KR102637535B1 - Method for producing high soluble cellulose using 4,6-alpha-glucanotransferase and alpha-amylase complex enzyme reaction - Google Patents

Abstract

The present invention relates to a method for producing highly soluble fiber using a complex enzymatic reaction of 4,6-alpha-glucanotransferase and alpha-amylase, and specifically, glycosyltransferase and hydrolytic enzymes using dextrin as a substrate. It relates to a method of producing highly soluble fiber through composite processing. The fiber produced by the production method of the present invention had α-1,6 bonds increased to 87%, the proportion of products with a degree of glucose polymerization exceeding 10 increased to 79%, and water solubility was improved.

Description

Method for producing high soluble cellulose using 4,6-alpha-glucanotransferase and alpha-amylase complex enzyme reaction}

The present invention relates to a method for producing highly soluble fiber using a complex enzymatic reaction of 4,6-alpha-glucanotransferase and alpha-amylase.

Dextrin is a general term for polysaccharides with a molecular weight smaller than starch. It refers to a wide range from high molecular weight that slightly decomposes starch to low molecular weight that does not show iodine-starch reaction. Soluble starch is also a type of dextrin. In the living body, a reaction occurs to produce dextrin from starch by saliva and bacteria in the small intestine. These are various hydrolysis products that occur in the intermediate stage from starch to maltose when starch is hydrolyzed with acid, heat, enzymes, etc. It is used as office glue, water-based paint, confectionery composition, and as an excipient for pharmaceuticals.

Meanwhile, glycosyltransferase is a general term for transferases involved in the transfer of sugar residues (glycosyl groups) and is involved in the synthesis and decomposition of sugars such as polysaccharides, oligosaccharides, and glycosides. In general, the specificity varies depending on the type and three-dimensional structure of the sugar directly involved in the bond subjected to enzyme action. Like maltose phosphorylase or cellobiose phosphorylase, it causes inversion of the anomeric structure in the glucosyl donor and transfer product. There is also something. Sugars or derivatives (such as sucrose and glycosyl phosphate) with a glycosidic bond at a relatively high energy level are often used as donors, and they function in saccharide biosynthesis within the living body. Depending on the transfer stage, 1) transglucosylase (glucosyl transferase): sucrosephosphorylase (a type of α-glucosyl transferase), etc., 2) transfructosylase: levansucrase, etc., 3) trans Ribosylase: Nucleoside phosphorylase, etc., 4) Transglucuronylase: Uridine diphosphate glucuronic acid transglucuronylase, etc.

Korean Patent No. 2167849 discloses a method for producing glucosyl stevioside using alpha-1,6 glycosyltransferase, but the complex enzyme of 4,6-alpha-glucanotransferase and alpha-amylase of the present invention There has been no disclosure regarding a method for producing highly soluble fiber using reaction.

The present invention was derived from the above needs, and the present invention provides a method for producing highly soluble fiber using a complex enzymatic reaction of 4,6-alpha-glucanotransferase and alpha-amylase, and the present invention The fiber produced by the manufacturing method had α-1,6 bonds increased to 87%, the proportion of products with a degree of glucose polymerization exceeding 10 increased to 79%, and water solubility was improved, thereby confirming the present invention. was completed.

In order to achieve the above object, the present invention includes the steps of (1) treating maltodextrin with 4,6-alpha-glucanotransferase (4,6αGT, 4,6-α-glucanotransferase) to induce a glycosyltransferase reaction; ; and

(2) After the glycosyltransferase reaction of step (1), a hydrolytic enzyme is added to form a non-reducing end of the α-1,4 bond, resulting in a glycosyltransferase reaction using the 4,6-alpha-glucanotransferase. It provides a method for producing highly soluble fiber comprising the step of further inducing.

Additionally, the present invention provides highly soluble fiber produced by the above production method.

Additionally, the present invention provides a food containing the high soluble fiber.

The present invention relates to a method for producing highly soluble fiber using a complex enzymatic reaction of 4,6-alpha-glucanotransferase and alpha-amylase, and specifically, glycosyltransferase and hydrolytic enzymes using dextrin as a substrate. It relates to a method of producing highly soluble fiber through composite processing. The fiber produced by the manufacturing method of the present invention has α-1,6 bonds increased to 87%, the proportion of products with a degree of glucose polymerization exceeding 10 increased to 79%, and water solubility is improved. will be.

