KR20070006963A - Enzymatic preparation of highly purified maltooligosaccharide - Google Patents

Abstract

A method for preparing maltooligosaccharide is provided to be able to easily and conveniently obtain linear maltohexaose, maltoheptaose, and maltooctaose with high purity at relatively low cost by using cyclodextrin ring opening reaction of amylase. To prepare an aliphatic maltooligosaccharide having a glucose molecule number of 6-9, amylase isolated from Pyrococcus furiosus is added to a substrate solution including cyclodextrin such as alpha-cyclodextrin, beta-cyclodextrin and gamma-cyclodextrin, and is reacted under pH of 4.0-6.0 at a temperature of 65-95 deg.C. In the method, the substrate solution is prepared by reacting a soluble starch with cyclodextrin glucanotransferase.

Description

Method for producing high purity maltooligosaccharides using enzymes {ENZYMATIC PREPARATION OF HIGHLY PURIFIED MALTOOLIGOSACCHARIDE}

FIG. 1 shows linear maltohexaose (G6, maltohexata), maltoheptaose (G7, maltoheptaose) and maltooctaose (G8) from starch using a novel heat resistant amylase isolated from Pyrococcus puriosus. It is a process chart to manufacture.

FIG. 2 is a process chart for preparing chain maltoheptaose, maltooctase and maltononaose (G9) by transglycosylation reation of Pyrococcus puriosus amylase.

3 is a vector map of pETPFTA-6h.

4 is a vector map of pRARE.

5 is E. coli Electrophoresis photographs of the purification steps of the pyrococcus puriosus amylase produced in BL21 (DE3).

6 is a result of analysis of matrix associated laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) of purified amylase recombinant protein.

Figure 7 shows the enzyme activity according to various pH conditions of amylase recombinant protein.

8 shows the pH stability of amylase recombinant protein.

Figure 9 shows the activity according to the temperature of amylase recombinant protein.

10 is a graph showing the temperature stability of amylase recombinant protein.

Figure 11 shows the DSC (Differential Scanning Calorimetry) results of the amylase recombinant protein.

12 is a photograph analyzed by thin layer chromatography (Thin Layer Chromatography, TLC) of the new heat-resistant amylase isolated from Pyrococcus puriosus to alpha-, beta-, gamma-cyclodextrin.

Figure 13 is a photograph of the new heat-resistant amylase isolated from Pyrococcus puriosus to beta-cyclodextrin and the sample taken according to the reaction time and analyzed by TLC.

FIG. 14 is a high performance anion exchange chromatography after removing unreacted beta-cyclodextrin by using hydrophobic column chromatography of a reaction solution in which beta-cyclodextrin was reacted with a novel heat resistant amylase isolated from Pyrococcus puriosus. -Performance Anion Exchange Chromatography (HPAEC).

FIG. 15 shows thin-film chromatography of alpha-, beta-, and gamma-cyclodextrin concentrates obtained by reacting cyclodextrin glucanotransferase with soluble starch, followed by reaction with new heat resistant amylases isolated from Pyrococcus puriosus. It was analyzed by Thin Layer Chromatography (TLC).

FIG. 16 is a thin-film chromatography (Thin Layer Chromatography, TLC) by adding a new heat resistant amylase isolated from Pyrococcus puriosus to a solution containing alpha, beta- and gamma-cyclodextrin and glucose in each. ) Is a picture analyzed.

17 is a vector map for the production of cyclodextrin glucanotransferase according to Example 6.

[TECHNICAL FIELD OF THE INVENTION]

The present invention relates to a method for preparing a linear or branched maltooligosaccharide having 6 to 9 glucose molecules in high purity from a substrate containing cyclodextrin using a novel heat resistant amylase isolated from Pyrococcus puriosus.

[Private Technology]

Maltooligosaccharides are functional sugars widely used in the food field, such as protein denaturation effect, masking effect of food and soft texture. Malto-oligosaccharides include maltose (G2, maltose), maltotriose (G3, maltotriose), maltotetraose (G4, maltotetraose) and maltopentaose (G5, maltopentaose), maltohexaose (G6, maltohexaose), maltoheptaose ( G7, maltoheptaose), maltooctaose (G8, maltooctaose), maltononaose (G9, maltononaose) and the like.

Acid hydrolysis and enzyme-based methods are mainly used to produce maltohexaose (G6), maltoheptaose (G7) and maltooctaose (G8) from starch. The method using the enzyme is a method for producing maltohexaose and maltoheptaose by applying alpha-amylase derived from microorganisms to starch. However, when prepared by the above method, the yield of maltohexaose and maltoheptaose is low, and glucose (G1, glucose), maltose (G2, maltose), maltotriose (G3, maltotriose), maltotetraose (G4, maltotetraose) ) And maltopentaose (G5, maltopentaose) are produced in large quantities, and the separation and purification process using column chromatography and organic solvent are required to remove them. It is very difficult to obtain maltohexaose and maltoheptaose of US Pat. No. 5,739,024; E. Ben Messaoud et al. Enzyme Microb . Technol . 34: 662-666. 2004.

In addition, in the case of maltooctaos, a method of enzymatic synthesis by applying cyclomaltodextrinase (CyclomaltoDextrinase, CDase) has been reported (Uchida et al. Carbohydr . Res . 287: 271-274. 1996). . However, maltose, maltotriose, maltotetraose and maltopentose are produced in a large amount in the reaction process, resulting in a low production yield and a purification process for removing by-products.

Therefore, there is a need for the development of a method capable of producing high purity maltohexaose, maltoheptaose, maltooctase and maltononaose without the production of by-products.

In order to solve the problems of the prior art, an object of the present invention is to provide a cyclodextrin by using a very fast ring opening rate for the cyclodextrin of the heat-resistant amylase compared to the hydrolysis rate of maltooligosaccharide produced by ring opening It is to provide a method for producing a malto oligosaccharide having a molecular weight of 6 or more glucose from a substrate containing a high purity cyclodextrin without generating other small sugars from the.

In order to achieve the above object, the present invention is a matrix solution containing cyclodextrin, 0.04-0.1 unit (enzyme titer) / mg (cyclodextrin substrate amount) separated from Pyrococcus furiosus amylase The present invention relates to a method for producing a linear maltooligosaccharide having 6 to 8 glucose molecules by reacting at a pH of 4.0 to 6.0 and a temperature of 65 to 95 ° C.

In addition, the present invention is added to the substrate solution further comprising cyclodextrin and glucose, the amylase in 0.2-0.5 unit (enzyme titer) / mg (cyclodextrin substrate amount), pH 4.0 to 6.0 and temperature 65 to 95 It is related with the method of manufacturing the chain malto oligosaccharide of 6-9 glucose molecules by making it react at degreeC.

In addition, the present invention may be obtained by reacting the cyclodextrin-containing substrate solution with cyclodextrin glucanotransferase to soluble starch.

Hereinafter, the present invention will be described in detail.

