linked" means that the linked DNA sequence is in contact, and in the case of a secretory leader, is in contact and present within the reading frame. However, enhancers do not need to touch. Linking of these sequences is performed by ligation (linking) at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers according to conventional methods are used.

The recombinant microorganism according to the present invention may be prepared by inserting the gene into a chromosome of the microorganism or introducing the recombinant vector into a host microorganism according to a conventional method.

In the present invention, the host microorganism is genus Agrobacterium , Aspergillus , Acetobacter , Aminobacter , Agromonas , Acidphilium , Bulleromyces , Bullera , Brevundimonas , Cryptococcus , Chionosphaera , Candida , Cerinosterus , Escherichia , Exisophiala in, Exobasidium in, Fellomyces in, Filobasidium genus, Geotrichum genus, Graphiola genus, Gluconobacter genus, Kockovaella in, Curtzmanomyces in, Lalaria in, Leucospoidium genus, Legionella genus, Psedozyma in, Paracoccus genus, Petromyc genus, Rhodotorula genus, Rhodosporidium genus, Rhizomonas in, Rhodobium in, Rhodoplanes genus Rhodopseudomonas genus Rhodobacter genus Sporobolomyces, A Spridobolus genus Saitoella genus Schizosaccharomyces genus Sphingomonas genus Sporotrichum genus, Sympodiomycopsis in, Sterigmatosporidium in, Tapharina genus Tremella genus, Trichosporon genus, Tilletiaria in, Tilletia genus, Tolyposporium in, Tilletiposis in, Ustilago genus, Udenlomyce in, Xanthophilomyces in, Xanthobacter Genus, Paecilomyces genus, Acremonium genus, Hyhomonus genus, Rhizobium genus, etc. can be exemplified, but is not limited thereto.

Recombinant microorganisms having a glucan sucrase production ability according to the present invention may be characterized in that the Leuconostoc Lactis EG001-derived gene or a recombinant vector containing the gene is inserted into the chromosome of the host microorganism. .

In one embodiment of the present invention, the recombinant E. coli strain [BL21 (DE3) / pET22b (+)] to which a recombinant vector containing a glucan sucrase gene derived from leuconostock lactis EG001 is introduced is cultured and cultured. It was confirmed that glucan sucrase can be produced in large quantities by crushing and preparing a glucan sucrase coenzyme solution and then purifying it.

That is, in order to produce a large amount of glucan sucrase derived from leukonostock lactis EG001, a recombinant vector (pET22b (+)) containing the glucan sucrase gene was introduced into E. coli BL21 (DE3). The transformed recombinant E. coli strain [BL21 (DE3) / pET22b (+)] was then shaken in LB liquid medium (100 μg / ml ampicillin added) to induce overexpression of the enzyme. The overexpression-induced culture was centrifuged after crushing with an ultrasonic crusher, and then an enzyme solution showing activity was obtained by using Ni-NTA affinity chromatography. Purified enzyme was confirmed by SDS-PAGE.

Therefore, in another aspect, the present invention relates to a method for producing glucan sucrase, wherein the glucan sucrase is recovered from the cultured microorganism after culturing the recombinant microorganism.

The optimum pH of glucan sucrase derived from Leukonostalk lactis EG001 prepared by the above method is 4.5 to 6, preferably 5, and the optimum temperature is 25 ~ 35 ℃, preferably about 30 ℃ do. In addition, the glucan sucrase may exhibit residual activity even in the range of 10 ~ 45 ℃.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1. Isolation and Screening of Strains

Kimchi lactic acid bacteria were collected to find new glucan sucrase. 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 ℃. Phenol-sulfuric acid was performed to isolate strains with high glucan content from collected Kimchi lactic acid bacteria (Meulenbeld and Hartmans Biotechnol. And Bioeng., 70 (4), 2000).

After the kimchi lactic acid bacteria were incubated, 1 ml of 75% ethanol was added to 100 µl of the culture solution, followed by precipitating and drying, 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 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 ° C. water bath, the tube was cooled at room temperature and then absorbed at 490 nm using a spectrophotometer. At this time, glucose was used as a standard.

Example 2. Leuconostoc lactis Gene glucan sucrase gene isolation and recombinant vector construction from EG001 strain

Strains with high glucan content selected in Example 1 were incubated in LB broth (100 µg / ml ampicillin), and then chromosomal DNA was extracted. In order to know the nucleotide sequence of the sample strain, extracted chromosomal 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).