Figure 1 shows the glycosyltransferase (4,6-alpha-glucanotransferase) and hydrolytic enzyme (alpha-amylase) treatment times and conditions.
Figure 2 shows the results of ¹ H-NMR analysis of fiber produced according to the conditions of the production method of the present invention. (a) is the ¹ H-NMR analysis result of maltodextrin, the substrate used in the glycosyltransferase reaction, (b) is the ¹ H-NMR analysis result of the product after only the glycosyltransferase reaction was performed for 24 hours, and (c) ) is the result of ¹ H-NMR analysis of the product after performing only the glycosyltransferase reaction for 72 hours, and (d) is the result of 1 H-NMR analysis of the product after performing only the glycosyltransferase reaction for 72 hours, followed by treatment with alpha-amylase for an additional glycosyltransfer reaction This is the result of ¹ H-NMR analysis of the product after performing.
Figure 3 is the result of high-performance anion exchange chromatography analysis to confirm the degree of polymerization of the reaction product (cellulose) prepared by the production method of the present invention. The results are according to conditions A to G in Figure 1, and G1 to G9 at the top are It shows the degree of glucose polymerization.
Figure 4 shows the results of ¹ H-NMR analysis confirming the ratio of α-1,4 bond, α-1,3 bond, and α-1,6 bond of the reaction product prepared by the method of the present invention for 72 hours. (a) is a case of using a 5% G10 substrate, (b) is a case of using a 20% G10 substrate, and (c) is a case of using a 30% G10 substrate.

The present invention includes the steps of (1) treating maltodextrin with 4,6-alpha-glucanotransferase (4,6αGT, 4,6-α-glucanotransferase) to induce a glycosyltransferase reaction; and

(2) 24 to 48 hours after the glycotransferase reaction of step (1), a hydrolytic enzyme is added to form the non-reducing end of the α-1,4 bond to the 4,6-alpha-glucanotransferase. It relates to a method for producing highly soluble fiber comprising the step of further inducing a glycosyltransferase reaction.

In general, alpha-glucanotransferase is an enzyme that transfers glucose units from the non-reducing end of the donor molecule to the non-reducing end of the recipient molecule. It hydrolyzes the long branches of amylose and amylopectin, and at the same time hydrolyzes the hydrolyzed low molecular weight glucan. It is an enzyme that reorganizes amylopectin branches by carrying out glycotransferase to α-1,4 and α-1,6 bonds. 4,6-α-glucanotransferase of the present invention is (1→4)-α-D-gluco- Oligosaccharides can produce linear α-glucans with consecutive (α1→6) glucosidic linkages. The enzyme cleaves the non-reducing glucose component of (1→4)-α-D-gluco-oligosaccharide and attaches this glucose component to the non-reducing end of another glucan chain through a (α1→6) bond. By repeating this enzymatic reaction, α-glucan with a continuous (α1→6) bond can be produced. In the method according to one embodiment of the present invention, the 4,6-α-glucanotransferase may consist of the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

The maltodextrin may have a degree of glucose polymerization of 2 to 20, but is not limited thereto.

The hydrolytic enzyme in step (2) may be alpha-amylase, but is not limited thereto.

The 4,6-alpha-glucanotransferase includes (1) a gene encoding a maltose binding protein and a gene encoding a 4,6-α-glucanotransferase protein derived from Lactobacillus reuteri . Preparing a recombinant vector;

(2) transforming the prepared recombinant vector into E. coli; and

(3) isolating and purifying the maltose-binding protein fusion 4,6-α-glucanotransferase enzyme from the transformed E. coli; but is not limited thereto.

The reaction temperature may be 37-42°C, and the pH of the reaction may be 4.7-5.3, but is not limited thereto.

Additionally, the present invention relates to highly soluble fiber produced by the above production method.

The highly soluble fiber has an α-1,6 bond of 80% or more, preferably 83 to 87%, a glucose polymerization degree of 10 or more, and a ratio of 55 to 80%, preferably 77 to 79%, with respect to water. The solubility may be 60-65%, but is not limited thereto.