The present invention relates to amylase isolated from Pyrococcus furiosus , wherein the amylase is an enzyme having excellent heat resistance.

Amylase according to the present invention comprises the amino acid sequence of SEQ ID NO: 2, and has a molecular weight of about 77,163 Da.

The amylase of the present invention is very excellent in thermal stability and pH stability, stable at 60 to 95 ℃, and has a stability at pH 3 to 11. In addition, amylase has enzymatic activity at pH 4-6 at a temperature of 65 to 95 ° C. Preferably the range having enzymatic activity is at a temperature of 85 to 90 ° C. and a pH of 4 to 4.5. The amylases do not require calcium ions for thermal stability unlike alpha-amylases.

The base sequence encoding the amylase of the present invention may be a base sequence encoding the amino acid sequence of SEQ ID NO: 2, for example, may be SEQ ID NO: 1. The base sequence is shown in Tables 1a to 1b (SEQ ID NO: 1) and Tables 2A to 2C (SEQ ID NO: 2).

The amylases of the present invention can be prepared by separation or genetic engineering from Pyrococcus puriosus. In particular, amylases can be easily produced from eukaryotic cells derived from prokaryotes, eukaryotes or eukaryotes. For example, the present invention provides an amylase production method.

The production method of amylase comprises the steps of: (a) inserting a DNA sequence encoding amylase isolated from Pyrococcus furiosus into a vector to prepare a recombinant vector; (b) introducing the recombinant vector into a host cell to prepare a transformant; (c) culturing the transformant to produce amylase.

The production method of amylase can be easily prepared by a conventional known method, which will be apparent to those skilled in the art.

In the step (a), the sequence encoding the amylase is inserted into a conventional expression vector, such as a pET-22b (+) vector (promoter: T7, transcription terminator: T7, selection marker: ampicillin resistance gene) and recombination. Vectors may be prepared, but the expression vector may be selected and used according to the type of host cell. In one embodiment of the present invention, a pETPFTA-6h vector having a DNA cleavage map of FIG. 3 was prepared.

Step (b) is a step of transforming, the host cell may be a prokaryote, eukaryote or eukaryote-derived cells, such as E. coli, lactic acid bacteria, yeast, mold, etc., but is not limited thereto. The transformation method can be easily carried out by a conventional known method.

Step (c) is a step of culturing the transformant, it is possible to select a suitable medium according to the host cell, the culture conditions can also be applied differently depending on the host cell. Escherichia prepared in one embodiment of the present invention coli BL21 (DE3) / pETPFRA-6h, pRARE was deposited on December 3, 2004 at the Korea Institute of Bioscience and Biotechnology, Eerun-dong, Yuseong-gu, Daejeon, Korea, and received the accession number KCTC 10738BP. Escherichia coli BL21 (DE3) / pETPFRA-6h, pRARE (KCTC 10738BP) are incubated at 30-37 ° C in LB medium with ampicillin and when the absorbance is 0.6 at 600nm, IPTG is added at a final concentration of 0.1-10 mM, By incubation for 5 hours, a large amount of amylase recombinant protein can be produced.

In addition, the production method of amylase may further comprise the step of purifying the amylase recombinant protein from the transformant cells after step (c). The purification step may include the step of crushing the transformant cells, taking the supernatant to heat treatment, and taking the supernatant to perform nickel affinity chromatography.

In addition, in the present invention, six codons (AUA, AGG, AGA, CUA, CCC, GGA), which are not commonly found in E. coli, in step (b) of the above steps, may be independently PRARE plasmid encoding (chloramphenicol resistance) can be transformed into the host cell at the same time as the recombinant vector prepared in step (a). In this case, the production rate of amylase recombinant protein is increased by 30 to 40% compared to the case where only the recombinant vector of step (a) is transformed alone.

The amylase production method, as compared to the production method using the parent strain has an effect of reducing the microbial culture cost by lowering the culture temperature can also increase the productivity of the enzyme.

Amylases of the present invention include enzymatic properties that degrade α-1,4 glucoside and α-1,6 glucoside bonds. In other words, amylase is maltooligosaccharides, soluble starch, cyclodextrin, pullulan, acarbose and para-nitrophenyl maltopentaoside ( p NPG5, p -nitrophenyl maltopentaoside). Acts on a substrate selected from the group consisting of) to break down α-1,4 glucoside and α-1,6 glucoside bonds, thereby reducing glucose, maltose, maltotetraose, maltotriose, pannose, acarbiocin-glucose And a sugar compound selected from the group consisting of para-nitrophenyl glucoside ( p NPG1, p- nitrophenyl glucoside), para-nitrophenyl maltoside ( p NPG2, p- nitrophenyl maltoside) and mixtures thereof. Examples of the malto oligosaccharides include maltotriose (G3, maltotriose), maltotetraose (G4, maltotetraose), maltopentase (G5, maltopentaose), maltohexaose (G6, maltohexaose), maltoheptaose (G7, maltoheptaose) And maltooctaose (G8), and the like, and cyclodextrins include, but are not limited to, alpha-cyclodextrin, beta-cyclodextrin, and gamma-cyclodextrin.

In particular, the amylases of the present invention ring-open cyclodextrin to form maltooligosaccharide, wherein the formed maltooligosaccharide is hydrolyzed. At this time, the ring opening rate of the cyclodextrin is 8 to 9 times faster than the hydrolysis rate of malto oligosaccharides, by controlling the reaction time can be easily obtained ring-opened malto oligosaccharides before hydrolysis after ring opening. For example, the amylase isolated from the heat-resistant pyrococcus puriosus of the present invention has a reaction rate as shown in Table 3 below for ring cleavage and maltoheptaose hydrolysis of beta-cyclodextrin. The reaction rate for beta-cyclodextrin is about 8.6 times higher than that for maltoheptaose.

temperament Reaction Rate (mmol / min) Relative reaction rate Beta-cyclodextrin β-CD 499.9 8.6 Maltoheptaose (G7) 58.3 1.0

Specifically, the method for producing maltooligosaccharide ring-opened from cyclodextrin is to produce the ring-opened maltooligosaccharide by adding the amylase to the cyclodextrin solution, and then reacting at a pH of 4.0 to 6.0 and a temperature of 65 to 95 ° C. In addition, the reaction time, that is, the reaction time between amylase and cyclodextrin (0.04-0.1 unit, enzyme titer / mg, amount of cyclodextrin substrate) may be 10 to 60 minutes.

In the present invention, the substrate solution for amylase may be a substrate solution containing cyclodextrin. In addition, the substrate solution may be a mixed solution containing cyclodextrin and glucose. The cyclodextrin may be selected from the group consisting of alpha-cyclodextrin (G6), beta-cyclodextrin (G7) and gamma-cyclodextrin (G8). In the case of using only cyclodextrin as a substrate solution, the alpha-cyclodextrin (G6), beta-cyclodextrin (G7) and gamma-cyclodextrin (G8) were each represented by alpha-1, Maltohexaose, maltoheptaose, and maltooctase linked to four bonds can be produced. In addition, in the case of using a substrate solution containing glucose and cyclodextrin, the reduction end of maltooligosaccharide prepared by ring opening of cyclodextrin by the ring-opening action of the amylose of Pyrococcus puriosus and another active sugar transfer action. The glucose can be transferred to alpha-1,4 or alpha-1,6 to prepare a cyclodexmaltooligosaccharide obtained by reacting a chain-stranded glucanotransferase having a glucose molecule number of 6 to 9 with garganese starch.

