KR101523517B1 - Method for producing modified starch using branching enzyme from Vibrio vulnificus and uses thereof - Google Patents

Abstract

The present invention relates to a method for producing modified starch by using a branching enzyme derived from Vibrio vulnificus and to a use thereof. More specifically, the method for producing modified starch comprises a step of allowing a branching enzyme to react with amylose, and amylopectin. The present invention further provides modified starch produced by the method, food produced by using the modified starch, and a composition for producing modified starch, containing the branching enzyme or a gene, amylose, and amylopectin which encode the branching enzyme as active ingredients.

Description

[0001] The present invention relates to a method for producing modified starch using a branching enzyme derived from Vibrio vulnificus and a method for producing the modified starch using branching enzyme from Vibrio vulnificus and uses thereof.

The present invention relates to a method for producing modified starch using a branching enzyme derived from Vibrio vulnificus and a use thereof, and more particularly to a method for producing modified starch using a branching enzyme and amylose and amylopectin A method for producing modified starch, a modified starch prepared by the method, a food prepared using the modified starch, and a gene encoding the branching enzyme or a gene encoding the modified starch, amylose and amylopectin, As an active ingredient, to a composition for producing modified starch.

Starch is a storage polysaccharide contained in plants such as grains, papers, etc., and is a major source of human carbohydrates. Starch is divided into nutritionally rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS). The hydrolyzable starch is a starch which is completely digested in the small intestine but has a slow digestion rate and the indigestible starch is a starch which is not digested in the small intestine but fermented by microorganisms in the large intestine.

Many studies have been conducted on the physiological advantages of the lipid-digestible starch and the indigestible starch. The starch has a health effect of slowly increasing the postprandial blood glucose level, while the indigestible starch is a protein of the colon cancer, hyperglycemia and hypercholesterolemia And prevent the accumulation of fat.

On the other hand, foods that rapidly increase blood sugar levels cause excessive insulin secretion and repetition, resulting in insulin resistance that does not function properly even if insulin is secreted by overload of the pancreas. However, And diabetic patients. In general, physical treatment, chemical treatment, and enzymatic treatment are used alone or in combination to increase the fatigueability and indigestible content of starch. Among them, enzymatic treatment is environmentally friendly, safe and health-oriented treatment for consumers. In addition, since only a specific reaction is performed, it is possible to induce only the desired reaction, and the yield is high, the byproduct of the reaction is small, and the purification for use after the treatment is easy. Therefore, it would be required to develop a technique for increasing the degree of the starch's branching by using an enzymatic reaction.

Korean Patent No. 1214923 discloses a process for producing a starch which is capable of hydrolyzing or hydrolyzing starch having improved thermal stability and a starch prepared therefrom. In Korean Patent No. 1400966, A method for producing modified starch using a branching enzyme derived from Vibrio vulnificus as in the present invention and its use have not been disclosed.

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned needs. In the present invention, a gene encoding a branching enzyme protein derived from Vibrio vulnificus is expressed in E. coli in a large amount, As a result of the treatment with amylopectin, it was confirmed that a modified starch having a lipidizing property was produced, thereby completing the present invention.

In order to solve the above problems, the present invention provides a method for producing modified starch, which comprises reacting a branching enzyme with amylose and amylopectin.

The present invention also provides a modified starch prepared by the above method.

The present invention also provides a food prepared using the modified starch.

The present invention also provides a composition for producing modified starch comprising the branching enzyme or a gene coding therefor and amylose and amylopectin as an active ingredient.