Additionally, the present invention relates to a food containing the high soluble fiber.

The food of the present invention can be used by adding the high-soluble fiber prepared in the present invention as is or with other foods or food ingredients, and can be used appropriately according to conventional methods. In addition, there are no particular restrictions on the type of food, but health functional foods, health foods, or health supplements can be manufactured using the high-soluble fiber. In the present invention, health food or health supplement refers to food that provides bioregulatory functions by including physiologically active ingredients in addition to nutritional functions.

Hereinafter, the present invention will be described in more detail using examples. These examples are only for illustrating the present invention in more detail, and it is obvious to those skilled in the art that the scope of the present invention is not limited thereto.

Example 1. Expression and purification using MBP (maltose binding protein)

The size of the 4,6αGT protein is 101kD, and in order to increase expression in the soluble fraction, the malE gene that expresses MBP (maltose binding protein) was used to express it in the soluble fraction, and the protein was expressed in the soluble fraction using amylose affinity chromatography. The degree of purification was improved using a purification method. The 4,6αGT gene derived from Lactobacillus reuteri was subcloned into the pMAL_TEV_XhoⅠ vector containing the malE gene expressing MBP into the NdeⅠ and HindⅢ restriction enzyme sites. Using E. coli as a host, the culture was cultured at 37°C using LB medium to an OD value of 0.6 to 0.8, then 0.1mM of IPTG was added and cultured at 30°C for 6 hours. After culturing, the cells were harvested, disrupted, and centrifuged to obtain the supernatant, and the protein fused with MBP was separated and purified. Purification was performed using a column made using amylose resin, taking advantage of the affinity between MBP and amylose. Put 1 ml of amylose resin into the column, stabilize it by flowing 5 ml of 50mM Tris (pH 7.5) buffer, load 5 ml of sample, and wash by loading 5 ml of 50mM Tris (pH 7.5) buffer again. Then, 5 ml of elution buffer containing 10mM maltose in 50mM Tris was loaded to elute the fusion protein attached to the amylose resin. The size of the protein was 142 kD, expressed in the soluble fraction, and purified using amylose affinity chromatography.

Example 2. Production of highly soluble fiber through complex enzyme treatment

The 4,6-αGT reaction was performed using DE 10 maltodextrin called PureDex produced by Daesang Co., Ltd. as a substrate. Maltodextrin was selected because it has higher water solubility than starch and has low viscosity at high concentrations, making it easy to carry out enzymatic reactions. Preferably, maltodextrin with a degree of polymerization equal to or greater than that of maltose can be used.

In order to confirm the effect of the complex enzyme treatment of the present invention, four types of enzyme reactions were constructed as follows.

① Reaction 1: When treating only 4,6-αGT,

② Reaction 2: When the enzyme is inactivated after reaction by treatment with 4,6-αGT and treated with alpha-amylase,

③ Reaction 3: When the enzyme is inactivated after the enzyme reaction by treatment with 4,6-αGT, treated with alpha-amylase, and then treated with 4,6-αGT again,

④ Reaction 4: The experiment was divided into cases where 4,6-αGT was treated, enzymatic reaction was followed by alpha-amylase treatment, and the reaction results were compared.

In reaction 1, 30% (w/v) maltodextrin, 5mM calcium chloride, and 0.05% (w/v) sodium azide as substrates were dissolved in 50mM sodium acetate buffer solution (pH 5.0). . Per 1 g of substrate, 0.5 U of 4,6-αGT was treated to induce a glycosyltransferase reaction at 40°C for 72 hours.

In Reaction 2, after Reaction 1, the 4,6-αGT was sterilized at 100°C for 10 minutes to inactivate, and the reaction mixture was purified using a 0.45㎛ filter.

Afterwards, it was treated with the hydrolytic enzymes Termamyl and Novamyl alpha-amylase, respectively, and hydrolyzed at 40°C under pH 5.0 conditions.

In reaction 3, after reaction 2, 0.5 U of 4,6-αGT was added per 1 g of substrate to induce a glycosyltransferase reaction at 40°C for 72 hours.