The substrate solution including the cyclodextrin may be a solution in which cyclodextrin is dissolved in a solvent, or may be a cyclodextrin solution.

The cyclodextrin glucanotransferase may use a known enzyme. The cyclodextrin glucanotransferase of the present invention can be prepared by isolation or genetic engineering method from the strain producing the electrical enzyme, and can be used to purchase a commercially available enzyme. The method for producing the cyclodextrin glucanotransferase can be easily prepared by a common known method, which will be apparent to those skilled in the art. Commercially available enzymes include Toruzym sold by Novozymes.

Examples of cyclodextrin glucanotransferases usable in the present invention include alkalophilic Bacillus sp. The cyclodextrin glucanotransferase gene of SEQ ID NO: 5 obtained from I-5 may be an enzyme solution obtained by cloning after purification of protein expression in E. coli MC1061, and the enzyme may have an optimum temperature and pH of 60 ° C. and 6.0, respectively. to be. Specific examples of the method for producing the cyclodextrin glucanotransferase, (a) alkalophilic Bacillus sp. Preparing a recombinant vector by inserting the CGTase encoding DNA sequence (SEQ ID NO: 5) isolated from I-5 into a vector, (b) introducing the recombinant vector into a host cell to prepare a transformant, (c) ) The step of culturing the transformant to produce a CGTase. In step (a), the sequence encoding the CGTase may use a conventional expression vector. For example, an amyR2 promoter was used as the pBR322 vector and an ampicillin resistance gene was used as a selection marker. Step (b) is a transforming step, wherein the host cell may be a prokaryote, eukaryote or eukaryote-derived cell, for example, E. coli, lactic acid bacteria, yeast, mold, etc., but is not limited thereto. It can be performed easily by a conventional well-known method. Escherichia in step (c) The transformant transformed with the pR2CGT plasmid using coli MC1061 as a host cell can be cultured in LB medium containing ampicillin for 15 hours at 37 ° C. to produce a large amount of CGTase recombinant protein. In addition, the prepared enzyme can be purified in the same manner as the general CGTase purification method, for example, by transforming the transformant cells, taking the supernatant can be purified by performing beta cyclodextrin affinity chromatography. The cyclodextrin glucanotransferase gene nucleotide sequence of SEQ ID NO: 5 is shown in Tables 4a to 4b.

Maltooligosaccharide production method ring-opened from the cyclodextrin of the present invention, the step of performing a hydrophobic column chromatography of the reactant can be further carried out. Through the hydrophobic column chromatography, the unreacted cyclodextrin is selectively bound to the resin due to the hydrophobic property inside the cyclodextrin ring, and the ring-opened maltooligosaccharide is eluted without binding. Therefore, it is possible to easily separate only the ring-opened maltooligosaccharide, and also beta-cyclodextrin bound for reuse of the unreacted substrate can be eluted with 90 ℃ distilled water (Fig. 1).

Hydrophobic column chromatography can be performed using a column filled with hydrophobic resin, and water, for example distilled water, can be used as the column solvent. The hydrophobic resin may be a commercially available hydrophobic resin, and examples thereof include, but are not limited to, butyl sepharose, amberlite XAD-4 HP, and XAD-7 HP.

The method for producing a linear malto oligosaccharide having 6-8 glucose molecules from the cyclodextrin according to the present invention can easily prepare high purity maltohexaose, maltoheptaose and maltooctaose. Each maltooligosaccharide can be used not only for expensive diagnostic reagent raw materials used for measuring amylose activity in blood but also for chemical reagents.

In addition, the method for producing a chain malto oligosaccharide having 6 to 9 glucose molecules from cyclodextrin and glucose can easily produce high purity maltohexaose, maltoheptaose, maltooctaose and maltononaose. Mixed concentrate containing the malto oligosaccharides can be widely used in food as a new reduced rice material.

Hereinafter, examples of the present invention will be described. The following examples are only for illustrating the present invention and the present invention is not limited to the following examples.

Example  1: heat resistance Pyrococcus From Puriosus Amylase  gene Cloning  And Amylase  production

1-1. Amylase Gene Cloning

Pyrococcus furiosus ) The chromosomal DNA of DSM 3638 was obtained from Dr. AM Grunden of North Carolina State University (Raleigh, NC).

In order to isolate the heat-resistant amylase gene, a forward primer PFTA-Fnd (SEQ ID NO: 3) and a reverse primer PFTA-Rxo (SEQ ID NO: 4) were prepared, respectively, and a polymerase chain reaction (PCR) was performed on Pyrococcus puriosus chromosomal DNA. ) To obtain a PCR product of about 2.0 kb having a nucleotide sequence of SEQ ID NO: 1. The PCR product was treated with restriction enzymes NdeI and XhoI and ligated with a pET-22b (+) expression vector treated with the same restriction enzyme to prepare pETPFTA-6h. 3, the vector map of pETPFTA-6h is briefly shown. The pETPFTA-6h vector contains an amylase gene and contains eight additional amino acid residues (Leu-Glu-6His) at the 3 'end of the amylase gene. The base sequence of the primer (SEQ ID NO: 3,4) used in the experiment is shown in Table 5.

SEQ ID NO: Sequence 3 caattgctac acatatgtat aagctcg 4 gcggatgggt actcgaggag gcaccacct

For transformation, E. coli MC1016, a host cell, was inoculated into 5 ml LB liquid medium and incubated for 12 hours at 37 ° C. 1.0 ml of the culture was inoculated into 50 ml of fresh LB liquid medium to have an absorbance of 0.5 at 600 nm. Incubate until. The cells were recovered by centrifugation (7000 xg, 5 minutes) at 4 ℃ centrifugation at 4 ℃ and suspended with 0.75 ml of the transformation solution I (50 mM CaCl2) and left for 30 minutes in ice. Thereafter, the cells were recovered by centrifugation (4 ° C., 6000 × g, 2 minutes), and the recovered cells were suspended in 0.15 ml of transformation solution II (100 mM CaCl 2) and left in ice for 30 minutes. 500 ng of the ligated solution was mixed in 0.15 ml of the suspension and left for 1 hour in ice, followed by thermal shock at 42 ° C. for 2 minutes. 0.8 ml of LB liquid medium was added thereto, followed by incubation at 37 ° C for 1 hour, and plated on LB agar medium containing ampicillin (final concentration of 100 μg / ml) was first selected for strains showing resistance.

