KR101112732B1 - Novel Sucrose Phosphorylase Derived from Leuconostoc lactis - Google Patents

Abstract

Leuconostoc lactis EG001 ( Leuconostoc The present invention relates to a novel sucrose phosphorylase derived from lactis EG001, and more specifically, to lactic acid bacterium leuconosstock lactis EG001 ( Leuconostoc ) isolated from kimchi. sucrose phosphorylase derived from lactis EG001) and a gene encoding the same.

Leuconostoke lactis EG001 according to the present invention ( Leuconostoc The new sucrose phosphorylase derived from lactis EG001) can be used in cosmetics and food applications because it can enhance the safety and efficacy of ingredients by transferring glycosides to produce glycosides.

Sucrose, kimchi, lactic acid bacteria, enzyme, gene, recombinant, vector

Description

Novel Sucrose Phosphorylase Derived from Leuconostoc lactis}

The present invention is leukonostock lactis EG001 (Leuconostoc lactis A novel sucrose phosphorylase derived from EG001), and more specifically, the lactic acid bacterium leukonostock lactis EG001 (isolated from kimchi)Leuconostoc lactis EG001) from sucrose phosphorylase and the gene encoding the same.

Sucrose Phosphorylase EC 2.4.1.7 is an enzyme belonging to the glycosyl hydrolases family 13 (α-amylase family). Sucrose and inorganic phosphates are converted to D-fructose. The enzyme is involved in the conversion of α-D-glucose-1-phosphate (G1P) and its reverse reaction to the glycosyl transfer reaction in addition to phosphorolysis. Catalyzes (John J. Mieyal et al ., The enzymes ., 7: 515-532, 1972).

Sucrose phosphorylase is Pseudomonas saccharophila), P. puterfaciens, flow Stock Kono (Leuconostoc mesenteroides), Streptococcus (Streptococcus mutans), Bifidobacterium (Bifidobacterium adolescentis DSM20083) It was found to be present in a variety of bacteria. The catalysis mechanism of sucrose phosphorylase was revealed through the third structure analysis of sucrose phosphorylase isolated from the genus Bifidobacterium adolescentis . Catalytic-site residues of sucrose phosphorylase of the genus Bifidobacterium adolescentis were identified as catalytic nucleophile Asp ¹⁹⁶ , catalytic acid / base Glu ²³⁷ and Asp ²⁹⁵ . On the basis of this, pairwise suquence alignment was performed to match the catalytic residues of sucrose phosphorylase of L. mesenteroides and sucrose phosphorylase of Bifidobacterium adolescentis . (Christiane Goedl et al ., Cabohydrate Research , 343: 2032-2040, 2008).

In general, the glycolysis reaction proceeds from a specific donor to a number of structurally diverse receptors. Sucrose, α-D-glucose-1-phosphate (G1P), and glucose-1-fluorise are the only sugars in sucrose phosphorylase. It is known that it can act as a donor, but sugars and sugar alcohols are known as receptors, and in addition, it is reported that glucose groups of sucrose can be transferred to phenol compounds such as catechin and alcohol compounds such as benzene alcohol. Biosci . Biotechnol . Biochem ., 56: 2011-2014, 1992).

Sucrose phosphorylase can be converted into glycosides by transferring sugars to catechin, α-arbutin, and ascorbic acid, and the function of the product by applying such glycosides to cosmetics or food fields. However, it is expected that the stability can be improved, and development of sucrose phosphorylase having activity under various physical conditions is required.

Accordingly, the present inventors have made efforts to develop a new sucrose phosphorylase. As a result, the present inventors cloned the gene of sucrose phosphorylase from lactic acid bacterium Leukonostoc lactis EG001 isolated from kimchi, and sequenced it and thereby. After determining the amino acid sequence of the enzyme to be translated from, it was confirmed that the novel enzyme to complete the present invention.

A main object of the present invention is to provide a sucrose phosphorylase derived from Leuconostoc lactis EG001, a gene encoding the sucrose phosphorylase, and a recombinant vector containing the gene, isolated from kimchi. have.

