JPH10150986A - Super thermostable 4-alpha-glucanotransferase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new enzyme, comprising a super thermostable 4-α-glucanotransferase having the optimal temperature close to the boiling point of water, having excellent saccharide transfer actions under high- temperature conditions and useful for industrial production, etc., of high-purity maltooligosaccharides, etc. SOLUTION: This new super thermostable 4-α-glucanotransferase has the optimal pH about 6.0-8.0, the optimal temperature of about 100 deg.C at pH7.5, remains at least >=90% activities after heat treatment at pH7.5 and 100 deg.C for 30min, further actions on transfer of one or several glucose units in α-1,4-glucan to the α-1,4-glucan through 1,4-glucoside bonds and excellent saccharide transfer actions under high-temperature conditions and is useful for industrial preparation of high-purity maltooligosaccharide and synthesis, etc., of various heterooligosaccharides. The enzyme is obtained by providing a DNA fragment containing a gene capable of producing the 4-α-glucanotransferase from a KOD-1 strain (FERM P-15007) and then using a gene recombination technique.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a 4-α-glucanotransferase, particularly a hyperthermostable 4-α-glucanotransferase derived from a hyperthermophilic bacterium and a 4-α-glucanotransferase gene encoding this enzyme. DNA fragments containing. An expression vector incorporating a DNA fragment containing the 4-α-glucanotransferase gene was prepared, and host cells transformed with this expression vector were cultured to produce 4-α-glucanotransferase.
By expressing the glucanotransferase gene, it becomes possible to mass-produce the target enzyme under industrially advantageous conditions. Furthermore, it becomes possible to produce an enzyme with increased stability under high-temperature conditions, for example, by site-directed mutagenesis.

[0002]

2. Description of the Related Art 4-α-glucanotransferase is widely distributed in the natural world, and has been used in higher plants such as potatoes, potatoes, mungbeans, carrots, tomatoes and barley seeds, Escherichia coli, Bacillus subtilis, P.
seudomonas stutzeri, Streptococcus bovis, Streptoc
Bacteria such as occus mitis and Thermotoga maritima and its presence in higher animals such as bovine plasma and rat liver have been observed. 4-α-glucanotransferase is α-
One or several glucose units in 1,4-glucan (donor substrate) are transferred to an acceptor substrate (glucose or α-1,4-glucan) while forming an α-1,4-glucoside bond. The molecular weight is larger or smaller than the original substrate molecule, and the substrate polymerization degree is redistributed. Examples of applications of 4-α-glucanotransferase include the preparation of high-purity maltooligosaccharides (high-purity maltriose action to obtain glucose and a series of maltooligosaccharides with high purity), and the preparation of high-purity methyl α-D-maltooligoside (methyl α-
Using D-maltooligoside as an acceptor substrate to prepare methyl α-D-maltoside, methyl α-D-maltotrioside, methyl α-D-maltotetraoside, etc.)
Was made.

In recent years, it has been reported that cyclic amylose (consisting of 17 to several hundred glucose units) is produced from amylose by intramolecular glycosyltransfer reaction of 4-α-glucanotransferase derived from potato. Since this cyclic amylose is expected to have a different inclusive action from cyclodextrin, which is a cyclic oligosaccharide composed of 6 to 8 glucose units, it is expected to be used in a wide range of fields such as the chemical, pharmaceutical and food industries. Is done.

However, since amylose and starch liquids serving as substrates have high viscosities and have a problem of mass transfer at low temperatures, it is desirable to use them at the highest possible temperature. However, the optimal temperature of most of the known 4-α-glucanotransferases is low. Exceptionally Thermo
Although the enzyme derived from toga maritima has an optimum temperature around 70 ° C., supply of industrial 4-α-glucanotransferase that can react under higher temperature conditions is desired.

[0005] Enzymes having high heat resistance and high optimum temperature are generally isolated from hyperthermophilic bacteria. 1972, Broc
Since the isolation of the thermoacidophilic bacterium Sulfolobus from the hot springs of Yellowstone by K. et al., a number of hyperthermophilic bacteria have been isolated. Hyperthermophilic bacteria are microorganisms having an optimum growth temperature of 80 ° C. or higher, and the enzymes produced by these bacteria are expected to be extremely excellent in heat resistance. fact,
The heat inactivation temperature of enzymes produced by hyperthermophilic bacteria is often higher than the maximum temperature at which the bacteria can grow, and some enzymes have an optimal temperature of 100 ° C. or higher. Therefore, the problem of contamination of harmful microorganisms during the reaction can be solved by setting the temperature to high. Such a hyperthermostable enzyme is also highly useful from an industrial application point of view, and its search, research, and development are being widely promoted worldwide. Furthermore, the physiological functions of various oligosaccharides or hetero-oligosaccharides are currently attracting attention,
These oligosaccharides or heterooligosaccharides are highly pure,
There is a demand for a technology for efficiently manufacturing.

Thermococcus litor, a hyperthermophilic archaeon
alis-derived 4-α-glucanotransferase is known (Matsuzawa et al., Novo Nordisk Enzyme Symposium, Nov.
ember 1, 1996). Although this enzyme has a molecular weight of about 85 kDa and an optimal temperature of about 90 ° C., it is considered that sucrose and cellobiose cannot be used as receptors, and thus it is not possible to sufficiently provide various oligosaccharides and heterooligosaccharides. Can be

[0007]

SUMMARY OF THE INVENTION The present invention provides a 4-α-glucanotransferase having excellent thermostability, obtaining a DNA fragment containing a 4-α-glucanotransferase gene, An object of the present invention is to produce and provide enzymes using breeding by genetic manipulation. Furthermore, the purpose is to produce various hetero-oligosaccharides.

[0008]

According to the present invention, there is provided a 4-α-glucanotransferase having the following physicochemical properties: (1) the optimum pH is around 6.0 to 8.0; (2) ) The optimum temperature at pH 7.5 is around 100 ° C; and (3) at least 90 after heat treatment at pH 7.5, 100 ° C for 30 minutes.
% Or more activity remains; and (4) transferring one or several glucose units in α-1,4-glucan to α-1,4-glucan via α-1,4-glucosidic bond.

In a preferred embodiment, the present invention provides
α-glucanotransferase is D-glucose,
Α-1,4-using xylose, isomaltose, D-cellobiose, sucrose, or D-fucose as a receptor
Transfer one or several glucose units in the glucan.

[0010] In a preferred embodiment, the 4-α-glucanotransferase is obtained from a hyperthermophilic strain KOD-1.

According to the present invention, it has the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence containing one or more deletions, additions or substitutions of amino acids in the sequence, and comprises 4-α-glucano 4- with transferase activity
An α-glucanotransferase is provided.

According to the present invention, it has an amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence containing one or more deletions, additions or substitutions of amino acids in the sequence, and has the following physicochemical properties: 4-α-glucanotransferase having: (1) an optimum pH is around 6.0-8.0; (2) an optimum temperature at pH 7.5 is around 100 ° C .; (3) pH 7. 5.After heat treatment at 100 ℃ for 30 minutes, at least 90
% Or more activity remains; and (4) transferring one or several glucose units in α-1,4-glucan to α-1,4-glucan via α-1,4-glucosidic bond.

In a preferred embodiment, the present invention provides
α-glucanotransferase is D-glucose,
Α-1,4-using xylose, isomaltose, D-cellobiose, sucrose, or D-fucose as a receptor
Transfer one or several glucose units in the glucan.

According to the present invention, there is provided a DNA fragment encoding the 4-α-glucanotransferase.

According to the present invention, there is provided an expression vector containing the DNA fragment.

According to the present invention, there is provided a host cell transformed with the expression vector.

According to the present invention, there is provided a method for producing 4-α-glucanotransferase, comprising culturing the transformed host cell.