In reaction 4, 30% (w/v) maltodextrin, 5mM calcium chloride, and 0.05% (w/v) sodium azide as substrates were dissolved in 50mM sodium acetate buffer solution (pH 5.0). . Per 1 g of substrate, 0.5 U of 4,6-αGT was treated to induce a glycosyltransferase reaction at 40°C for 72 hours, and as shown in Figure 1, alpha-amylase enzyme was activated at the beginning, 24 hours, or 48 hours after the reaction. was processed to simultaneously perform a saccharide transfer reaction and a hydrolysis reaction.

As a result, as shown in Table 1 below, the reaction results of ③ 4,6-αGT treatment to inactivate the enzyme after the enzyme reaction, alpha-amylase treatment, and then again 4,6-αGT treatment and ④ 4,6- After the enzyme reaction with αGT treatment, it was confirmed by ¹ H-NMR analysis that the α-1,6 bond ratio of the reaction product was 83-84% in the reaction results of alpha-amylase treatment (FIG. 2).

Proportion of alpha-glucan produced according to 4,6-αGT reaction by condition α-1,4 binding ratio (%) α-1,6 binding ratio (%) Substrate (Puredex) 96 4.0 ① Product of Puredex treated with 4,6-αGT 66 34.0 ② Puredex product treated with 4,6-αGT and then inactivated -> alpha-amylase treated 36.1 63.9 ③ Inactivation after 4,6-αGT treatment -> alpha-amylase treatment -> 4,6-αGT treated Puredex product 16 84 ④ 4,6-αGT treatment and enzymatic reaction -> Alpha-amylase treated Puredex product 17.0 83.0

Example 3. Confirmation of polymerization degree of reaction product (fibrin) using high-performance anion exchange chromatography analysis

In Reaction 4, the degree of polymerization of the reaction product was found to be optimal when treated with alpha-amylase 24 to 48 hours after the reaction, and through high-performance anion exchange chromatography analysis, the glycotransfer reaction was induced and 24 hours later. When treated with alpha-amylase, it was confirmed that more than 90% of reaction products with a degree of polymerization of 2 to 9 were obtained, and among them, reaction products with a degree of polymerization of 4 to 9 were found at about 80%, based on the total. It was confirmed that 72% of reaction products with a degree of polymerization of 4 to 9 were obtained (Figure 3).

Example 4. Measurement of glucose content

To measure the hydrolytic activity of the improved 4,6-α-glucanotransferase (4,6-α-GT) according to the degree of substrate polymerization, maltotetraose (G4), maltohexaose; Hydrolysis reaction was performed using G6), maltooctaose (G8), and maltodecaose (G10) as substrates. Afterwards, the inactivated reactant was diluted, filtered using a 0.2㎛ hydrophilic membrane filter, and then analyzed by high-performance anion exchange chromatography.

[High-performance anion exchange chromatography analysis conditions]

Column: CarboPac ^TM PA-1 column (4×250 mm, Dionex) composed guard column (4×50mm, Dionex)

Eluent: 0.15 M NaOH buffer, 0.15 M NaOH with 0.6 M NaOAc.

Sample concentration: 0.05mg/ml

Flow rate: 1.0 mL/min

Injection volume: 20㎕

The peak areas of glucose in the reactant and standard material were obtained from the chromatogram obtained from high-performance anion exchange chromatography analysis, the glucose content of the reactant was determined using the glucose concentration of the standard material, and the amount of glucose produced compared to the reaction substrate was compared.

As a result, the glucose content depending on the substrate was shown in Table 3 below.

Glucose production after 4,6-α-GT hydrolysis according to substrate concentration Substrate concentration (%) Glucose production (mg/g substrate) 24h 48h 72h G4 10% 194 204 206 20% 178 180 168 30% 152 172 184 G6 10% 102.9 105.9 109.9 20% 68.4 74.1 80.3 30% 68.0 66.1 62.9 G8 10% 144 138 158 20% 100 100 90 30% 98 100 88 G10 5% 132 120 136 20% 102 96 88 30% 68 82 62

Example 4. Measurement of polymerization degree distribution

To evaluate the degree of polymerization distribution of the synthesized isomaltooligosaccharide, it was analyzed by size exclusion chromatography (SEC). Isomaltooligosaccharides and G4, G6, G8, G10, and G12 used as standards were dissolved in water to a concentration of 0.5 mg/ml and then filtered through a 0.2㎛ hydrophilic membrane filter.