Another object of the present invention is to provide a recombinant microorganism transformed with a recombinant vector or in which the sucrose phosphorylase gene is inserted on a chromosome.

Another object of the present invention is leukonostoc lactis EG001 ( Leuconostoc lactis EG001) or culturing the recombinant microorganism to produce sucrose phosphorylase; And it provides a method for producing sucrose phosphorylase comprising the step of recovering the produced sucrose phosphorylase.

In order to achieve the above object, the present invention is a sucrose phosphorylase represented by the amino acid sequence of SEQ ID NO: 1 derived from Leuconostoc lactis EG001, a gene encoding the sucrose phosphorylase, the Provided is a recombinant vector containing the gene.

The present invention also provides a recombinant microorganism transformed with a recombinant vector or in which the sucrose phosphorylase gene is inserted on a chromosome.

The invention also relates to leuconostock lactis EG001 ( Leuconostoc lactis EG001) or culturing the recombinant microorganism to produce sucrose phosphorylase; And it provides a method for producing sucrose phosphorylase comprising the step of recovering the produced sucrose phosphorylase.

Leuconostoke lactis EG001 according to the present invention ( Leuconostoc The new sucrose phosphorylase derived from lactis EG001) can be used in cosmetics and food applications because it can enhance the safety and efficacy of ingredients by transferring glycosides to produce glycosides.

In one aspect, the invention relates to sucrose phosphorylase represented by the amino acid sequence of SEQ ID NO: 1.

In the present invention, "Sucrose Phosphorylase" is an important enzyme related to the metabolite of sucrose and other metabolites, and sucrose phosphatase is a sucrose fructose and α-D-glucose-1-. It catalyzes the reaction to convert to phosphate (α-D-glucose-1-phosphate, G1P).

The sucrose phosphorylase of the present invention exhibited an optimum activity at 45 ° C and an optimum pH of 7, and was found to have high residual activity at 40 ° C or lower.

In another aspect, the present invention relates to a gene encoding the sucrose phosphorylase.

In the present invention, the gene of sucrose phosphorylase is composed of 1455 bases, and the enzyme encoded from the gene has been identified as having a molecular weight of about 58 kDa and consisting of 484 amino acid sequences. In addition, the amino acid sequence of the enzyme is NCBI BLAST search results, Leuconostoc It was found to be a novel sucrose phosphorylase with homology of 89%, 78% and 63%, respectively, with the sucrose phosphorylase of citreum KM20, Leuconostoc mesenteroides and Lactobacillus johnsonii NCC 533.

In the present invention, the gene may be characterized by being represented by the nucleotide sequence of SEQ ID NO: 2.

In the present invention, the gene may be characterized as derived from Leuconostoc lactis EG001.

In another aspect, the present invention relates to a recombinant vector containing a gene encoding sucrose phosphorylase.

By "vector" is meant a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA in a suitable host. The vector may be a plasmid, phage particles or simply a potential genomic insert. 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 currently the most commonly used form of vectors, in the context of the present invention "plasmids and vectors" are sometimes used interchangeably. In the present invention, it is preferable to use a plasmid vector.

As the method for inserting the gene on the chromosome of the host cell in the present invention can be used a commonly known genetic engineering method, for example retrovirus vector, adenovirus vector, adeno-associated virus vector, herpes simplex virus vector , Poxvirus vectors, lentiviral vectors or non-viral vectors.