[0018]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.

The present invention relates to a DNA fragment encoding 4-α-glucanotransferase having excellent heat resistance, a DNA fragment encoding 4-α-glucanotransferase, an expression vector containing the DNA fragment, and a host cell transformed with the expression vector. , And a method for producing 4-α-glucanotransferase.

As used herein, "4-α-glucanotransferase" is defined as an enzyme that acts on maltodextrin and transfers its glycosyl group to another dextrin molecule via an α-1,4-glucosidic bond. Is done.

A hyperthermophilic bacterium that can be used to obtain the 4-α-glucanotransferase of the present invention is defined as a microorganism that grows at 80 ° C. or higher. Examples of hyperthermophiles include Pyrococcus, Thermotoga, Dictyoglomus, Th
genus ermus, Theromococcus, Desulfurococcus, Pyrod
Bacteria belonging to the genus ictium. A preferred hyperthermophilic bacterium is a thermostable thiol protease-producing bacterium strain KOD-1 (Appl. Enviro
n. Microbiol. 60 (12), 4559-4566 (1994)). KOD-
One strain has been deposited with the Institute of Biotechnology and Industrial Technology, National Institute of Industrial Science, and its accession number is FERM P-15007. This KOD-1 strain was initially classified as belonging to the genus Pyrococcus as described in the above-mentioned literature, but was subsequently input to DNASIS (manufactured by Hitachi Software Engineering Co., Ltd.) by accumulating knowledge. Comparison of the sequence of 16S rRNA using the registered data of GenBank (registered trademark) R91.0 October, 1995 + Daily Update shows that the KOD-1 strain is Pyrococ.
It has been suggested that it is more closely related to Thermococcus than to Cus.

A method for screening the gene for 4-α-glucanotransferase of the present invention will be described.

Almost all α-amylases are known to have a common conserved region of homology. In contrast, α-amylase derived from the hyperthermophilic bacterium Pyrococcus furiosus (Laderman KH et al., J. Bio. Chem. 268)
Vol. 32, 24402-24407, 1993) and Dictyoglomu
α-amylase derived from s thermophilus (Fukusumi S.
Et al., Eur. J. Biochem. 174, 15-21 1988) do not have their conserved regions of homology.

The present inventors have studied P-furiosus α-amylase (hereinafter abbreviated as Pf α-amylase) in order to study α-amylase which does not have these common conserved regions of homology. And the homology with D-thermophilus α-amylase were examined, and a primer was prepared based on this homology. As a result of screening genes of hyperthermophilic bacteria using DNA fragments amplified by PCR using these primers as a probe, the gene encoding Pfα-amylase homologous protein was successfully cloned. After purifying the recombinant Pf α-amylase homologous protein, the enzymatic properties were examined. As a result, the Pf α-amylase homologous protein was found to have a thermostable 4-α-glucanotransferase activity different from α-amylase. Was found to have.

In the following, D.ther
A case where a probe is prepared in consideration of homology with α-amylase of mophilum will be described, but is not limited thereto.

First, homology search of the amino acid sequence of Pf α-amylase is performed using a commercially available program, for example, DNASIS (Hitachi Software Engineering Co., Ltd.).
Homology with D-thermophilum α-amylase is found. Particularly, a region having a high homology is selected, and a primer for amplifying the region is prepared. In the case of the present invention, P.
amino acid sequence from positions 214 to 220 of furiosus (AspAspGlyG
luLysPheGly) and the DNA sequences corresponding to the amino acid sequence at positions 354 to 360 (AsnAspAlaTyrTrpHisGly) were synthesized as forward and reverse primers (mixed primer of SEQ ID NO: 3 and mixed primer of SEQ ID NO: 4), respectively. The synthesis of the primer DNA can be performed by a method known to those skilled in the art.

On the other hand, a chromosome is obtained from a
Extract the DNA. In the present invention, Appl.Environ.Microbio
Chromosomal DNA was extracted from the KOD-1 strain cultured according to the method described in l. 60 (12), 4559-4566 (1994) according to a conventional method.

PCR is performed using the above primers and chromosomal DNA. For PCR, for example, a commercially available PCR kit can be used according to the manufacturer's instructions. PCR conditions can vary depending on the characteristics of the prepared primers, but it is easy for those skilled in the art to set optimal conditions. The PCR-amplified fragment is recovered by a conventional method, for example, digoxigenin dUTP
Can be labeled by using a random prime kit using, and can be used as a labeled probe.

On the other hand, KOD-1 chromosomal DNA is cut with an appropriate restriction enzyme and an appropriate vector DNA, for example, pUC18 or
A plasmid is prepared by integrating the vector into a vector such as pUC19, and Escherichia coli is transformed to prepare a gene library. At this time, the approximate size of the restriction enzyme fragment that hybridizes with the labeled probe is determined using a standard hybridization method, and a gene library is prepared using the restriction enzyme fragment of this size, whereby the labeling is performed. A gene library enriched with restriction enzyme fragments that hybridize with the probe can be prepared.

This gene library is screened with the above labeled probe. Standard hybridization methods can be used for screening. The resulting clone is isolated. For the nucleotide sequence, for example, a Kilo-sequence deletion kit (Takara Shuzo), AutoRe
The ad Sequencing Kit (dideoxy method: manufactured by Pharmacia) or the like can be used, and the sequence can be determined using ALF DNA Sequencer II (manufactured by Pharmacia).

Whether the obtained clone produces 4-α-glucanotransferase can be determined by the following method.

First, a clone selected from the library is cultured, and the cells are disrupted according to a conventional method to obtain a supernatant.
The enzyme may be purified after heat treatment (eg, 85 ° C., 10 minutes) and ammonium sulfate precipitation, using column chromatography, eg, hydrophobic chromatography, anion exchange chromatography, etc., alone or in combination.

The reaction characteristics of the purified enzyme may be determined, for example, by converting the purified enzyme into a substrate (eg, soluble starch) in a buffer having a predetermined pH (eg, 100 mM Tris-HCl buffer (pH 7.5)).
At a predetermined temperature (for example, 90 ° C.) for a predetermined time (0,
(5, 10, 15, 30 and 60 minutes), and the enzyme activity is measured by the iodine-starch method and the Somogyi-Nelson method.

In the iodine-starch method, 15 μl of an iodine solution (4% KI, 1.25% iodine) is added to 60 μl of the reaction solution to form a color, and then 1 ml of distilled water is added, followed by addition of A _600.
The absorbance at is measured. Absorbance at reaction time 0 min is 100
Is defined as the blue value (%). Normally, the amylose contained in starch forms a spiral molecule consisting of six glucose residues in one turn, and one molecule of iodine enters the space, giving it a unique blue color (maximum absorption wavelength λmax = 650nm).
give. Since the absorption maximum wavelength of the complex of amylose and iodine shifts to the longer wavelength side as the amylose chain is longer, the change in amylose chain length of starch can be measured by measuring the absorbance.

In the Somogyi-Nelson method, copper oxide generated from Cu ²⁺ by a reducing sugar in a weak alkaline copper solution is reacted with hymolybdate in sulfuric acid, reduced to molybdenum blue, and its absorbance (500 nm) By measuring, the reducing sugar can be quantified.

The decomposition reaction of a substrate (for example, soluble starch) by α-amylase or the redistribution of substrate by 4-α-glucanotransferase, or the cyclodextrin generation reaction by cyclodextrin glucanotransferase (CGTase) is carried out by iodine. -It can be measured as a decrease in blue value by measuring the enzyme activity by the starch method. At this time, since the substrate decomposition reaction by α-amylase involves an increase in reducing sugars, the activity can also be detected by activity measurement by the Somogyi-Nelson method. However, the reaction of 4-α-glucanotransferase or CGTase is
When the activity is measured by the gyi-Nelson method, almost no increase in reducing sugar is detected.