In the present invention, a gene coding for a branching enzyme protein derived from Vibrio vulnificus was expressed in E. coli in a large amount, separated and purified and reacted with amylose and amylopectin. As a result, the modified starch Was confirmed to be produced. The modified starch, which is produced by specifically modifying the existing starch molecular structure by treating the branching enzyme of the present invention, significantly inhibits the digestion rate by the body's starch hydrolyzing enzyme and prevents rapid increase of the blood glucose level upon ingestion and is slowly digested It is long-lasting and can supply sugar to the body continuously and has high water-solubility. Therefore, this characteristic is applied as an ideal carbohydrate such as a patient-customized food for diabetic and obese patients or a functional food for weight control, and the specific inherent sugar-binding property of the novel novel branching enzyme (VvBE) It can be used for production.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic view of the Vibrio vulnificus of the present invention Derived branching enzyme (VvBE, Vibrio vulnificus branching enzyme coding genes. (Amp is an ampicillin resistance gene)
Figure 2 shows the purification results of VvBE derived from Vibrio vulnificus of the present invention. (M is a protein size marker, 1 is soluble fraction of cell extract, 2 is insoluble fraction of cell extract, 3 is Ni-NTA purification)
Figure 3 shows the results of measuring the activity of VvBE according to the temperature and pH of the present invention.
4 shows the reaction product production process by VvBE of the present invention.
FIG. 5 shows the result of analyzing the modified amylose using HPAEC after the VvBE treatment of the present invention. (G1 to G7 std is a mixture of G1 to G7, G3 is maltotriose, G4 is maltotetraose, G5 is maltopentaose, G6 is maltohexaose, G7 is maltoheptaose, Al is amylose, VvBE is Vibrio vulnificus Kus branching enzyme)
FIG. 6 shows the results of analysis of modified amylopectin using HPAEC after VvBE treatment of the present invention. (G1 to G7 std is a mixture of G1 to G7, G3 is maltotriose, G4 is maltotetraose, G5 is maltopentaose, G6 is maltohexaose, G7 is maltoheptaose, AP is amylopectin, VvBE is Vibrio vulnificus Kus branching enzyme)
FIG. 7 shows the results of comparing the digestion rate of modified starch and amylopectin produced by treating VvBE of the present invention with amylopectin using AMG (amyloglucosidase).
FIG. 8 shows the results of comparing the digestion rate of modified starch produced by treating VvBE of the present invention with amylopectin and general amylopectin using HPA (human pancreatic amylase).
FIG. 9 shows the results of comparing the digestion rate of modified starch produced by treating VvBE of the present invention with amylopectin and general amylopectin using PPA (porcine pancreatic amylase).

In order to achieve the above object, the present invention provides a method for producing modified starch, which comprises reacting a branching enzyme with amylose and amylopectin.

Generally, the branching enzyme is a glycoconjugate that acts on α-1,4 glucosidic bonds of amylose, amylopectin and glycogen to transfer 6-7 units of glucose residues at the non-reducing end to the 6-positions in the molecule or between molecules The enzyme is an enzyme that produces amylopectin or glycogen-type branched polysaccharide from amylose-type polysaccharide by the action of this enzyme.

In a method according to an embodiment of the present invention, the branching enzyme may be composed of the amino acid sequence of SEQ ID NO: 1 derived from Vibrio vulnificus , but is not limited thereto. The scope of the branching enzyme according to the present invention includes a protein having an amino acid sequence represented by SEQ ID NO: 1 and a functional equivalent of the protein. "Functional equivalent" means at least 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably at least 70% or more, more preferably 90% or more, Refers to a protein having a homology of at least 95% and exhibiting substantially the same physiological activity as the protein represented by SEQ ID NO: 1. "Substantially homogenous physiological activity" means an activity of producing a fatigueable modified starch.

In a method according to one embodiment of the present invention, the branching enzyme derived from Vibrio vulnificus may be produced by the following steps, but is not limited thereto:

Preparing a recombinant vector comprising a gene encoding a branching enzyme derived from Vibrio vulnificus ;

Transforming the prepared recombinant vector into E. coli; And

Isolating and purifying the branching enzyme from the transformed E. coli.

The gene encoding the branching enzyme derived from the Vibrio vulnificus of the present invention may preferably be composed of the nucleotide sequence of SEQ ID NO: 2. In addition, homologues of the nucleotide sequences are included within the scope of the present invention. Specifically, the gene has a nucleotide sequence having a sequence homology of 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more, with the nucleotide sequence of SEQ ID NO: 2 . "% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >

In the method according to an embodiment of the present invention, the modified starch may be 20-30% on the basis of the total modified starch, but the present invention is not limited thereto.

In the method according to one embodiment of the present invention, the modified starch may have a water solubility of 30% or more, preferably 35 to 45%, and most preferably 39% .