[SEC analysis conditions]

Column: YMC-Pack DIOL 60 column (4.6×300 mm)

Eluent: water

Detector: refractive index (RI) detector

Sample concentration: 0.5mg/ml

Flow rate: 0.3 mL/min

Injection volume: 20㎕

The SEC analysis results were classified into three peak areas: Degree of Polymerization DP less than 4, DP greater than 4, DP less than 10, and DP greater than 10, and the distribution of each degree of polymerization was calculated using each peak area, as shown in Table 4 below. When using a substrate of 30% G10 as described in, the rate of DP>10 was found to be 79%.

Degree of polymerization distribution (%) = each peak area/total peak area × 100

Degree of polymerization (%) depending on substrate concentration Substrate concentration (%) Degree of polymerization (%) DP>10 4<DP<10 DP<4 G4 10% 55.4 27.3 17.3 20% 56.0 29.1 14.8 30% 51.6 31.5 16.9 G6 10% 63.4 21.9 14.7 20% 60.8 25.7 13.5 30% 68.9 27.2 3.9 G8 10% 64.8 13.0 22.2 20% 71.9 16.2 11.9 30% 71.7 17.4 10.9 G10 5% 61.7 21.0 17.3 20% 77.2 12.5 10.3 30% 79.0 12.4 8.6

Meanwhile, as a result of ¹ H-NMR analysis of each product, it was confirmed that the α-1,6 bond of the product was increased to 87% after the hydrolysis reaction of the substrate with more than 95% of the α-1,4 bond. When 5% G10 substrate was used, the ratio of α-1,4 bond, α-1,3 bond, and α-1,6 bond was 12:1:87, and when 20% G10 substrate was used, α-1,4 bond, α-1,3 bond, and α-1,6 bond ratio were found to be 12:1:87. The ratio of -1,4 bond, α-1,3 bond, and α-1,6 bond was found to be 11:2:87, and when 30% G10 substrate was used, α-1,4 bond, α- The ratio of 1,3 bonds and α-1,6 bonds was found to be 13:2:85 (Figure 4).

Example 5 . Analysis of soluble sugar content using phenol-sulfuric acid method

It was confirmed that the solubility in water of the fiber produced according to the method of the present invention was significantly higher than that produced under other conditions (Table 4).

Solubility in water of fiber produced by glycosyltransfer reaction with dextrin Solubility in water (%) Puredex ^TM (4.03% α-1,6) 25.2 ① 4,6-αGT treatment (34.0% of α-1,6) 38.4 ② After 4,6-αGT treatment, inactivation -> alpha-amylase treatment
(63.9% α-1,6) 52.0 ④ 4,6-αGT treatment to enzymatic reaction -> alpha-amylase treatment
(83.0% α-1,6) 62.9