Under the following conditions, (i) maltooligosaccharide or glucose, or (ii) soluble starch or soluble starch and glucose, the oligosaccharide formation pattern is examined by thin layer chromatography (TLC).

Substrate: (i) maltooligosaccharide or glucose (final 1.3%) or (ii) soluble starch (final 2.
0%) or soluble starch and glucose (final 40m
M) pH: 7.5 (100 mM Tris-HCl buffer) Temperature: 90 ° C. Action time: 60 minutes The reaction solution (3 μl) on which the enzyme was allowed to act under the above conditions was subjected to thin layer chromatography, for example, silica gel 60 TLC plate (Merck). And developed with, for example, isopropyl alcohol: acetone: water-4: 4: 2 at room temperature for about 240 minutes, and then a coloring solution (aniline 4 ml, diphenylamine 4 g,
200 ml of acetone, 30 ml of 85% phosphoric acid)
Heat at 05 ° C for 30 minutes to develop color.

When glucose and maltooligosaccharide of each degree of polymerization are used as a substrate, it acts on maltose to maltoheptaose and produces various maltooligosaccharides including maltooligosaccharide having a higher degree of polymerization than the oligosaccharide used for the substrate and glucose. When seen, it is shown to have maltooligosyltransferase activity. Further, when various maltooligosaccharides are produced using maltose as a substrate, it is also shown that the glucosyl group has been transferred.

Thus, the 4-α-glucanotransferase gene can be screened.

The isolated gene may be used as is or one or more of the functional equivalents may be modified so as to encode a functional equivalent modified so as not to impair the properties of 4-α-glucanotransferase. Modifications such as deletions, additions, or substitutions may be made. The 4-α-glucanotransferase of the present invention is the same as the amino acid sequence of SEQ ID NO: 2 except that the amino acid sequence of SEQ ID NO: 2 has been modified by one or more deletions, additions, or substitutions. And various functional equivalents which exhibit enzymatic activity. Therefore, the DNA sequence encoding the 4-α-glucanotransferase of the present invention is not limited to the DNA sequence of SEQ ID NO: 1,
All DNA sequences that can code for the amino acids of SEQ ID NO: 2,
And all DNA sequences that can encode a functional equivalent of the amino acid SEQ ID NO: 2.

The isolated gene can be expressed as follows. When the isolated DNA sequence encoding 4-α-glucanotransferase does not have a transcription control sequence such as an SD sequence or a promoter sequence, the DNA sequence should be introduced into a vector having an SD sequence or a promoter sequence. Can be expressed by When the isolated gene itself has a transcription regulatory sequence and a translation regulatory sequence, it can be used and expressed by inserting it into a multicopy plasmid in the form of the isolated gene as it is. The expression vector used in the present invention may be a commercially available expression vector or an expression vector derived from a commercially available expression vector,
Any expression vector that expresses 4-α-glucanotransferase in a host may be used. In order to express the isolated gene in a large amount, it is preferable that the gene be operably linked in-frame under the control of the transcription control sequence and the translation control sequence of the high expression vector. Any suitable host cell is transformed with the vector thus obtained.

As a method for producing the enzyme encoded by the isolated gene, there is a method of culturing transformed host cells under appropriate conditions and purifying the desired enzyme from the culture. . The medium used for the culture is
For example, the medium may be a medium commonly used for growing microorganisms, such as an LB medium and a 2 × YT medium, or may be a specific medium suitable for host cells. The culture can be performed continuously or batchwise. The culture conditions for culturing such a microorganism to produce the 4-α-glucanotransferase of the present invention are not particularly limited as long as the microorganism grows.

[0044] The produced protein is purified using any known method. After the culture, 4-α-glucanotransferase is recovered from the resulting culture by a conventional method. For example, the cells are separated by centrifugation or filtration of the culture, for example, after crushing the cells using an ultrasonic crusher and a French press, centrifugation is performed to obtain a supernatant. The desired enzyme protein can be precipitated by conventional means, for example, salting-out method, solvent precipitation method (for example, ethanol, acetone, etc.), or by ultrafiltration (for example, Diaflow membrane YC, manufactured by Amicon). Concentrate to obtain 4-α-glucanotransferase. In the salting-out method and the solvent precipitation method, 4-α-glucanotransferase can be precipitated, filtered, centrifuged and desalted, and then used as a lyophilized powder.
Further, 4-α-glucanotransferase obtained above is subjected to ordinary enzyme purification means such as salting out, solvent precipitation, isoelectric point precipitation, electrophoresis, ion exchange chromatography, gel filtration, affinity chromatography, and crystallization. By appropriately combining these, a crude enzyme or a purified enzyme having an improved specific activity can be obtained.

The enzymological and physicochemical properties of the enzyme found to have 4-α-glucanotransferase activity can be determined as follows.

(1) Method for measuring enzyme activity The method for measuring enzyme activity is as follows: 225 μl of a reaction mixture (3 mg maltotriose, 100 mM Tris-HCl buffer (pH
7.5), and the test enzyme solution) are incubated at 90 ° C for 10 minutes; after stopping the reaction in ice water, the amount of produced glucose is measured using a glucose sensor (manufactured by Kyoto Daiichi Kagaku). . Under these reaction conditions, the amount of enzyme that produces 1 μmol of glucose per minute is defined as 1 unit (U).

(2) Optimum pH Buffers at each pH containing a substrate (eg, maltotriose), for example, sodium acetate buffer (pH 4.0 to 5.5), ME
S-NaOH buffer (pH 5.5 to pH 7.0), Tris-HCl buffer (pH 7.
0-9.0), or glycine-NaOH buffer (pH 9.0-1.0)
4-α-glucanotransferase is added to the mixture at a predetermined temperature (for example, 90 ° C.) for a predetermined time (for example, 30 minutes).
Perform a reaction, determine the activity from the amount of reducing sugars generated,
It can be determined by examining the relative activity at each pH.

(3) Optimum temperature For example, using maltotriose as a substrate, at a predetermined pH (for example, pH 7.5) for a predetermined time (for example, 10 minutes), at each temperature (for example, 70, 80, 90, (100 and 110 ° C.), 1 U of 4-α-glucanotransferase is reacted, the activity is determined from the amount of reducing sugar generated, and the activity at the optimal temperature is reduced by 10
It can be determined by examining the relative activity at each temperature assuming 0%.

(4) Temperature stability A 4-α-glucanotransferase is added to a predetermined buffer (for example, Tris-HCl buffer (pH 7.5)), and each temperature (for example, 70, 80, 90, 100, and 110 ° C) for a predetermined time (for example, 30 minutes), and after cooling with ice, for example,
Temperature stability can be measured by measuring residual activity using maltotriose as a substrate.

(5) Estimation of Molecular Weight For example, after electrophoresis of 4-α-glucanotransferase of the present invention and a molecular weight marker protein on a SDS-polyacrylamide gel having a polyacrylamide concentration of 12% under the same conditions, By staining and comparing the mobility of 4-α-glucanotransferase and the molecular weight marker, the molecular weight of 4-α-glucanotransferase can be estimated.

As the molecular weight marker, various proteins whose molecular weights are known can be used. Such proteins include, for example, phosphorylase b
(Mw: 94,000), bovine serum albumin (Mw: 67,000), ovalbumin (Mw: 43,000), carbonic anhydrase (Mw: 30,0)
00), and trypsin inhibitor (Mw: 20,100). Protein staining can be performed, for example, using Coomassie Brilliant Blue R-250 (CBB).

