JPH05227955A - New heat-resistant sucrose oligo saccharide-producing enzyme and its use - Google Patents

Abstract

PURPOSE:To provide the subject new enzyme capable of effectively, stably and mass producing a sucrose oligo saccharide consisting mainly of theanderose and isomelezitose. CONSTITUTION:A new heat-resistant sucrose oligo saccharide-producing enzyme. The action: the production of 1 mole of a new alpha-D-glucopyranoside from 1 mole of alpha-D-glucopyranoside and 1 mole of a glucosyl group acceptor by a new transfer reaction, etc.; substrate specificity: the main products of the transfer reaction are theanderose and/or isomelezitose, when both the a-D- glucopyranoside and the glucosyl group acceptor are sucrose, etc.; the optimal pH: 5-6; the optimal temperature: 75 deg.C at a pH of 6; the isoelectric point: pI4.6; mol.wt. 65000 (SDS-PAGE), etc. The enzyme is obtained by isolating the enzyme from a bacterium (e.g. Bacillus stearotheramophilus AIK90-30 strain) producing the enzyme, recombining the enzyme with a vector, transforming a microorganism with the vector, culturing the transformed microorganism, and subsequently collecting the enzyme from the cultured product.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention uses a novel thermostable sugar oligosaccharide-forming enzyme, a gene thereof, and a recombinant vector containing the gene, which enables efficient and stable mass production of sugar oligosaccharides. The present invention relates to a method for producing the enzyme and a method for using the enzyme.

[0002]

BACKGROUND OF THE INVENTION Theanderose (also called isomaltosylfructoside, G16GF) in which glucose is α-1,6 linked to glucose in sucrose and isomeretitose (GF61G) in which glucose is α-1,6 linked to fructose in sucrose are known. , Is a trace oligosaccharide contained in honey and is considered to be closely related to the flavor of honey.

Since these oligosaccharides selectively promote the growth of bifidobacteria, which are useful intestinal bacteria, they are expected to have an intestinal regulating action, and also produce carious fungi, Streptococcus mutans and Sobrinus. It inhibits glucosyl transferase (insoluble glucan synthase), and acid production by these dental caries is weak, so it is expected to have a dental caries preventive effect.

It is also known that fructose, which is a constituent monosaccharide of sucrose, has high sweetness and excellent taste, does not serve as a substrate for insoluble glucan synthesis by glucosyltransferase, and inhibits insoluble glucan synthesis similarly to theanderdelose and isomeretitose. Has been.

Therefore, by transferring the glucose moiety which causes caries generation to another sucrose molecule, theanderose and isomeretitose having the above-mentioned preferable functions are produced, and the resulting mixture of sugars (suclooligosaccharide). Is an excellent sugar product containing theandelose, isomeretitose, and fructose liberated as a result of the transfer reaction.

This sucrose oligosaccharide production reaction is most efficient by a glucosyltransferase reaction between sucrose molecules using sucrose as a glucosyl donor and an acceptor. For this purpose, various glucosyl group transfer reactions are performed. Enzymes have been used.

[0007]

This enzymatic reaction progresses more efficiently as the substrate sucrose concentration is increased, but a high-concentration sucrose solution has a high viscosity at room temperature, and the uniformity of the reaction solution is maintained by stirring. There was a problem in the handling in the manufacturing process, such as a decrease in the flow velocity during liquid transfer and clogging of the piping.

If this enzymatic reaction is carried out at a high temperature, it is thought that in addition to solving these problems, it is possible to shorten the reaction process time by increasing the reaction rate and prevent contamination of various bacteria in the manufacturing process. Requires a highly thermostable enzyme. Further, if these enzymes are immobilized on an industrial scale and repeatedly used at high temperature for a long period of time, enzymes having higher thermostability are required.

The research group of the inventors of the present invention has previously found microorganisms that produce such thermostable glucosyltransferase, and transfer sucrose using these microorganisms and isomaltosylfructoside and And / or patent applications relating to novel food materials containing isomeretitose (Japanese Patent Laid-Open No. Hei 4
-30788, 430796, 4-307
71).

However, since the enzyme productivity of these microorganisms is low and the activity varies depending on the culture, it is difficult to industrially produce saccharide oligosaccharides. It was not possible to completely purify the enzyme.

Therefore, the inventors of the present invention diligently made efforts to improve and stabilize the enzyme productivity of the microorganism, but could not achieve the purpose for the following reasons. That is, in the course of research, Bacillus stearothermophilus A, which has been deposited with the Institute of Microbial Technology, Institute of Industrial Science and Technology for the above application.
Since the IK90-30 strain was found to consist of at least two or more kinds of microorganisms, it was attempted to isolate these microorganisms from each other and isolate only the sucrose oligosaccharide-producing enzyme-producing bacterium. However, this sucrose-oligosaccharide-producing enzyme-producing bacterium is very easy to die by itself, and because it is in a parasitic or symbiotic relationship with other microorganisms, the sucrose-oligosaccharide-producing enzyme-producing bacterium itself could not be purely isolated.

[0012]

The inventors of the present invention aimed to stably improve the productivity of the sucrose-oligosaccharide-forming enzyme from the symbiotic group of microorganisms by a genetic engineering technique. By isolating only the gene encoding Escherichia coli and transforming it with Escherichia coli, a transformant having the ability to produce a sucrose-oligosaccharide-forming enzyme several hundred times to 2,000 times that of the parent strain is obtained. The present invention has been completed by clarifying the enzyme itself.

That is, the first object of the present invention is to provide an industrially-available succharooligosaccharide-forming enzyme which can be stably obtained in a large amount by applying a genetic engineering technique. The second object of the present invention is to provide a novel gene encoding a sucrose oligosaccharide-forming enzyme, a vector incorporating the gene, and a transformant producing the enzyme. Further, a third object of the present invention is to provide a method for producing a saccharide oligosaccharide-forming enzyme, which comprises culturing the transformant, extracting the enzyme from the culture, and purifying the enzyme.
Furthermore, another object of the present invention is to provide a method for producing succlo-oligosaccharide using the enzyme.

The saccharide oligosaccharide-forming enzyme of the present invention is obtained by isolating a gene encoding the enzyme from a microorganism that produces a thermostable glucosyltransferase, for example, Bacillus stearothermophilus AIK90-30 strain, and using it as a suitable vector. It is obtained by transforming an appropriate host microorganism with this and separating and obtaining it from the culture solution of the microorganism. Bacterial DN from the AIK90-30 strain
To isolate A, for example, the Saito-Miura method (Saito,
H. and Miura, K., Biochem. Biophys. Acta, 72, 619
Page, 1963) and other known methods can be used. In addition, a known method using a restriction enzyme can be used to create a gene library.