Non-viral delivery methods for inserting the sucrose phosphorylase gene onto chromosomes include cell perforation, lipofection, microinjection, ballistic methods, virosomes, liposomes, immunoliposomes, polyvalent cations or lipids: nucleic acid conjugates, naked DNA, Artificial viron and chemically-promoted DNA influx. Sonoporation, for example methods using the Sonitron 2000 system (Rich-Mar), can also be used for the delivery of nucleic acids. Other representative nucleic acid delivery systems are Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Maryland). And BTX Molesular Syetem (Holliston, Mass.). Lipofection methods are described in US Pat. No. 5,049,386, US Pat. No. 4,946,787, and US Pat. No. 4,897,355 and lipofection reagents are commercially available, for example, Transfectam ^™. And Lipofectin ^™ . Suitable cations or neutral lipids for effective receptor-recognition lipofection of polynucleotides include lipids from Felgner (WO91 / 17424 and WO91 / 16024) and can be delivered to cells via in vitro introduction and to target tissues via in vivo introduction. have. Methods for preparing lipid: nucleic acid complexes, including target liposomes, such as immunolipid complexes, are well known in the art (Crystal, Science ., 270: 404-410, 1995; Blaese et. al ., Cancer Gene Ther ., 2: 291-297, 1995; Behr et al., Bioconjugate Chem ., 5: 382389, 1994; Remy et al ., Bioconjugate Chem ., 5: 647-654, 1994; Gao et al ., Gene Therapy ., 2: 710-722, 1995; Ahmad et al ., Cancer Res ., 52: 4817-4820, 1992; US Patent No. 4,186,183; US Patent No. 4,217,344; US Patent No. 4,235,871; US Patent No. 4,261,975; US Patent No. 4,485,054; US Patent No. 4,501,728; US Patent No. 4,774,085; US Patent No. 4,837,028; US Patent No. 4,946,787).

The affinity of retroviruses can be altered by integrating with outer envelope proteins, thereby expanding the type of target cell. Lentiviral vectors are retroviral vectors that generate high viral titers by transducing or infecting non-dividing cells. The target tissue determines the retroviral gene persistence system. Retroviral vectors contain cis acting long terminal repeats that can pack 6-10 kb outer sequences. Minimal cis acting LTRs sufficient for replication and packaging of the vector can be used to integrate the therapeutic gene into target cells for permanent transgene expression. Widely used retroviral vectors include murine leukemia virus (MuLV), gibbon leukemia virus (GaLV), monkey immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combination viruses thereof (Buchscher et al. al ., J. Virol ., 66: 2731-2739, 1992; Johann et al ., J. Virol ., 66: 1635-1640 1992; Sommerfelt et al ., Virol ., 176: 58-59, 1990; Wilson et al ., J. Virol ., 63: 2374-2378, 1989; Miller et al ., J. Virol ., 65: 2220-2224, 1991; PCT / US94 / 05700).

Transient expression of sucrose phosphorylase proteins is more common with adenovirus-based systems, and adenovirus-based vectors cause high efficiency transduction in many cells but do not require cell division. The vector allows for high titers and high levels of expression and can be produced in large quantities in a simple system. Adeno-associated virus (AAV) vectors are also used to transduce into cells with target nucleic acids, for example for the production of nucleic acids and peptides in vitro and for gene therapy in vivo and in vitro (West et). al ., Virology., 160: 38-47, 1987; US Patent No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy ., 5: 793-801, 1994; Muzyczka, J. Clin . Invest ., 94: 1351, 1994), the construction of recombinant AAV vectors is already known (US Pat. No. 5,173,414; Tratschin et al ., Mol . Cell . Biol ., 5: 3251-3260, 1985; Tratschin, et. al ., Mol . Cell . Biol ., 4: 20722081, 1984; Hermonat & Muzyczka, PNAS., 81: 6466-6470, 1984; Samulski et al ., J Virol ., 63: 038223828, 1989). In clinical trials, gene transfer methods using at least six viral vectors are used, which complement the defective vector by inserting the gene into a helper cell line that generates transgenes. pLASN and MFG-S are examples of retroviruses used in clinical trials (Dunbar et al ., Blood ., 85: 3048-305, 1995; Kohn et al ., Nat . Med ., 1: 1017-102, 1995; Malech et al ., PNAS ., 94: (22) 12133-12138, 1997), PA317 / pLASN was the first therapeutic vector used in gene therapy (Blaese et. al ., Science ., 270: 475-480, 1995) The transduction efficiency of MFG-S packaging vector was 50% or higher (Ellem et al., Immunol Immunother ., 44 (1): 10-20, 1997; Dranoff et al ., Hum . Gene Ther ., 1: 111-2, 1997).

