KR100494813B1 - Mutant Gene of Dextransucrase, Recombinant Vector comprising the Mutant Gene, Microorganism Transformed by the Recombinant Vector, and the Dextransucrase produced from the Microorganism - Google Patents

Abstract

The present invention relates to a modified dextran sucrase gene comprising a modified dextran sucrase gene, a recombinant vector comprising the gene, a microorganism transformed with the recombinant vector, and an amino acid sequence produced from the microorganism. ,

A modified dextran sucrase gene consisting of the DNA sequence of SEQ ID NO: 1 and synthesizing dextran having a high ratio of branched bonds, a recombinant vector comprising the modified dextran sucrase gene, and the recombinant vector. Microorganisms producing dextran having a high proportion of transformed branches (DSRB742CK, Accession No .: KACC 95013), and produced from the transformed microorganism, consisting of the amino acid sequence of SEQ ID NO: 2, Modified dextran sucrase is provided that can synthesize dextran having a bond.

The mutant strains of the present invention exhibit increased dextran production ability, and in particular, have the property of synthesizing dextran having many branch bonds, and are very useful for synthesizing dextran, which can be mainly used as a material useful for medicine, food, cosmetics, etc. Do.

Description

Modified dextran sucrase gene, recombinant vector containing the gene, microorganisms transformed with the recombinant vector, and dextran sucrase produced from such microorganisms (Mutant Gene of Dextransucrase, Recombinant Vector comprising the Mutant Gene, Microorganism Transformed by the Recombinant Vector, and the Dextransucrase produced from the Microorganism}

The present invention provides a modified dextran sucrase gene for synthesizing dextran having a high ratio of branched bonds, a recombinant vector comprising the gene, a microorganism transformed with the recombinant vector, and an amino acid sequence produced from the microorganism. It relates to a modified dextran sucrase comprising. In particular, the modified dextran sucrase gene of the present invention is obtained by directly irradiating the gene with radiation having a specific wavelength range.

Thousands of enzymes in living organisms have evolved to process very efficiently the complex and varied reactions necessary to maintain their functions. Therefore, in order to actually use the enzyme industrially, it is necessary to improve the function of the enzyme in accordance with the purpose of the reaction.

To improve the properties of enzymes, traditional protein engineering based on relevant information such as the structure of the enzyme and directed evolution method to improve the function of the enzyme in the desired direction without specific information about the enzyme This can be utilized. Especially for enzymes that do not have accurate information about the structure or function of the protein, directional evolution is very efficient in improving the enzyme's function in the desired direction. Methods for generating mutant genes can be used, and methods for generating such mutant genes include chemical mutagenesis, error-prone PCR (mutagenic PCR), and cassette mutagenesis. ), DNA shuffling, homologous recombination, etc. are mainly developed and used.

The directional evolution method using genes developed and used abroad is a molecular genetic random mutation method used to mutate a gene part when there is no information on which part of an enzyme occurs. Random mutations using radiation have long been used industrially to induce mutations in microorganisms themselves. However, in conventional methods of mutation induction using radiation, the subject is the microorganism itself, and no direct research has been conducted to isolate the gene.

There have been many studies on the effects of radiation on living organisms in medicine, food-related biochemistry, and molecular genetics, but most of them use the ultraviolet light of several electron volts to directly irradiate the microorganisms, check the effects on genetic material, It was to induce mutations. The impact of higher energy light on dielectric materials has been largely performed since the development of radiation accelerators. Recently, experiments on the effect of DNA on the nucleotide sequence using radiation generated from a radiation accelerator have been performed.

Hieda et al. Investigated the plasmid DNA using five photon energies near 2.1 keV and confirmed that the covalent closed circular form of DNA changed into open and linear DNA forms. Phosphorus has been shown to absorb photons by the K-shell of phosphorus and the possibility of DNA cleavage by auger events [Hieda, K. DNA damage induced by vacuum and soft X-ray photons from synchrotron radiation. Int. J. Radiat. Biol. 66 (1994), 561-567. The spores of Bacillus subtilis were used to analyze changes in DNA sequence after irradiation in the 50-300 nm region, and the degree of absorption of high-energy radiation to microorganisms was measured near the K shell. Research has also been conducted to identify the effect on cells [Wehner, J. and Horneck, G. Effects of vacuum UV and UVC radiation on dry Escherichia coli plasmid pUC 19. J. Photochem. Photobiol. 30 (1995) 171-177.

Meanwhile, the Gram-positive group, Leuconostoc mesenteroides , produces an enzyme (dextranuckrase; dextransucrase) that biosynthesizes dextran, a polysaccharide in which glucose is mainly linked to α-1 → 6. The mechanism by which enzymes biosynthesize dextran using sugar is as follows.

n sucrose →

(Glucose) _nmw + (nm) fructose + m _lucrose + w glucose

(n 〉〉 m or w)