The DNA fragment thus obtained can be incorporated into a plasmid by a conventional method and introduced into a host to obtain the transformant. As the host cell, Escherichia coli (Escherichia coli) or Bacillus subtilis can be used, and as the vector, pUC18, p that can replicate in E. coli
P that can be replicated in B. subtilis, such as BR322
Known plasmids such as UB110, pE194 and pC194 can be used.

The α-glucosidase activity of the transformant thus obtained is measured, and a strain having a high enzyme activity is selected to obtain the desired transformant. The E. coli (pUSU334 / W3110) strain, which is one of the obtained transformants, was Escherichia coli.
Escherichia coli SAM 1954 was named, and on February 14, 1992, the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology, Micro Engineering Lab. No. 3749 (FERM BP-3
749) has been deposited.

The nucleotide sequence of the gene encoding the sucrose oligosaccharide-forming enzyme of the present invention can be determined by a conventional method using a transformant plasmid exhibiting α-glucosidase activity. Moreover, the amino acid sequence of the enzyme can be deduced from these base sequences. The nucleotide sequence of the gene encoding the thus-obtained sugar oligosaccharide-forming enzyme and the amino acid sequence of the sugar-oligosaccharide-producing enzyme deduced therefrom are shown in the sequence listing below. In the present specification, an amino acid sequence having homology includes amino acid substitutions, deletions, additions, etc. in a part of the sequence, but those having the same function as the peptide represented by the original amino acid sequence. Say.

In order to produce the suctooligosaccharide-forming enzyme of the present invention, a host cell transformed with the plasmid containing the succharooligosaccharide-producing enzyme gene obtained as described above is treated with a suitable carbon source, nitrogen source and Culture using a medium containing a trace amount of metal element, then extract the bacterial cell components from the obtained bacterial cells by a known method, and further ordinary ammonium sulfate fractionation method, ion exchange chromatography, gel filtration chromatography, etc. The protein may be purified using the protein purification method described in.
Since the enzyme is thermostable, a large amount of a crude enzyme solution containing the enzyme can be easily prepared by expressing the enzyme in a large amount in the strain and then heat-treating the obtained culture. ..

The thus-obtained saccharide oligosaccharide-forming enzyme had the following physicochemical properties. (1) Action Transfer reaction: α-D-glucopyranoside (Glc-X)
1 mol of new α-D-glucopyranoside (Glc-Y) and 1 mol of (X) are produced from 1 mol and 1 mol of the glucosyl group acceptor (Y). Hydrolysis: 1 mol of α-D-glucopyranoside (Gl
c-X) is hydrolyzed to produce 1 mol of (X) and 1 mol of glucose. (Here, both X and Y represent sugar or aglycone) (2) Substrate specificity Transfer reaction: α-D-glucopyranoside (Glc-X)
The main products of the transfer reaction in the case where both of the glucosyl group acceptor and the glucosyl group acceptor (Y) are sucrose are theanderose and / or isomeretitose. Hydrolysis: Sucrose and p-nitrophenyl-α
It hydrolyzes -D-glucopyranoside, but not p-nitrophenyl-β-D-glucopyranoside. (Here, X and Y are the same as above) (3) Optimum reaction pH: pH 5.0 to 6.0 Optimum reaction temperature: 75 ° C (pH 6.0) (4) pH stability: pH 5.0 to 12.0 (55 ℃, 1
Stable for 0 minutes Thermal stability: Stable at 65 ° C (pH 7.2, 10 minutes) (5) Isoelectric point: pI 4.6 (isoelectric focusing) (6) Molecular weight: Approximately 65,000 (SDS-) PAGE) About 240,000 (gel filtration)

A purified enzyme may be used to produce a sucrose-oligosaccharide using the above-described sucrose-oligosaccharide-producing enzyme of the present invention. However, as long as the enzyme reaction is not hindered, for example, a crude enzyme obtained as described above is used. A partially purified enzyme purified by an enzyme solution, ammonium sulfate fractionation, gel filtration chromatography or the like can also be used. Alternatively, an enzyme immobilized on an insoluble carrier such as Sepharose can be used.

Regarding the enzymatic reaction for producing sucrose oligosaccharides, it is desirable that the sucrose concentration is as high as possible, but considering the viscosity of the sugar solution, it is preferably about 1 to 2 g / ml. The enzymatic reaction proceeds in the range of pH 5.0 to pH 12.0, but the optimum pH of the enzyme is taken into consideration, and the pH is 5.0.
It is preferable to keep it between ~ 6.0. The enzyme reaction proceeds at room temperature to 85 ° C, but it is preferable to keep the reaction temperature at about 65 to 75 ° C in consideration of the stability of the enzyme and the optimum temperature. The amount of added enzyme is 1 for 1 g of sucrose.
-10 PNP units (1 PNP unit is 1 μmol per minute)
P-nitrophenol (hereinafter, abbreviated as PNP) of (showing an enzyme titer of releasing enzyme) is desirable. The enzyme may be added all at once, or may be added in several batches while monitoring the reaction by high performance liquid chromatography or the like. A reaction time of about 1 to 4 days is sufficient, but the end time of the reaction can be determined while monitoring the reaction.

After completion of the reaction, the enzyme is removed or inactivated by heating, if necessary, and the reaction-completed solution is treated with an ion exchange resin column,
The desired sucrose oligosaccharide can be produced by treating with activated carbon or the like and then concentrating it.

[0023]

INDUSTRIAL APPLICABILITY According to the present invention, it becomes possible to stably obtain a large amount of a thermostable enzyme capable of producing sucrose-oligosaccharide, and the industrial production of sucrose-oligosaccharide mainly comprising theanderose and / or isomeretitose using the enzyme. Is what makes it possible.

[0024]

EXAMPLES Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 Acquisition of sucrose oligosaccharide-forming enzyme (α-glucosidase) gene from Bacillus AIK90-30 strain: Step 1: Isolation of chromosomal DNA Isolation of chromosomal DNA was carried out by Saito and Miura (Saito, Miura).
H. and Miura, K., Biochem. Biophys. Acta, 72, 619
Page, 1963). That is, 1.7 liters of a liquid medium (pH 7.0) containing 5% sucrose, 0.1% yeast extract and 0.5% polypeptone was used to centrifuge cells of Bacillus AIK90-30 strain that had been shake-cultured at 60 ° C. , And suspended in a 25% sucrose solution, added EDTA and lysozyme,
Incubate for 30 minutes, then pronase E
1% S containing 200 μg / ml and NaCl 0.2M
The DS solution was added, stirred gently and incubated overnight at 37 ° C.