Thus, the method according to one embodiment of the present invention preferably comprises the step of reacting amylose and amylopectin with a branching enzyme comprising the amino acid sequence of SEQ ID NO: 1 derived from Vibrio vulnificus (DP) of 1 to 5 is 20 to 30% based on the total modified starch, and has a water solubility of 35 to 45%.

The present invention also provides a method for producing a modified starch, which is produced by the above process, wherein the short branch ratio of Degree of Polymerization (DP) is 20 to 30% based on the total modified starch and has a water solubility of 35 to 45% It provides starch.

The present invention also provides a food prepared using the modified starch.

The food of the present invention can be used as it is or in combination with other food or food ingredients, and can be suitably used according to conventional methods. There is no particular limitation on the kind of the food. The modified starch may be used to produce a functional food, a health food or a health supplement. Functional foods, health foods, or health supplements refer to foods that provide biocontrol functions, including biologically active ingredients, in addition to nutritional functions.

The present invention also provides a composition for producing modified starch comprising a branching enzyme or a gene coding therefor and amylose and amylopectin as an active ingredient.

The composition for producing modified starch according to the present invention comprises, as an active ingredient, a branching enzyme comprising the amino acid sequence of SEQ ID NO: 1 derived from Vibrio vulnificus of the present invention, or a gene encoding the same, and amylose and amylopectin. When a gene encoding a branching enzyme is used, the gene may be expressed and expressed in a host cell such as Escherichia coli, followed by separation and purification, followed by reaction with amylose and amylopectin to form a branch It may also produce Mars modified starch.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited thereto.

vibrio Bulfinicus  Branching Enzyme ( VvBE , Vibrio vulnificus branching  enzyme Cloning

The seawater microbe, Vibrio The subcloning of the branching enzyme encoding gene derived from E. coli V. vulnificus MO6-24 / O was performed in restriction enzymes Nde I and Pst I of the p6xHis119 cloning vector for Escherichia coli expression. Escherichia coli was used as a host and cultured in a solid LB medium containing ampicillin antibiotics. The cultured colonies were cultured in a liquid LB medium for 15 hours to extract plasmids, and it was confirmed that the clones were cloned normally through gene sequence analysis. The overall cloning procedure is shown in Fig.

VvBE ≪ / RTI >

For expression of the branching enzyme, a recombinant plasmid was introduced into Escherichia coli , The cells were transformed into MC1061 and cultured in a solid LB medium containing ampicillin at 37 DEG C for 15 hours to isolate the colonies. The colonies were cultured in a liquid LB medium containing ampicillin, and then centrifuged to harvest the cells and perform cell disruption. After centrifugation, the supernatant was filtered, and the cell extract was obtained. The recombinant proteins expressed in the form of six His-tag for purification were dialyzed (50 mM tris-HCl, pH 7.5) after purification using Ni-NTA (Ni ^{2 +} -nitrrilotriacetate) affinity column chromatography. Then, SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) was performed to confirm the protein purification. The results of the whole process are shown in FIG.

Example  One. VvBE ( Vibrio vulnificus branching enzyme ) ≪ / RTI & pH  Explore Optimal Conditions

The enzyme reversal proceeded to the Lugols method using amylose as substrate. To determine optimal pH and temperature conditions, 50 mM sodium acetate buffer (pH 5.5-6.0), 50 mM sodium phosphate buffer (pH 6.5-8.5) Maximal branching activity was sought at various temperatures. The titration was repeated two times for one sample, and one unit means the amount of enzyme required to reduce 1 mg of amylose per minute.

The optimum reaction conditions for VvBE were 30 ° C and pH 7.5 as shown in FIG. The enzyme showed a sharp decrease in titer at a temperature of 30 ° C or higher and showed no activity at temperatures above 45 ° C, indicating that the stability at high temperature was not high. At pH 6.5-8.0, the activity was over 80% and the activity was maximal at pH 7.5.

