KR20020002588A - Method for production of isomaltooligosaccharides - Google Patents

Abstract

PURPOSE: Provided is a method for producing maltooligosaccharides or isomaltooligosaccharides using maltose-producing amylase and alpha-glucanotransferase, thereby shortening enzyme reaction time and increasing the production yields of isooligosaccharides. CONSTITUTION: Isooligosaccharides are produced by adding maltose-producing amylase and alpha-glucanotransferase sequently or simultaneously into liquid dextrin, then reacting them. Wherein maltose-producing amylase can be additionally added while adding alpha-glucanotransferase.

Description

Method for production of isomaltooligosaccharides

The present invention relates to a method for producing isomaltooligosaccharide.

Recently, the physiological properties of various oligosaccharides and the physiological effects obtained thereby have been reported. The function of maltooligosaccharides depends on their size and binding form. Branched maltooligosaccharides are mainly linked by α-1,4-glycosidic bonds and at least one α-1,6-glycosidic Α-D-glucopyranoase oligomer having a bond means an oligomer consisting of isomaltose, isomaltotriose, panose and 4-5 glucose residues.

Branched oligosaccharides are softer than sugar and can lower the viscosity and water activity relatively, thus preventing microbial contamination. In addition, when ingested, less metabolic energy than sugar and can improve the intestinal microflora and has the effect of preventing tooth decay.

In the process for producing isomaltooligosaccharide from starch, starch degrading enzymes such as amylase and flulanease and sugar transfer enzymes are used. Thus, the method for preparing isomaltooligosaccharide is a time-consuming process.

Amylase hydrolyzes most α- (1,4) -glucosidic bonds in starch to produce glucose, maltooligosaccharides, and α-limiting dextrins, and the remaining α- (1,6) -glucosidic bonds Is hydrolyzed by pullulanase. Therefore, dextrin does not remain in the starch hydrolysis process using amylase and flulanase. This method can obtain the desired final product in higher yield from starch.

Glucose reactions using amylase and other related enzymes are used to produce various oligosaccharides. For example, liquefied starch liquefied with starch hydrolase, such as α-amylase or beta-amylase, is treated with maltogenase, beta-amylase and pullulanase to primary glycosylation, and mainly glucose and maltose are α- ( 1,6)-Process of transglucosidase, which is glycosylated with glycosylated bonds, is used to produce isooligosaccharides by secondary glycosylation. In this method, about 72 hours for primary glycosylation and about 2 times glycosylation are used. It takes 48 hours.

Korean Patent Application 96-25135 describes a method for producing maltooligosaccharides using maltose-producing amylase. When maltose-producing amylase is used, the glycosylation is completed in about 12 hours without distinguishing between primary and secondary saccharification to obtain malto-oligosaccharide, but characteristics of maltose-producing amylase having both hydrolytic activity and sugar-transfer activity Due to this, the isomaltooligosaccharide content cannot be sufficiently increased above a certain level.

The present invention provides a method for producing isomaltooligosaccharide with a short enzyme reaction time.

Figure 1 shows the sequence of F4 and MSI-344 used in the preparation of the vector pGNX4F4M used in the present invention.

Figure 2 shows the comparison of the production yield of isomaltooligosaccharides according to the concentration of BSMA.

Figure 3 shows the comparison of the yield of isomaltoligosaccharides according to the concentration of ThMA.

Figure 4 shows the change in the production yield of isomaltoligosaccharide upon simultaneous treatment of ThMA and α-glucanotransferase.

The present invention relates to a method for producing isomaltooligosaccharide.

The method of the present invention consists in producing isomaltooligosaccharide by acting maltose-producing amylase and α-glucanotransferase on liquefied starch. In the method of the present invention, maltose-producing amylase and α-glucanotransferase may be simultaneously applied or sequentially applied.

Therefore, the method of the present invention consists in reacting liquefied starch by applying maltose-producing amylase and α-glucanotransferase simultaneously.

Another method of the present invention consists in reacting liquefied starch by applying maltose-producing amylase, followed by applying α-glucanotransferase.

Another method of the present invention consists in reacting liquefied starch by applying maltose-producing amylase, then inactivating the maltose-producing amylase and applying it to α-glucanotransferase.

In the process of the invention, maltose amylase may be further added if necessary when the α-glucanotransferase is added to the starch.