Recombinant adeno-associated virus vectors (rAAV) are promising alternative gene delivery systems based on defective and nonpathogenic type 2 parvovirus adeno-associated viruses. All vectors are derived from plasmids with AAV 145 bp inverted terminal repeats flanking the transgene expression cassette. Efficient gene delivery and stable transgene delivery due to integration into the genome of transduced cells is a great advantage of the vector system (Wagner et. al ., Lancet ., 351: 9117-17023, 1998; Kearns et al ., Gene Ther., 9: 748-55, 1996). Replication deficient recombinant adenovirus vectors (Ad) can be produced at high titers and infect many cells. Most adenovirus vectors are delivered to human 293 cells where the transgene is designed to replace the Ad E1a gene, the Ad E1b gene, and the Ad E3 gene, and in fact provides a gene function where the replication defective vector is removed in the trans. Ad vectors can be transduced into many forms of in vivo tissue, including non-dividing, differentiated cells found in liver, kidney and muscle. Conventional Ad vectors have a large carrying capacity. Examples of use of Ad vectors in clinical trials include polynucleotide therapy for anti-tumor immunization via intramuscular injection (Sterman et al. al ., Hum . Gene Ther., 7: 1083-9, 1998).

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.

In another aspect, the present invention relates to a recombinant microorganism transformed with a recombinant vector or wherein the sucrose phosphorylase gene is inserted on a chromosome.

In another aspect, the present invention comprises the steps of culturing the recombinant microorganism to produce sucrose phosphorylase; And it relates to a method for producing sucrose phosphorylase comprising the step of recovering the produced sucrose phosphorylase.

In the present invention, the recombinant microorganism transformed with the sucrose phosphorylase gene was cultured in an LB liquid medium to produce endoglucanase, the cells were recovered from the culture solution, and the supernatant was recovered to recover the enzyme. . The supernatant was subjected to chromatography through a Ni-NTA agarose carrier to recover endoglucanase.

Cultivation of the recombinant strain according to the present invention can be carried out according to well-known methods, conditions such as the culture temperature and time, the pH of the medium can be appropriately adjusted. Recovery of cellulase from the culture medium of the recombinant strain may be carried out using conventional separation techniques, for example, salting out, molecular weight cutting, chromatography, affinity chromatography, distillation, electrodialysis, pervaporation, solvent extraction, reaction extraction, and the like. In general, a combination of these may be used to separate materials of high purity.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited to these examples.

Example  1: Screening and Isolation of Strains

To isolate strains with high sucrose phosphorylase activity in kimchi, dilute solutions of kimchi were prepared using 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 inoculated at 30 ℃ Lactobacillus was collected.

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 and cooling the tube at room temperature, the absorbance was measured at 490 nm using a spectrophotometer, and the sucrose hydrolyzation and glycosylation activity was excellent, and sucrose phosphorylase activity was observed. This excellent EG001 strain was selected and classified into leuconostock lactis through 16S rRNA analysis.

Example  2: leukonostock Lactis EG001  ( Leuconostoc lactis EG001 ) Sucrose from strain Phosphorylase  Gene isolation

Leukonostock lactis EG001 ( Leuconostoc lactis EG001) strain was shaken in a liquid medium used for strain selection, and chromosomal DNA was isolated from the recovered cells. Encodes sucrose phosphorylase Some sequences of genes were amplified using degenerate PCR. Oligonucleotide primers were selected from highly conserved sites by comparing the amino acid sequences of various strains of sucrose phosphorylase.

PCR was used to isolate the sucrose phosphorylase gene. The primers of the following nucleotide sequence 3 and nucleotide sequence 4 were prepared and PCR was performed.