Example  2. VvBE ( Vibrio vulnificus branching enzyme ) Characterization

Table 1 below shows the characteristics of VvBE that establishes optimum conditions. Concentration of protein was measured at the optimum conditions as above. Protein concentration was measured by repeating 2 times, 1 time, 2 times, 5 times and 10 times dilution of proteins using Bradford method. The specific activity (U / mg) increased from 0.2 to 1.7 after concentration and dialysis of the purified Ni-NTA affinity column.

As a result of purification of VvBE according to the present invention Purification step The degree of inactivity (U / mg ) yield(%) Refining diagram Cell extract 0.2 100 One Ni-NTA 1.7 23.7 10

Example  3. Bio - LC Enzyme Reaction product  Pattern analysis

(One) Amylose , Modification of amylopectin and starch

In order to analyze the reaction pattern of VvBE of the present invention, amylose and amylopectin substrates were prepared, respectively, and then the enzyme was treated to study the changes. Amylose and amylopectin and VvBE were reacted in a shaking incubator at 30 ° C for 4 hours under optimal conditions. The reaction was stopped by heating in boiling water to inactivate the enzymes, followed by centrifugation to remove proteins. The supernatant was decanted, treated with absolute ethanol to precipitate, and then the transformed product was dried by centrifugation.

(2) Isoamylase ( Isoamylase ) After processing Reaction product  analysis

Completely dried samples were reacted with isoamylase (40 units / mg) for 40 and 72 hours. Then, the reaction was stopped by inactivating the enzyme by heating and centrifugation, and the supernatant was taken and filtered using a filter. Analysis was performed using HPAEC (High-Performance Anion-Exchange Chromatography) equipped with CarboPac PA-1 (anion-exchange column) (FIG.

(3) HPAEC Modified substrate analysis using

The results of analysis of reaction products produced after treating amylose and amylopectin of each substrate modified by VvBE according to the present invention with isoamylase were analyzed using HPAEC as shown in Figs. 5 and 6. The peak observed after the treatment with isoamylase can be regarded as a result of decomposition of the α-1,6-bond. Amylose has only α-1,4-linkage and does not show a peak even when it reacts with isoamylase. However, it has been shown that a short chain (side chain) is produced in a substrate modified with VvBE. The peak of DP (Degree of Polymerization) 10 or higher was relatively smaller than that of the conventional substrate, the short branches of DP 5 or less increased sharply, and the shape of distribution was gentle around DP 5 and 6.

(4) derived from other microorganisms Branching  Enzyme ( branching enzyme )

Table 2 shows the results of comparing the reaction pattern of VvBE with other microorganism-branching enzymes. When reacted with the substrate, VvBE exhibited a unique glycoconjugate characteristic that produced clusters containing 27% or more of short branching fractions of 5 or less, compared with other branching enzymes with high branching frequency near DP10.

The result of comparing this microorganism-derived branching enzyme with the corresponding pattern GBE temperament Chain length distribution (%) DP? 5 DP 6-12 DP 13-18 DP≥19 - Natural corn amylopectin 0.49 ± 0.09 27.3 ± 0.89 32.9 ± 0.96 39.3 ± 1.76 V. vulnificus Amylose 27.4 ± 0.23 60.2 + - 3.07 11.3 ± 2.72 1.17 ± 0.58 Amylopectin 23.6 ± 0.14 54.7 ± 6.10 16.6 ± 2.62 5.10 ± 3.35 D. geothermal Amylose 4.77 ± 0.11 51.6 ± 1.49 23.1 ± 1.04 20.5 ± 2.59 E. coli Amylose 1.18 ± 0.02 63.2 ± 0.96 30.7 ± 1.47 4.99 ± 0.98 B. subtilis Amylose 2.12 + 0.04 56.3 ± 1.93 25.6 ± 2.08 16.0 + - 1.75

Example  4. SEC - RI Enzyme Reaction product  Molecular weight analysis

In order to analyze the reaction pattern of VvBE of the present invention, amylose and amylopectin substrates were prepared, respectively, and the molecular structure of the starch was studied by treatment with enzyme. After reacting the substrate with the substrate at 30 ° C for 4 hours, the reaction was stopped by heating in boiling water at 100 ° C for 5 minutes to remove the inactivated protein by centrifugation. The supernatant was treated with absolute ethanol to remove the bovine sugars, the enzyme was precipitated, and the precipitate was dried by centrifugation.