The first selected strains were inoculated in 5 ml of LB liquid medium containing ampicillin, incubated at 37 ° C. for 12 hours, and centrifuged to obtain cells. Plasmids were isolated from the cells recovered by the plasmid separation kit and digested with NdeI and XhoI restriction enzymes to confirm that the amylase gene of about 2.0 kb was contained.

1-2. Transformant Preparation

Ampicillin resistant strains were selected after transforming the pETPFTA-6h vector prepared above with E. coli BL21 (DE3). Selected transformant I was inoculated in 2.5 L of LB liquid medium containing ampicillin and incubated at 37 ° C., and when the absorbance was 0.6 at 600 nm, IPTG was added to a final concentration of 1 mM and incubated for 3 hours.

Also for efficient overexpression, the pRARE plasmid (chloramphenicol resistance) of FIG. 4, which independently encodes six codons (AUA, AGG, AGA, CUA, CCC, GGA), which are not commonly seen in E. coli , is recombinantly expressed plasmid pETPFTA- After co-transfection with 6 h, strains showing ampicillin and chloramphenicol (final concentration 34 μg / ml) resistance were selected as transformant II. The pRARE contains a tRNA gene encoding six codons (AUA, AGG, AGA, CUA, CCC, GGA) and includes a clonamphenicol resistance gene and a replicon (P15A ori).

1-3. refine

The two transformants selected above were cultured and centrifuged (4 ° C, 7,000 xg, 30 minutes) to recover the cells, and 50 mM Tris-HCl buffer solution containing 300 mM NaCl and 10 mM imidazole. It was suspended in (pH 7.5) and then ultrasonically ground. The supernatant was collected by centrifugation, heat-treated at 70 ° C. for 20 minutes, and then centrifuged (10,000 × g, 30 minutes) to obtain a supernatant. The supernatant was purified by injection into Ni-NTA affinity chromatography.

5 is E. coli Electrophoresis photographs of the purification steps of the pyrococcus puriosus amylose produced in BL21 (DE3), M is the size marker, lane 1 is E. coli The supernatant obtained by the ultrasonic grinding of BL21 (DE3) / pETPFTA-6h transformant, lane 2 is E. coli The supernatant obtained by ultrasonic grinding of BL21 (DE3) / pETPFTA-6h / pRARE transformant, lane 3 is the sample of “2” heat-treated at 70 ° C. for 20 minutes, and lane 4 is Ni-NTA affinity chromatography. The amylase recombinant protein was purified to and lane 5 is a reaction of the amylase recombinant protein of lane 4 with a soluble starch as a substrate.

As shown in Figure 5, E. coli The BL21 (DE3) / pETPFTA-6h transformant had a lower expression rate of amylase recombinant protein than the E. coli BL21 (DE3) / pETPFTA-6h / pRARE transformant. Inefficient expression of E. coli in archaea genes is known to be due to the large use of AGA, Arginine of AGG, Isoleucine of AUA, and Glycine codons of GGA, which are rarely used in E. coli cells. In the amylase gene of the present invention, 38 out of 40 for arginine, 17 out of 47 for isoleucine, and 23 out of 44 for glycine are E. coli. The expression of amylase recombinant protein is thought to be low in the BL21 (DE3) / pETPFTA-6h transformant. In order to increase protein expression in E. coli cells, pRARE plasmids containing these codons were introduced into E. coli simultaneously with the pETPFTA-6h vector. As a result, E. coli as confirmed in FIG. The expression of amylase recombinant protein was significantly increased in BL21 (DE3) / pETPFTA-6h / pRARE transformants.

In addition, the amylase recombinant protein was efficiently purified through heat treatment (lane 3 of FIG. 5) and Ni-NTA affinity chromatography (lane 4 of FIG. 5), and showed a molecular weight of about 70,000 Da on SDS-PAGE. When the soluble starch was reacted with a substrate and stained with an iodide solution, the enzyme titer was shown at the same position (lane 5 in FIG. 5).

FIG. 6 shows MALDI-TOF MS (matrix associated laser desorption ionization-time-of-flight mass spectrometry) analysis of purified amylase recombinant protein, which is almost the same as the theoretical molecular weight 77,149 Da inferred from the amino acid sequence of amylase. The molecular weight was 77,163 Da.

Example  2: Pyrococcus Puriosus  origin Amylase  characteristic

2-1. Enzyme titers

150 μl of 1 (w / v)% beta-cyclodextrin substrate solution dissolved in 50 mM sodium acetate buffer (pH 4.5) was added to 120 μl of the same buffer, followed by heat treatment at 90 ° C. for 5 minutes, and then diluted appropriately. 30 μl of purified amylase was added thereto and reacted at 90 ° C. for 10 minutes. Then, 3,5-dinitrosalicylic acid (DNS) was added to stop the reaction, and the mixture was boiled for 5 minutes to measure absorbance at 575 nm. It was. The absorbance was set to 1 unit of the amount of enzyme that produces 1 mol of maltose per minute compared to the maltose standard curve.

2-2. pH characteristics

To determine the optimal pH of the enzyme, the relative titers for the beta-cyclodextrin solutions of various pH ranges were measured.

50 mM sodium citrate buffer (pH 3.0-4.0), 50 mM sodium acetate buffer (pH 4.0-6.0), 50 mM sodium phosphate buffer (pH 6.0-7.5) and 50 mM Tris-HCl buffer (pH 7.5-8.0) respectively Into 150 μl of 1 (w / v)% beta-cyclodextrin substrate solution, 120 μl of each buffer solution was heat-treated at 90 ° C. for 5 minutes, and 30 μl of purified amylase was added thereto and reacted at 90 ° C. for 10 minutes. Relative enzyme activity was measured.

Figure 7 shows the enzyme activity according to various pH conditions of the amylase recombinant protein, amylase had an optimum titer at pH 4.5 and maintained a titer of 50% or more at pH 4.0 to 6.0.

In addition, in order to measure the pH stability of amylase, the enzyme solution (final concentration 0.1 mg / ml) was left in 0.1 M Britton-Robinson buffer at various pH ranges for 24 hours at 37 ° C, and then remained. The enzyme titer was 120 μl of the same buffer in 150 μl of 1 (w / v)% beta-cyclodextrin substrate solution dissolved in 50 mM sodium acetate buffer (pH 4.5), followed by heat treatment at 90 ° C. for 5 minutes, and the amyl 30 μl of the raise was added and reacted at 90 ° C. for 10 minutes to measure relative enzyme activity.

Figure 8 shows the pH stability of the amylase recombinant protein, it was confirmed that it is maintained in a stable state in a wide pH range of pH 3.0 to 11.0.

2-3. Temperature characteristic

To determine the optimal temperature of the amylase recombinant protein, 120 μl of the same buffer was added to 150 μl of 1 (w / v)% beta-cyclodextrin substrate solution dissolved in 50 mM sodium acetate buffer (pH 4.5) at 55 ° C. After heat treatment at various temperatures ranging from 110 ° C. for 5 minutes, 30 μl of purified amylase was added and reacted at each temperature for 10 minutes to determine relative enzyme activity.