Next, an equal amount of phenol was added, and the mixture was slowly stirred and centrifuged, and then the upper layer was recovered with a Komagome pipette and subjected to phenol / chloroform extraction and chloroform extraction. DNA generated by adding twice the amount of cold ethanol
The precipitate was collected by winding it on a glass rod. Obtained D
NA was dissolved in 10 mM Tris-HCl buffer (pH 8.0) containing 1 mM EDTA to a final concentration of NaCl2.
00 mM, RNase 100 μg / ml was added, and the mixture was incubated at 37 ° C. for 1 hour, then phenol / chloroform extraction and ethanol precipitation were performed in the same manner as above, and the obtained DNA precipitate was dissolved in the above Tris-hydrochloric acid buffer solution. 1 ml of a preparation having a DNA concentration of 2 mg / ml was prepared.

Step 2: Preparation of library The DNA thus prepared was partially digested with Sau3AI, electrophoresed on 0.7% agarose, and a 6-9 kb DNA fragment was excised from the gel to recover the DNA fragment. .. This 6-9 kb DNA fragment was completely digested with BamHI and then ligated with the vector pUC18 treated with alkaline phosphatase (CAP), and the method of Mandel Higa (Mandel, M. and Higa, A., J. Mol. B.
iol., 53, 154, 1970) using E. coli JM10
Transformed into 9.

Since the obtained transformant harbors the ampicillin resistance gene in pUC18, Bactopeptone 1.6%, Bacto-Yeast Extract 1.0.
%, NaCl 0.5%, pH 7.4, ampicillin 5
0 μg / ml, 0.3 mM isopropyl-1-thio-β
Grow as white colonies on 2xYT agar containing -D-galactoside (IPTG) and 0.03% 5-chloro-4-bromo-3-indolyl-β-D-galactoside (X-gal).

Step 3: Selection of transformants White colonies of the transformants thus obtained were replicated in the same 2xYT agar medium and dissolved in 10 mM sodium phosphate buffer (pH 7.2) to give 20 mM p. −
Nitrophenyl-α-D-glucopyranoside (PNP-Gl
c) Overlaid with 0.7% agar containing solution and incubated at 60 ° C. According to this method, the transformant expressing the sucrose-oligosaccharide-forming enzyme decomposes PNP-Glc to produce p-nitrophenol, so that it can be isolated as a yellow-colored colony on the medium. As a result of the above selection, four strains No. 31, 51, 64 and 71 were isolated. After subjecting the disrupted cell suspensions of these cultured cells to heat treatment at 65 ° C for 1.5 hours, it was confirmed that the target enzyme gene was expressed by measuring the PNP-Glc degrading activity and the saccharide oligosaccharide synthesis activity. confirmed.

Step 4: Subcloning Recombinant plasmids contained in positive clones No. 31, 51, 64 and 71, pUSU31 and pUSU, respectively.
A restriction enzyme map was prepared for 51, pUSU64 and pUSU71 (Fig. 1). By comparing the restriction enzyme maps, it was found that each of these plasmids had inserted DNA fragments of different lengths having a common part with each other. A more detailed restriction map was created for pUSU71 having the shortest inserted DNA fragment. Based on this restriction map, insert DN using restriction enzyme site
The A fragment was reduced. Insert DN in pUSU71
A DNA fragment (about 2.9 kb) obtained by cutting at the HindIII site in the middle of the A fragment and the XbaI site at the multicloning site was the HindIII of pUC19.
/ XbaI site to obtain pUSU267. In addition, S at the multiple cloning site of pUSU71
pUSU334 having about 2.3 kb Bacillus AIK90-30 strain-derived inserted DNA fragment deleted between the SmaI site and the SmaI site in the inserted DNA fragment was obtained (FIG. 1). Escherichia coli containing pUSU267 and pUSU334 had sucrose oligosaccharide-forming enzyme activity.

Example 2 Determination of nucleotide sequence of sucro-oligosaccharide-forming enzyme (α-glucosidase) gene: pUSU33 obtained in Example 1
The 2.3 kb inserted DNA fragment of 4 was cloned into phage M13 in both directions to prepare double-stranded DNA (RF). This is from 289 of "Tsukusei Chemistry Experiment Course, Volume 1, Gene Research Method II" (published by Tokyo Kagaku Dojin 1986).
By the method described on page 305, double-stranded DNA in which a deletion was introduced in one direction was prepared by reacting with E. coli exonuclease III. The double-stranded DNA thus obtained was transformed into Escherichia coli JM109 to prepare a phage clone in which a deletion was inserted in one direction.

Double-stranded DNA was prepared from each phage clone, the degree of deletion was examined from the restriction enzyme cleavage pattern, and single-stranded phage DNA was prepared from an appropriate clone. Using these single-stranded phage DNAs as templates, the dideoxy method (Sanger, F., et al., Proc. Natl. Acad. Sc
i. USA, 74, 5463, 1977). The entire nucleotide sequence of the 2.3 kb DNA insert (3.1 kb) was determined by connecting the nucleotide sequences of the clones.

The determined nucleotide sequence consists of 2320 bases and includes the entire region of the α-glucosidase gene. In the nucleotide sequence, it begins at the 393-395th TTG and ends at the 2154-2156th TGA 176
There is an open reading frame corresponding to the α-glucosidase gene consisting of 1 base pair (see Sequence Listing). The translation initiation codon is usually ATG, but TT
Examples starting with G are also known. The reason why the 393rd to 395th TTGs were used as the start codon was that A was preceded by 9 base pairs.
This is due to the existence of a ribosome binding site consisting of GGAGG and the fact that the N-terminal amino acid sequence of the α-glucosidase shown in Example 6 matches the amino acid sequence deduced from the DNA base sequence over 29 amino acid residues.

From the above results, the α-glucosidase gene had a molecular weight of about 586 amino acids and a molecular weight of about 69,000.
It was revealed that it encodes the protein of.

Example 3 Expression of sucrose oligosaccharide-forming enzyme (α-glucosidase): The α-glucosidase activity in the E. coli recombinant containing the α-glucosidase gene obtained in Example 2 was measured. The activity was measured by adding 0.1 ml of enzyme solution to 0.6 ml of 10 mM PNP-Glc solution dissolved in 10 mM sodium phosphate buffer (pH 7.2) and reacting at 55 ° C for 1 minute with a spectrophotometer. The amount of PNP released per hit was measured as the absorbance at 405 nm over time. The enzyme activity is 1 μm per minute under the above measurement conditions.
The enzyme titer for releasing PNP of ol was defined as 1 PNP Unit.