This reaction is an irreversible reaction, and the main products are high molecular weight (1X10 ⁷ ~ 1X10 ⁸ Da) of dextran and fructose, and trace amounts of rucrose (5-0-α-D-glucopyranosyl-D-fructopyranose) and glucose Produced. Different luconosestock mesentroids produce different types of dextran sucrase, and all of the dextrans synthesized by these enzymes have branched bonds. Of the branch bonds discovered so far, the main bonds are α1 → 3 and α1 → 2 and α1 → 4 structures are also reported. Until now, all of the known Lukonostok bacteria had to add sugar to the enzyme-producing substrate to produce dextran sucrose. Recently, however, Kim and Robyt have found unique dextran without the addition of sugar to the substrate from various species of Luconosestock mesenteroids (eg NRRL B-5l2F, B-1142, B-1355, B-742, B-1299). Constitutive mutant strains producing sucrase were developed (Kim, D, Robyt, JF (1995b) Enz. Microb. Technol. 17, 689). That is, even when glucose and fructose, but not sugar, are used as a carbon source, dextran sucrase is produced during growth, and when it is produced in LM medium containing sugar, the glucan sucrase is obtained in the state of being combined with the produced dextran. In comparison, it is possible to obtain an enzyme only of a protein that is not contaminated with dextran. One mutant bacterium, B-512FMC, was developed from B-512 (F) dextran-producing bacteria, which are commercially used in many fields. The dextran synthesized by this enzyme is 95% α1 like the enzyme of the parent strain. → 6 and 5% of the α1 → 3 bonds synthesize a branching bond. Altenan, a polysaccharide linked by alternating glucose with α1 → 3 and α1 → 6 linkages, has low viscosity and excellent solubility in water, and is not decomposed by α1 → 6 synthase, resulting in food additives and extenders. It is a substance that has many uses. Of the glucans sucrases of three B-742 mutants (B-742C, B-742CA, and 5-742CB), B-742CA synthesizes dextran having α1 → 4 structures and about 50% of B-742CB. It produces glucan sucrase which synthesizes a material having a high structure ratio of α 1 → 3 structures. B-1299CB mutant glucan sucrase synthesizes a dextran substance having 35% α1 → 2 structure.

However, there is still a need for the development of microbial strains that improve the genes of useful industrial enzymes such as dextran and have new properties, and in particular, the modification of synthesizing dextran having a high ratio of branch bonds by direct irradiation of the gene with radiation. No studies have been carried out or published to produce dextransukraase genes.

It is therefore an object of the present invention to provide a modified dextran sucrase gene that synthesizes a high proportion of dextran having branched bonds, which is obtained by directly irradiating a gene with specific radiation.

Another object of the present invention is to provide a recombinant vector comprising the modified dextran sucrase gene.

Still another object of the present invention is to provide a microorganism transformed with the recombinant vector.

Another object of the present invention is to provide a modified dextran sucrase enzyme produced from the transformed microorganism.

According to the first aspect of the present invention, there is provided a modified dextran sucrase gene, which consists of the DNA sequence of SEQ ID NO: 1 and synthesizes dextran having a high proportion of branched bonds.

According to a second aspect of the present invention, there is provided a recombinant vector ( pdsrB742CK ) comprising the modified dextran sucrase gene.

According to a third aspect of the present invention, there is provided a microorganism (KACC 95013) producing dextran having a high ratio of branched bonds transformed with the recombinant vector.

According to the fourth aspect of the present invention, a modified dex produced from the transformed microorganism (KACC 95013), consisting of the amino acid sequence of SEQ ID NO: 2, and capable of synthesizing dextran having a high ratio of branched bonds Transucrase is provided.

Hereinafter, the present invention will be described in detail.

The present inventors directly irradiated the dextran sucrase gene ( dsrB742 : GeneBank Accession No. AF294469) with radiation having a specific wavelength range to obtain a modified dextran sucrase gene, and also the modified dextran sucrase. By transforming the Aze gene into Escherichia coli strain DH5, a new microbial strain was obtained, which was characterized by synthesizing dextran having a large number of branch bonds. The strain was named 'DSRB742CK' and was used as an agricultural microorganism. It was deposited in a preservation center (accession number: KACC 95013).

Radiated light that can be used to generate the modified dextran sucralase genes of the present invention generally include UVA, UVB, UVC, X-rays, VUV (vacuum ultraviolet) and ultrasoft X-rays that are easily obtained and easy to use. Among them, VUV and low energy ultra-soft X-rays are electromagnetic waves between UV and X-rays, and the energy range is 10 eV-5.0 keV, which is useful in the present invention, and VUV is particularly preferable.

With UV, for example, the binding of a molecule is broken by the energy of the UV, and the electrons that fall off have a low energy level that does not affect other molecules around it, so that it affects only the UV-irradiated area directly. For example, when irradiating VUV 10-100 times higher than UV, the electrons that are released have enough energy to affect the binding of surrounding molecules, which may affect the binding of the irradiation site. VUV is also light whose wavelength of impact (or how much photons are absorbed by the material) is maximal compared to other wavelengths. These characteristics make the mutation more likely than UV.

The dextran sucrase gene for irradiating radiation is prepared in advance in isolated form prior to irradiation with radiation. Isolation of the gene may then be isolated from, for example, Leuconostoc mesenteroides by conventional methods known in the art, and the gene may be cloned and prepared in the form of DNA inserted into the vector. Can be.

Radiation light irradiation can be carried out by a generally known method, for example, by radiating light to an aluminum target to generate scattered light having an energy of 10 eV-5.0 KeV, and then irradiating the gene in a box filled with helium. Lose.

By this method, the mutated parts of the gene that can be obtained from irradiation will vary, which may be repaired or repaired through the repair process after the gene has been transformed into a microorganism used as a host. Depending on the degree of re-mutation may occur during the repair process, the resulting mutant strains of the gene can be a variety and variety.

In the present invention, using the Leconostoc mesenteroides B-742 dextran sucrase gene ( dsrB742 ) in the form of a plasmid DNA inserted into the vector through mutation, and then sequencing the gene It was confirmed that the mutation occurs, the microbial strain transformed with the mutated gene increases the productivity of dextran sucrase, the enzyme is expressed constitutive (particularly synthesized dextran having a lot of branch bonds) It showed the characteristic.

Hereinafter, the present invention will be described in more detail with reference to the following examples.