Figure 9 shows the activity according to the temperature of amylase recombinant protein, showed an optimum titer at 90 ℃ and maintained a titer of about 80% at 75 to 92 ℃.

For temperature stability investigations, the enzyme solution (final concentration 0.1 mg / ml) dialyzed in 50 mM sodium acetate buffer (pH 5.0) was taken at different temperatures at different temperatures ranging from 85 ° C. to 100 ° C., and the remaining enzyme titer was 150 μl of the 1 (w / v)% beta-cyclodextrin substrate solution dissolved in 50 mM sodium acetate buffer (pH 4.5) was added to 120 μl of the same buffer, followed by heat treatment at 90 ° C. for 5 minutes, followed by reaction 30 The reaction was carried out for 10 minutes at 90 占 폚 and the relative enzyme activity was measured.

10 is a graph showing the temperature stability of the amylase recombinant protein, showing a very high thermal stability so that the titer does not decrease at all even after standing at 85 ℃ for 120 minutes, and after 60 minutes at 100 ℃ More than 30% of the titer was maintained. The half-life of amylase recombinant protein is 398 minutes at 90 ° C. and 114 minutes at 95 ° C.

2-4. Differential Scanning Calorimetry (DSC) Analysis

DSC was performed to analyze the melting temperature of amylase recombinant protein. Purified amylase recombinant protein was suspended in 50 mM sodium phosphate buffer (pH 7.0) at a concentration of 1 mg / ml, and the samples were analyzed at temperatures between 25 and 125 degrees Celsius at a rate of 1 degrees Celsius per minute.

FIG. 11 shows DSC results of the amylase recombinant protein, and the melting temperature of the amylase recombinant protein was very high at 104.3 ° C. and showed a small endothermic inflection point attributable to the monomer of the enzyme at 91.7 ° C. FIG.

Example  3: Cyclodextrin  ring Cleavage  Speed and G7 Comparison of Hydrolysis Rates of Oligosaccharides

The hydrolysis rates of the novel heat resistant amylases isolated from Pyrococcus puriosus against beta-cyclodextrin and maltoheptaose as substrates were measured, respectively.

120 μl of the same buffer was added to 150 μl of 1 (w / v)% substrate solution dissolved in 50 mM sodium acetate buffer (pH 4.5), followed by heat treatment at 90 ° C. for 5 minutes, and 30 μl of amylase of Example 1-3. (0.07 unit) was added, and 50 μl of the reaction sample was taken at 90 ° C. for 2 to 10 minutes, and the reaction was terminated by heating in boiling water for 15 minutes. The amount of hydrolysis of the substrate beta-cyclodextrin or maltoheptaose was measured using high-performance Anion Exchange Chromatography (HPAEC).

Table 6 shows the results of quantification after analyzing the ring cleavage rate and maltoheptaose hydrolysis rate of the beta-cyclodextrin using high performance anion exchange chromatography.

temperament Reaction Rate (mmol / min) Relative reaction rate Beta-cyclodextrin (β-CD) 499.9 8.6 Maltoheptaose (G7) 58.3 1.0

Example  4: From cyclodextrin Maltohexaos , Maltoheptaos  And Maltose Preparation of Taos

4-1. Preparation of Maltohexaose, Maltoheptaose and Maltooctase

To 450 µl of the substrate solution of 1 (w / v)% alpha-, beta- and gamma-cyclodextrin dissolved in 50 mM sodium acetate buffer (pH 4.5), 360 µl of the same buffer was added for 5 minutes at 90 ° C. After heat treatment, 90 μl (0.22 unit) of amylase of Example 1-3 was added and reacted at 90 ° C. for 10 to 60 minutes. After the reaction, the reaction solution was heated in boiling water for 15 minutes to terminate the reaction.

4-2. Thin Layer Chromatography (TLC) Analysis of Reaction Liquid

After the reaction in Example 4-1, the reaction solution was analyzed by using a thin layer chromatography.

Each sample was loaded in a 1 μl Whatman K5F silica gel TLC plate, developed once in a developing solution in which the ratio of isopropyl alcohol, ethyl acetate and distilled water was 3: 1: 1 (v / v / v), followed by drying to 0.3 After dipping into methanol containing% (w / v) N- (1-naphthyl) -ethylenediamine and 5% (w / v) sulfuric acid, the solution was developed for 10 minutes in an oven at 110 ° C.

FIG. 12 is a thin-film chromatography photograph showing the results of alpha-, beta-, gamma-cyclodextrin substrate degradation by novel heat resistant amylases isolated from Pyrococcus puriosus, where M is a maltooligosaccharide standard and lane 1 is Alpha-cyclodextrin was used as the substrate, lane 2 was beta-cyclodextrin as the substrate, and lane 3 was gamma-cyclodextrin as the substrate.

In FIG. 12, the novel heat resistant amylases isolated from Pyrococcus puriosus hydrolyzed the substrate alpha-cyclodextrin to produce maltohexaose as the main reaction product, and hydrolyzed beta-cyclodextrin to the main reaction product. Maltoheptaose was produced, and gamma-cyclodextrin was hydrolyzed to produce maltooctaose as the main reaction product. Therefore, it can be seen that the enzyme easily cleaves the ring structure of the cyclodextrin.

FIG. 13 is a thin-film chromatography photograph showing hydrolysis products over time after reacting beta-cyclodextrin as a substrate with a novel heat resistant amylase isolated from Pyrococcus puriosus, where M is a maltooligosaccharide standard and lanes. Lanes 1 to 4 were reacted for 10 minutes, 20 minutes, 40 minutes, and 60 minutes, respectively.

In Figure 13, amylase rapidly cleaved the ring structure of the substrate beta-cyclodextrin, maltoheptaose produced as the main reaction product can be seen that slowly hydrolyzed.

Therefore, since the ring cleavage rate and maltoheptaose hydrolysis rate of the cyclodextrin are significantly different, the ring structure of alpha-, beta-, gamma-cyclodextrin is cleaved by using the same, and maltohexaose, maltoheptaose and By generating a large amount of maltooctaose, and by reducing the enzyme titer and reaction time, it is possible to easily control the reaction conditions to minimize the hydrolysis of maltohexaose, maltoheptaose and maltooctaose.

Example  5: high purity Maltohexaos , Maltoheptaos  And Malto Octaos  Produce

5-1.Isolation and Purification of Maltohexaose, Maltoheptaose and Maltooctase

Maltoheptaose and maltooctase prepared by reacting at 90 ° C. for 10 minutes in Example 4-1 were separated and purified using hydrophobic column chromatography.

About 5 ml of butyl sepharose (butyl-separose) resin was charged to the column, and 20 ml of distilled water was flowed to stabilize the column, and the reaction solution obtained in Example 4-1 was injected. Due to the hydrophobic nature inside the cyclodextrin ring, unreacted cyclodextrin was selectively bound to the resin, and maltohexaose, maltoheptaose and maltooctase eluted without binding. The combined cyclodextrin is eluted with 90 ° C. distilled water for reuse of the unreacted substrate.