<110> The Industry & Academic Cooperation in Chungnam National University (IAC) <120> Method for producing highly soluble cellulose using 4,6-alpha-glucanotransferase and alpha-amylase complex enzyme reaction <130> PN21387 <150> KR 2020/0151441 <151> 2020-11-13 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 904 <212> PRT <213> Lactobacillus reuteri <400> 1 Met Gly Ile Asp Gly Lys Asn Tyr His Phe Ala Ser Asn Gly Gln Leu 1 5 10 15 Leu Gly Asn Leu Tyr Gly Lys Ile Val Asp Gly Lys Phe Asn Ile Tyr 20 25 30 Asp Ser Leu Ser Asn Lys Leu Ile Lys Thr Leu Asp Ser Gly Asp Trp 35 40 45 Glu Asn Met Ala Tyr Ser Gln Asp Ser Ser Ser Ile Asn Asn Thr Asp 50 55 60 Gly Tyr Leu Ser Tyr Ser Gly Trp Tyr Arg Pro Tyr Gly Thr Ser Gln 65 70 75 80 Asp Gly Lys Thr Trp Tyr Lys Thr Thr Ala Ser Asp Trp Arg Pro Leu 85 90 95 Leu Met Tyr Thr Trp Pro Ser Lys Asp Val Glu Ala Lys Phe Ile Lys 100 105 110 Tyr Phe Val Asp Asn Gly Tyr Thr Asn Thr Asp Tyr Gly Leu Thr Lys 115 120 125 Asp Asn Val Thr Asn Leu Ser Gln Asp Thr Asp Thr Gln Thr Leu Asn 130 135 140 Lys Tyr Ala Arg Asn Leu Arg Phe Val Ile Glu Lys Ser Ile Ala Ala 145 150 155 160 Asn Lys Ser Thr Gly Pro Leu Ala Asn Asp Ile Asn Lys Phe Met Leu 165 170 175 Thr Ile Pro Glu Leu Ser Ala Lys Ser Glu Leu Pro Val Glu Tyr Ser 180 185 190 Asn Gly Tyr Val Pro Asp Val Ser Gly Ser Ile Asp Asn Asn Gln Leu 195 200 205 Ile Phe Ile Asn Asn Asn Ser Asp Asn Gln Ala Lys Gly Asn Thr Ser 210 215 220 Tyr Ala Asp Ser Asn Tyr Arg Leu Met Asn Arg Thr Ile Asn Asn Gln 225 230 235 240 Thr Asn Asn Asp Asn Ser Asp Gln Ser Pro Glu Leu Leu Val Gly Asn 245 250 255 Asp Ile Asp Asn Ser Asn Pro Ala Val Gln Ala Glu Asn Phe Asn Trp 260 265 270 Glu Tyr Phe Leu Leu Asn Tyr Gly Lys Leu Met Lys Tyr Asn Ala Asp 275 280 285 Gly Asn Phe Asp Gly Phe Arg Val Asp Ala Ala Asp Asn Ile Asp Ala 290 295 300 Asp Val Leu Asp Gln Leu Gly Gln Leu Val Asn Asp Met Tyr His Thr 305 310 315 320 Lys Gly Asn Gln Glu Asn Ala Asn Asn His Leu Val Tyr Asn Glu Gly 325 330 335 Tyr His Ser Gly Ala Ala Arg Met Leu Asn Asp Lys Gly Asn Pro Glu 340 345 350 Leu Phe Met Asp Ala Gly Tyr Phe Tyr Thr Leu Glu Asn Val Leu Gly 355 360 365 Gln Ala Glu Asn Lys Arg Asp Asn Val Asn Asn Leu Ile Thr Asn Ser 370 375 380 Val Val Asn Arg Ala Asn Asp Ile Thr Glu Asn Thr Ala Thr Pro Asn 385 390 395 400 Trp Ser Phe Val Thr Asn His Asp Gln Arg Lys Asn Val Ile Asn Gln 405 410 415 Ile Ile Ile Asp Asn His Pro Asn Ile Pro Asp Ile Met Ala Asn Ser 420 425 430 Tyr Lys Ser Thr Tyr Ala Gln Lys Ala Trp Asp Glu Phe Tyr Ala Asp 435 440 445 Gln Ala Lys Ala Asp Lys Lys Tyr Ala Gln Tyr Asn Leu Pro Ala Gln 450 455 460 Tyr Ala Leu Leu Leu Ser Asn Lys Asp Thr Val Pro Gln Val Tyr Tyr 465 470 475 480 Gly Asp Leu Tyr Lys Glu Thr Asp Gln Tyr Met Lys Thr Lys Ser Met 485 490 495 Tyr Tyr Asp Ala Ile Thr Thr Leu Met Lys Ala Arg Gly Glu Phe Val 500 505 510 Asn Gly Gly Gln Thr Met Thr Lys Val Asn Asp Asn Leu Ile Thr Ser 515 520 525 Val Arg Tyr Gly Lys Gly Val Val Asp Val Ser Ser Asn Gly Thr Asp 530 535 540 Pro Leu Ser Arg Thr Thr Gly Met Ala Val Ile Val Gly Asn Asn Pro 545 550 555 560 Ser Met Ser Glu Gln Val Val Ala Ile Asn Met Gly Leu Ala His Ala 565 570 575 Asn Glu Gln Tyr Arg Asn Leu Ile Asp Ser Thr Ala Asp Gly Leu Thr 580 585 590 Tyr Asn Ser Asn Gly Ser Val Asn Pro Ser Val Leu Thr Thr Asp Ser 595 600 605 Lys Gly Ile Leu Arg Val Thr Val Lys Gly Tyr Ser Asn Pro Tyr Val 610 615 620 Ser Gly Tyr Leu Ser Val Trp Val Pro Leu Ile Asn Gly Thr Gln Asn 625 630 635 640 Ala Arg Thr Ser Ala Gln Glu Val Arg Asn Val Pro Gly Lys Val Phe 645 650 655 Thr Ser Asn Ala Ala Leu Asp Ser His Met Ile Tyr Glu Asp Phe Ser 660 665 670 Leu Phe Gln Pro Glu Pro Thr Thr Val Asn Glu His Ala Tyr Asn Val 675 680 685 Ile Lys Asp Asn Val Ala Leu Phe Asn Gln Leu Gly Ile Thr Asp Phe 690 695 700 Trp Met Ala Pro Ser Tyr Thr Pro Phe Asn Thr Ser Arg Tyr Asn Glu 705 710 715 720 Gly Tyr Ala Met Thr Asp Arg Tyr Asn Leu Gly Thr Ala Asp Asn Pro 725 730 735 Thr Lys Tyr Gly Asn Gly Glu Glu Leu Ser Asn Ala Ile Ala Ala Leu 740 745 750 His Gln Ala Gly Leu Lys Val Gln Glu Asp Leu Val Met Asn Gln Met 755 760 765 Ile Gly Phe Ser Thr Gln Glu Ala Val Thr Val Thr Arg Val Asp Arg 770 775 780 Asp Ala Lys Gln Leu Ser Val Asp Gly Gln Thr Phe Ala Asp Gln Ile 785 790 795 800 Tyr Phe Gly Tyr Thr Arg Gly Gly Gly Gln Gly Gln Gln Asp Tyr Gly 805 810 815 Gly Lys Tyr Leu Ala Glu Leu Lys Gln Lys Tyr Pro Asp Leu Phe Thr 820 825 830 Thr Lys Ala Ala Ser Thr Gly Val Ala Pro Asp Pro Asn Thr Arg Ile 835 840 845 Thr Glu Trp Ser Ala Lys Tyr Glu Asn Gly Thr Ser Leu Gln Asn Val 850 855 860 Gly Ile Gly Leu Ala Val Lys Met Pro Asn Gly Tyr Tyr Ala Tyr Leu 865 870 875 880 Asn Asp Gly Asn Asn Lys Ala Phe Ala Thr Thr Leu Pro Asp Ala Ile 885 890 895 Ser Ser Ala Asp Tyr Tyr Ala Asn 900