Example 1 Gene Preparation

As a gene to be mutated in the present embodiment, Leuconostoc mesenteroides B-742CB dextran sucrase gene ( dsrB742 : GeneBank Accession No. AF294469) was used. This gene is a 6.1 kb DNA fragment with an open reading frame (ORF) consisting of 4,536 bp bases. The estimated amino acid sequence is the stop codon at position 5,223 from the start codon (ATG) at base 698. It is a structure located up to (TAA). The amino acid of the structural gene consists of 1,508, and the molecular weight is 168.6 kDa by calculation. This gene was used as plasmid DNA cloned in pGEM 3Zf (-) (Promega, USA). 2 shows a restriction map of the vector into which the gene is inserted. That is, a 6.193 kb insert containing the entire dextran sucrase gene was cloned into the Pst I restriction site of pGEM-3Zf (−).

Example 2 (radiation light irradiation)

X-rays of LIGA white beam (9C1) of the radiation accelerator (31, Pohang Accelerator Research Institute, Nam-gu, Pohang-si, Gyeongsangbuk-do) were incident on an aluminum target to generate scattered light with an energy of 1.47 keV, and in a box filled with helium Leuconostoc mesenteroides B-742CB dextransukraase DNA ( dsrB742 ) obtained in Example 1 was irradiated for various times from 5 seconds to 30 minutes.

Example 3 (mutation production with increased productivity of dextran sucrase)

1) Mutation screening and sequencing

Compare the irradiated DNA with non radiation irradiation on DNA electrophoresis was identified structural changes, and the irradiation was carried out for gene transfection in Escherichia coli car (Escherichia coli) DH5 strain. This was plated in LB [0.5% (w / v) NaCl, 0.5% (w / v) yeast extract, 1% (w / v) trypton] solid medium to which 2% (w / v) sugar was added and at 37 ° C. After incubation for 8 hours, it was transferred to 28 ℃ and incubated overnight. The colonies produced after transformation were first screened with colonies with more viscous viscosity than the control in sugar-added LB solid medium, and this time, the supernatant obtained through liquid culture was reacted with sugar to produce sugar decomposition and dextran. Stronger mutations were finally screened. At this time, the control group was compared using the DSRB742 clone (Accession No.-KACC 95014). In addition, the mutant was separated from the plasmid DNA, and confirmed the changed part through sequencing.

The results of sequencing of the genetic variants obtained by irradiation with light were shown in SEQ ID NO: 1 and the putative amino acid sequence in SEQ ID NO: 2, and also shown in Figures 1A-1D together with the base sequence and the putative amino acid sequence.

DNA sequencing results in the lack of three bases at the beginning of the structural gene (ORF) in the structural gene region, where the start codon starts at the 31st amino acid missing 30 amino acids. That is, it lacks 30 amino acids at the N-terminus. Except for this part, the base of the structural gene was not changed, and it was found that three bases were changed in the promoter part which is the front part of the structural gene.

2) Characterization of enzymes produced by mutations

① Polysaccharide recovery and characterization: Selected E. coli colonies were cultured in LB liquid medium to which sugar was added, followed by centrifugation (8000 rpm) to separate enzymes from supernatant. The obtained enzyme solution was reacted with 200 mM sugar (pH 5.2) and 1: 1 to confirm that the sugar was completely decomposed, and then polysaccharide was recovered through ethanol precipitation. After washing three times with distilled water to remove the residual residue, it was recovered by reprecipitation. In order to identify the unit components of the polysaccharide produced by the recombinant E. coli , an acid hydrolysis reaction was performed with 1 M HCl, followed by TLC.

In addition, dextran was prepared at a concentration of 1% and penicillium ( Penicillium) dextranase (Sigma, USA) 60 U / ml of 37 ℃, 16 hours of reaction was confirmed the reaction product. Dextranase decomposes dextran to produce D-glucose, isomaltose, isomaltotriose and high-branched oligosaccharides (3 ³ -isomaltosyl isomaltotetraose and 3 ³ -D-glucopyranosyl-isomaltopentaose). At this time, the degradation product of dextran by dextranase was confirmed by TLC. The developing solvent was nitromethane: 1-propanol: water in a ratio of 2: 5: 1.5 (v / v / v).

② After culturing the selected clone in glucose or sugar-added LB medium, the enzyme is separated from the supernatant obtained, and the polysaccharide obtained by the reaction with sugar is recovered. Then, NMR ( ¹³ C Nuclear Magnetic Resonance) is confirmed to confirm the structure. Analysis was performed. Polysaccharide NMR spectra were measured at ¹³ C (125 MHz) and proton (500 MHz) using a Bruker AMX-500.

These analytical measurement results are shown in FIGS. 3-5. Through this analysis, when the enzyme supernatant cultured in glucose medium was reacted with sugar, a mutant in which polysaccharide dextran was formed was obtained, which was named DSRB742CK.

As shown in FIG. 3, after acid hydrolysis of dextran using enzymes obtained from the original and modified genes, glucose is mainly detected, and the enzyme produced by the original and modified genes is derived from sugar. Dextran, the polysaccharide to make, is a substance synthesized with glucose.

As shown in Fig. 4, the resistance to degradation of dextranase is increased by 2.2 times that of the original bacteria in the mutant, that is, the newly produced gene seems to synthesize dextran having many branch bonds.

As shown in FIG. 5, ¹³ C-NMR analysis of the polysaccharide recovered from the enzyme supernatant obtained by culturing the mutant DSRB742CK in a medium to which sugar was added again with sugar, showed that the chemical shift of the polysaccharide recovered from DSRB742CK was A signal was formed that was not present in the dextran of the original strain, indicating α (1 → 3). Therefore, it can be seen that the dextran produced by the mutant DSRB742CK using sugar has more branched bonds than the dextran of the original strain.