5-2. Purity analysis

The eluate obtained in Example 5-1 was analyzed using high performance anion exchange chromatography.

The sample was diluted 100-fold with ultrapure water, filtered through a 0.45 μm thin film filter paper, and injected into 20 μl, and the flow rate was 1 ml per minute. HPAEC analysis was performed using a GP40 gradient pump, ED40 electrochemical detector, and CarboPac PA1 from Dionex (USA). 150 mM caustic soda solution was used as solvent A. After 40 minutes of dissolving mM sodium acetate as the solvent B, the concentration gradient was linearly adjusted so that the solvent B became 0 to 40% for 40 minutes.

Figure 14 shows the analysis of the purity of the isolated maltoheptaose, unreacted beta-cyclodextrin can be seen that the hydrophobic column chromatography is relatively simply removed, only high-purity maltoheptaose is easily separated and purified. The purity of maltoheptaose to total oligosaccharides contained was about 90%.

Table 7 shows the composition of the eluate of Example 5-1, analyzed by high performance anion exchange chromatography.

Furtherance (%) Glucose     0.6 maltose     0.3 Malto trios     1.1 Maltotetraos     1.3 Maltopentaos     0.5 Maltohexaos     1.4 Unknown sugar compound     6.1 Maltoheptaos    88.7

Example  6: using enzyme From starch solution Maltohexaos , Maltoheptaos  And preparation of maltooctaose concentrate

400 µl of the same buffer was added to 500 µl of 4 (w / v)% soluble starch substrate solution dissolved in 50 mM sodium acetate buffer (pH 6.0) and heat-treated at 60 ° C for 5 minutes.

Cyclodextrin glucanotransferase gene having the gene sequence of SEQ ID NO: 5 in the heat-treated solution was produced and purified by genetic engineering method, and 50 μl of cyclodextrin glucanotransferase at a concentration of 1 mg / ml was added. Reaction produced alpha-beta- and gamma-cyclodextrins. In order to produce the enzyme, Alkaline Bacillus sp. The cyclodextrin glucanotransferase gene of SEQ ID NO: 5 from I-5 was cloned into the pBR322 vector with the ampicillin resistance gene using the amyR2 promoter and the selection marker (see FIG. 17), and LB with ampicillin added in E. coli MC1061. By incubating at 37 ° C. for 15 hours in a medium, a large amount of CGTase recombinant protein can be produced. After 15 hours of incubation at 37 ° C. in LB medium to which ampicillin was added by protein expression and purification to produce a large amount of CGTase recombinant protein, transformed cells were disrupted, supernatant was taken, and beta cyclodextrin affinity chromatography was performed. It was used by purification.

After the reaction, the reaction solution was heated in boiling water for 5 minutes to terminate the reaction. 50 µl (3 units) of amylase of Example 1-3 was added to the obtained reaction solution and reacted at 85 ° C. for 60 minutes to cleave the ring structure. After the reaction, the reaction solution was heated in boiling water for 15 minutes to terminate the reaction. The reaction solution was analyzed using anion exchange chromatography.

FIG. 15 shows that alpha-, beta-, and gamma-cyclodextrin concentrates obtained by reacting cyclodextrin glucanotransferase with soluble starch are reacted with new heat resistant amylases isolated from Pyrococcus puriosus to maltohexaose, Anion exchange chromatography picture showing the results of the preparation of maltoheptaose and maltooctaose, (A) is a cyclodextrin concentrate before amylase reaction, (B) is maltohexaose, maltose produced after amylase reaction Concentrate of heptanes and maltooctaose.

In FIG. 15, it is found that the novel heat resistant amylases isolated from Pyrococcus puriosus effectively hydrolyze alpha-, beta- and gamma-cyclodextrins to produce maltohexaose, maltoheptaose and maltooctaose concentrates. Can be.

Example  7: A former transition  Preparation of Special Oligosaccharide Mixtures by Reaction

10 (w / v)% alpha-cyclodextrin and 30 (w / v)% glucose solution, 10 (w / v)% beta-cyclodextrin and 30 (w) dissolved in 50 mM sodium acetate buffer (pH 4.5) 25 μl (4.5 unit) of the novel heat resistant amylases of Examples 1-3, respectively, in 1 ml of 10% (w / v)% gamma-cyclodextrin and 30 (w / v)% glucose solution After the reaction for 60 minutes at 80 ℃ put the reaction solution in boiling water for 15 minutes to terminate the reaction. The reaction solution was analyzed by using a thin layer chromatography.

FIG. 16 is a thin-film chromatography photograph showing the product resulting from the sugar transition reaction of alpha-, beta- and gamma-cyclodextrin and glucose by novel heat resistant amylases isolated from Pyrococcus puriosus, where M is malto Oligosaccharide is a standard material, lane 1 uses alpha-cyclodextrin and glucose as substrate, lane 2 uses beta-cyclodextrin and glucose, and lane 3 uses gamma-cyclodextrin and glucose as substrate.

In Fig. 16, the novel heat resistant amylases isolated from Pyrococcus puriosus cleavage the ring structure of alpha-, beta-, gamma-cyclodextrin and glucose at the reduced end of the resulting maltooligosaccharides alpha-1,4 and A special oligosaccharide mixture A (linear maltohexaose, maltoheptaose, and side chain maltoheptaose in which glucose at the reducing end is linked to alpha-1,6), specially undergoes a sugar transition to alpha-1,6. Oligosaccharide Mixture B (Straight maltoheptaose, Maltooctase and Side chain maltooctaose with reduced terminal glucose connected to alpha-1,6) and Special Oligosaccharide Mixture C (Straight maltooctaose, Maltononose and Reduction) Terminal glucose is effectively produced side chain maltononases linked by alpha-1,6).

As described above, the present invention provides a method for preparing linear maltohexaose, maltoheptaose and maltooctaose using cyclodextrin ring-opening reaction of amylase, and linear maltononaose and branched maltoheptaose, maltose including the same. By providing an octaos and mal tonanos mixed concentrate, high purity straight maltohexaose, maltoheptaose and maltooctaose can be easily and simply and inexpensively prepared. Maltoheptaose and maltooctase can be used for diagnostic medical reagents and chemical reagents. In addition, linear maltononases and branched maltoheptaose, maltooctase and maltononaose mixed concentrates containing them can be used in food.