In the present invention, liquefied starch means that the starch is liquefied with starch hydrolase, such as α-amylase. Therefore, liquefied starch can be prepared by liquefying starch by adding starch hydrolase to the starch slurry, and commercially available liquefied starch can also be used.

Maltogenic amylase according to the present invention is hydrolyzed starch to produce mainly maltose, and to break down pullulan to produce mainly pannose, amylose having a specific enzymatic properties that strongly degrade cyclodextrin It means raise. Examples of such maltose-producing amylase include amylase produced from Bacillus stearothermophilus (KFCC-10896) described in Korean Patent Application 96-25135 (hereinafter referred to as BSMA), Bacillus licheniformis (ATCC 27811) described in US Patent 5,583,039, and Korean Patent Thermus sp. Amylases (hereinafter referred to as ThMA) isolated from (IM6501 KCTC 0527BP), or enzymes having substantially the same properties as those, and enzymes having substantially the same properties as those produced in genetic recombination technology. Expression vector used in the present invention was made with reference to the method described in Korean Patent Application No. 1999-17920, the patent application is included in the content of the present specification.

The α-glucosidyltransferase used in the present invention means a glycotransferase that transfers maltooligosaccharides to small sugars while forming α- (1,4) -glucosidyl bonds. Although the kind of (alpha)-glucosidyl transferase is not specifically limited, A process heat resistant (alpha)-glucosidyl transferase is preferable.

Enzyme activity measurement method and the medium used in the present invention is as follows.

The active unit (CU) of maltose-producing amylase was added to 0.5 ml of enzyme solution, 0.4 ml of 50 mM sodium citrate buffer solution at pH 6.0 and 0.5 ml of 1% (w / v) β-cyclodextrin solution, followed by 30 minutes at 55 ° C. After stopping the reaction by adding the DNS solution and boiled for 5 minutes to develop the color was measured by measuring the absorbance at 575nm, it is determined as 1 unit when the difference in absorbance is 1.0.

The active unit (AU) of α-glucanotransferase is mixed with 0.05% amylose, 0,05% maltose solution dissolved in 50 mM Tris / HCl buffer (pH 7.5) at 60 ° C. After 0 min and 15 min of the reaction, 0.1 ml of each was mixed with 1 ml of a 0.02% iodide / potassium iodide solution and the absorbance was measured at 620 nm. The amount of enzyme when the measured absorbance difference becomes 1 is expressed by 1 unit.

LB medium (10 g / l bacto-tryptone, 5 g / l yeast extract, 10 g / l NaCl)

R medium (Reisenberg _{medium; KH 2 PO 4 13.3 g /} l, (NH 4) 2 HPO 4 4.0 g / l, citric acid _{0.17 g / l, MgSO 4 .7H} 2 O 0.22 g / l, glucose 20 g / l, Trace element solution 10ml / l)

Isomaltooligosaccharide analysis in the present invention was carried out by HPLC method according to the following conditions.

DP (polymerization) analysis conditions

Instrument: HPLC (waters)

Column: Biorad Aminex-HPX42A Column (Biorad; 7.8x300 cm)

Detector: RI Detector

Column temperature: 80 ℃

Mobile phase: distilled water

Flow rate: 0.4ml / min

Basis Polymerization Condition

Instrument: HPLC (waters)

Column: Carbohydrate analysis column (waters; length diameter)

Detector: RI Detector

Column temperature: 35 ℃

Mobile phase: acetonitrile / water (65:35 (w / w))

Flow rate: 2.0ml / min

Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited by Examples.

Example 1 Expression Vector Preparation

PGNX4F4M was prepared by referring to the method described in Korean Patent Application No. 1999-17920.

In other words, E.coli HM174 (DE3) / pGNX2 deposited as KCTC 0486BP in the Korean Collection for Type Cultures, located at 52, Eeun-dong, Yuseong-gu, Daejeon, Korea. The expression vector pGNX2 was isolated according to the method described in Molecular cloning a laboratory manual 2nd (Sambrook et al., Cold Spring Habor laboratory Press, 1989). PGNX2F4M was prepared by referring to Example 3 and FIG. 4 of Korean Patent Application No. 1999-17920. That is, the 3 'end of F4 (see Fig. 1) was cut with Ssp I, and the 5' end of MSI-344 (see Fig. 1) was cut with Sma I, and F4 and MSI-344 were connected to obtain a DNA construct F4M. A fragment obtained by cutting the F4M by Nde I and Bam HI, and cloning the insert into pGNX2 cut into Nde I and Bam HI to prepare a plasmid pGNX2F4M.