First, each E. coli recombinant was treated with glucose.
0.1%, yeast extract 0.5%, polypeptone 1%, N
In a liquid medium consisting of aCl 0.5% (pH 7.2),
After shaking culture at 37 ° C, 10 ml of the culture solution was added to 2 ml.
The cells were inoculated into 400 ml of the same medium contained in a 1-volume Erlenmeyer flask and cultured with shaking at 37 ° C. for 3 hours. 10 at this point
After adding 1 ml of 0 mM IPTG solution and shaking culture for further 1 day, the cells were collected and washed with 10 mM sodium phosphate buffer (pH 7.2). Next, the washed cells were treated with an appropriate amount of 10 mM sodium phosphate buffer (pH
After suspending in 7.2) and crushing the cells with glass beads for 3 minutes, an enzyme solution was prepared by centrifugation.

Table 1 summarizes the amount of cultured cells and enzyme activity in various E. coli recombinants.

As is clear from this result, the inserted DNA
The fragment was designated as pUSU71, pUSU267, pUSU334.
The enzyme production was improved by shortening it.
In addition, by changing the host Escherichia coli from JM109 to W3110, 4.2 PNP Unit per 1 ml of the culture solution was obtained.
The amount of enzyme produced is about 200 times that of JM109 (pUSU71) and about 2 times that of the parent strain (Bacillus).
We have succeeded in remarkably improving it by 000 times.

Example 4 A sucrose oligosaccharide-forming enzyme produced by a transformant was used.
Sucrose oligosaccharide production: Step 1: Preparation of enzyme solution from transformant Escherichia coli, which is a sucrose oligosaccharide-producing bacterium, is used as seed culture.
(PUSU334 / W3110) to glucose 0.1
%, Yeast extract 0.5%, polypeptone 1%, NaC
l After inoculation in a liquid medium consisting of 0.5%,
The cells were shake-cultured for one day. Then, add 1.6 l of this culture solution to 3
0l yeast extract 0.1%, polypeptone 1%, NH_Four
Cl 0.1%, Na₂HPO_Four 1.52%, KH₂PO_Four 
0.3%, Na₂ SO_Four 0.01%, NaCl 0.3%,
MgCl₂ 50 with liquid medium consisting of 0.008%
Inoculate a 1-liter jar fermenter for 200 r at 37 ° C
Aeration and agitation culture was performed at pm, 0.5 vvm for 19 hours.
It was After culturing, collect the cells by centrifugation and deionize water.
The cells were washed with to obtain a microbial cell having a wet weight of 118 g.

The obtained microbial cells were suspended in 1 liter of 10 mM sodium phosphate buffer (pH 7.2), crushed by bead crushing at low temperature, and the microbial cell residue was removed by centrifugation to remove the crude enzyme solution. Was prepared. The resulting crude enzyme solution is 60 ° C
Heat treatment for 30 minutes, centrifuge and remove supernatant 1,520.
ml was obtained. After adding 11.55 g of ammonium sulfate to this, 60.8 ml of 3% polyethyleneimine solution was added, and the mixture was allowed to stand at room temperature for 30 minutes and then centrifuged to obtain supernatant 1.5.
60 ml was obtained. Then add ammonium sulfate 4
74.24 g was added and the precipitate was collected by centrifugation.

The obtained precipitate was dissolved in 80 ml of 10 mM sodium phosphate buffer (pH 7.2) and desalted by gel filtration using Sephadex G-25 to obtain the activity 812 PNP Unit. / Ml of enzyme solution (220 ml) was prepared and stored frozen at -20 ° C.

Step 2: Preparation of sucrose oligosaccharide solution 10 kg of sucrose was added to 20 mM acetate buffer (pH 5.
5) Dissolved in 10 l, added 2 PNP Units per gram of sucrose to the above enzyme solution, and reacted at 65 ° C. for 1 day to give: theanderose 7.5%, isomeretitose 7
%, Sucrose 71%, isomaltose 0.7%, fructose 10.3% and glucose 3.5%.

Furthermore, 2 PNPs / g of sucrose
An enzyme solution of Unit was added, the mixture was reacted at 65 ° C for 2 days, and then heated at 95 ° C for 10 minutes. 16 l of the heat-treated reaction solution thus prepared was used as a cation exchange resin (Diaion SK-1B; manufactured by Mitsubishi Kasei) 0.8 l, anion exchange resin (Diaion WA-30; manufactured by Mitsubishi Kasei) 1.2 l and activated carbon. (Take call; manufactured by Takeda Pharmaceutical Co., Ltd.) Decolorization and desalting were performed with 150 g. Diatomaceous earth (Radiolite # 100; Showa Kagaku Kogyo Co., Ltd.) is used to remove activated carbon by filtration to obtain tetrasaccharide oligosaccharides 5.4%, theanderose 13.6%, isomeretitose 12.6%, sucrose 32.8%. , A sugar oligosaccharide solution having a sugar composition of isomaltose 3.5%, fructose 23.4% and glucose 8.7% was prepared. This was concentrated to a sugar concentration of 75% (W / W) to obtain 12.5 kg of a sucrose oligosaccharide solution.

During this enzymatic reaction, the reaction products were analyzed by high performance liquid chromatography under the following conditions. Column: Asahi Pack NH2P-50 (Asahipak NH2
P-50; 4.6 mmID x 250 mmL) Elution solvent: Acetonitrile-water (70:30) Elution rate: 1.0 ml / min Detection means: Differential refractometer (Shodex SE-61)

Example 5 Complete purification of sucrose-oligosaccharide-forming enzyme (α-glucosidase) that produces a sucrose-oligosaccharide solution: The enzyme solution obtained in Example 4 was treated with 50 mM sodium phosphate buffer (pH).
It was subjected to Sepabeads FP-DA 13 (manufactured by Mitsubishi Kasei Co., Ltd.) column chromatography equilibrated in 7.2),
After the flow-through fraction was eluted, it was eluted with sodium chloride. The active fraction was subjected to ammonium sulfate fractionation, dialysis, and membrane concentration, followed by high performance liquid chromatography gel filtration chromatography (TSKgel G3000 SW; manufactured by Tosoh Corporation), ion exchange chromatography (DEAE-Toyopea).
rl 650S; Tosoh Corporation), hydrophobic chromatography (Butyl-Toyopearl 650S; Tosoh Corporation) and hydroxyapatite chromatography (HCA P-20).
05; manufactured by Mitsui Toatsu Co., Ltd., and electrophoretically purified single purified enzyme preparation (specific activity 27.6 PNP Unit / A2)
80, total activity 4,576 PNP Unit) was obtained.