[SEQ ID NO 3]: 5'-GAYAAYWSNAAYCCNRYNGTNC-3 '

[SEQ ID NO 4]: 5'-ADRTCNCCRTARTANAVNYKNG-3 '

PCR amplification was performed using a PCR reaction solution containing 2U Taq polymerase (Solgent), ₂ μl of 10-fold polymerase buffer (including MgCl ₂ ), 1 μl of 10 mM dNTP, 2 μl of each primer (200 uM), and 100 ng of template DNA (chromosome DNA). Μl) was prepared and performed using a gene amplifier.

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 min extension at 72 ° C. and maintained at 4 ° C. The obtained amplified fragment was cloned into the pGEM T-easy plasmid to determine the nucleotide sequence, and confirmed to have homology with a part of glucan sucrase through NCBI.

 An inverse PCR method was used to determine the overall sequence of the novel glucans sucrase gene. The primers of SEQ ID NOs: 5 and 6 were prepared from the nucleotide sequences obtained as a result of the Degenerate PCR.

[SEQ ID NO 5]: 5'-CACGATAGTGAAGTGCAAACG-3 '

[SEQ ID NO 6]: 5'-ATCGTTGGCAGTAATCGAGC-3 '

After chromosomal DNA was treated with Hind III restriction enzyme for PCR amplification, 500 ng of chromosomal DNA treated with restriction enzyme was used for self-ligation.

PCR amplification was performed using a PCR reaction solution containing 2U EF Taq polymerase (Solgent), 5 μl of 10-fold polymerase buffer (including MgCl ₂ ), 1 μl of 10 mM dNTP, 2.5 μl of each primer (20 mM), and 25 ng of template DNA (chromosomal DNA). 50 μl) was prepared and performed using a gene amplifier.

The PCR reaction was denatured at 95 ° C. for 3 minutes, followed by denaturation at 95 ° C. for 30 seconds, annealing at 50 ° C. for 1 minute, and extension at 72 ° C. for 1 minute, 30 times. And finally a 10 min extension at 72 ° C. and maintained at 4 ° C. The obtained amplified fragment was cloned into the pGEM T-easy plasmid to determine the nucleotide sequence, and confirmed some sequences of glucan sucrase. The confirmed sequence was combined with the nucleotide sequence obtained by performing degenerate PCR, and 2 ^nd inverse PCR was performed after ^preparing the primers of SEQ ID NOs: 7 and 8 to know the remaining nucleotide sequences.

[SEQ ID NO 7]: 5'-ATAGCTTAGCACCAACAACAGAAC-3 '

[SEQ ID NO 8]: 5'-GTTTCGCTGGCCTTGTTCA-3 '

For PCR amplification, chromosomal DNA was treated with Nco I restriction enzyme, and then 500 ng of restriction enzyme treated chromosomal DNA was used for self-ligation.

PCR amplification was performed using a PCR reaction solution containing 2U EF Taq polymerase (Solgent), 5 μl of 10-fold polymerase buffer (including MgCl ₂ ), 1 μl of 10 mM dNTP, 2.5 μl of each primer (20 mM), and 25 ng of template DNA (chromosomal DNA). 50 μl) was prepared and performed using a gene amplifier.

The PCR reaction was denatured at 95 ° C. for 3 minutes, followed by denaturation at 95 ° C. for 30 seconds, annealing at 50 ° C. for 1 minute, and extension at 72 ° C. for 1 minute, 30 times. And finally a 10 min extension at 72 ° C. and maintained at 4 ° C. The obtained amplified fragment was cloned into pGEM T-easy plasmid to request nucleotide sequence determination, and further confirmed some sequences of glucan sucrase. The identified sequences were combined into 1 ^st inverse PCR results.

Degenerate PCR, the first inverse PCR, and the second inverse PCR were performed to confirm the gene sequence of about 2.4 kb in the sequence of the glucan sucrase gene of about 4.5 kb. However, to secure the rest of the sequence, the fosmid library was prepared using the Copycontrol Fosmid Library Production Kit (the fosmid library was manufactured according to Epicentre's manual), and the colony PCR was performed to confirm that the insert was properly inserted. Fosmid was then purified. The primers of SEQ ID NOs: 9 and 10 were prepared before performing Colony PCR.

[SEQ ID NO 9]: 5'-ACTGTGGCTTTCAACACCCA-3 '

[SEQ ID NO 10]: 5'-TGTGCGCGCATCTTGGGTA-3 '

100 ng of fosmid library was diluted in D.W. for PCR amplification.

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 at 94 ° C. for 1 minute, and then denatured at 94 ° C. for 30 seconds, and repeated annealing at 68 ° C. for 15 minutes 30 times. 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. As a result, the entire nucleotide sequence of 4.5kb was confirmed.