To make plasmid pGNX3, pGNX2F4M was first cleaved with Bsp HI, and fragments cut from only one Bsp HI locus were cut with Bam HI. To create fragments containing T7 and rrnBT1T2 terminators, 132 bp fragments cut with Bam HI and Eco RV were isolated from pET11a (Novagen), and 488 bp fragments isolated from ptrc99a (Pharmacia) with Hin cII and Bsp HI. Connected with. Plasmid pGNX3F4M was prepared by digesting the fragments with Bam HI and Bsp HI and linking them with the previously made vector.

In order to make plasmid pGNX4F4M, plasmid pETACc was cleaved with Xba I and Alw N I to isolate 3502 bp fragment, which was then cleaved with pGNX3F4M with Xba I and Alw NI to connect 2405 bp fragment isolated to prepare pGNX4F4M. . Plasmid pETACc was prepared as follows. The plasmid pET21c (Novagen) was digested with XbaI, then peeled-in (filling-in) and digested with BglII to isolate 5375 bp DNA fragments.The plasmid pKK223-3 (Pharmacia) was digested with Eco RI, then peeled-in and Bam HI was isolated to 259bp DNA fragment containing the tac promoter was isolated. These two DNA fragments were linked to obtain plasmid pETACc.

Example 2. Enzyme Production

1) BSMA manufacture

E. coli / pSG18 was incubated at 37 ° C. in LB-Epicillin medium with Accession No. KFCC-10896, described in Korean Patent Application 1996-25135, to separate plasmid pSG18 according to the method described in the reference of Sambrook et al. From this, the BSMA gene (NCI Accession No .: U50744) was isolated. Using the isolated BSMA gene and the primers below, a BSMA gene was produced having a restriction enzyme Nde I cleavage site at 5 'end and a restriction enzyme Bam HI cleavage site at 3' end by polymerase chain reaction (PCR) reaction. Parts cut the gene with restriction enzymes Nde I and that a complete cut with Bam HI, was cloned into a plasmid pGNX4F4M cut with restriction enzymes Nde I and Bam HI to obtain a pGNX4-BSMA.

primer

BMNdeI 5'-CCC CATATG TTCAAAGAAGCC-3 '

BMBmI 5'-CCC GGATCC TTATAACCAATCTTCG-3 '

Transformants obtained by transforming pGNX4-BSMA into E. coli TG1 were incubated at 30 ° C. in a 5 L fermenter containing 1.5 L of R medium to which cassanoic acid was added. When the culture medium was OD ₆₆₀ 80, 1mM IPTG was added to induce enzyme expression, followed by further incubation for 5 hours. The cells were recovered, and the recovered cells were suspended in 50 mM Tris-HCl buffer at pH 7.5 and the cells were disrupted by a homogenizer. Polyethyleneimine 0.5% (w / v) was treated to remove the nucleic acid. The supernatant was collected by centrifugation at 12,000 g for 30 minutes, dialyzed with 50 mM Tris-HCl buffer at pH 7.5, and concentrated with a Pelicon Ultrafiltration Kit (Millipore, USA) with a cutoff of 10,000. The concentrate was partitioned with 20% (w / v) ammonium sulfate and left at 4 ° C. for 2 hours, followed by centrifugation. The resulting supernatant was partitioned with 60% (w / v) ammonium sulfate and left for 4 hours at 4 ° After centrifugation, the supernatant was removed. The pellet obtained by centrifugation was suspended in 50 mM Tris-HCl buffer at pH 7.5, then dialyzed, and concentrated with a Pelicon Ultrafiltration Kit (Millipore, USA) to obtain partially purified BSMA.