Claims (8)

(1) treating maltodextrin with 4,6-alpha-glucanotransferase (4,6αGT, 4,6-α-glucanotransferase) to induce a glycosyltransferase reaction; and
(2) 24 to 48 hours after the glycolysis reaction of step (1), alpha-amylase is added to form the non-reducing end of the α-1,4 bond to form the 4,6-alpha-glu A step of additionally inducing a glycosyltransferase reaction by carnotransferase; manufacturing highly soluble fiber having more than 80% of α-1,6 bonds and a ratio of 55 to 80% of the glucose polymerization degree of 10 or more. method. The method of claim 1, wherein the maltodextrin has a degree of glucose polymerization of 2 to 20. delete The method of claim 1, wherein the 4,6-alpha-glucanotransferase comprises (1) a maltose binding protein coding gene and a 4,6-α-glucanotransferase protein derived from Lactobacillus reuteri . Preparing a recombinant vector containing the coding gene;
(2) transforming the prepared recombinant vector into E. coli; and
(3) isolating and purifying the maltose-binding protein fusion 4,6-α-glucanotransferase enzyme from the transformed E. coli. The method of claim 1, wherein the reaction temperature is 37 to 42°C and the pH of the reaction is 4.7 to 5.3. It is manufactured by the method of any one of claims 1, 2, 4, and 5, and has an α-1,6 bond of 80% or more and a glucose polymerization degree of 10 or more in a ratio of 55 to 80%. Highly soluble fiber. delete Food containing the high-soluble fiber of paragraph 6.