SEQ ID NO: 5'-TTYGAYTTYATGATHAAYCAYAT-3 '

Where Y is T / U or C, H is A or C or T / U

SEQ ID NO: 5'-NAGRTTNGCNCCNACYTTRTA-3 '

Where N is A or G or C or T / U, R is G or A, Y is T / U or C

The reaction solution for PCR was composed of 100ng chromosomal DNA, 5μM primer, 200nM dNTP, 2U Taq polymerase (Solgent), 1x Taq DNA polymerase buffer (MgCl ₂ ) in 20μl and used for PCR amplification. The PCR reaction was denatured at 95 ° C. for 3 minutes, followed by 45 seconds of denaturation at 95 ° C., annealing at 50 ° C., and 1 minute extension at 72 ° C. for 30 times. Finally a 10 minute extension was performed at 72 ° C. and maintained at 4 ° C. The obtained amplified fragment was cloned into pGEM-Teasy plasmid to request nucleotide sequence determination, and it was confirmed that it was a partial sequence of sucrose phosphorylase gene.

Inverse PCR was used to determine the overall sequence of the sucrose phosphorylase gene. Primers (SEQ ID NOs: 5, 6) were prepared from some sequences of sucrose phosphorylase identified from the PCR.

SEQ ID NO: 5'-ATGACTTTGCGTTGCCATTGATTAC-3 '

SEQ ID NO: 5'-GTTGGTCACCGAATGTGTTCCA-3 '

For PCR amplification, the chromosomal DNA was treated with various restriction enzymes, and then 500 ng of restriction enzyme-treated chromosomal DNA was self-ligation. The reaction solution for PCR was composed of 25ng chromosomal DNA, 2μM primer, 200nM dNTP, 2U r Taq polymerase (Takara), 1x Taq DNA polymerase buffer (MgCl ₂ ) and used for PCR amplification. . The PCR reaction was denatured at 94 ° C. for 3 minutes, followed by 30 seconds of denaturation at 94 ° C., annealing at 60 ° C., and extension of 2 minutes 30 seconds at 72 ° C. for 30 minutes. Finally a 10 minute extension was performed at 72 ° C. and maintained at 4 ° C. The obtained amplified fragment was cloned into the pGEM-Teasy plasmid, and the base sequence was determined to confirm the entire sequence (SEQ ID NO: 2) of the sucrose phosphorylase gene.

Example  3: sucrose Phosphorylase  Recombinant vector containing genes

The sucrose phosphorylase gene was prepared by forward primers from the gene sequences identified above and inserted into XhoI and BamHI, respectively.

SEQ ID NO: 7: 5'-GACTCGAGATGAAAAAGCTCACCAATGAAGTG-3 '

SEQ ID NO: 8'-TAGGATCCTTAAGCCTGTGTGTTGAAAATTTC-3 '

PCR amplification solution for amplifying the gene was 100ng chromosomal DNA, 0.2μM primer, 200nM dNTP, 2U Taq polymerase (Solgent), 1x Taq DNA polymerase (MgCl ₂₎ Including buffer) was used to amplify the PCR. The PCR reaction was performed for 5 minutes at 95 ° C, followed by 30 seconds of denaturation at 95 ° C, 30 seconds of annealing at 55 ° C, and 2 minutes extension at 72 ° C. Finally a 10 minute extension was performed at 72 ° C. and maintained at 4 ° C. The PCR product obtained by the above method was cloned into the pGEM T-easy vector and treated with NcoI-NotI to subcloning the XhoI-BamHI site of the expression vector pET302 / NT-his to construct a recombinant vector.