Example 6 Examination of physicochemical properties of a sucrose-oligosaccharide-forming enzyme (α-glucosidase) that produces a sucrose-oligosaccharide solution: The single purified enzyme preparation obtained in Example 5 was as follows. The physicochemical properties were examined.

(1) Molecular Weight Phast-Gel Electrophoresis Device (Pharmacia LKB)
The molecular weight by SDS polyacrylamide gel electrophoresis using Biotechnology) was about 65,000. Further, the molecular weight was about 240,000 by gel filtration by high performance liquid gel chromatography using a TSKgel G3000SW column (manufactured by Tosoh Corporation).

(2) Isoelectric point Phast-Gel electrophoresis apparatus (Pharmacia LKB
The isoelectric point by the isoelectric focusing method using Biotechnology) was pI 4.6.

(3) Optimum pH 10 mM sodium acetate buffer (pH 4.5 to 5.5)
And 10 mM sodium phosphate buffer (pH 5.5
~ 8.5) 50 mM sucrose solution 0.95
The amount of glucose produced by adding 0.05 ml of the enzyme solution to ml and allowing it to act at 55 ° C. for 10 minutes was measured by the glucose oxidase method, and the activity at pH 5.5 was relatively shown as 100. The optimum pH was around pH 5 to 6 as shown in FIG.

10 mM dissolved in the same buffer
The enzyme solution (0.05 ml) was added to p-nitrophenyl-α-D-glucopyranoside solution (0.65 ml), and the mixture was stirred at 55 ° C for 1 hour.
After operating for 0 minutes, 1M sodium carbonate solution 0.3
Also in the endpoint measurement method in which the reaction was stopped by adding ml to measure the amount of released PNP, the optimum pH was around pH 5 to 6 as shown in FIG.

(4) pH stability range After being left in buffer solutions of various pHs at 55 ° C. for 10 minutes, they were immediately cooled with ice and the residual activity was measured by the following initial velocity method. As shown in FIG. 4, it was stable in the pH range of 5.0 to 12.0. The buffers were sodium acetate buffer (pH 3.5 to 6.0), sodium phosphate buffer (pH 5.5 to 8.5), glycine-NaOH buffer (pH 8.5 to 11.5). )It was used. Residual activity is 10
0.1 ml of enzyme solution was added to 0.6 ml of 10 mM p-nitrophenyl-α-D-glucopyranoside solution dissolved in mM sodium phosphate buffer (pH 7.2), and 5
While reacting at 5 ° C., the amount of PNP released per minute was measured with a spectrophotometer over time.

(5) Optimum temperature and temperature stability range The optimum temperature was examined by the measuring method using sucrose of Example 6 (3). As shown in FIG. 5, the activity was highest at 75 ° C. under the reaction conditions of pH 6.0 and 10 minutes, and the optimum temperature was estimated to be 75 ° C. The temperature stability range is 10m
After keeping the enzyme in M sodium phosphate buffer (pH 7.2) at various temperatures for 10 minutes, it was ice-cooled and its residual activity was measured by the above method. The result is 65 ° C as shown in FIG.
Was stable until.

(6) Effect of various inhibitors on enzyme activity The effect of metal ions and chemical reagents / sugars on enzyme activity was measured at 0.1 m using the measurement system shown in Example 6 (4).
Various metal ions with M concentration and chemical reagents with 1 mM concentration
Enzyme activity was measured by adding sugars. As a result, Table 2
, Copper ion, lead ion, and silver ion have relative activities of 68%, 67%, and 19%, respectively.
Was observed, but the phenomenon of remarkable activation due to addition of the compound was not observed.

[0054] * Abbreviations in compounds indicate the following. AcO: Acetyloxy group IAA: Monoiodoacetic acid DTNB: 5,5′-dithiobis (2-nitrobenzoic acid) DTT: Dithiothreitol EDTA: Ethylenediaminetetraacetic acid EGTA: Ethylene glycol bis (β-aminoethyl ether) tetraacetic acid

(7) N-terminal amino acid sequence The N-terminal amino acid sequence of the sucrose oligosaccharide-forming enzyme was analyzed using a gas phase sequencer PSQ-1 manufactured by Shimadzu Corporation.
It was decided as follows. Ser-Thr-Ala-Leu-Thr-Gln-Thr-Ser-Thr-Asn -Ser-Gln-Gln-Ser-Pro-Ile-Arg-Arg-Ala-Trp -Trp-Lys-Glu-Ala-Val- Val-Tyr-Gln-Ile-This amino acid sequence was in agreement with the amino acid sequences 1 to 29 shown in the sequence listing.

(8) C-Terminal Amino Acid The C-terminal amino acid of the sucrose oligosaccharide-forming enzyme is hydrazine-decomposing method (“Biochemistry Experimental Course, Volume 1, Protein Chemistry II” (1976, Tokyo Kagaku Dojin), 186-193).
Page) to determine histidine.

(9) Action and Substrate Specificity In the case of using p-nitroglycosides as the substrate, the hydrolysis activity of the sucrose oligosaccharide-forming enzyme was determined in Example 6
According to the method described in (4), when a disaccharide is used,
The measurement was carried out according to the method described in Example 6 (3), and the measurement results are shown in Table 3 as the relative activity when the decomposition rate of p-nitrophenyl-α-D-glucopyranoside was 100%.

[0058]

As shown in Table 3, in addition to sucrose (73.5%), this enzyme contained maltose (39.0%), trehalose (25.5%) and p-nitrophenyl-α.
-D-maltopyranoside (3.4%) was decomposed.

[0060]

INDUSTRIAL APPLICABILITY According to the present invention, a thermostable enzyme capable of producing sucrose-oligosaccharide can be stably produced in a large amount, and by using the sucrose-oligosaccharide-producing enzyme, theanderose and / or isomeletitose are the main components. It became possible to industrially produce sucrose oligosaccharides.