[0052]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

Example 1 Cloning of Pf α-amylase homologous gene from KOD1 strain (1) Design of primers The amino acid sequence of α-amylase of the hyperthermophile P. furiosus was changed to D
A homology search was performed using the NASIS program, and it was found that D-thermophilum α-amylase had a homology of 41.9%. Therefore, in order to amplify a genomic DNA sequence encoding a portion corresponding to the amino acid sequence from position 214 to position 360 of P. furiosus, particularly in the region of high homology, the amino acid sequence from position 214 to position 220 of P. furiosus was A forward primer of SEQ ID NO: 3 was designed from the genomic DNA sequence encoding the amino acid at position (AspAspGlyGluLysPheGly), and the amino acid at positions 354 to 360 of the amino acid sequence (AsnA
A reverse primer of SEQ ID NO: 4 was designed from the genomic DNA sequence encoding spAlaTyrTrpHisGly (see FIG. 1).

(2) Preparation of Chromosomal DNA from KOD-1 Strain The KOD-1 strain used in the present invention was prepared using the method described in the aforementioned report (Appl. Environ.
Microbiol. 60 (12), 4559-4566 (1994)).
216 marine broth medium (2216 marine broth 18.7 g / L,
PIPES 3.48 g / L, CaCl ₂・ H ₂ O 0.725 g / L, 0.4 mL 0.2% resazurin, 475 mL artificial seawater (NaCl 28.16 g / L, KCl 0.7 g / L
L, MgCl ₂・ 6H ₂ O 5.5 g / L, MgSO ₄・ 7H ₂ O 6.9 g / L), distilled water 500 mL, pH 7.0, final concentration 400 μM Na ₂ S ・ 9H ₂ O, sulfur 10 g
/ L) was inoculated into 1,000 ml and cultured in a 2 liter fermenter. During culturing, the inside of the fermenter was replaced with nitrogen gas, the internal pressure was maintained at 0.1 kg / cm ² with the same gas, and the cultivation temperature was 85 ±
The cells were cultured at 1 ° C for 14 hours. The cultivation was performed by stationary cultivation, and aeration and agitation of nitrogen gas were not performed during the culture.

The cells obtained by the culture were centrifuged (7000 rp).
m, 10 minutes, 5 ° C), 1 g of which was suspended in 10 ml of A solution (50 mM Tris-HCl, 50 mM EDTA, pH 8.0) and centrifuged (8,000 rpm, 5 minutes, 4 ° C). After collection, 3ml
Was suspended in A solution containing 15% sucrose, and kept at 37 ° C. for 30 minutes, and 3 ml of A solution containing 1% N-lauryl sarcosine was added. Add 5.4 g of cesium chloride and 10 mg / m
l of ethidium bromide solution (300 μl), 55,000 rpm, 16
Ultracentrifugation was performed at 18 ° C for a time to fractionate chromosomal DNA. After removing ethidium bromide from the obtained chromosomal DNA fraction by n-butanol extraction, a TE solution (10 mM Tris-HCl,
(0.1 mM EDTA, pH 8.0) was dialyzed overnight to obtain chromosomal DNA.

(3) Preparation of Probe by PCR PCR was performed using the primer obtained in the above (1) and the chromosomal DNA obtained in the above (2). New PCR reaction solution
-Vent DNA polymerase from England BioLabs was used according to the manufacturer's instructions. The PCR conditions were 30 cycles of 94 ° C for 2 minutes, 55 ° C for 2 minutes, and 72 ° C for 1 minute. When the reaction solution after the PCR reaction was run on a 1.5% agarose gel, a PCR amplified fragment of about 500 bp was confirmed. The PCR-amplified fragment was recovered from the agarose gel using GeneClean Kit manufactured by Bio 101, and the recovered PC
The R amplified fragment was labeled using a random prime kit (Boehringer Mannheim) using digoxigenin dUTP, and used as a probe.

(4) Including Pf α-amylase homologous gene
Search for DNA fragment (i) Southern hybridization The chromosomal DNA obtained by the above (1) was converted into BamHI and HindII.
Complete digestion was performed with any of the restriction enzymes I, KpnI, PstI, SacI, or XbaI. In this DNA cleavage, a restriction enzyme was added at a ratio of 10 units to 1 μg of the DNA, and the reaction was carried out according to the manufacturer's instructions. When the chromosomal DNA cleaved with each of the above restriction enzymes was subjected to Southern hybridization using the probe obtained in (3) above, chromosome D cleaved with HindIII was obtained.
A single positive band of about 3 kbp was detected in NA.

(Ii) Library Construction and Screening The chromosomal DNA cut with HindIII was electrophoresed on a 1% agarose gel, and fragments having a size of about 2 kbp to about 4 kbp were agarose-purified using a GeneClean kit manufactured by Bio101. Recovered from gel. Vector DNA pUC18 or pUC19 was digested with HindIII. The recovered DNA fragment was mixed with either pUC18 or pUC19 digested with HindIII to a weight ratio of about 1: 1 and ligated using a DNA ligation kit (Takara Shuzo Co., Ltd.) to thereby allow recombination. A plasmid was obtained. The ligation method followed the instruction manual of the ligation kit.

Next, the above recombinant plasmid was added to Escherichia
The cells were added to competent cells of E. coli JM109 strain, left on ice for 30 minutes, and kept at 42 ° C for 2 minutes. Then this 1.
5 ml of a 1% tryptone, 0.5% yeast extract, 0.5% NaCl medium (hereinafter referred to as “LB medium”) was inoculated at 37 ° C.
For 1 hour. This culture was treated with 100 μg / ml ampicillin, 200 μg / ml 5-bromo-4-chloro-3-indolyl-β-D
-Galactopyranoside (X-gal), 24 μg / ml isopropyl
Escherichia coli that forms ampicillin-resistant and white colonies by plating on LB agar medium containing -β-D-thiogalactopyranoside (IPTG) and culturing at 37 ° C overnight
About 1300 transformants were obtained.

With respect to the obtained transformant, the above (1)
Colony hybridization was performed using the probe obtained in the above. As a result, 17 positive clones were obtained.

(5) Confirmation of Cloned DNA Fragment Plasmid DNA was extracted from the positive clone obtained in (4) by the following method. That is, the transformed strain was inoculated into 500 ml of LB medium containing 100 μg / ml ampicillin,
After shaking culture at 37 ° C. overnight, the cells were collected by centrifugation. Bacteria cells were buffered (50 mM glucose, 25 mM Tris-HCl, 10 mM EDT
A, pH 8.0), add 2 ml of a 50 mg / ml lysozyme solution, place on ice for 5 minutes, and then add SDS solution (0.2
(N NaOH, 1% SDS) was added and placed on ice for 10 minutes.
Further, 15 ml of 3 M potassium acetate (pH 4.8) is added, and 30 ml of ice is added.
Each minute, the supernatant was obtained by centrifugation, and the plasmid DNA was recovered by phenol extraction and ethanol precipitation. The recovered plasmid DNA was dissolved in 8 ml of a TE solution, RNaseA was added thereto at a concentration of 20 μg / ml, the mixture was incubated at 37 ° C. for 1 hour, and the plasmid DNA was recovered again by phenol extraction and ethanol precipitation. Dissolved in the solution. The obtained plasmid DNA was digested with Hin under the conditions described in (2).
cut with dIII, perform 1% agarose gel electrophoresis,
When the size of the inserted DNA fragment on the plasmid DNA was measured using λ / HindIII digest-φX174 / HaeIII digest (manufactured by Toyobo) as a size marker, the insertion of the same approximately 3.1 kbp HindIII fragment in all 17 positive clones was performed. Was confirmed.

(6) Determination of Base Sequence of Cloned DNA Fragment To determine the base sequence of the cloned DNA fragment, a plasmid for base sequence determination was constructed by the stepwise deletion method, and the base sequence was determined by dideoxy. Performed by the method.
For the stepwise deletion method, use a deletion kit for kilosequencing (Takara Shuzo). For the dideoxy method, use AutoRead.
ALFD using Sequencing Kit (Pharmacia)
The nucleotide sequence was determined by NA Sequencer II (Pharmacia). In addition, the method followed each instruction manual.