<110> SEOUL NATIONAL UNIVERSITY INDUSTRY FOUNDATION <120> ENZYMATIC PREPARATION OF HIGHLY PURIFIED MALTOOLIGOSACCHARIDE <130> dpp20050174kr <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 1962 <212> DNA <213> Pyrococcus furiosus <400> 1 atgtataagc tcgtcagttt cagagatagc gagatctttg gaagagttgc agaggtggaa 60 ttttccctaa taagagaagg aagctatgca tacctccttg gtgactttaa tgcatttaat 120 gagggaagtt ttagaatgga gcaagaagga aagaattgga agattaaaat cgcccttcca 180 gaaggggtat ggcactatgc tttttcaata gatgggaaat tcgtcttaga tccagataat 240 cctgaaagaa gagtatatac tagaaagggt tacaaattcc acagagaagt taatgttgca 300 agaattgtca aaagtgacga tttggttttc catactcctt ctctcctgta tctgtatgaa 360 atttttggga gggttcacgt tcttttaagg acacaaaagg gggtaataaa aggggcaact 420 ttcctggggg agaagcatgt tcctatgagg aagaatgctt ctgatgagct atttgattac 480 tttgaggtta tcgtagaagg aggagataag aggttaaatt attcatttga agtattaaca 540 atggaaggag cgaaatttga gtatggtcaa tttaaggcga gacccttcag tatcgaattt 600 ccaacctggg ttattgacag ggtattctat caaataatgc cagacaaatt tgctaggtct 660 aggaaaattc aaggaattgc ctatccaaaa gataaatatt ggggaggaga tctaatcgga 720 attaaggaaa agatagacca ccttgtgaac cttggaataa atgcaatata tttaactcca 780 atcttcagtt ccctcacata tcatggatat gacatagttg attatttcca cgttgcaagg 840 agacttggag gagacagggc ctttgtggat ttgttatctg agcttaaaag gtttgatata 900 aaggttatcc ttgatggggt atttcaccat acgagctttt tccacccata tttccaagat 960 gttgtaagga agggggaaaa tagcagcttt aaaaactttt acaggattat taaatttcca 1020 gtagtctcaa aagaatttct tcaaatcctc cattcaaaat cttcctggga agaaaagtac 1080 aaaaagataa aatcccttgg ctggaattac gagagctttt tctcagtttg gataatgccg 1140 agattaaacc acgataatcc aaaggttaga gagtttatca agaatgtgat tcttttctgg 1200 acaaataagg gggtcgatgg atttagaatg gatgttgctc atggggttcc tccagaagtg 1260 tggaaagaag ttagagaagc attgccaaaa gaaaagtact taattggaga agttatggat 1320 gatgcaaggt tatggctgtt tgataaattc catggagtca tgaactatcg gctctatgat 1380 gcaatcttaa ggttttttgg atatgaggag attacagccg aggaattctt aaatgaacta 1440 gagctcttaa gctcgtatta tggcccagca gagtatctaa tgtataattt tttagacaat 1500 catgatgttg agagattttt ggatatagtt ggggataaga ggaagtacgt gtgtgctttg 1560 gtcttcttga tgacttacaa aggtattcca tctctcttct acggagatga aatagggttg 1620 agagggatta atctccaagg aatggaatct tccagagcac caatgttgtg gaatgaagaa 1680 gagtgggatc aaaggatttt ggagataaca aagacactag taaagattag gaaaaacaat 1740 aaagccctcc tttttggtaa ttttgtcccg gtgaagttta aaagaaaatt catggtatac 1800 aagagggaac acatgggaga gagaactatc gttgccatta actactcaaa ttcacgcgtt 1860 aaagaactag gaataacaat tcctgaatat tcgggcgtga taattaatga agataaagtg 1920 aaattgataa agtacctcga gcaccaccac caccaccact ga 1962 <210> 2 <211> 653 <212> PRT <213> Pyrococcus furiosus <400> 2 Met Tyr Lys Leu Val Ser Phe Arg Asp Ser Glu Ile Phe Gly Arg Val   1 5 10 15 Ala Glu Val Glu Phe Ser Leu Ile Arg Glu Gly Ser Tyr Ala Tyr Leu              20 25 30 Leu Gly Asp Phe Asn Ala Phe Asn Glu Gly Ser Phe Arg Met Glu Gln          35 40 45 Glu Gly Lys Asn Trp Lys Ile Lys Ile Ala Leu Pro Glu Gly Val Trp      50 55 60 His Tyr Ala Phe Ser Ile Asp Gly Lys Phe Val Leu Asp Pro Asp Asn  65 70 75 80 Pro Glu Arg Arg Val Tyr Thr Arg Lys Gly Tyr Lys Phe His Arg Glu                  85 90 95 Val Asn Val Ala Arg Ile Val Lys Ser Asp Asp Leu Val Phe His Thr             100 105 110 Pro Ser Leu Leu Tyr Leu Tyr Glu Ile Phe Gly Arg Val His Val Leu         115 120 125 Leu Arg Thr Gln Lys Gly Val Ile Lys Gly Ala Thr Phe Leu Gly Glu     130 135 140 Lys His Val Pro Met Arg Lys Asn Ala Ser Asp Glu Leu Phe Asp Tyr 145 150 155 160 Phe Glu Val Ile Val Glu Gly Gly Asp Lys Arg Leu Asn Tyr Ser Phe                 165 170 175 Glu Val Leu Thr Met Glu Gly Ala Lys Phe Glu Tyr Gly Gln Phe Lys             180 185 190 Ala Arg Pro Phe Ser Ile Glu Phe Pro Thr Trp Val Ile Asp Arg Val         195 200 205 Phe Tyr Gln Ile Met Pro Asp Lys Phe Ala Arg Ser Arg Lys Ile Gln     210 215 220 Gly Ile Ala Tyr Pro Lys Asp Lys Tyr Trp Gly Gly Asp Leu Ile Gly 225 230 235 240 Ile Lys Glu Lys Ile Asp His Leu Val Asn Leu Gly Ile Asn Ala Ile                 245 250 255 Tyr Leu Thr Pro Ile Phe Ser Ser Leu Thr Tyr His Gly Tyr Asp Ile             260 265 270 Val Asp Tyr Phe His Val Ala Arg Arg Leu Gly Gly Asp Arg Ala Phe         275 280 285 Val Asp Leu Leu Ser Glu Leu Lys Arg Phe Asp Ile Lys Val Ile Leu     290 295 300 Asp Gly Val Phe His His Thr Ser Phe Phe His Pro Tyr Phe Gln Asp 305 310 315 320 Val Val Arg Lys Gly Glu Asn Ser Ser Phe Lys Asn Phe Tyr Arg Ile                 325 330 335 Ile Lys Phe Pro Val Val Ser Lys Glu Phe Leu Gln Ile Leu His Ser             340 345 350 Lys Ser Ser Trp Glu Glu Lys Tyr Lys Lys Ile Lys Ser Leu Gly Trp         355 360 365 Asn Tyr Glu Ser Phe Phe Ser Val Trp Ile Met Pro Arg Leu Asn His     370 375 380 Asp Asn Pro Lys Val Arg Glu Phe Ile Lys Asn Val Ile Leu Phe Trp 385 390 395 400 Thr Asn Lys Gly Val Asp Gly Phe Arg Met Asp Val Ala His Gly Val                 405 410 415 Pro Pro Glu Val Trp Lys Glu Val Arg Glu Ala Leu Pro Lys Glu Lys             420 425 430 Tyr Leu Ile Gly Glu Val Met Asp Asp