The completely dried samples were diluted to 50% DMSO (dimethyl sulfoxide) and filtered to prepare samples, which were then analyzed using SEC (Size Exclusion Chromatography). Dextran and pullulan were used as standards for molecular weight standards. The results of the analysis of enzyme reaction products are shown in Table 3 below.

The reaction products of VvBE and amylose were found to have higher molecular weight than the conventional molecular weight. This leads to molecular weight increase by molecular oligomer formation by intermolecular transglycosylation reaction while branching. In the case of amylopectin, the molecular weight of the enzyme reaction product was much smaller than that of VvBE in that it reacted with VvBE to form a cluster-like form through intermolecular transglycosylation in the state of branching of the original polymer. This is because it is converted into a product.

Analysis of change in molecular weight of modified starch produced with SEC standard substance and VvBE Standard material Elution time (min) Elution volume (ml) Molecular weight (Da) Dextran 10.597 5.3 6100000 p-800 14.406 7.2 805000 p-400 15.106 7.6 366000 p-200 15.747 7.9 200000 p-100 16.246 8.1 113000 p-50 16.819 8.4 48800 p-20 17.336 8.7 21700 p-10 17.812 8.9 10000 p-5 18.1 9.1 6200 Analytical sample Natural amylose 16.804 8.4 48591 Natural amylopectin 10.88 5.4 5321769 Modified amylose 10.079 5 8557631 Modified amylopectin 17.183 8.6 26814

Example  5. Solubility analysis of enzyme reaction product

Amylopectin (5%) and VvBE (5%) were reacted at 30 ℃ and the reaction product was modified by ethanol precipitation more than 2 times. Amylopectin (20%) and enzyme reaction products (60%) were dissolved in distilled water in an excess amount and centrifuged to measure the content of sugar dissolved in the supernatant by phenol-sulfuirc acid method (Table 4).

Existing amylopectin showed a water solubility of 0.16%, whereas the enzyme reaction product showed 39%. This is because amylopectin has been transformed into small molecular clusters due to the glycation reaction by VvBE.

Analysis of change in molecular weight of modified starch produced with SEC standard substance and VvBE sample Total sugar (mg / ml) Water solubility (%) Amylopectin 1.6 0.16 Modified starch 390.4 39

Example  6. Digestion rate analysis of enzyme reaction product

To determine the digestion characteristics of modified starches produced with amylopectin and VvBE, digestion rates were measured using amyloglucosidase (AMG), human pancreatic amylase (HPA) and porcine pancreatic amylase (PPA) as three enzymes. AMG (pH 4.5 and 50 ° C), HPA (pH 7.0 and 37 ° C) and PPA (pH 7.4 and 37 ° C) were reacted with the substrate at optimal conditions and the glucose and reducing sugar ) Was analyzed by GOPOD and DNS methods to confirm the extent of degradation (FIGS. 7, 8, and 9).

 The digestion rate of amylopectin and denatured starch to the substrate was measured. As a result, it was confirmed that the digestion rate of all three enzymes was reduced by more than 2 times (Table 5).

Digestion rate comparison result Digestive enzyme Digestion rate (mg / ml · min) Amylopectin Modified starch AMG 0.024 0.012 HPA 0.045 0.02 PPA 0.013 0.005

Example  7. VvBE Application of modified starch

The modified starch, which is produced by specifically modifying the existing starch molecular structure using the VvBE of the present invention, is remarkably inhibited by the digestion rate by the body's starch hydrolyzing enzyme, thereby preventing rapid increase in blood glucose level upon ingestion, Is able to supply the body with sugar continuously for a long time and has high water solubility. Therefore, this characteristic is applied as an ideal carbohydrate such as patient-customized food for diabetic and obesity patients and functional food for weight control, and at the same time, specific characteristics of the novel VvBE can be utilized in the production of useful materials in the starch industry.