As a result, the base sequence of the inserted DNA fragment was as shown in SEQ ID NO: 1 in the sequence listing. Also obtained DN
The 1973 bp open reading frame found in the A fragment contained the amino acid sequence (653 amino acid residues, calculated molecular weight 76,693) shown in SEQ ID NO: 2 in the sequence listing. The homology search of this amino acid sequence was performed using DNASIS software and Genebank DNA database. Pyrococcus furiosus and Dictyoglomus thermop
Only the α-amylase of hilum has significant homology (6
8.3% and 36.0%) (Fig. 1). This confirmed that the target Pfα-amylase homologous gene was isolated.

Example 2 Construction of Pfα-amylase Homologous Gene Expression Plasmid In order to overexpress Pfα-amylase homologous protein, lac of pUC19 plasmid was used.
Hin immediately downstream of the Shine-Dalgarno (SD) sequence of the Z gene
The dIII site was designed. The primer of SEQ ID NO: 5 (corresponding to positions 454 to 435) was designed to include the original HindIII site of pUC19, and the primer of SEQ ID NO: 6 (including the HindIII site immediately downstream of the SD sequence and the SD sequence) 476th to 495th). PCR was performed using the primers of SEQ ID NO: 5 and SEQ ID NO: 6 and pUC19, and the amplification product was digested with HindIII. The obtained digest was ligated to obtain pUC19-Δ25 in which 25 nucleotides at positions 451 to 475 of the pUC19 plasmid had been deleted.

The plasmids obtained in pUC19-Δ25 and Example 1 (5) were digested with HindIII. The Pfα-amylase homologous gene was recovered from the digested plasmid according to the above (4) (ii). HindIII of pUC19-Δ25
The digest and the Pfα-amylase homologous gene were mixed at a ratio of about 1: 5, and both were ligated using a DNA ligation kit (Takara Shuzo). In this way, the Pf α-amylase homologous gene is replaced with pUC19-Δ25
A pUKA plasmid ligated in the same direction as the acZ gene was obtained.

Using the pUKA plasmid, E. coli JM109 strain was transformed and positive clones were selected in accordance with Example 1. The positive clone obtained in the main culture was cryopreserved for preparing a Pfα-amylase homologous protein in Example 3 below.

Example 3 Purification of Pf α-Amylase Homologous Protein Produced by Hyperthermophilic Bacteria The positive clone obtained in Example 2 was supplemented with L to which 100 μg / ml ampicillin was added.
Shaking culture was carried out at 37 ° C. overnight in a B medium. Using this culture solution, shaking culture was further performed at 37 ° C. in an LB medium supplemented with 100 μg / ml ampicillin until the absorbance at 660 nm became 0.4. Next, IPTG was added to a final concentration of 1 mM, and shaking culture was further performed at 37 ° C. for 16 hours. After completion of the culture, the culture solution (about 1,000 ml) was centrifuged at 8,000 g for 10 minutes, and the precipitate was washed with 100 mM Tris-HCl buffer (pH
Washing and resuspension were performed at 8.0). The suspension was sonicated and then centrifuged at 15,000 g for 30 minutes to remove the supernatant, which was used as a crude extract. The crude extract was heat-treated at 85 ° C. for 10 minutes and centrifuged again at 15,000 g for 30 minutes to remove denatured proteins. The supernatant is used to form an 80% saturated ammonium sulfate solution, kept at 4 ° C. overnight, and then 27,000
A precipitate was obtained by centrifugation at g for 30 minutes. 25 mM Tris-HCl containing 10% saturated ammonium sulfate
Phenyl S dissolved in buffer (pH 8.0) and equilibrated with 25 mM Tris-HCl buffer (pH 8.0) in 10% saturated ammonium sulfate
It was passed through an FPLC system (Pharmacia) packed with uperose HR 5/5 (Pharmacia) and eluted with a linear gradient (10% to 0%) of ammonium sulfate at a flow rate of 0.3 ml / min. PORs obtained by pooling the eluted fractions having a protein with a molecular weight of about 77,000 and equilibrating with 100 mM Tris-HCl buffer (pH 8.0)
OS HQ columns (PerSeptive Biosystems, Framingham, M
A), and the column is subjected to 100 mM Tris-HCl buffer (pH 8.0)
And a linear gradient of NaCl at a flow rate of 1 ml / min (0
(0.5 M), it was possible to purify a single band electrophoretically (FIG. 2).

Example 4 Characterization of Pf α-Amylase Homologous Protein Produced by Hyperthermophilic Bacteria Using the obtained purified enzyme, the enzymatic properties and physical chemistry of the Pf α-amylase homologous protein of the present invention were obtained. Properties were examined as follows.

(1) Measurement of enzymatic reaction characteristics Purified Pf α-amylase homologous protein was added to 100 mM Tris-HCl
Soluble starch in buffer (pH 7.5) at 90 ° C with 0,
The enzyme was allowed to act for 5, 10, 15, 30, and 60 minutes, and the enzyme activity was measured by the iodine-starch method and the Somogyi-Nelson method (FIG. 3). For reference, the same enzyme activity was measured for α-amylase of B. subtilis (manufactured by Nagase Seikagaku Corporation) and cyclodextrin glucanotransferase (CGTase) of B. macerans (manufactured by Amano Pharmaceutical Co., Ltd.). .
Iodine - The Starch method, the reaction solution 15μl iodine solution (4% KI, 1.25% iodine) in 60μl is added for color development, then distilled water was added 1 ml, to measure the absorbance at A _600. The ratio of the absorbance when the absorbance at the reaction time of 0 minute is defined as 100 is defined as the blue value (%). In FIG. 3, ○ indicates Pf
Blue value of α-amylase homologous protein, ● indicates generation of reducing sugar by α-amylase homologous protein, Δ indicates B. subtilis
Indicates the blue value of α-amylase of B. subtilis, the production of reducing sugar by α-amylase of B. subtilis, □ indicates the blue value of CGTase of B. macerans, and the production of reducing sugar by CGTase of B. macerans . For the Pf α-amylase homologous protein, a decrease in blue value (starch degradation) was confirmed, but only a trace amount of reducing sugar was detected.

Next, the purified Pf α-amylase homologous protein is allowed to act on (i) maltooligosaccharide or glucose, or (ii) soluble starch or soluble starch and glucose under the following conditions, and the oligosaccharide production pattern is determined by thin-layer chromatography. Investigation was performed by lithography (TLC).

Substrate: (i) maltooligosaccharide or glucose (1.3% final) or (ii) soluble starch (2.
0%), or soluble starch and glucose (final 40
mM) pH: 7.5 (100 mM Tris-HCl buffer) Temperature: 90 ° C Action time: 60 minutes Enzyme addition amount: (i) 0.15 unit, (ii) 0.05 unit The Pf α-amylase homologous protein was allowed to act under the above conditions. The reaction solution (3 μl) was spotted on a silica gel 60 TLC plate (manufactured by Merck), developed with isopropyl alcohol: acetone: water-4: 4: 2 at room temperature for about 240 minutes, and then a color developing solution (aniline 4 ml, diphenylamine 4 g, acetone) 200m
1, a mixture of 85 ml of 85% phosphoric acid) and heated at 105 ° C. for 30 minutes to develop color.