Example  4: recombinant sucrose Phosphorylase  Expression and Purification

For overproduction of sucrose phosphorylase, a recombinant plasmid was introduced into Escherichia coli [BL21 (DE3)] to construct a transformant, and the resulting transformant was transferred to LB liquid medium containing 100 µg / ml ampicillin. Shake culture at 200 rpm for 16 hours at ℃. After shaking the transformant cultured in a fresh LB liquid medium at 1% concentration and shake culture. In addition, when the absorbance value was 0.5 at 600 nm, IPTG was added so as to be 0.01 mM, followed by shaking culture at 180 ° C. for 16 hours at 20 ° C. to induce excessive production of enzyme. The culture was centrifuged to recover the precipitated cells and washed with 0.85% NaCl. 100 mM Tris-HCl (pH 8.0) added with 1 mM PMSF and 10 mM imidazole was added to the recovered cells and disrupted by ultrasonication. The supernatant was recovered by centrifugation, and the enzyme was purified. The supernatant was chromatographed by passing through a Ni-NTA agarose carrier. The eluate containing 250 mM imidazole, 300 mM NaCl, 20 mM tris-HCl (pH 8.0) during the elution of the enzyme was confirmed by SDS-PAGE for purification for the enzymatic activity fraction (FIG. 1). The purified enzyme was concentrated with Amicom Ultra-15 (Millipore, 50K NMWL device), and then dialyzed in 100 mM Tris-HCl (pH 8.0) solution containing 20% glycerol and protease inhibitor cocktail (Roche) for 12 hours at 4 ° C. Was performed.

Standard enzymatic reaction of purified sucrose phosphorylase was carried out to 100 mM sucrose, 50 mM potassium phosphate (pH 7.0), and a properly diluted purified enzyme to a final volume of 100 μl. Thereafter, heat was added at 100 ° C. for 5 minutes to terminate the reaction. 100 μl of DNS solution was added to the finished enzyme reaction solution, followed by heating at 100 ° C. for 5 minutes. After cooling for 5 minutes on ice, 1 ml of distilled water was added, and then the absorbance was measured at 575 nm. Unit (U) of the enzyme shows the number of μmol of fructose produced per minute when sucrose is broken down (Table 1).

Leukonostock lactis EG001 ( Leuconostoc activity and recovery rate of the sucrose phosphorylase purification step derived from lactis EG001) strain Total vol. Μl (ml) Total protein (mg) Total activity (U) Specific activity (U / mg) Yield
(%) Purification (Fold) Culture suprernatant 7.5 30 9.6 x 10 ² 32 100 One Ni-NTA affinity chromatography 0.42 3.5 4.4 X 10 ² 126 45 3.9

Example  5: purified sucrose Phosphorylase  Biochemical properties

Leukonostock lactis EG001 ( Leuconostoc The effects of reaction temperature and pH were investigated by DNS method to determine the optimal reaction conditions of sucrose phosphorylase purified from lactis EG001) strain (Gail Lorenz Miller, Analytical). Chemistry , 31 (3): 426-428). The enzymatic reaction of purified sucrose phosphorylase was reacted with 100 mM sucrose, 50 mM Potassium phosphate (pH 7.0), and a properly diluted purified enzyme to a final volume of 100 μl. Thereafter, heat was added at 100 ° C. for 5 minutes to terminate the reaction. 100 μl of DNS solution was added to the reaction solution, and heat was applied at 100 ° C. for 5 minutes. Then, the mixture was cooled on ice for 5 minutes, 1 ml of DW was added, and the absorbance was measured at 575 nm.

In order to determine the optimum reaction temperature, the relative activity was measured at intervals of 5 ° C. in the range of 25˜80 ° C. using purified enzyme, and it was found that the optimum reaction temperature was 45 ° C. (FIG. 2A). In addition, different buffers were used in the standard enzyme reaction to determine the optimal pH of the purified enzyme. The buffer used for determination of the optimal pH was 50 mM citric acid / Na ₂ HPO ₄ buffer (pH 5.0-6.5), 50 mM sodium phosphate buffer (pH 6.5-8.0), and Tris-Cl buffer (pH8.0-9.0). It was. Investigation of the relative activity in each pH buffer showed maximum activity at pH 7 (FIG. 2B). In addition, the remaining activity was measured in the range of 25 ~ 50 ℃ to investigate the stability to heat. As a result of confirming the residual activity after 1 hour at each designated temperature, it was confirmed that the residual activity remained high at 40 ° C. or lower (FIG. 2C).

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

1 is a diagram showing the SDS-PAGE results of sucrose phosphorylase purified from recombinant E. coli.