[Sequence Listing] SEQ ID NO: 1 Sequence Length: 2320 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence Type: Genomic DNA Source: Organism: Bacillus stearothermophilus Strain Name : AIK90-30 strain Sequence characteristics: Characteristic symbol: RBS Location: 379. . 384 Method by which the characteristics were determined: S Sequence characteristics: Characteristic symbols: CDS Location: 396. . 2153 Method of characterizing features: E arrangement 1 CC CGG GCT GTT CAC GGC GGC CGT ATT CAC GTT TGT CGC GAG CTG GAC 48 CGA GTT TTT CAT GGC GTT GGT GTT TAA CTC GTC GAG TGC GAT GCG AAC 96 CAT CCC GGT CGG GAT TGC ACT GTT CAG CAG CGA ATA CAA CGT GCC ATA 144 CGG GGA CAT CTT CGC CGG TTC CGT GGT CTC CAT CCT GCC CAT TGT CGT 192 GTT GGT CAT CCT GTT CCG CAA GTG GAT TGT CTC TGG CCT CAC GGC GGG 240 CGC GGT GAA AGG CTG ACC GCT GGG CCG CCC CAA GCC GCT TGT CCG TGC 288 GCG GGG ATG AAG CCA GTT TCG CCC GTC CCG CGG CTG TGC ACC GGT GAG 336 CGG CAG ACA AAG ACG CCG ACG ACT GGA AAC AGG TTG AGC AAA AAG GAG 384 GCA TCG ACA TTG TCC ACC GCC TTG ACG CAG ACC TCA ACG AAC TCG CAG 1 Ser Thr Ala Leu Thr Gln Thr Ser Thr Asn Ser Gln 432 CAA TCG CCC ATT CGC CGG GCG TGG TGG AAG GAA GCG GTC GTA TAC CAG 13 Gln Ser Pro Ile Arg Arg Ala Trp Trp Lys Glu Ala Val Val Tyr Gln 480 ATT TAT CCG AGA TCC TTC ATG GAC TCG AAT GGC GAC GGT ATC GGC GAT 29 Ile Tyr Pro Arg Ser Phe Met Asp Ser Asn Gly Asp Gly Ile Gly Asp 528 TTG CGC GGC ATC CTC TC G AAG CTG GAC TAC CTC AAA CTG CTC GGC GTG 45 Leu Arg Gly Ile Leu Ser Lys Leu Asp Tyr Leu Lys Leu Leu Gly Val 576 GAT GTG TTG TGG CTG AAT CCC ATC TAC GAC TCG CCG AAT GAC GAC ATG 61 Asp Val Leu Trp Leu Asn Pro Ile Tyr Asp Ser Pro Asn Asp Asp Met 624 GGC TAT GAC ATC CGC GAC TAC TAC AAG ATT ATG GAA GAG TTC GGG ACG 77 Gly Tyr Asp Ile Arg Asp Tyr Tyr Lys Ile Met Glu Glu Phe Gly Thr 672 ATG GAG GAC TTT GAG GAG CTG CTG CGC GAG GTG CAC GCC CGC GGC ATG 93 Met Glu Asp Phe Glu Glu Leu Leu Arg Glu Val His Ala Arg Gly Met 720 AAG CTG GTC ATG GAC CTG GTG GCC AAC CAT ACC TCA GAC GAG CAC CCG 109 Lys Leu Val Met Asp Leu Val Ala Asn His Thr Ser Asp Glu His Pro 768 TGG TTC ATC GAG TCA CGC TCC TCG CGC GAC AAC CCG TAC CGC GAC TGG 125 Trp Phe Ile Glu Ser Arg Ser Ser Arg Asp Asn Pro Tyr Arg Asp Trp 816 TAC ATT TGG CGC GAT CCG AAA GAC GGC CGC GAG CCG AAC AAC TGG TTG 141 Tyr Ile Trp Arg Asp Pro Lys Asp Gly Arg Glu Pro Asn Asn Trp Leu 864 TCG TAT TTC AGC GGA TCG GCC TGG GAA TAC GAC GAG CGC ACG GGG CAG 157 Ser T yr Phe Ser Gly Ser Ala Trp Glu Tyr Asp Glu Arg Thr Gly Gln 912 TAC TAC CTG CAC TTG TTC AGC CGC AGG CAG CCG GAC TTG AAC TGG GAA 173 Tyr Tyr Leu His Leu Phe Ser Arg Arg Gln Pro Asp Leu Asn Trp Glu 960 AAC CCA AAG GTT CGC GAG GCC ATC TTC GAG ATG ATG CGG TTC TGG CTC 189 Asn Pro Lys Val Arg Glu Ala Ile Phe Glu Met Met Arg Phe Trp Leu 1008 GAC AAA GGG ATA GAT GGC TTC CGC ATG GAT GTC ATC AAC GCC ATC GCC ATC 205 Asp Lys Gly Ile Asp Gly Phe Arg Met Asp Val Ile Asn Ala Ile Ala 1056 AAG GCC GAG GGT CTG CCG GAC GCG CCG GCG CGT CCC GGA GAG CGT TAC 221 Lys Ala Glu Gly Leu Pro Asp Ala Pro Ala Arg Pro Gly Glu Arg Tyr 1104 GCT TGG GGC GGC CAG TAC TTT CTC AAT CAG CCG AAG GTC CAC GAG TAC 237 Ala Trp Gly Gly Gln Tyr Phe Leu Asn Gln Pro Lys Val His Glu Tyr 1152 CTG CGC GAG ATG TAT GAT AAG GTG CTC TCT CAT TAC GAC ATC ATG ACC 253 Leu Arg Glu Met Tyr Asp Lys Val Leu Ser His Tyr Asp Ile Met Thr 1200 GTC GGC GAG ACC GGC GGT GTG ACG ACA AAG GAT GCA CTC TTG TTT GCC 269 Val Gly Glu Thr Gly Gly Val Thr Thr Lys Asp Ala Leu Leu Phe Ala 1248 GGT GAA GAC CGG CGC GAG CTG AAC ATG GTC TTC CAG TTT GAA CAC ATG 285 Gly Glu Asp Arg Arg Glu Leu Asn Met Val Phe Gln Phe Glu His Met 1296 GAC ATC GAT GCG ACC GAC GGC GAC AAG TGG CGG CCG CGG CCG TGG CGA 301 Asp Ile Asp Ala Thr Asp Gly Asp Lys Trp Arg Pro Arg Pro Trp Arg 1344 TTG ACC GAG TTG AAA ACC ATC ATG ACG CGG TGG CAA AAT GAT TTG TAC 317 Leu Thr Glu Leu Lys Thr Ile Met Thr Arg Trp Gln Asn Asp Leu Tyr 1392 GGC AAG GCC TGG AAC AGC CTG TAC TGG ACC AAC CAT GAC CAG CCC CGC 333 Gly Lys Ala Trp Asn Ser Leu Tyr Trp Thr Asn His Asp Gln Pro Arg 1440 GCG GTG TCG CGG TTT GGC AAC GAC GGT CCC TAC CGC GTG GAG TCA GCC 349 Ala Val Ser Arg Phe Gly Asn Asp Gly Pro Tyr Arg Val Glu Ser Ala 1488 AAG ATG CTT GCC ACC GTC CTG CAC ATG ATG CAG GGG ACC CCC TAC ATC 365 Lys Met Leu Ala Thr Val Leu His Met Met Gln Gly Thr Pro Tyr Ile 1536 TAC CAG GGC GAG GAA ATC GGC ATG ACG AAC TGT CCG TTT GAC TCT ATT 381 Tyr Gln Gly Glu Glu Ile Gly Met Thr Asn Cys Pro Phe Asp Ser Ile 1584 GAC GAG TAC CGG GAC GTC GAG ATC CAC AAC CTG TGG CGG CAC CGT GTG 397 Asp Glu Tyr Arg Asp Val Glu Ile His Asn Leu Trp Arg His Arg Val 1632 ATG GAA GGC GGC CAG GAC CCT GCC GAG GTG CTG CGC GTC ATC CAG CTC 413 Met Glu Gly Gly Gln Asp Pro Ala Glu Val Leu Arg Val Ile Gln Leu 1680 AAA GGG CGG GAC AAC GCG CGC ACG CCC ATG CAG TGG GAC GAT TCA CCG 429 Lys Gly Arg Asp Asn Ala Arg Thr Pro Met Gln Trp Asp Asp Ser Pro 1728 AAC GCC GGG TTC ACC ACC GGT ACG CCG TGG ATC AAG GTG AAC CCA AAC 445 Asn Ala Gly Phe Thr Thr Gly Thr Pro Trp Ile Lys Val Asn Pro Asn 1776 TAT CGC GAG ATT AAC GTC AAG CAG GCC CTG GCG GAC CCG AAC TCG ATT 461 Tyr Arg Glu Ile Asn Val Lys Gln Ala Leu Ala Asp Pro Asn Ser Ile 1824 TTC CAT TAC TAC CGC CGC CTG ATT CAG TTG CGC AAA CAG CAT CCC ATC 477 Phe His Tyr Tyr Arg Arg Leu Ile Gln Leu Arg Lys Gln His Pro Ile 1872 GTG GTG TAC GGC AAG TAT GAC CTG ATT CTG CCG GAT CAC GAG GAG ATT 493 Val Val Tyr Gly Lys Tyr Asp Leu Ile Leu Pro Asp His Glu Glu Ile 1920 TGG GCT TAC ACG CGC ACG CTG GGA GAC GAG CGC TGG CTG ATT GTG GCC 509 Trp Ala Tyr Thr Arg Thr Leu Gly Asp Glu Arg Trp Leu Ile Val Ala 1968 AAC TTC TTT GGC GGC ACT CCG GAG TTC GAG CTG CCG CCG GAA GTC CGG 525 Asn Phe Phe Gly Gly Thr Pro Glu Phe Glu Leu Pro Pro Glu Val Arg 2016 TGC GAG GGC GCA GAG CTG GTG ATT GCC AAC TAC CCG GTG GAC GAT TCG 541 Cys Glu Gly Ala Glu Leu Val Ile Ala Asn Tyr Pro Val Asp Asp Ser 2064 GAA GCG GGC GGT CCA GCG GCC GCT GGG GCA CCT CAC CGC TTC CGT CTG 557 Glu Ala Gly Gly Pro Ala Ala Ala Gly Ala Pro His Arg Phe Arg Leu 2112 CGC CCG TAC GAA TGC CGC GTC TAC CGG CTG CTC GGC TGG CAC TGA CAG 573 Arg Pro Tyr Glu Cys Arg Val Tyr Arg Leu Leu Gly Trp His 2160 GCG ATT GGG CGG GTG CGC GCA CGG GCC TGC GCA AGA GGG AGG TCG GTA 2208 CGA TGA AGG CGG TCT ACA TTG AGG CCT ATG GTG GCA GGG AAC AGC TCA 2256 AGT ACG GTG ATC TCC CGA AGC CTG AGC TTC GTC CCC GTC ATC ACG TGC TCA 2304 TCG AGG TGC ACG CCG CC