<110> Chungnam national university Industry-Academic Cooperation Foundation <120> Method for producing modified starch using branching enzyme from          Vibrio vulnificus and uses thereof <130> PN15045 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 742 <212> PRT <213> Vibrio vulnificus <400> 1 Met Arg Ile Val Phe Thr Thr Leu Ala Val Trp Arg Gly Lys Lys Gly   1 5 10 15 Leu Lys Lys Leu Val Lys Ser Lys Val Asn Gln Met Phe Glu Lys Leu              20 25 30 Ser Gln Ala Ala Cys Ser Glu Pro Phe Ala Phe Leu Gly Pro Phe Ile          35 40 45 Asp Pro Thr Gln Gly Ala Leu Arg Val Trp Met Pro Gly Ala Thr Gly      50 55 60 Val Ala Leu Val Leu Glu Gly Gln Pro Arg Ile Ala Leu Glu Arg Glu  65 70 75 80 Lys Glu Ser Ala Phe Ile Leu Lys Ala Asp Leu Asn Leu His Leu Thr                  85 90 95 His Tyr Gln Leu Ala Ile Asp Trp Asn Gly Val Glu Gln Leu Ile Asp             100 105 110 Asp Pro Tyr Gln Tyr His Gly Ile Tyr Ala Glu Tyr Asp Asp Leu His         115 120 125 Thr Pro Lys Thr Met Tyr Gln His Met Gly Ser Gln Phe Met Thr Leu     130 135 140 Glu Arg Asp Gly Lys Ser Ser Ser Gly Ile Arg Phe Leu Val Tyr Ala 145 150 155 160 Pro His Ala Thr Ala Val Ser Leu Val Gly Cys Phe Asn Asp Trp Asp                 165 170 175 Gly Arg Arg His Pro Met Gln Arg Leu Asp Tyr Gly Ile Trp Gly Leu             180 185 190 Phe Ile Pro Gly Leu Thr Glu Gly Val Ser Tyr Lys Phe Glu Met Lys         195 200 205 Gly Pro Lys Gly Glu Gly Leu Pro His Lys Ala Asp Pro Trp Gly Phe     210 215 220 Tyr Ala Glu Gln Tyr Pro Ser Phe Ala Ser Val Thr Tyr Asp His Ala 225 230 235 240 Arg Tyr Gln Trp Gln Asp Ala Gln Trp Gln Thr Arg Pro Val Thr Glu                 245 250 255 Lys Arg Lys Glu Ala Leu Ser Phe Tyr Glu Leu His Ala Gly Ser Trp             260 265 270 Lys Arg Asn Glu Gln Gly Glu Phe Leu Asn Tyr Arg Glu Leu Ala Ala         275 280 285 Glu Leu Val Pro Tyr Leu Val Asp Met Gly Tyr Thr His Val Glu Leu     290 295 300 Met Pro Val Ser Glu His Pro Phe Tyr Gly Ser Trp Gly Tyr Gln Pro 305 310 315 320 Val Gly Leu Phe Ala Pro Thr Ser Arg Tyr Gly Ser Pro Asp Asp Phe                 325 330 335 Lys Phe Phe Val Asp Ala Cys His Gln Ala Gly Ile Gly Val Val Leu             340 345 350 Asp Trp Val Pro Ala His Phe Pro Ser Asp Asp His Gly Leu Ala Asn         355 360 365 Phe Asp Gly Thr Pro Leu Phe His Asp Pro Asp Pro Arg Arg Gly Trp     370 375 380 His Gln Asp Trp Asn Ser Phe Ile Tyr Asp Leu Gly Arg Glu Gln Val 385 390 395 400 Arg Arg Phe Leu Val Ser Asn Ala Leu Tyr Trp Phe Glu Gln Phe His                 405 410 415 Ile Asp Gly Ile Arg Val Asp Ala Val Ala Ser Met Leu Tyr Leu Asp             420 425 430 Tyr Ser Arg Ser His Gly Gln Trp Ile Pro Asn Met Asp Gly Gly Asn         435 440 445 Glu Asn Tyr Asp Ala Ile Ala Thr Leu Lys Trp Met Asn Glu Glu Val     450 455 460 Tyr Lys Tyr Phe Pro Asn Ala Met Thr Ile Ala Glu Glu Ser Thr Ala 465 470 475 480 Phe Pro Gly Val Ser Ala Pro Thr Phe Met Gly Gly Leu Gly Phe Gly                 485 490 495 Phe Lys Trp Asn Met Gly Trp Met His Asp Ser Leu Ser Tyr Ile Lys             500 505 510 Glu Glu Pro Val His Arg Lys Tyr His His Asn Thr Leu Thr Phe Pro         515 520 525 Leu Val Tyr Ala His Ser Glu Asn Tyr Val Leu Ser Leu Ser His Asp     530 535 540 Glu Val Val Tyr Gly Lys Gly Ser Ile His Asn Lys Met Pro Gly Asp 545 550 555 560 Glu Trp Gln Gln Thr Ala Asn Leu Arg Ala Tyr Phe Gly Tyr Met Tyr                 565 570 575 Gly Gln Pro Gly Lys Lys Leu Asn Phe Met Gly Ala Glu Ile Gly Gln             580 585 590 Thr Ala Glu Trp Asn His Asp