2 is Leukonostactactis EG001 ( Leuconostoc lactis EG001) is a graph showing the biochemical properties of sucrose phosphorylase isolated from strains.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Novel Sucrose Phosphorylase Derived from Leuconostoc lactis <130> P09-B124 <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 484 <212> PRT <213> Leuconostoc lactis <400> 1 Met Lys Lys Leu Thr Asn Glu Val Met Leu Ile Thr Tyr Ala Asp Ser   1 5 10 15 Leu Gly Lys Asn Leu Lys Glu Leu Asp Gln Val Leu Ser Asn Glu Leu              20 25 30 Asp Gly Val Val Gly Gly Val His Val Leu Pro Phe Phe Pro Ser Thr          35 40 45 Gly Asp Arg Gly Phe Ala Pro Glu Glu Tyr Glu Thr Val Asp Pro Lys      50 55 60 Phe Gly Asp Trp Ser Asp Ile Asp Lys Leu Ala Glu Lys Tyr Tyr Leu  65 70 75 80 Met Phe Asp Phe Met Leu Asn His Ile Ser Pro Gln Ser Lys Tyr Phe                  85 90 95 Gln Asp Phe Leu Glu Lys Lys Glu Asp Ser Glu Tyr Tyr Asp Met Phe             100 105 110 Leu Lys Tyr Ser Glu Phe Trp Pro Glu Asn Arg Pro Thr Ala Glu Asp         115 120 125 Ile Asp Leu Ile Tyr Lys Arg Lys Asp Lys Ala Pro Phe Ala Pro Val     130 135 140 Thr Phe Ala Asp Gly Thr Thr Asp Gln Val Trp Asn Thr Phe Gly Asp 145 150 155 160 Gln Gln Met Asp Leu Asp Val Thr Lys Pro Val Thr Lys Gln Phe Ile                 165 170 175 Lys Asp Ser Leu Ile Asn Leu Ala Arg His Gly Ala Ser Val Ile Arg             180 185 190 Leu Asp Ala Phe Ala Tyr Ala Ile Lys Lys Leu Asp Thr Asn Asp Phe         195 200 205 Phe Val Glu Pro Glu Ile Trp Asp Val Leu Asn Glu Val Arg Glu Ile     210 215 220 Leu Ala Ala Glu Gly Ser Glu Val Leu Pro Glu Ile His Glu His Tyr 225 230 235 240 Lys Ile Val Arg Lys Ile Thr Asp His Asp Met Tyr Ser Tyr Asp Phe                 245 250 255 Ala Leu Pro Leu Ile Thr Leu Tyr Ser Leu Tyr Ser Gly Lys Thr Asn             260 265 270 Arg Leu Ala Asp Trp Leu Arg Gln Ser Pro Met Lys Gln Phe Thr Thr         275 280 285 Leu Asp Thr His Asp Gly Ile Gly Val Val Asp Ala Lys Asp Leu Leu     290 295 300 Thr Gln Glu Glu Thr Asp Phe Thr Thr Glu Glu Met Tyr Lys Val Gly 305 310 315 320 Ala Asn Val Lys Lys Ile Phe Ser Ser Ala Ala Tyr Asn Asn Leu Asp                 325 330 335 Ile Tyr Gln Ile Asn Thr Thr Tyr Tyr Ser Ala Leu Gly Asn Asp Asp             340 345 350 Ala Ala Tyr Leu Leu Ala Arg Ala Thr Gln Ile Phe Ala Pro Gly Ile         355 360 365 Pro Gln Val Tyr Tyr Val Gly Leu Leu Ala Gly Glu Asn Asp Leu Glu     370 375 380 Leu Leu Glu Ser Thr Lys Glu Gly Arg Asn Ile Asn Arg His Tyr Tyr 385 390 395 400 Asp Leu Glu Glu Ile Ala Glu Thr Val Lys Arg Pro Val Val Ser Asn                 405 410 415 Leu Phe Asp Leu Leu Arg Phe Arg Asn Thr Ser Ser Ala Phe Asp Leu             420 425 430 Asp Gly Glu Ile Lys Val Glu Asn Asn Gly Asp Asn Asp Leu Thr Ile         435 440 445 Thr Arg Thr Ser Ala Asp Gly Gln Thr Thr Ala Val Leu Thr Ala Asp     450 455 460 Phe Ala Thr Lys Gln Phe Lys Val Thr Glu Asn Asp Lys Glu Ile Phe 465 470 475 480 Asn Thr Gln Ala                 <210> 2 <211> 1455 <212> DNA <213> Leuconostoc lactis <400> 2 atgaaaaagc tcaccaatga agtgatgctc attacatatg ctgactcact tggtaagaat 60 ttgaaggaat tggatcaagt gttgtcaaat gaattggatg gtgttgttgg tggtgtccac 120 gttttgccat tcttcccatc aacaggcgac cgtggctttg cccctgaaga atatgaaaca 180 gttgatccaa agtttgggga ctggtctgac attgacaagt tagctgagaa gtattacttg 240 atgtttgact tcatgttgaa ccacatttca ccacaatcaa agtatttcca agacttctta 300 gaaaagaagg aagactcaga atactatgac atgttcttga agtattctga attctggcct 360 gaaaaccgtc caacagctga agacattgat ttgatttata agcgtaagga taaggcacca 420 tttgcgcccg taacatttgc ggacggcaca acagaccaag tttggaacac attcggtgac 480 caacaaatgg atttggatgt cacaaagcca gtgacaaagc aattcatcaa ggattcattg 540 attaaccttg cccgtcatgg tgcgtcagtg attcgtttgg atgcctttgc ttacgctatc 600 aagaagttgg atacaaacga cttcttcgtt gaaccagaaa tctgggacgt tttgaacgaa 660 gtacgtgaaa tcttggcagc agaaggttct gaagttttgc cagaaatcca tgaacactac 720 aagattgtgc gcaagattac ggatcacgac atgtattcat atgactttgc gttgccattg 780 attacgttgt actcacttta ttcaggtaag acaaaccgtt tggctgactg gttgcgtcaa 840 agcccaatga agcaatttac aacattggat acacacgatg gtattggtgt tgttgatgcc 900 aaggacttgt tgacacaaga agaaacagac tttacaacgg aagaaatgta caaggttggg 960 gctaacgtga agaagatctt ctcatcagca gcctacaata acttggatat ttaccaaatt 1020 aatacaactt attactcagc tttgggtaat gatgatgccg cttacttgct agcacgtgca 1080 acacaaatct ttgcaccagg tatcccacaa gtttattacg ttggtttgtt ggcaggtgaa 1140 aacgaccttg aattgttgga aagcacaaag gaaggccgta acatcaaccg tcactattac 1200 gaccttgaag aaattgctga aacagttaag cgtccagttg tgtcaaactt gtttgacttg 1260 cttcgcttcc gtaacacaag ctcagccttt gatcttgatg gtgagattaa ggttgaaaac 1320 aacggtgaca atgatttgac aattacacgt acatcagctg atggtcaaac aactgccgtt 1380 ttgacagctg actttgcgac aaagcaattc aaagtcactg aaaatgataa agaaattttc 1440 aacacacagg cttaa 1455 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ttygayttya tgathaayca yat 23 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 nagrttngcn ccnacyttrt a 21 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 atgactttgc gttgccattg attac 25 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 gttggtcacc gaatgtgttc ca 22 <210> 7 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 gactcgagat gaaaaagctc accaatgaag tg 32 <210> 8 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 taggatcctt aagcctgtgt gttgaaaatt tc 32  

Claims (8)

Sucrose phosphorylase derived from Leuconostoc lactis EG001 represented by the amino acid sequence of SEQ ID NO: 1. The gene encoding the sucrose phosphorylase of claim 1. The gene according to claim 2, wherein the gene is represented by a nucleotide sequence of SEQ ID NO: 2. delete Recombinant vector containing the gene of claim 2. Recombinant microorganism transformed with the recombinant vector of claim 5. The recombinant microorganism in which the sucrose phosphorylase gene of claim 2 is inserted on a chromosome. Culturing the recombinant microorganism of claim 6 or 7, producing sucrose phosphorylase; And recovering the produced sucrose phosphorylase.

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