Next, the reaction solution of the Pf α-amylase homologous protein was subjected to thin layer chromatography (FIG. 4).
In FIG. 4, (+) and (−) represent: addition of enzyme and addition of no enzyme. (+ G1) is glucose added,
(-G1) indicates that glucose was not added. Also, G1
G7 represents G1: glucose, G2: maltose, G3: maltotriose, G4: maltotetraose, G5: maltopentaose, G6: maltohexaose, and G7: maltoheptaose, respectively. In lane M, a mixture of G1 to G7, which are standard maltooligosaccharides, was passed. As a result, the production of low-molecular oligosaccharides and cyclodextrins was not confirmed from the soluble starch treated with the Pf α-amylase homologous protein (FIG. 4B). When a product obtained by using glucose and maltooligosaccharides of each degree of polymerization as a substrate is examined by thin-layer chromatography, maltooligosaccharides that act on maltoheptaose from maltose and have a higher degree of polymerization than the oligosaccharide used for the substrate are obtained. The production of various maltooligosaccharides and glucose was observed (FIG. 4A).

The results of this experiment showed that the enzyme has maltooligosyl transfer activity. Furthermore, the results of producing various maltooligosaccharides using maltose as a substrate indicate that the present enzyme can transfer glucosyl groups. To determine if glucose is an acceptor substrate,
When the present enzyme was allowed to act in the coexistence of soluble starch and glucose, various maltooligosaccharides were produced that were not observed when soluble starch was used alone (FIG. 4B). This indicated that glucose could act as an acceptor substrate.

As a result, the enzyme of the present invention has the activity of transferring maltooligosaccharide or maltosyl group or glucosyl group from malto-oligosaccharides of maltose or higher and soluble starch to various maltooligosaccharides containing glucose as donor substrates.
-Glucanotransferase.
The purified enzyme sample prepared in Example 3 had a 4-α-glucanotransferase activity of 16.5 U / mg, and a total activity of 31.2 U.

The gene encoding this enzyme was isolated based on the α-amylase gene of hyperthermophilic bacteria.
The obtained gene encoded a protein having a 4-α-glucanotransferase activity different from α-amylase.

(2) Examination of sugars that can serve as receptors The sugars that can serve as receptors for the 4-α-glucanotransferase of the present invention were examined. Soluble starch (final 2.0
%) And D-xylose, isomaltose, D-cellobiose, sucrose, and D-fucose as receptors were added to a final concentration of 40 mM. The reaction conditions are as follows. pH: 7.5 (100 mM Tris-HCl buffer) Temperature: 90 ° C. Action time: 60 minutes Enzyme addition amount: 0.05 unit After the reaction was completed, the reaction product was subjected to thin-layer chromatography (FIG. 5). In FIG. 5, (+) and (-)
These represent addition and no addition of the enzyme, respectively. Lanes 1 to 6 show the results obtained by sequentially adding no receptor, D-xylose, isomaltose, D-cellobiose, sucrose, and D-fucose. Lane M is a mixture of G1 to G7, which are standard maltooligosaccharides, as markers. As shown in FIG. 5, compared to the case where the receptor was not added (lane 1), in all cases where the above receptor was added, the formation of sugars of various degrees of polymerization was observed. Therefore, the 4-α-glucanotransferase of the present invention can be obtained from these D-xylose, isomaltose,
-It has been found that cellobiose, sucrose and D-fucose can be receptors. The generated oligosaccharides are important for studying the bioactive functions of these oligosaccharides.

(3) Optimum pH 150 μl of 100 mM sodium acetate buffer (pH 4.0 to 5.5) containing 1% maltotriose at each pH, 100 mM MES-NaOH buffer (pH 5.5 to pH 7.0), 100 mM Tris-HCl buffer (pH 7.0 ~
9.0) or 100 mM glycine-NaOH buffer (pH 9.0-1
0.0) was added with 1 U of 4-α-glucanotransferase, and the mixture was reacted at 90 ° C. for 30 minutes. 10 activities at optimal pH
FIG. 6 shows the relative activity at each pH when the concentration was set to 0%.

As is clear from FIG. 6, the 4-
The optimal pH of α-glucanotransferase is around 6.0-8.0.

(4) Optimum temperature Using maltotriose as a substrate, 1 U of 4-α-glucanotransferase was reacted at pH 7.5 for 10 minutes at each temperature to make the activity at the optimum temperature 100%. The relative activity at each temperature is shown in FIG. 7 (A).

As is clear from this figure, the 4-α-
The optimal temperature of glucanotransferase is around 100 ° C.

(5) Temperature stability 1 U of 4-α-glucanotransferase was added to 100 mM Tris-HCl buffer (pH 7.5), heat-treated at each temperature for 30 minutes, ice-cooled, and then maltotriose. Was used as a substrate to measure residual activity. FIG. 7B shows the result.

As is clear from this figure, the 4-α-
Glucanotransferase remained at least 90% or more in activity even after heat treatment at 100 ° C. and pH 7.5 for 30 minutes.

(6) SDS-polyacrylamide gel electrophoresis The molecular weight of the 4-α-glucanotransferase of the present invention was estimated by SDS-polyacrylamide gel electrophoresis at a polyacrylamide concentration of 12% (FIG. 2). In FIG. 2, Lane 1 is the extract before the crude purification, Lane 2 is the extract after the crude purification by heat treatment at 85 ° C. for 10 minutes, and Lane 3 is the Phenyl
The active fraction after Superose purification, lane 4 shows the active fraction after POROS HQ purification, and lane 5 shows the molecular weight marker, and the molecular weight is indicated by KDa on the right. As standard proteins, phosphorylase b (Mw: 94,000), bovine serum albumin (Mw: 67,000), ovalbumin (Mw: 4)
3,000), carbonic anhydrase (Mw: 30,000), and trypsin inhibitor (Mw: 20,100), electrophoresed under the same conditions as the 4-α-glucanotransferase of the present invention, and stained with Coomassie brilliant blue R-250 (CBB). And used as a molecular weight marker. As a result, a band of 4-α-glucanotransferase of the present invention was observed at around 77,000.

As described above, the 4-α-glucanotransferase of the present invention exhibited excellent glycosyltransferase activity under high temperature conditions, and exhibited high stability under the same conditions. Therefore, it was revealed that the 4-α-glucanotransferase of the present invention is a 4-α-glucanotransferase having excellent heat resistance and having glycosyl transfer ability under high temperature conditions.

[0085]

As described in detail above, 4-α-glucanotransferase exhibiting excellent glycosyltransferase activity under high temperature conditions has been obtained. The present 4-α-glucanotransferase is an excellent thermostable 4-α-glucanotransferase having a glycosyl transfer ability under high temperature conditions, and an enzyme useful for industrially producing various oligosaccharides or cyclic amylose. It is.

In addition, a DNA fragment containing the 4-α-glucanotransferase producing gene of the present invention was obtained. This D
By using the NA fragment, it is possible to breed a microorganism capable of high production of 4-α-glucanotransferase.
The way to efficiently produce α-glucanotransferase has been opened. Furthermore, this DNA fragment was synthesized by a site-directed mutagenesis method or the like to increase the stability of 4-α-
It can also be a material for creating glucanotransferase.