2) ThMA manufacture

E. coli / pThMA119 deposited with accession number KCTC-0528BP described in Korean Patent Application 1998-63956 was incubated at 37 ° C. in LB-empicillin medium, and the plasmid pThMA119 was prepared according to the method described in the reference of Sambrook et al. Separated. Using the reported ThMA coding DNA sequence (NCI Accession No .: AF060204) and the primers described below, the ThMA gene was obtained by polymerase chain reaction, and then cleaved with Nde I and Hin dIII to generate a Somers maltose-producing amylase (ThMA) gene. Was separated.

primer

TMNdeI

5'-GGAGGAAAAAG CATATG AGGAAAGAAGCC-3 '

TMHdIII

5'-AAAGGCA AAGCTT TGGTTGGCGGAGACG-3 '

The obtained ThMA gene was cloned into pGNX4F4M cut with Nde I and Hin dIII to obtain plasmid pGNX4-ThMA. The transformed cells obtained by transforming pGNX4-ThMA to E. coli TG1 were cultured in the same manner as the BSMA transformants, and the recovered cells were suspended in 50 mM Tris-HCl buffer at pH 7.5, and the cells were homogenized. Destroyed. Heat treated for 20 minutes in a 60 ℃ water bath, and treated with polyethyleneimine 0.5% (w / v) to remove the nucleic acid and centrifuged for 30 minutes at 12,000g to remove the pellets. The supernatant was dialyzed with 50 mM Tris-HCl buffer at pH 7.5 and concentrated with a Pelicon Ultrafiltration Kit (Millipore, USA) with a cutoff of 10,000 to obtain partially purified ThMA.

3) α-glucanotransferase production

Using the chromosome of the deposited strain Thermotoga maritima MSB8 JCM10099 (Japan Collection of Microorganisms) as a template, and using the following primers based on the reported α-glucanotransferase gene sequence (NCI Accession No .: Z50813) Polymerase chain reaction (PCR) yielded a DNA fragment containing the α-glucanotransferase gene.

primer

TmAGT5: 5'-GACGGCTCAGAAGTTCTACAATACC-3 '

TmAGT3: 5'-CCCGCGAAGTCTTCAAGAGGAACG-3 '

The DNA fragment containing the obtained α-glucanotransferase gene was cloned into pUC119 digested with Hin cII to obtain pα-GT119.

After PCR using the following primers based on the pα-GT119 completely cut with Nde I and cut the α- glue transfer Kano raised gene comprising a DNA fragment obtained by partial cleavage with Hin dIII to Nde I and Hin dIII was cloned in pGNX4F4M The plasmid pGNX4-αGT was obtained.

primer

TGTNd: 5'-CAAGGAGGATAGA CATATG ATAGGCTATCA G -3 '

Reverse primer (sequencing): 5'-AGCGGATAACAATTTCACACAGGA-3 '

The transformed cells obtained by transforming pGNX4-αGT into E. coli TG1 were cultured in the same manner as the BSMA transformants, and the recovered cells were suspended in 50 mM Tris-HCl buffer at pH 7.5 and the cells were homogenized. Destroyed. Heat treated for 20 minutes in an 80 ℃ water bath, and treated with 0.5% polyethylenimine (w / v) to remove the nucleic acid. The supernatant was removed by centrifugation at 12,000g for 30 minutes, washed once, dialyzed with 50 mM Tris-HCl buffer at pH 7.5, concentrated with a Pelicon Ultrafiltration Kit (Millipore, USA) with a cutoff of 10,000. Purified α-glucanotransferase was obtained.

Comparative example. Branch oligosaccharide production using maltose-produced amylase

Branched oligosaccharide production reactions were performed using liquefied corn syrup (Brix33, DE22).

BSMA was added to liquefied corn syrup 100, 200, and 400 CU / g substrate, respectively, and the yield of branched oligosaccharides over time was measured while saccharifying at 50 ° C. for 65 hours. The result is shown in FIG.

Using ThMA instead of BSMA, the experiment was conducted under the same conditions except that the saccharification temperature was 55 ° C., and the results are shown in FIG. 3.

Example 3. Branch Oligosaccharide Production Using BSMA and α-Glucanotransferase

BSMA 200 CU / g substrate produced in Example 2 and α-glucanotransferase were added to liquefied corn syrup (Brix33, DE22) 3000, 6,000, and 12,000 AU / g substrate, respectively, and 0-50 hours at 55 ° C. Reacted. Malto oligosaccharides and isomaltooligosaccharides produced over time are analyzed and the results are shown in Table 1.