Mutant strain of the present invention shows an increased dextran production capacity than the existing L econostoc mesentroides B-742CB strain, the enzyme is expressed constitutive (especially dextran having a lot of branch bonds) It has the property to synthesize. Accordingly, dextran produced by these mutant strains can be used as a useful material for medicine, food, cosmetics, and the like.

Figure 1a-1d-nucleotide sequence and estimated amino acid sequence of the gene variant (DSRB742CK) obtained by the method of the present invention

(The numbers on the left represent the nucleotide and amino acid numbers, the bold font in italics indicates the estimated sugar binding site, and the underlined bold font indicates the glucosyl group transfer site.)

Figure 2-Recombinant vector ( pdsrB742CK ) comprising a gene variant obtained by the method of the present invention

Figure 3-TLC analysis photos after acid hydrolysis of dextran produced by the genetic variant (DSRB742CK) obtained by the method of the present invention

Mn, maltodextrin series; IMn, isomaltodextrin series; F, fructose; Lanes 1 and 2, before and after acid hydrolysis of DSRB742 dextran, before and after acid hydrolysis of DSRB742CK dextran

G1: glucose, G2: maltose, G3: maltotriose, G4: maltotetraose, G5: maltopentaose, G6: maltohexaoz, G7: maltoheptaose, G8: maltooctase, IM2: isomaltose, IM3: isomaltotriose, IM4: isomaltotetraose, IM5: isomaltopentaose

Figure 4-TLC analysis result after dextranase hydrolysis of dextran produced by the gene variant (DSRB742CK) obtained by the method of the present invention

Mn: maltodextrin series, IMn: isomaltoxtrin series, D: penicillium dextranase, lanes 1 and 2: before and after dextranase treatment of dextran from native strain (DSRB742), lanes 3 and 4 : Before and after dextranase treatment of dextran from gene variant (DSRB742CK)

G1: glucose, G2: maltose, G3: maltotriose, G4: maltotetraose, G5: maltopentose, G6: maltohexaoz, G7: maltoheptaose, G8: maltooctase, IM2: isomaltose, IM3: isomaltotriose, IM4: isomaltotetraose

BOS1 and BOS2: oligosaccharides with branched bonds (branched)

HMW: high molecular weight oligosaccharide

Figure 5 - the original strain (DSRB742) and genetic variants ¹³ C-NMR analysis of the photo-dextran produced in (DSRB742CK)

(A) DSRB742 dextran produced by dextran sucrase of the original strain produced in sugar medium

(B) DSRB742CK dextran produced by dextran sucrase of gene variants produced in sugar medium