Asp Gln Leu Gln Trp Phe Leu Leu Asp         595 600 605 Phe Pro Arg His Gln Gly Val Gln Ala Leu Thr Arg Asp Leu Asn His     610 615 620 Leu Tyr Arg Asn Glu Ala Ala Leu His Asp Gln Asp Cys Ile Pro Ala 625 630 635 640 Gly Phe Glu Trp Arg Leu Gln Asp Ala Ala Glu Gln Ser Ile Ile Ala                 645 650 655 His Glu Arg Ile Ser Glu Ala Gly Glu Arg Ile Leu Val Val Ser Asn             660 665 670 Phe Thr Pro Val Pro Arg Asp Glu Phe Arg Leu Gly Val Pro Asn Lys         675 680 685 Gly Arg Tyr Gln Leu Leu Leu Asn Thr Asp Asp Ser Lys Tyr Ala Gly     690 695 700 Ser Gly Tyr Glu Val Val Val Asp Ala Lys Ser Glu Ala Val Val Ser 705 710 715 720 Glu Asp Leu Ala Gln Ser Ile Val Leu Arg Leu Pro Pro Leu Ser Thr                 725 730 735 Leu Phe Tyr Lys Leu Val             740 <210> 2 <211> 2229 <212> DNA <213> Vibrio vulnificus <400> 2 atgaggattg tgttcacaac gttggctgtt tggcgaggga aaaaaggttt gaaaaaatta 60 gtaaaaagta aagtgaatca gatgtttgag aaattgtctc aagccgcttg ttcagagccg 120 tttgcatttt taggtccatt tattgaccca acacaagggg cactgcgcgt ctggatgccg 180 ggtgcaacgg gggtggcatt ggtattagaa ggccaaccac gtatcgcgct tgagcgagaa 240 aaagagagtg ccttcattct caaagccgat ctcaatctgc atcttaccca ttatcaattg 300 gcgatcgatt ggaatggcgt cgagcaactt atcgatgacc cataccaata ccacggtatc 360 tacgcggagt atgacgatct ccacacgccg aaaaccatgt atcaacacat gggctcacag 420 tttatgacgc ttgagcgcga cggcaaatca atctcaggta tccgtttctt ggtgtatgcg 480 ccgcatgcga ccgcagtgag tctcgtaggc tgtttcaatg attgggatgg ccgacgtcat 540 ccaatgcagc gccttgatta cggcatttgg ggtttattca ttccggggtt aaccgaaggg 600 gtgagctaca aattcgaaat gaaagggccg aagggggaag gcttaccaca caaagccgac 660 ccttggggtt tctacgccga gcaataccca tcttttgctt ccgtcacgta cgatcacgct 720 cgttaccagt ggcaagacgc ccagtggcaa actcgcccag tgaccgagaa acgcaaagaa 780 gcgctctctt tctacgagtt gcatgctggt tcttggaagc gcaacgagca aggtgagttc 840 ctgaattatc gcgaactggc cgccgagttg gtgccctatt tggtggatat gggctacacc 900 catgttgaac tgatgcctgt ctctgagcac ccgttctacg gctcttgggg ttaccagcct 960 gtgggtctct ttgcgccaac tagccgctat ggttcacccg atgatttcaa attcttcgtc 1020 gatgcgtgtc accaagcggg cattggtgtg gtattggatt gggtgcctgc gcacttccca 1080 tcggacgatc acggcttggc caattttgat ggtacaccat tgttccacga cccagaccca 1140 cgccgtggtt ggcaccaaga ctggaactcg tttatctacg atcttggccg cgagcaagta 1200 cgccgtttct tggtttctaa cgctctgtat tggtttgagc agttccatat cgatggtatt 1260 cgggtggatg cggtggcatc gatgctgtat ctcgattatt cacgcagcca tggccagtgg 1320 atcccgaaca tggatggcgg caacgaaaac tacgatgcga ttgccaccct aaaatggatg 1380 aatgaggaag tgtacaaata cttcccgaat gcgatgacga ttgcggaaga gtcgactgct 1440 ttcccgggcg tgtccgcacc gacctttatg ggcggtttgg gctttggctt taagtggaat 1500 atggggtgga tgcacgatag tctttcttac atcaaagaag agcctgttca tcgcaaatac 1560 cccacaata ccctcacgtt cccactcgtt tacgctcaca gtgagaacta cgtcttgtca 1620 ctttctcacg atgaagtggt atacggcaaa ggttctattc acaacaaaat gccaggtgat 1680 gagtggcagc aaaccgccaa tttacgtgcg tattttggtt acatgtatgg tcaaccgggc 1740 aaaaaactca actttatggg cgcggaaatc ggccaaacgg cagagtggaa tcacgatgac 1800 caactgcaat ggttcttgct cgatttccca cgtcaccaag gcgtgcaagc cttgacgcgt 1860 gatctcaacc acttgtatcg aaatgaagcg gcgctgcacg atcaagattg catcccagcc 1920 ggttttgagt ggcgtttaca agatgctgcc gagcagagca ttattgctca tgagcgtatc 1980 agtgaagcag gcgagcgcat tctggtagtg agtaacttta ccccagtgcc acgtgatgaa 2040 ttccgcttag gcgtgccaaa caaaggccgc taccaattgc tgctcaacac cgacgacagc 2100 aaatacgcag gcagtggcta cgaggttgtg gtggacgcca agagcgaagc ggtggtcagt 2160 gaagatttgg ctcagtcgat tgtgttgcgc ctaccaccac tgtcgacatt gttctacaag 2220 ctggtctaa 2229