Ala Arg Leu Trp Leu Phe Asp         435 440 445 Lys Phe His Gly Val Met Asn Tyr Arg Leu Tyr Asp Ala Ile Leu Arg     450 455 460 Phe Phe Gly Tyr Glu Glu Ile Thr Ala Glu Glu Phe Leu Asn Glu Leu 465 470 475 480 Glu Leu Leu Ser Ser Tyr Tyr Gly Pro Ala Glu Tyr Leu Met Tyr Asn                 485 490 495 Phe Leu Asp Asn His Asp Val Glu Arg Phe Leu Asp Ile Val Gly Asp             500 505 510 Lys Arg Lys Tyr Val Cys Ala Leu Val Phe Leu Met Thr Tyr Lys Gly         515 520 525 Ile Pro Ser Leu Phe Tyr Gly Asp Glu Ile Gly Leu Arg Gly Ile Asn     530 535 540 Leu Gln Gly Met Glu Ser Ser Arg Ala Pro Met Leu Trp Asn Glu Glu 545 550 555 560 Glu Trp Asp Gln Arg Ile Leu Glu Ile Thr Lys Thr Leu Val Lys Ile                 565 570 575 Arg Lys Asn Asn Lys Ala Leu Leu Phe Gly Asn Phe Val Pro Val Lys             580 585 590 Phe Lys Arg Lys Phe Met Val Tyr Lys Arg Glu His Met Gly Glu Arg         595 600 605 Thr Ile Val Ala Ile Asn Tyr Ser Asn Ser Arg Val Lys Glu Leu Gly     610 615 620 Ile Thr Ile Pro Glu Tyr Ser Gly Val Ile Ile Asn Glu Asp Lys Val 625 630 635 640 Lys Leu Ile Lys Tyr Leu Glu His His His His His His                 645 650 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 caattgctac acatatgtat aagctcg 27 <210> 4 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gcggatgggt actcgaggag gcaccacct 29 <210> 5 <211> 2154 <212> DNA <213> Bacillus sp. <400> 5 atgaggaggt atagtatgaa aagatttatg aaactaacag ccgtatggac actctggtta 60 tccctcacgc tgggcctctt gagcccggtc cacgcagccc cggatacctc ggtatccaac 120 aagcagaatt tcagcacgga tgtcatatat cagatcttca ccgaccggtt ctcggacggc 180 aatccggcca acaatccgac cggcgcggca tttgacggat catgtacgaa tcttcgctta 240 tactgcggcg gcgactggca aggcatcatc aacaaaatca acgacggtta tttgaccggc 300 atgggcatta cggccatctg gatttcacag cctgtcgaga atatctacag cgtgatcaac 360 tactccggcg tccataatac ggcttatcac ggctactggg cgcgggactt caagaagacc 420 aatccggcct acggaacgat gcaggacttc aaaaacctga tcgacaccgc gcatgcgcat 480 aacataaaag tcatcatcga ctttgcaccg aaccatacat ctccggcttc ttcggatgat 540 ccttcctttg cagagaacgg ccgcttgtac gataacggca acctgctcgg cggatacacc 600 aacgataccc aaaatctgtt ccaccattat ggcggcacgg atttctccac cattgagaac 660 ggcatttata aaaacctgta cgatctggct gacctgaatc ataacaacag cagcgtcgat 720 gtgtatctga aggatgccat caaaatgtgg ctcgacctcg gggttgacgg cattcgcgtg 780 gacgcggtca agcatatgcc attcggctgg cagaagagct ttatgtccac cattaacaac 840 tacaagccgg tcttcaactt cggcgaatgg ttccttggcg tcaatgagat tagtccggaa 900 taccatcaat tcgctaacga gtccgggatg agcctgctcg atttcccgtt tgcccagaag 960 gcccggcaag tgttcaggga caacaccgac aatatgtacg gcctgaaagc gatgctggag 1020 ggctctgaag tagactatgc ccaggtgaat gaccaggtga ccttcatcga caatcatgac 1080 atggagcgtt tccacaccag caatggcgac agacggaagc tggagcaggc gctggccttt 1140 accctgactt cacgcggtgt gcctgccatc tattacggca gcgagcagta tatgtctggc 1200 gggaatgatc cggacaaccg tgctcggatt ccttccttct ccacgacgac gaccgcatat 1260 caagtcatcc aaaagctcgc tccgctccgc aaatccaacc cggccatcgc ttacggttcc 1320 acacaggagc gctggatcaa caacgatgtg atcatctatg aacgcaaatt cggcaataac 1380 gtggccgttg ttgccattaa ccgcaatatg aacacaccgg cttcgattac cggccttgtc 1440 acttccctcc cgcagggcag ctataacgat gtgctcggcg gaattctgaa cggcaatacg 1500 ctaaccgtgg gtgctggcgg tgcagcttcc aactttactt tggctcctgg cggcactgct 1560 gtatggcagt acacaaccga tgccacagct ccgatcaacg gcaatgtcgg cccgatgatg 1620 gccaaggcag gggtcacgat tacgattgac ggccgcgctt cggctcggca aggaacggtt 1680 tacttcggta caacggcagt cactggcgcg gacatcgtag cttgggaaga tacacaaatc 1740 caggtgaaaa tcctgcgggt ccctggcggc atctatgata tcagagttgc caacgcagcc 1800 ggagcagcca gcaacatcta cgacaatttc gaggtgctga ccggagacca ggtcaccgtt 1860 cggttcgtaa tcaacaatgc cacaacggcg ctgggacaga atgtgttcct cacgggcaat 1920 gtcagcgagc tgggcaactg ggatccgaac aacgcgatcg gcccgatgta taatcaggtc 1980 gtctaccaat acccgacttg gtattatgat gtcagcgttc cggcaggcca aacgattgaa 2040 tttaaattcc tgaaaaagca aggctccacc gtcacatggg aaggcggcgc gaatcgcacc 2100 ttcaccaccc caaccagcgg cacggcaacg gtgaatgtga actggcagcc ttaa 2154  

Claims (6)

To the substrate solution containing cyclodextrin, amylase isolated from Pyrococcus furiosus was added to the substrate solution, and reacted at a pH of 4.0 to 6.0 and a temperature of 65 to 95 ° C to form 6 to 9 glucose molecules. Method for producing a chain maltooligosaccharide. The method of claim 1, wherein the substrate solution further comprises glucose. The method according to claim 1 or 2, wherein the substrate solution containing cyclodextrin is a cyclodextrin solution obtained by reacting cyclodextrin glucanotransferase with soluble starch. The method of claim 1, wherein the cyclodextrin is selected from the group consisting of alpha-cyclodextrin, beta-cyclodextrin, and gamma-cyclodextrin. The method of claim 1 or 2, wherein the amylase is amylase derived from Pyrococcus furiosus comprising the amino acid sequence of SEQ ID NO: 2. The method of claim 1, wherein the reaction time of amylase and cyclodextrin is 10 to 60 minutes.

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