[Brief description of drawings]

FIG. 1 is a drawing showing restriction enzyme maps of plasmids having different lengths of various inserted DNA fragments, into which DNA fragments containing a sucrose oligosaccharide synthase gene have been inserted.

FIG. 2 is a drawing showing the optimum reaction pH of the sucrose oligosaccharide-forming enzyme of the present invention when sucrose is used as a substrate. In the figure, ○ indicates sodium acetate buffer and ● indicates sodium phosphate buffer.

FIG. 3 is a drawing showing the optimum reaction pH of the sucrose oligosaccharide-forming enzyme of the present invention when measured by the endpoint method using p-nitrophenyl-α-D-glucopyranoside as a substrate. In the figure, ○ and ● are the same as in FIG.

FIG. 4 pH of sucrose oligosaccharide-forming enzyme of the present invention
Drawing which shows a stable range. In the figure, ○ indicates sodium acetate buffer, ● indicates sodium phosphate buffer, and □ indicates glycine-Na.
OH buffer is shown.

FIG. 5 is a drawing showing the optimum reaction temperature of the sucrose oligosaccharide-forming enzyme of the present invention.

FIG. 6 is a view showing the temperature stability range of the sucrose oligosaccharide-forming enzyme of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical indication C12P 19/18 7432-4B // (C12N 15/54 C12R 1:07) (72) Inventor Shibano Yuuji 1-1-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka Suntory Ltd. Research Laboratories

Claims (6)