Claims (10)

Comprising reacting amylose and amylopectin with a branching enzyme consisting of the amino acid sequence of SEQ ID NO: 1 derived from Vibrio vulnificus . delete The method according to claim 1, wherein the branching enzyme derived from Vibrio vulnificus is produced by the following steps:
Preparing a recombinant vector comprising a gene encoding a branching enzyme derived from Vibrio vulnificus ;
Transforming the prepared recombinant vector into E. coli; And
Isolating and purifying the branching enzyme from the transformed E. coli. [2] The modified starch according to claim 1, wherein the modified starch has a short branching ratio of 1 to 5 of Degree of Polymerization (DP) of 20 to 30% based on the total modified starch. The process for producing modified starch according to claim 1, wherein the modified starch has a water solubility of 35 to 45%. The method according to claim 1, comprising the step of reacting amylose and amylopectin with a branching enzyme consisting of the amino acid sequence of SEQ ID NO: 1 derived from Vibrio vulnificus , wherein the short starch ratio of 1 to 5 is 20 to 30% based on the total modified starch, and the water soluble starch has a water solubility of 35 to 45%. 7. The method according to any one of claims 1 to 6, wherein the short branch ratio of Degree of Polymerization (DP) 1-5 is 20-30% based on total modified starch, 35-45% &Lt; / RTI &gt; by weight of the modified starch. A food manufactured using the modified starch of claim 7. A composition for producing modified starch comprising a branching enzyme comprising the amino acid sequence of SEQ ID NO: 1 derived from Vibrio vulnificus or a gene coding therefor and amylose and amylopectin as an active ingredient. The modified starch according to claim 9, wherein the modified starch has a short branching ratio of 1 to 5 (DP) of 20 to 30% based on the total modified starch, and has a water solubility of 35 to 45% / RTI &gt;

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