Analysis of Oligosaccharides Produced by Simultaneous Treatment with BSMA and α-Glucanotransferase Substrate (0hr) BSMA (CU / ml) + α-GT (AU / ml) 200 + 3,000 200 + 6,000 200 + 12,000 14h 20h 36h 50h 14h 20h 36h 50h 14h 20h 36h 50h MO G1 4.2 10.2 10.9 11.7 11.1 10.4 10.8 12.5 11.2 10.5 11.0 12.3 12.3 G2 13.8 11.0 11.5 11.2 12.4 11.5 11.4 12.1 12.0 11.5 11.2 12.5 13.0 G3 16.4 3.1 2.6 2.7 3.3 3.5 2.7 2.5 3.3 3.3 3.0 2.6 3.2 G4 10.9 1.6 1.4 1.4 1.7 1.7 1.3 1.4 1.5 1.6 1.4 1.3 1.5 G5 17.6 0.6 0.4 0.4 0.5 0.5 0.4 0.5 0.5 0.4 0.4 0.3 0.5 G6 14.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 > G7 22.6 7.4 6.9 5.8 6.0 7.0 6.0 5.7 5.6 6.8 6.2 5.0 4.6 IMO B2 0.0 6.8 8.0 9.6 8.7 6.5 8.2 9.1 9.6 6.9 8.9 10.2 10.2 B3 0.0 19.5 23.2 25.2 25.7 19.4 23.8 25.8 26.2 19.5 23.6 27.2 27.3 B4 0.0 27.8 24.7 22.5 21.0 27.5 35.0 20.7 20.5 27.7 24.3 19.6 18.3 B5 0.0 5.6 5.2 4.4 4.9 5.8 4.9 4.8 5.0 6.0 4.8 4.8 4.9 B6 0.0 6.3 5.2 5.1 4.8 6.3 5.5 4.6 4.7 5.8 5.1 4.3 4.2 IMO total content 0.0 66.0 66.3 66.8 65.1 65.5 67.4 65.0 66.0 65.9 66.7 66.1 64.9 MO: maltooligosaccharide IMO: isomaltooligosaccharide

Example 4 Branch Oligosaccharide Production Using BSMA and α-Glucanotransferase

BSMA 100 CU / g substrate was added to the liquefied corn syrup (Brix33, DE22) and reacted at 50 ° C. for 20 hours. BSMA was inactivated and the sample was divided into four flasks. Add only BSMA 50 CU / g substrate to one flask, BSMA 50 CU / g substrate + α-Glucanotransferase 3000 AU / g substrate, BSMA 50 CU / g substrate + α-glucano A transferase 6000 AU / g substrate, BSMA 50 CU / g substrate + α-glucanotransferase 12000 AU / g substrate were added and reacted at 50 ° C. The produced maltooligosaccharides and isomaltooligosaccharides are analyzed and the results are shown in Table 2.

Analysis of Oligosaccharides Produced by Simultaneous Treatment with BSMA and α-Glucanotransferase Substrate (0h) BSMA (100 CU / ml) BSMA (CU / ml) + α-GT (AU / ml) 50 50 + 3,000 50 + 6,000 50 + 12,000 14h 20h 36h 50h 36h 50h 36h 50h 36h 50h MO G1 4.2 7.3 6.7 8.7 9.3 8.4 9.9 8.8 10.5 8.7 10.7 G2 13.8 17.7 17.6 16.0 15.8 13.7 13.3 13.4 12.8 14.5 14.2 G3 16.4 14.0 14.1 9.8 9.1 8.6 6.4 6.7 4.5 7.3 5.3 G4 10.9 7.5 7.3 3.2 2.6 2.3 1.5 1.5 0.7 1.8 1.2 G5 17.6 2.8 2.6 1.0 0.7 0.7 0.4 0.5 0.3 0.5 0.2 G6 14.0 1.5 1.5 0.4 0.0 0.6 0.0 0.0 0.0 0.0 0.0 > G7 22.6 16.4 16.8 12.1 13.8 8.1 9.1 7.2 7.6 7.3 7.4 IMO B2 0.0 1.0 0.9 2.2 3.3 3.3 4.9 4.0 6.4 3.8 5.4 B3 0.0 3.6 3.5 7.9 9.3 10.9 14.1 15.2 18.9 14.0 16.9 B4 0.0 11.9 12.3 18.1 18.2 21.7 22.7 23.2 22.0 23.5 22.6 B5 0.0 7.5 7.9 11.0 8.7 10.8 9.4 10.7 8.9 10.8 10.5 B6 0.0 8.7 8.9 9.6 9.4 8.9 8.4 8.8 7.4 7.9 7.6 IMO total content 0.0 32.9 33.5 48.8 48.9 57.6 59.5 61.9 63.6 60.0 61.0 MO: maltooligosaccharide IMO: isomaltooligosaccharide