<110> KIM, doman <120> Mutant Gene of Dextransucrase, Recombinant Vector comprising the Mutant Gene, Microorganism Transformed by the Recombinant Vector, and the Dextransucrase produced from the Microorganism <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 6193 <212> DNA <213> Artificial Sequence <220> <223> Mutant of Leuconostoc mesentroides B-742CB dextransucrase gene (dsrB742CK) <400> 1 ctgcagctaa actcacttta actattgctg gttttttgag aaaactaatc aaccacgtcg 60 caccataatt taaaaaacca ctagcaaaca ctaataaagg tatatcgata ggtaatggtt 120 tttgaagtat catgtccaaa tacagtatga tgacaatagg tgttaaaaca aaatacagac 180 gaccaatggc cggactgata taataaaaca ccaaaatagc gagtaaataa tataaataga 240 tattaccaaa tactaattta gagacagata ataagctata tattgatggc aaaaacacca 300 caaccgtaat caattgccaa accaacgtat taggtttctc cagaaaattt aaaattgtca 360 atactttgat gcctaagcca ataaccattg tcgataatat caaggtacta attattaccg 420 tccacatagg tcctcccaaa atccacacat tgcagtaaaa aacgacacta acactacaca 480 ttttatcata taaattacat aaaacatatt aagaagaatc ccgtgatata gcgctcttat 540 caactaaaaa agcgagcggc ccattatttt atcactttat aaatattgtt tattttatat 600 ggtaaagaat tggtaatcgt ttgtgaaatg atataagttc gtaattttat agcttataat 660 tcatttgttt caaagaataa tcaccaatgg aggagagaat gtttgattaa gagagaaatg 720 tacgaaaaaa gctctacaag tctggtaaga gttgggttat tgggggactc attttatcga 780 caattatgct gtctatgacc gctacttcac aaaatgttaa tgcagatagc acaaacacag 840 tgacggataa gtcagttact gtctccaata attcgaatac aaccaatcaa cacgatactg 900 tcgttgacaa acaaacgata cctgtcaaaa atgaccaaac aacacaacaa atcgccgcaa 960 atgccaccca agcagaaaaa gtaaaagcat cagacacaac gactgatacg caaaagcaag 1020 ctgaaacggc aaacaacact aacaaggatt cgatagataa tctcaccaag cagttgccgg 1080 ctgttacacc aacagctaat caaaaaactg gttatctgga aaaagatggt aaatggtact 1140 atgtaaccag tgataacaca cttgctaagg ggttgactac tgttgacaac cacaagcagt 1200 attttgacaa caatggcgtg caggcaaaag gccaattcgt taccgataac agtaaaacat 1260 actatctcga tcctaactcc ggtaacgcag taaccgggat acaacaaatt ggctcacaaa 1320 cattagcctt caatgacaac ggtgaacaag tttttgctga tttctataca gcgccagatg 1380 gcaaaactta ttattttgac gataaaggac aagcaactat tggtctaaag gccattaatg 1440 ggcacaatta ttacttcgat agtttgggac aactaaaaaa aggatttacc ggtgtcattg 1500 acggtcaagt acgctatttt gatcaagaat caggacaaga ggtatcaaca accgactcac 1560 aaatcaaaga aggtttaact tctcagacaa cagactatac agcacataat gccgttcaca 1620 gcaccgatag cgctgatttc gacaatttta atggttattt gaccgcttct tcatggtatc 1680 gccctaaaga tgttttaaga aatggtcaac actgggaagc aacaacagct aatgacttcc 1740 ggcccattgt gtcagtttgg tggcctagca agcaaacaca agtaaattac ctaaactaca 1800 tgtctcaaat gggactcatt gacaatcgtc agatgttctc gctaaaagac aatcaagcca 1860 tgttgaatat tgcttgcaca acagtccaac aagcaattga aacaaaaatc ggtgtggcta 1920 atagtacagc atggcttaaa acagccattg atgatttcat tcgtacacag ccacaatgga 1980 acatgtcgag tgaagatccc aaaaatgatc atttacaaaa cggcgctttg actttcgtca 2040 acagtccatt gacaccagat actaactcta atttcagact attaaatcgc acaccaacaa 2100 accagacagg tgtgccaaaa tatacaattg atcaatctaa gggtggtttt gaactcttac 2160 tcgctaatga tgtagacaac tctaatcctg ttgtgcaagc tgagcagtta aattggttac 2220 actatttgat gaattttggt agcattacag caaacgattc tgctgctaat tttgatggga 2280 tacgtgtcga tgctgtcgat aatgttgacg ctgatttact ccagattgca gcagattatt 2340 tcaaagctgc ttatggtgtt gataaaaatg acgcaacagc aaatcaacat ctttcaattc 2400 ttgaagattg gagccataac gaccctgaat acgtgaagga ttttggtaat aatcaactca 2460 caatggatga ttacatgcat acccagttaa tctggtcctt gactaaagat atgcgtatgc 2520 gtggtaccat gcaacgcttc atggactatt acctcgtcaa tcgcaatcac gatagtaccg 2580 aaaacactgc cattccaaat tacagctttg ttcgcgcaca cgatagtgaa gtacaaacag 2640 tcattgctca aattatttct gagttacatc ccgacgtaaa aaatagtttg gcaccaacag 2700 cagaccagct agccgaagcc tttaaaattt ataataacga tgaaaaacag gcggataaga 2760 aatatacaca atacaacatg cctagcgcct atgcgatgct gttaactaat aaagatacag 2820 taccgcgcgt ttattatggt gatttataca ccgatgatgg tcaatatatg gcaaataagt 2880 ccccttattt tgatgccatc aacggcttgc taaagtcacg tatcaaatat gttgctggtg 2940 gtcagtcaat ggctgttgat caaaacgata tcctgacaaa tgttcgttat ggtaaaggtg 