[Claims] 1. A novel thermostable sugar oligosaccharide-forming enzyme having the following physicochemical properties. (1) Action Transfer reaction: α-D-glucopyranoside (Glc-X) 1
1 mol of new α-D-glucopyranoside (Glc-Y) and 1 mol of (X) are produced from the mol and 1 mol of the glucosyl group acceptor (Y). Hydrolysis: 1 mol of α-D-glucopyranoside (Glc
-X) is hydrolyzed to produce 1 mol (X) and 1 mol glucose. (Here, both X and Y represent a sugar or an aglycone.) (2) Substrate specificity Transfer reaction: When both α-D-glucopyranoside (Glc-X) and glucosyl group acceptor (Y) are sucrose The main products of the rearrangement reaction are thenderose and / or
Or isomeletitose. Hydrolysis: Sucrose and p-nitrophenyl-α-
It hydrolyzes D-glucopyranoside, but not p-nitrophenyl-β-D-glucopyranoside.
(Here, X and Y are the same as above) (3) Optimum reaction pH: pH 5.0 to 6.0 Optimum reaction temperature: 75 ° C (pH 6.0) (4) pH stability: pH 5.0 to 12.0 (55 ℃, 1
Stable for 0 minutes Thermal stability: Stable at 65 ° C (pH 7.2, 10 minutes) (5) Isoelectric point: pI 4.6 (isoelectric focusing) (6) Molecular weight: Approximately 65,000 (SDS-) PAGE) About 240,000 (gel filtration) 2. A alphabet following formula (I) STATTSTNSSRRAWWKAVVYYRSMDSNGDGGDRGSKDYKGVDVWNYDSNDDMGYDRDYY KMGTMDRVHARGMKVMDVANHTSDHWSRSSRDNYRDWYWRDKDGRNNWSYSGSAWYDRTG YYHSRRDNWNKVRAMMRWDKGDGRMDVNAAKAGDAARGRYAWGGYNKVHYRMYDKVSHYD MTVGTGGVTTKDAAGDRRNMVHMDDATDGDKWRRWRTKTMTRWNDYGKAWNSYWTNHDRA VSRGNDGYRVSAKMATVHMMGTYYGGMTNCDSDYRDVHNWRHRVMGGDAVRVKGRDNART MWDDSNAGTTGTWKVNNYRNVKAADNSHYYRRRKHVVYGKYDDHWAYTRTGDRWVANGGT VRCGAVANYVDDSAGGAAAGAHRRRYCRVYRGWH (I) (wherein represents the following amino acid.
A: alanine, C: cysteine, D: aspartic acid,
E: Glutamic acid, F; Phenylalanine, G; Glycine, H; Histidine, I; Isoleucine, K; Lysine,
L: leucine, M: methionine, N: asparagine,
P; Proline, Q; Glutamine, R; Arginine, S;
The novel thermostable sucrose oligosaccharide-forming enzyme according to claim 1, which has an amino acid sequence represented by serine, T; threonine, V; valine, W; tryptophan, Y; tyrosine) or homologous thereto. 3. A gene encoding an amino acid sequence represented by the formula (I) of claim 2 or a polypeptide having homology thereto. Wherein the following formula (II) TCCACCG CCTTGACGCA GACCTCAACG AACTCGCAGC AATCGCCCAT TCGCCGGGCG TGGTGGAAGG AAGCGGTCGT ATACCAGATT TATCCGAGAT CCTTCATGGA CTCGAATGGC GACGGTATCG GCGATTTGCG CGGCATCCTC TCGAAGCTGG ACTACCTCAA ACTGCTCGGC GTGGATGTGT TGTGGCTGAA TCCCATCTAC GACTCGCCGA ATGACGACAT GGGCTATGAC ATCCGCGACT ACTACAAGAT TATGGAAGAG TTCGGGACGA TGGAGGACTT TGAGGAGCTG CTGCGCGAGG TGCACGCCCG CGGCATGAAG CTGGTCATGG ACCTGGTGGC CAACCATACC TCAGACGAGC ACCCGTGGTT CATCGAGTCA CGCTCCTCGC GCGACAACCC GTACCGCGAC TGGTACATTT GGCGCGATCC GAAAGACGGC CGCGAGCCGA ACAACTGGTT GTCGTATTTC AGCGGATCGG CCTGGGAATA CGACGAGCGC ACGGGGCAGT ACTACCTGCA CTTGTTCAGC CGCAGGCAGC CGGACTTGAA CTGGGAAAAC CCAAAGGTTC GCGAGGCCAT CTTCGAGATG ATGCGGTTCT GGCTCGACAA AGGGATAGAT GGCTTCCGCA TGGATGTCAT CAACGCCATC GCCAAGGCCG AGGGTCTGCC GGACGCGCCG GCGCGTCCCG GAGAGCGTTA CGCTTGGGGC GGCCAGTACT TTCTCAATCA GCCGAAGGTC CACGAGTACC TGCGCGAGAT GTATGATAAG GTGCTCTCTC ATTACGACAT CATGACCGTC GGCGAGACCG GCGGTGTGAC GACAAAGGAT GCACTCTTGT TTGCCGGTGA AGACCGGCGC GAGCTGAAC A TGGTCTTCCA GTTTGAACAC ATGGACATCG ATGCGACCGA CGGCGACAAG TGGCGGCCGC GGCCGTGGCG ATTGACCGAG TTGAAAACCA TCATGACGCG GTGGCAAAAT GATTTGTACG GCAAGGCCTG GAACAGCCTG TACTGGACCA ACCATGACCA GCCCCGCGCG GTGTCGCGGT TTGGCAACGA CGGTCCCTAC CGCGTGGAGT CAGCCAAGAT GCTTGCCACC GTCCTGCACA TGATGCAGGG GACCCCCTAC ATCTACCAGG GCGAGGAAAT CGGCATGACG AACTGTCCGT TTGACTCTAT TGACGAGTAC CGGGACGTCG AGATCCACAA CCTGTGGCGG CACCGTGTGA TGGAAGGCGG CCAGGACCCT GCCGAGGTGC TGCGCGTCAT CCAGCTCAAA GGGCGGGACA ACGCGCGCAC GCCCATGCAG TGGGACGATT CACCGAACGC CGGGTTCACC ACCGGTACGC CGTGGATCAA GGTGAACCCA AACTATCGCG AGATTAACGT CAAGCAGGCC CTGGCGGACC CGAACTCGAT TTTCCATTAC TACCGCCGCC TGATTCAGTT GCGCAAACAG CATCCCATCG TGGTGTACGG CAAGTATGAC CTGATTCTGC CGGATCACGA GGAGATTTGG GCTTACACGC GCACGCTGGG AGACGAGCGC TGGCTGATTG TGGCCAACTT CTTTGGCGGC ACTCCGGAGT TCGAGCTGCC GCCGGAAGTC CGGTGCGAGG GCGCAGAGCT GGTGATTGCC AACTACCCGG TGGACGATTC GGAAGCGGGC GGTCCAGCGG CCGCTGGGGC ACCTCACCGC TTCCGTCTGC GCCCGTACGA ATGCCGCGTC TACCGGCTGC salt represented by TCGGCTGGCA C (II) The gene according to claim 3, which has a base sequence. 5. A host cell transformed with the recombinant vector containing the gene according to claim 3 is cultured, and a thermostable sucrose oligosaccharide-forming enzyme is collected from the culture. A method for producing the novel thermostable sucrose oligosaccharide-forming enzyme described in 1. 6. A method for producing a sucrose-oligosaccharide containing theanderose and / or isomeretitose as a main component, which comprises using the novel thermostable sucrose-oligosaccharide-forming enzyme according to claim 1.