Example 5 Branched Oligosaccharide Production Using BSMA and α-Glucanotransferase

BSMA 100 CU / g substrate was added to the liquefied corn syrup (Brix33, DE22) and reacted at 50 ° C. for 20 hours. Samples were divided into four flasks, one flask without the addition of α-glucanotransferase, and the remaining three flasks inactivated BSMA and added the α-glucanotransferase 3,000, 6,000 and 12,000 AU / g substrate, respectively. After the reaction was continued at 50 ℃. The produced maltooligosaccharides and isomaltooligosaccharides are analyzed and the results are shown in Table 3.

Analysis of Oligosaccharides Produced in α-Glucanotransferase Treatment after BSMA Inactivation Substrate (0h) BSMA (100 CU / ml) α-GT (AU / ml) 3,000 after BSMA deactivation 6,000 after BSMA deactivation 12,000 after BSMA inactivation 14h 20h 36h 50h 36h 50h 36h 50h 36h 50h MO G1 4.2 7.3 6.7 6.6 8.1 6.5 7.7 6.5 7.6 6.4 7.5 G2 13.8 17.7 17.6 17.7 16.7 16.0 15.6 15.6 15.0 15.3 14.6 G3 16.4 14.0 14.1 14.5 13.6 12.5 11.8 12.6 10.8 12.5 12.1 G4 10.9 7.5 7.3 7.1 6.2 9.0 8.0 9.2 8.5 9.4 8.1 G5 17.6 2.8 2.6 2.6 1.9 3.1 2.5 3.2 2.7 3.4 2.8 G6 14.0 1.5 1.5 1.3 0.7 2.2 1.8 2.1 1.7 2.3 2.0 > G7 22.6 16.4 16.8 17.1 14.2 16.5 13.6 16.5 13.8 16.8 14.4 IMO B2 0.0 1.0 0.9 0.9 3.0 0.8 2.0 1.0 2.8 1.3 3.1 B3 0.0 3.6 3.5 3.6 4.9 3.1 4.5 3.1 4.7 3.1 5.2 B4 0.0 11.9 12.3 11.8 13.6 12.9 15.1 12.6 14.7 11.7 12.9 B5 0.0 7.5 7.9 7.9 8.2 8.3 8.6 8.4 8.5 8.3 8.5 B6 0.0 8.7 8.9 9.1 9.0 9.0 8.9 9.3 9.1 9.4 9.0 IMO total content 0.0 32.9 33.5 33.5 38.7 34.1 39.1 34.4 39.8 33.8 38.7 MO: maltooligosaccharide IMO: isomaltooligosaccharide

Example 6 Branched Oligosaccharide Production Using ThMA and α-Glucanotransferase

To two flasks, ThMA 200 CU / g substrate, ThMA 200CU / g substrate + α-glucanotransferase 6000AU / g substrate were added to liquefied corn syrup (Brix33, DE22), respectively, and reacted at 55 ° C. for 0-50 hours. Two different flasks were reacted with liquefied corn syrup (Brix33, DE22) with ThMA 200 CU / g substrate for up to 20 hours, and one α-glucanotransferase with 6000AU / g substrate and the other with ThMA. After inactivation, the α-glucanotransferase 6000AU / g substrate was added and reacted at 55 ° C. for 50 hours. The produced maltooligosaccharides and isomaltooligosaccharides are analyzed and the results are shown in FIG. 4.

By the production method of the present invention, it is possible to shorten the production time of isomaltooligosaccharide and to improve the yield of isomaltooligosaccharide.

Claims (3)

A method for producing isomaltooligosaccharide, comprising reacting liquefied starch by adding maltose-producing amylase and α-glucanotransferase. The method for producing isomaltooligosaccharide according to claim 1, wherein the maltose-producing amylase and α-glucanotransferase are added sequentially. The method for producing isomaltooligosaccharide according to claim 2, wherein the α-glucanotransferase further comprises adding maltose-producing amylase and reacting the same.