3000 ccatgagtgt gacagatagc ggtaatgcag acacacgaac acaaggtatt ggtgtgattg 3060 tcagtaataa agaaaatctg gccttaaaat caggcgacac ggtgacatta cacatgggtg 3120 ccgctcacaa aaatcaagca ttcagattat tattagggac aactgctgac aatttgtctt 3180 attatgataa tgacaacgcc ccagtaaagt acaccaatga tcagggcgat ttaatttttg 3240 ataatactga aatctatggt gtccgtaacc cgcaagtctc tggcttctta gctgtttggg 3300 tgcctgttgg ggctgacagc catcaagacg cgcgtacttt gtctgacgac acagcccatc 3360 atgatggcaa aaccttccac tcaaatgctg ctttagattc tcaggttatt tacgaaggtt 3420 tttcaaattt ccaagctttt gccacaaaca ctgaagacta tacaaatgct gtcattgcaa 3480 aaaatggtca gttattcaaa gattggggta tcacaagttt ccagttggca ccacaatatc 3540 gttcaagcac cgataccagt ttcttagatt caattatcca aaatggttat gcctttacag 3600 atcgttatga tttaggctac ggtacaccaa caaaatatgg cacagttgac cagttacgcg 3660 atgccatcaa ggctctgcac gcaaatggca tccaagcaat cgctgactgg gtacccgacc 3720 aaatttataa tttaccgggt caagaattag cgaccgtcac ccgaacaaac tcttatggtg 3780 ataaagacac taactcagat attgatcagt cactatatgt catacaaagt cgtggtggtg 3840 gtaaatacca agcacagtat ggcggtgcct tcttatccga tatccagaaa aaatatccag 3900 cacttttcga aacaaaacaa atttctacag ggctacctat ggatcctagt cagaaaataa 3960 cagaatggtc tggtaaatac tttaatggct caaatattca aggcaaaggg gctggctatg 4020 tcttgaaaga cagtggtacc gatcaatact ataaggttac atcaaacaat aataatcgtg 4080 acttcttgcc aaaacaatta acagatgact tatctgaaac cggatttgtc cgcgataaca 4140 ttggtatggt ctattacaca ctgagtggct atctagctcg aaacaccttt atacaagatg 4200 ataatggcaa ttattattac tttgatagca ccggccatct cgttactggc ttccagaata 4260 ttaataacca tcactatttc ttcctaccaa acggtattga actcgttcaa tctttcttac 4320 agaatgctga cggttcaacg atttattttg accaaaaagg gcgtcaagta tttaatcaat 4380 acattactga ccaaaccggg accgcctatt acttccagaa tgatggcaca atggtcactt 4440 ctggcttcac tgaaatcgat ggtcataagc aatacttcta caagaacggc acacaagtca 4500 aagggcaatt tgtatcagac actgatggtc acgttttcta cttagaagct ggtaacggca 4560 acgtggcgac acaaagattt gcacaaaata gtcaaggtca gtggttctat ttgggtaatg 4620 atggcattgc cttgactggt ttgcaaacaa tcaatggtgt tcaaaattat ttctacgccg 4680 atggtcatca aagtaagggt gattttatta cgatacaaaa tcacgtatta tatactaacc 4740 cactaactgg cgctataacg acaggtatgc aacaaattgg tgacaagatt tttgtctttg 4800 acaatacggg caacatgttg accaatcaat actatcaaac actagatggc caatggttac 4860 atttaagcac tcaaggtcca gcagacactg gtttggtaaa cattaatggt aatttgaaat 4920 atttccaagc taatggtcgg caagtgaaag gtcaatttgt gactgatcct atcacgaacg 4980 tgagttatta tatgaatgcc actgatggtt cggcagtatt taatgactac tttacctatc 5040 aaggccaatg gtatttaacg gatagtaatt atcaactcgt caaaggattt aaagttgtta 5100 ataataagct acaacatttt gatgaaataa caggcgtaca aactaaatca gctcatatca 5160 tcgttaataa tcgaacatac attttcgatg accaaggtta ctttgtctca gtcgcttaac 5220 taaaaaaagc ctactcaatt aagagtgggc ttttttaatt taagaagtat aaggcaatta 5280 aagcaaccaa cgccggtaat ccttgtacca caagaatttt ccgagctgat gttatgctgc 5340 catatatcgc tgcaattaag atatagagca tttcaaattg taacatcagc acttgttgcg 5400 gaccatgaat gaaaaacatg gtcaccaata tgagtactcc gaataaaccg ttgtatattc 5460 cctgattagc taataacacc ttcacttctg gcatttttaa aacagggggt tctaattcaa 5520 aggacttagc ttgctgtgca actgagccca accatttcta tcatcatgat aatgattgct 5580 tcaatagcaa ctaacccggg taataatttg tgcgatcatt ttatacggtc tctttcttta 5640 tttctaccac aatcttacca cgcgcatgat gtgtttcgct acgctgatga gctgcacgca 5700 taccttctgt tgttagcgga taaatactat cgaccacaat ttgtaattga ttttttgtaa 5760 tataatcagc gagtgtggtt aaatctttac cattagtatc caaccaaccg ggttgtaatt 5820 gttttgttag gtgtctgctg ttgttgagcc gttaactgcg cagaaattgt caccaaatgg 5880 ccgccatctt ttaaaatatg aataccatta tcgatgtccc caaccatatc aaatacagca 5940 tcgtaatcag acaaaacatc ctgtatttgg tatgcgtggt aatcaatcac ttgatctgcc 6000 cccagtgacg caacaaaatc gtgattaact tgactcgctg ttgttgctac ataagtcccc 6060 attaacttgg cgagttgaat ggcataaata ccaacgccac cagcgccagc ttgtattaac 6120 accttgtgcc ctgctttaac ttgtagtttt tccaacattt gtagcgctgt tagggctgct 6180 aatggtactg cag 6193 <210> 2 <211> 1477 <212> PRT <213> Artificial Sequence <220> <223> Mutant of Leuconostoc mesentroides B-742CB dextransucrase (dsrB742CK) <400> 2 Met Leu Ser Met Thr Ala Thr Ser Gln Asn Val Asn Ala Asp Ser Thr 1 5 10 15 Asn Thr Val Thr Asp Lys Ser Val Thr Val Ser Asn Asn Ser Asn Thr 20 25 30 Thr Asn Gln His Asp Thr Val Val Asp Lys Gln Thr Ile Pro Val Lys 35 40 45 Asn Asp Gln Thr Thr Gln Gln Ile Ala Ala Asn Ala Thr Gln Ala Glu 50 55 60 Lys Val Lys Ala Ser Asp Thr Thr Thr Asp Thr Gln Lys Gln Ala Glu 65 70 75 80 Thr Ala Asn Asn Thr Asn Lys Asp Ser Ile Asp Asn Leu Thr Lys Gln 85 90 95 Leu Pro Ala Val Thr Pro Thr Ala Asn Gln Lys Thr Gly Tyr Leu Glu 100 105 110 Lys Asp Gly Lys Trp Tyr Tyr Val Thr Ser Asp Asn Thr Leu Ala Lys 115 120 125 Gly Leu Thr Thr Val Asp Asn His Lys Gln Tyr Phe Asp Asn Asn Gly 130 135 140 Val Gln Ala Lys Gly Gln Phe Val Thr Asp Asn Ser Lys Thr Tyr Tyr 145 150 155 160 Leu Asp Pro Asn Ser Gly Asn Ala Val Thr Gly Ile Gln Gln Ile Gly 165 170 175 Ser Gln Thr Leu Ala Phe Asn Asp Asn Gly Glu Gln Val Phe Ala Asp 180 185 190 Phe Tyr Thr Ala Pro Asp Gly Lys Thr Tyr Tyr Phe Asp Asp Lys Gly 195 200 205 Gln Ala Thr Ile Gly Leu Lys Ala Ile Asn Gly His Asn Tyr Tyr Phe 210 215 220 Asp Ser Leu Gly Gln Leu Lys Lys Gly Phe Thr Gly Val Ile Asp Gly 225 230 235 240 Gln Val Arg Tyr Phe Asp Gln Glu Ser Gly Gln Glu Val Ser Thr Thr 245 250 255 Asp Ser Gln Ile Lys Glu Gly Leu Thr Ser Gln Thr Thr Asp Tyr Thr 260 265 270 Ala His Asn Ala Val His Ser Thr Asp Ser Ala Asp Phe Asp Asn Phe 275 280 285 Asn Gly Tyr Leu Thr Ala Ser Ser Trp Tyr Arg Pro Lys Asp Val Leu 290 295 300 Arg Asn Gly Gln His Trp Glu Ala Thr Thr Ala Asn Asp Phe Arg Pro 305 310 315 320 Ile Val Ser Val Trp Trp Pro Ser Lys Gln Thr Gln Val Asn Tyr Leu 325 330 335 Asn Tyr Met Ser Gln Met Gly Leu Ile Asp Asn Arg Gln Met Phe Ser 340 345 350 Leu Lys Asp Asn Gln Ala Met Leu Asn Ile Ala Cys Thr Thr Val Gln 355 360 365 Gln Ala Ile Glu Thr Lys Ile Gly Val Ala Asn Ser Thr Ala Trp Leu 370 375 380 Lys Thr Ala Ile Asp Asp Phe Ile Arg Thr Gln Pro Gln Trp Asn Met 385 390 395 400 Ser Ser Glu Asp Pro Lys Asn Asp His Leu Gln Asn Gly Ala Leu Thr 405 410 415 Phe Val Asn Ser Pro Leu Thr Pro Asp Thr Asn Ser Asn Phe Arg Leu 420 425 430 Leu Asn Arg Thr Pro Thr Asn Gln Thr Gly Val Pro Lys Tyr Thr Ile 435 440 445 Asp Gln Ser Lys Gly Gly Phe Glu Leu Leu Leu Ala Asn Asp Val Asp 450 455 460 Asn Ser Asn Pro Val Val Gln Ala Glu Gln Leu Asn Trp Leu His Tyr 465 470 475 480 Leu Met Asn Phe Gly Ser Ile Thr Ala Asn Asp Ser Ala Ala Asn Phe 485 490 495 Asp Gly Ile Arg Val Asp Ala Val Asp Asn Val Asp Ala Asp Leu Leu 500 505 510 Gln Ile Ala Ala Asp Tyr Phe Lys Ala Ala Tyr Gly Val Asp Lys Asn 515 520 525 Asp Ala Thr Ala Asn Gln His Leu Ser Ile Leu Glu Asp Trp Ser His 530 535 540 Asn Asp Pro Glu Tyr Val Lys Asp Phe Gly Asn Asn Gln Leu Thr Met 545 550 555 560 Asp Asp Tyr Met His Thr Gln Leu Ile Trp Ser Leu Thr Lys Asp Met 565 570 575 Arg Met Arg Gly Thr Met Gln Arg Phe Met Asp Tyr Tyr Leu Val Asn 580 585 590 Arg Asn His Asp Ser Thr Glu Asn Thr Ala Ile Pro Asn Tyr Ser Phe 595 600 605 Val Arg Ala His Asp Ser Glu Val Gln Thr Val Ile Ala Gln Ile Ile 610 615 620 Ser Glu Leu His Pro Asp Val Lys Asn Ser Leu Ala Pro Thr Ala Asp 625 630 635 640 Gln Leu Ala Glu Ala Phe Lys Ile Tyr Asn Asn Asp Glu Lys Gln Ala 645 650 655 Asp Lys Lys Tyr Thr Gln Tyr Asn Met Pro Ser Ala Tyr Ala Met Leu 660 665 670 Leu Thr Asn Lys Asp Thr Val Pro Arg Val Tyr Tyr Gly Asp Leu Tyr 675 680 685 Thr Asp Asp Gly Gln Tyr Met Ala Asn Lys Ser Pro Tyr Phe Asp Ala 690 695 700 Ile Asn Gly Leu Leu Lys Ser Arg Ile Lys Tyr Val Ala Gly Gly Gln 705 710 715 720 Ser Met Ala Val Asp Gln Asn Asp Ile Leu Thr Asn Val Arg Tyr Gly 725 730 735 Lys Gly Ala Met Ser Val Thr Asp Ser Gly Asn Ala Asp Thr Arg Thr 740 745 750 Gln Gly Ile Gly Val Ile Val Ser Asn Lys Glu Asn Leu Ala Leu Lys 755 760 765 Ser Gly Asp Thr Val Thr Leu His Met Gly Ala Ala His Lys Asn Gln 770 775 780 Ala Phe Arg Leu Leu Leu Gly Thr Thr Ala Asp Asn Leu Ser Tyr Tyr 785 790 795 800 Asp Asn Asp Asn Ala Pro Val Lys Tyr Thr Asn Asp Gln Gly Asp Leu 805 810 815 Ile Phe Asp