NCBI BlastP search results, the enzyme is Leuconostoc mesenteroides (AAN38835) of dextransucrase (DsrR), Leuconostoc mesenteroides ( CAB76565) of dextransucrase (DsrB) and Leuconostoc citreum, respectively 75% to glucosyltransferase in KM20 (YP_001727410), 70% and 70% of the of It was identified as a novel glucans sucrase with homology.

In order to mass produce glucan sucrase derived from the strain Leuconostoc lactis EG001 in Escherichia coli (DH5α), the isolated glucan sucrase gene was inserted into the Nco I- Not I site of pET-22b (+) as an expression vector and recombined. DNA was prepared. For PCR amplification of glucan sukrase, chromosomal DNA of Leuconostoc lactis EG001 strain was isolated, and a reverse primer of SEQ ID NO: 12, in which a forward primer of SEQ ID NO: 11 into which a NcoI site was inserted, and a Not I site, were inserted.

[SEQ ID NO 11]: 5'-AT CCATGG ATAGTAACCAAAACACAACTGGTT-3 '

[SEQ ID NO 12]: 5'-AT GCGGCCGC CATATTGACGAGATCACCGTTG-3 '

PCR amplification was carried out using a PCR reaction solution containing 2U Taq polymerase (Solgent), 5 μl of 10-fold polymerase buffer (including MgCl ₂ ), 1 μl of 10 mM dNTP, 5 μl of each primer (2 uM), and 100 ng of template DNA (chromosome DNA). Μl) was prepared and performed using a gene amplifier.

The PCR reaction was denatured at 95 ° C. for 5 minutes, followed by 30 seconds of denaturation at 95 ° C., 30 seconds of annealing at 55 ° C., and 2 minutes extension at 72 ° C. for 30 times. Finally a 10 minute extension was performed at 72 ° C. and maintained at 4 ° C. PCR products obtained by the above method was to then cloning to pGEM T-easy vector (Promega) by treatment with Nco I- Not I subcloning in pET-22b (+) expression vector of the Nco I- Not I site prepare a recombinant vector .

Example 3. Expression and Purification of Glucan Sucrase by Recombinant Escherichia Coli Culture

For overproduction of glucan sucrase, the recombinant vector prepared in Example 2 was introduced into Escherichia coli [BL21 (DE3)] (Novagen), and the resulting transformant was added with 100 µg / ml ampicillin. Shake culture was performed at 200 rpm for 16 hours at 37 ℃ in LB liquid medium. Thereafter, when the absorbance value was 0.5 at 600 nm, IPTG was added to 0.1 mM, followed by shaking culture at 200 rpm for 12 hours at 20 ° C. to induce excessive production of enzyme. The culture was centrifuged to recover the precipitated cells and washed twice with 0.85% NaCl. The recovered cells were added with 1 mM PMSF (Phenylmethylsulfonyl fluoride), protease inhibitor cocktail (Roche), and 100 mM Tris-Cl (pH7.4) added with 10 mM imidazole. The supernatant was purified by chromatography using Ni-NTA agarose.

Enzyme purification was confirmed by SDS-PAGE after enzymatic activity with an eluate containing 250 mM imidazole, 300 mM NaCl, and 20 mM Tris-Cl (pH 8.0) (CBB (Coomasie Brilliant Blue) staining (Fig. 1). ).

As a result, as shown in Figure 1, the purified glucan sucrase (lane 4) of 165kDa size was confirmed.

The purified enzyme was concentrated in Amicon Ultra-15 (Millipore, 50K NMWL device), and then placed in 100mM Tris-Cl (pH 7.4) solution containing 20% glycerol for dialysis at 4 ° C for 12 hours. Was performed.

In order to confirm the standard enzymatic reaction of purified glucan sucrase, 100 mM sucrose, 50 mM Sodium acetate (pH 5.2), 1 mM CaCl ₂ , and appropriately diluted purified enzyme were reacted to a final volume of 100 μl. Thereafter, heat was applied at 100 ° C. for 5 minutes to terminate the reaction. 100 μl of DNS (3,5-dinitrosalicylic acid) solution was added to the reaction solution, and heat was applied at 100 ° C. for 5 minutes. After cooling for 5 minutes on ice, 1 ml of DW was added and the absorbance was measured at wavelength 575 nm. The unit of enzyme (U) means the number of μmole of fructose produced per minute by sucrose degradation. Table 1 shows the results of the activity and recovery rate of the glucan sucrase purification step derived from Leuconostoc lactis EG001 strain.