[0087]

[Sequence list]

[0088]

[SEQ ID NO: 1] Sequence length: 2154 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: genomic DNA Origin Organism: Pyrococcus (Pyrococcus sp.) Strain: KOD-1 Characteristic of sequence Symbol for characteristic: CDS Location: 14..1972 Method for determining characteristic: S sequence AAGCTTGTCG GTG ATG GAG ATG GTG AAC TTC ATA TTT GGT ATC CAC AAC 49 Met Glu Met Val Asn Phe Ile Phe Gly Ile His Asn 1 5 10 CAT CAG CCC CTT GGA AAC TTT GGA TGG GTT ATG GAG AGC GCC TAC GAG 97 His Gln Pro Leu Gly Asn Phe Gly Trp Val Met Glu Ser Ala Tyr Glu 15 20 25 AGG TCC TAT AGG CCC TTC ATG GAG ACC CTT GAG GAG TAC CCT AAC ATG 145 Arg Ser Tyr Arg Pro Phe Met Glu Thr Leu Glu Glu Tyr Pro Asn Met 30 35 40 AAG GTG GCC GTT CAT TAC TCC GGC CCG CTC CTT GAG TGG ATA CGC GAC 193 Lys Val Ala Val His Tyr Ser Gly Pro Leu Leu Glu Trp Ile Arg Asp 45 50 55 60 AAC AAG CCA GAA CAC CTC GAT TTG CTC CGC TCG CTC GTC AAG AGG GGA 241 Asn Lys Pro Glu His Leu Asp Leu Leu Arg Ser Leu Val Lys Arg G ly 65 70 75 CAG CTT GAG ATA GTG GTG GCT GGC TTC TAC GAG CCC GTT CTT GCC TCG 289 Gln Leu Glu Ile Val Val Ala Gly Phe Tyr Glu Pro Val Leu Ala Ser 80 85 90 ATT CCG AAG GAA GAT AGG ATA GTC CAG ATC GAG AAG CTC AAG GAG TTC 337 Ile Pro Lys Glu Asp Arg Ile Val Gln Ile Glu Lys Leu Lys Glu Phe 95 100 105 GCC AGG AAT CTC GGC TAC GAA GCG AGG GGC GTC TGG CTC ACT GAG AGG 385 Ala Arg Asn Leu Gly Tyr Glu Ala Arg Gly Val Trp Leu Thr Glu Arg 110 115 120 GTC TGG CAG CCG GAG CTC GTG AAA AGC CTC CGC GCT GCT GGA ATA GAT 433 Val Trp Gln Pro Glu Leu Val Lys Ser Leu Arg Ala Ala Gly Ile Asp 125 130 135 140 TAC GTC ATA GTT GAC GAC TAC CAC TTC ATG AGC GCG GGC CTC TCA AAG 481 Tyr Val Ile Val Asp Asp Tyr His Phe Met Ser Ala Gly Leu Ser Lys 145 150 155 GAT GAG CTG TTC TGG CCG TAC TAC ACC GAG GAT GGC GGT GAA GTC ATA 529 Asp Glu Leu Phe Trp Pro Tyr Tyr Thr Glu Asp Gly Gly Glu Val Ile 160 165 170 ACG GTT TTC CCG ATA GAC GAG AAG CTC AGG TAT TTG ATC CCC TTC CGC 577 Thr Val Phe Pro Ile Asp Glu Lys Leu Arg Tyr Leu Ile Pro Phe Arg 175 180 185 CCC GTT GAT AAG ACC CTC GAA TAC CTA CAC TCC CTC GAT GAT GGT GAT 625 Pro Val Asp Lys Thr Leu Glu Tyr Leu His Ser Leu Asp Asp Gly Asp 190 195 200 GAG AGC AAG GTT GCG GTC TTC CAC GAT GAC GGT GAG AAG TTC GGG GTC 673 Glu Ser Lys Val Ala Val Phe His Asp Asp Gly Glu Lys Phe Gly Val 205 210 215 220 TGG CCC GGA ACC TAC GAG TGG GTC TAC GAG AAG GGC TGG CTC AGG GAG 721 Trp Pro Gly Thr Tyr Glu Trp Val Tyr Glu Lys Gly Trp Leu Arg Glu 225 230 235 TTC TTC GAT AGG GTT TCA AGC GAC GAG AGG ATA AAC CTC ATG CTC TAC 769 Phe Phe Asp Arg Val Ser Ser Asp Glu Arg Ile Asn Leu Met Leu Tyr 240 245 250 TCT GAA TAC CTC CAG AGG TTC AGG CCG CGC GGT CTG GTT TAC CTC CCG 817 Ser Glu Tyr Leu Gln Arg Phe Arg Pro Arg Gly Leu Val Tyr Leu Pro 255 260 265 ATA GCT TCA TAC TTC GAG ATG AGC GAG TGG AGC CTC CCG GCG AGG CAG 865 Ile Ala Ser Tyr Phe Glu Met Ser Glu Trp Ser Leu Pro Ala Arg Gln 270 275 280 GCC AAG CTC TTC GTG GAG TTC GTC GAA GAG CTA AAG AAA GAG AAC AAG 913 Ala Lys Leu Phe Val Glu Phe Val Glu Glu Leu Lys Lys Glu Asn Lys 285 290 295 300 300 TTC GAC CGC TAT CGC GTC TTC GTC CGC GGC GGA ATC TGG AAG AAC TTC 961 Phe Asp Arg Tyr Arg Val Phe Val Arg Gly Gly Ile Trp Lys Asn Phe 305 310 315 TTC TTC AAG TAC CCG GAG AGC AAC TAC ATG CAC AAG CGC ATG CTG ATG 1009 Phe Phe Lys Tyr Pro Glu Ser Asn Tyr Met His Lys Arg Met Leu Met 320 325 330 GTG AGT AAG GCC GTG AGA AAC AAC CCC GAG GCG AGG GAA TTC ATC CTC 1057 Val Ser Lys Ala Val Arg Asn Asn Pro Glu Ala Arg Glu Phe Ile Leu 335 340 345 AGG GCC CAG TGC AAC GAC GCC TAC TGG CAC GGC GTC TTC GGC GGT GTC 1105 Arg Ala Gln Cys Asn Asp Ala Tyr Trp His Gly Val Phe Gly Gly Val 350 355 360 TAC CTC CCG CAC CTC CGC AGG GCT GTC TGG GAG AAC ATA ATA AAG GCT 1153 Tyr Leu Pro His Leu Arg Arg Ala Val Trp Glu Asn Ile Ile Lys Ala 365 370 375 380 380 CAG AGC CAC GTT AAG ACG GGG AAT TTC GTG AGG GAC ATC GAC TTC GAC 1201 Gln Ser His Val Lys Thr Gly Asn Phe Val Arg Asp Ile Asp Phe Asp 385 390 395 GGC AGG GAC GAG GTC TTC ATC GAG AAC GAG AAC TTC TAC GCC GTC TTC 1249 Gly Arg Asp Glu Val Ph e Ile Glu Asn Glu Asn Phe Tyr Ala Val Phe 400 405 410 AAA CCA GCA TAC GGC GGA GCC CTC TTC GAG CTC TCC TCC AAG AGA AAG 1297 Lys Pro Ala Tyr Gly Gly Ala Leu Phe Glu Leu Ser Ser Lys Arg Lys 415 420 425 GCG GTC AAC TAC AAT GAC GTT CTC GCG AGG CGC TGG GAG CAC TAC CAC 1345 Ala Val Asn Tyr Asn Asp Val Leu Ala Arg Arg Trp Glu His Tyr His 430 435 440 GAG GTT CCA GAG GCC GCT ACA CCG GAA GAA GGT GGT GAA GGA GTC GCC 1393 Glu Val Pro Glu Ala Ala Thr Pro Glu Glu Gly Gly Glu Gly Val Ala 445 450 455 460 AGC ATC CAC GAG CTT GGA AAG CAG ATT CCC GAC GAG ATA AGG CGT GAG 1441 Ser Ile His Glu Leu Gly Lys Gln Ile Pro Asp Glu Ile Arg Arg Glu 465 470 475 CTT GCC TAC GAC AGC CAT CTA AGG GCA ATT CTG CAG GAC CAC TTC CTT 1489 Leu Ala Tyr Asp Ser His Leu Arg Ala Ile Leu Gln Asp His Phe Leu 480 485 490 490 GAG CCA GAG ACG ACG CTC GAT GAG TAC AGG CTG AGC CGC TAC ATC GAG 1537 Glu Pro Glu Thr Thr Leu Asp Glu Tyr Arg Leu Ser Arg Tyr Ile Glu 495 500 505 CTT GGG GAC TTC CTC ACC GGT GCT TAC AAC TTC AGC CTT ATT GAG AAC 1585 Leu Gly Asp Phe Leu Thr Gly Ala Tyr Asn Phe Ser Leu Ile Glu Asn 510 515 520 GGA ATA ACC CTT GAG CGC GAT GGA AGC GTT GCT AAG AGG CCA GCA CGT 1633 Gly Ile Thr Leu Glu Arg Asp Gly Ser Val Ala Lys Arg Pro Ala Arg 525 530 535 540 GTG GAA AAG TCG GTT AGG CTC ACC GAG GAC GGT TTC ATA GTG GAC TAC 1681 Val Glu Lys Ser Val Arg Leu Thr Glu Asp Gly Phe Ile Val Asp Tyr 545 550 555 ACC GTC AGA AGC GAT GCC AGA GCC CTC TTC GGC GTC GAG CTC AAC CTG 1729 Thr Val Arg Ser Asp Ala Arg Ala Leu Phe Gly Val Glu Leu Asn Leu 560 565 570 GCA GTC CAC AGC GTC ATG GAG GAG CCG GCC GAG TTC GAG GCA GAG AAG 1777 Ala Val His Ser Val Met Glu Glu Pro Ala Glu Phe Glu Ala Glu Lys 575 580 585 TTC GAG GTG AAC GAC CCC TAC GGC ATC GGA AAG GTC GAG ATA GAG CTC 1825 Phe Glu Val Asn Asp Pro Tyr Gly Ile Gly Lys Val Glu Ile Glu Leu 590 595 600 GAC AGA AGG GCA AAA GTC TGG AAG TAC CCA ATA AAG ACC CTC AGC CAG 1873 Asp Arg Arg Ala Lys Val Trp Lys Tyr Pro Ile Lys Thr Leu Ser Gln 605 610 615 620 620 AGT GAG AGT GGG TGG GAC TTC ATC CAG CAG GGC GTT A GC TAC ACC GTT 1921 Ser Glu Ser Gly Trp Asp Phe Ile Gln Gln Gly Val Ser Tyr Thr Val 625 630 635 CTA TTC CCT GTC GAG GGA GAA CTC AGG TTC AGA CTC CGC TTC AGG GAG 1969 Leu Phe Pro Val Glu Gly Glu Leu Arg Phe Arg Leu Arg Phe Arg Glu 640 645 650 CTC TGAACTTTAT TCTCTCCTTT GCCTTTTCCG CCTTCTGCTT CCCTTTCAGT 2022 Leu * GCTATCATAA ATGTTAGAGC CTCAACACTG TCTTCAAGGA GCAGACCCCT GTTGTCCTTC
2082 GCTATCAAGT ATCCAAATAC CAGCCCGAAA ATCGCCCCGG CGACCCAAAG CTCCAGGAGG 2142 AACTTTCCTA CG 2154