Asn Thr Glu Ile Tyr Gly Val Arg Asn Pro Gln Val Ser 820 825 830 Gly Phe Leu Ala Val Trp Val Pro Val Gly Ala Asp Ser His Gln Asp 835 840 845 Ala Arg Thr Leu Ser Asp Asp Thr Ala His His Asp Gly Lys Thr Phe 850 855 860 His Ser Asn Ala Ala Leu Asp Ser Gln Val Ile Tyr Glu Gly Phe Ser 865 870 875 880 Asn Phe Gln Ala Phe Ala Thr Asn Thr Glu Asp Tyr Thr Asn Ala Val 885 890 895 Ile Ala Lys Asn Gly Gln Leu Phe Lys Asp Trp Gly Ile Thr Ser Phe 900 905 910 Gln Leu Ala Pro Gln Tyr Arg Ser Ser Thr Asp Thr Ser Phe Leu Asp 915 920 925 Ser Ile Ile Gln Asn Gly Tyr Ala Phe Thr Asp Arg Tyr Asp Leu Gly 930 935 940 Tyr Gly Thr Pro Thr Lys Tyr Gly Thr Val Asp Gln Leu Arg Asp Ala 945 950 955 960 Ile Lys Ala Leu His Ala Asn Gly Ile Gln Ala Ile Ala Asp Trp Val 965 970 975 Pro Asp Gln Ile Tyr Asn Leu Pro Gly Gln Glu Leu Ala Thr Val Thr 980 985 990 Arg Thr Asn Ser Tyr Gly Asp Lys Asp Thr Asn Ser Asp Ile Asp Gln 995 1000 1005 Ser Leu Tyr Val Ile Gln Ser Arg Gly Gly Gly Lys Tyr Gln Ala Gln 1010 1015 1020 Tyr Gly Gly Ala Phe Leu Ser Asp Ile Gln Lys Lys Tyr Pro Ala Leu 1025 1030 1035 1040 Phe Glu Thr Lys Gln Ile Ser Thr Gly Leu Pro Met Asp Pro Ser Gln 1045 1050 1055 Lys Ile Thr Glu Trp Ser Gly Lys Tyr Phe Asn Gly Ser Asn Ile Gln 1060 1065 1070 Gly Lys Gly Ala Gly Tyr Val Leu Lys Asp Ser Gly Thr Asp Gln Tyr 1075 1080 1085 Tyr Lys Val Thr Ser Asn Asn Asn Asn Arg Asp Phe Leu Pro Lys Gln 1090 1095 1100 Leu Thr Asp Asp Leu Ser Glu Thr Gly Phe Val Arg Asp Asn Ile Gly 1105 1110 1115 1120 Met Val Tyr Tyr Thr Leu Ser Gly Tyr Leu Ala Arg Asn Thr Phe Ile 1125 1130 1135 Gln Asp Asp Asn Gly Asn Tyr Tyr Tyr Phe Asp Ser Thr Gly His Leu 1140 1145 1150 Val Thr Gly Phe Gln Asn Ile Asn Asn His His Tyr Phe Phe Leu Pro 1155 1160 1165 Asn Gly Ile Glu Leu Val Gln Ser Phe Leu Gln Asn Ala Asp Gly Ser 1170 1175 1180 Thr Ile Tyr Phe Asp Gln Lys Gly Arg Gln Val Phe Asn Gln Tyr Ile 1185 1190 1195 1200 Thr Asp Gln Thr Gly Thr Ala Tyr Tyr Phe Gln Asn Asp Gly Thr Met 1205 1210 1215 Val Thr Ser Gly Phe Thr Glu Ile Asp Gly His Lys Gln Tyr Phe Tyr 1220 1225 1230 Lys Asn Gly Thr Gln Val Lys Gly Gln Phe Val Ser Asp Thr Asp Gly 1235 1240 1245 His Val Phe Tyr Leu Glu Ala Gly Asn Gly Asn Val Ala Thr Gln Arg 1250 1255 1260 Phe Ala Gln Asn Ser Gln Gly Gln Trp Phe Tyr Leu Gly Asn Asp Gly 1265 1270 1275 1280 Ile Ala Leu Thr Gly Leu Gln Thr Ile Asn Gly Val Gln Asn Tyr Phe 1285 1290 1295 Tyr Ala Asp Gly His Gln Ser Lys Gly Asp Phe Ile Thr Ile Gln Asn 1300 1305 1310 His Val Leu Tyr Thr Asn Pro Leu Thr Gly Ala Ile Thr Thr Gly Met 1315 1320 1325 Gln Gln Ile Gly Asp Lys Ile Phe Val Phe Asp Asn Thr Gly Asn Met 1330 1335 1340 Leu Thr Asn Gln Tyr Tyr Gln Thr Leu Asp Gly Gln Trp Leu His Leu 1345 1350 1355 1360 Ser Thr Gln Gly Pro Ala Asp Thr Gly Leu Val Asn Ile Asn Gly Asn 1365 1370 1375 Leu Lys Tyr Phe Gln Ala Asn Gly Arg Gln Val Lys Gly Gln Phe Val 1380 1385 1390 Thr Asp Pro Ile Thr Asn Val Ser Tyr Tyr Met Asn Ala Thr Asp Gly 1395 1400 1405 Ser Ala Val Phe Asn Asp Tyr Phe Thr Tyr Gln Gly Gln Trp Tyr Leu 1410 1415 1420 Thr Asp Ser Asn Tyr Gln Leu Val Lys Gly Phe Lys Val Val Asn Asn 1425 1430 1435 1440 Lys Leu Gln His Phe Asp Glu Ile Thr Gly Val Gln Thr Lys Ser Ala 1445 1450 1455 His Ile Ile Val Asn Asn Arg Thr Tyr Ile Phe Asp Asp Gln Gly Tyr 1460 1465 1470 Phe Val Ser Val Ala 1475

Claims (5)

A modified dextran sucrase gene consisting of a DNA sequence of SEQ ID NO: 1, which synthesizes dextran having branched bonds having an alpha (1 → 3) of 11-20%. delete Recombinant vector comprising the modified dextran sucrase gene of claim 1. A microorganism (DSRB742CK, Accession No .: KACC 95013) which produces dextran having branched bonds having 11-20% of α (1 → 3) transformed with the recombinant vector of claim 3. A modified dextran sucra which can synthesize dextran produced from the transformed microorganism of claim 4, consisting of the amino acid sequence of SEQ ID NO: 2, and having branched bonds having an alpha (1 → 3) of 11-20%. Aze.