TABLE 1

Total vol.
(ml) Total
protein (mg) Total
activity (U) Specific activity (U / mg) Yield (%) Purification
(Fold) Culture
supernatant 4 12 6.8 x 10 ³ 569.4 100 One Ni-NTA affinity chromatography 1.3 3.25 4.2 x 10 ³ 1291.2 61 2.3

Example 4 Biochemical Properties of Purified Glucan Sucrase

In order to determine the optimal reaction conditions of glucans sucrase purified from the Leuconostoc lactis EG001 strain of Example 3, the effects of reaction temperature and pH were investigated by DNS method (Gail Lorenz Miller, Analytical Chemistry, 31 (3), 426). -428). Enzyme reaction was carried out in the same manner as in Example 3, using a purified enzyme to determine the optimum reaction temperature was measured relative activity at 5 ℃ interval in the range of 10 ~ 50 ℃. As a result, as shown in Figure 2, the optimum reaction temperature of the enzyme was 30 ℃, it was confirmed that the relatively high activity at 25 ℃ ~ 35 ℃.

In addition, different buffers were used in the standard enzyme reaction to determine the optimal pH of the purified enzyme. 50 mM sodium acetate buffer (pH 3-6), 50 mM sodium phosphate buffer (pH 6-8), and 50 mM Tris-Cl buffer (pH 8-9) were used for the determination of the optimal pH. As a result of investigating the relative activity in each pH buffer, as shown in FIG.

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 designated temperature, as shown in Figure 4, it was confirmed that the residual activity remains high at 30 ℃ or less.

As described above in detail the specific parts of the present invention, it is apparent to those skilled in the art that such specific description is merely a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

1 is a photograph showing the results of staining glucan sucrase recovered from a recombinant microorganism according to an embodiment of the present invention after SDS-PAGE, CBB (Coomasie Brilliant Blue) staining (M: molecular weight marker, lane 1: Whole cell without induction, lane 2: whole cell with induction, lane 3: cell extract, lane 4: enzyme purified by Ni-NTA affinity chomatography).

Figure 2 is a graph confirming the optimum temperature of glucan sucrase recovered from a recombinant microorganism according to an embodiment of the present invention.

Figure 3 is a graph confirming the optimal pH of glucan sucrase recovered from the recombinant microorganism according to an embodiment of the present invention (open square: pH 3-6, closed square: pH 6-8, open triangle: pH 8 ~ 9).

Figure 4 is a graph confirming the residual activity (heat stability) for the heat of the glucan sucrase recovered from the recombinant microorganism according to an embodiment of the present invention.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Glucansucrase Derived from Leuconostoc lactis and Method for          Preparing the Same <130> P09-B043 <160> 12 <170> KopatentIn 1.71 <210> 1 <211> 1500 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of glucansucrase <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> Artificial Sequence <220> <223> base sequcence of glucansucrase <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> PRT <213> Artificial Sequence <220> <223> primer <400> 3 Gly Ala Tyr Ala Ala Tyr Trp Ser Asn Ala Ala Tyr Cys Cys Asn Arg   1 5 10 15 Tyr Asn Gly Thr Asn Cys              20 <210> 4 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> primer <400> 4 Ala Asp Arg Thr Cys Asn Cys Cys Arg Thr Ala Arg Thr Ala Asn Ala   1 5 10 15 Val Asn Tyr Lys Asn Gly              20 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 cacgatagtg aagtgcaaac g 21 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 atcgttggca gtaatcgagc 20 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 atagcttagc accaacaaca gaac 24 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 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> primer <400> 11 atccatggat agtaaccaaa acacaactgg tt 32 <210> 12 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 atgcggccgc catattgacg agatcaccgt tg 32  

Claims (12)

Glucansucrase represented by the amino acid sequence of SEQ ID NO: 1. The glucan sucrase according to claim 1, wherein the optimum pH of the glucan sucrase is 4.5 to 6 and the optimum temperature is 25 to 35 ° C. The gene encoding the glucansucrase of claim 1. The gene according to claim 3, wherein the gene is represented by the nucleotide sequence of SEQ ID NO: 2. The gene according to claim 3 or 4, wherein the gene is derived from Leuconostoc Lactis EG001. Recombinant vector containing the gene of claim 3. delete Recombinant bacteria having a glucan sucrase generating ability, characterized in that the recombinant vector of claim 6 is inserted into the bacteria. delete delete The recombinant bacterium of claim 8, wherein the recombinant bacterium is E coli BL21 (DE3) / pET22b (+). After culturing the recombinant bacterium of claim 8, the method for producing glucan sucrase, characterized in that to recover the glucan sucrase (glucansucrase) from the cultured recombinant bacteria.

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