[0089]

[SEQ ID NO: 2] Sequence length: 653 Sequence type: amino acid Topology: linear Sequence type: protein Origin Organism name: Pyrococcus (Pyrococcus sp.) Strain name: KOD-1 sequence Met Glu Met Val Asn Phe Ile Phe Gly Ile His Asn His Gln Pro Leu 1 5 10 15 Gly Asn Phe Gly Trp Val Met Glu Ser Ala Tyr Glu Arg Ser Tyr Arg 20 25 30 Pro Phe Met Glu Thr Leu Glu Glu Tyr Pro Asn Met Lys Val Ala Val 35 40 45 His Tyr Ser Gly Pro Leu Leu Glu Trp Ile Arg Asp Asn Lys Pro Glu 50 55 60 His Leu Asp Leu Leu Arg Ser Leu Val Lys Arg Gly Gln Leu Glu Ile 65 70 75 80 Val Val Ala Gly Phe Tyr Glu Pro Val Leu Ala Ser Ile Pro Lys Glu 85 90 95 Asp Arg Ile Val Gln Ile Glu Lys Leu Lys Glu Phe Ala Arg Asn Leu 100 105 110 Gly Tyr Glu Ala Arg Gly Val Trp Leu Thr Glu Arg Val Trp Gln Pro 115 120 125 Glu Leu Val Lys Ser Leu Arg Ala Ala Gly Ile Asp Tyr Val Ile Val 130 135 140 Asp Asp Tyr His Phe Met Ser Ala Gly Leu Ser Lys Asp Glu Leu Phe 145 150 155 160 Trp Pro Tyr Tyr Thr Glu Asp Gly Gly Glu Val Il e Thr Val Phe Pro 165 170 175 Ile Asp Glu Lys Leu Arg Tyr Leu Ile Pro Phe Arg Pro Val Asp Lys 180 185 190 Thr Leu Glu Tyr Leu His Ser Leu Asp Asp Gly Asp Glu Ser Lys Val 195 200 205 Ala Val Phe His Asp Asp Gly Glu Lys Phe Gly Val Trp Pro Gly Thr 210 215 220 Tyr Glu Trp Val Tyr Glu Lys Gly Trp Leu Arg Glu Phe Phe Asp Arg 225 230 235 240 Val Ser Ser Asp Glu Arg Ile Asn Leu Met Leu Tyr Ser Glu Tyr Leu 245 250 255 Gln Arg Phe Arg Pro Arg Gly Leu Val Tyr Leu Pro Ile Ala Ser Tyr 260 265 270 Phe Glu Met Ser Glu Trp Ser Leu Pro Ala Arg Gln Ala Lys Leu Phe 275 280 285 Val Glu Phe Val Glu Glu Leu Lys Lys Glu Asn Lys Phe Asp Arg Tyr 290 295 300 Arg Val Phe Val Arg Gly Gly Ile Trp Lys Asn Phe Phe Phe Lys Tyr 305 310 315 320 Pro Glu Ser Asn Tyr Met His Lys Arg Met Leu Met Val Ser Lys Ala 325 330 335 Val Arg Asn Asn Pro Glu Ala Arg Glu Phe Ile Leu Arg Ala Gln Cys 340 345 350 Asn Asp Ala Tyr Trp His Gly Val Phe Gly Gly Val Tyr Leu Pro His 355 360 365 Leu Arg Arg Ala Val Trp Glu Asn Ile Ile Lys Ala Gl n Ser His Val 370 375 380 Lys Thr Gly Asn Phe Val Arg Asp Ile Asp Phe Asp Gly Arg Asp Glu 385 390 395 400 Val Phe Ile Glu Asn Glu Asn Phe Tyr Ala Val Phe Lys Pro Ala Tyr 405 410 415 Gly Gly Ala Leu Phe Glu Leu Ser Ser Lys Arg Lys Ala Val Asn Tyr 420 425 430 Asn Asp Val Leu Ala Arg Arg Trp Glu His Tyr His Glu Val Pro Glu 4435 440 445 Ala Ala Thr Pro Glu Glu Gly Gly Glu Gly Val Ala Ser Ile His Glu 450 455 460 Leu Gly Lys Gln Ile Pro Asp Glu Ile Arg Arg Glu Leu Ala Tyr Asp 465 470 475 480 Ser His Leu Arg Ala Ile Leu Gln Asp His Phe Leu Glu Pro Glu Thr 485 490 495 495 Thr Leu Asp Glu Tyr Arg Leu Ser Arg Tyr Ile Glu Leu Gly Asp Phe 500 505 510 Leu Thr Gly Ala Tyr Asn Phe Ser Leu Ile Glu Asn Gly Ile Thr Leu 515 520 525 Glu Arg Asp Gly Ser Val Ala Lys Arg Pro Ala Arg Val Glu Lys Ser 530 535 540 540 Val Arg Leu Thr Glu Asp Gly Phe Ile Val Asp Tyr Thr Val Arg Ser 545 550 555 560 Asp Ala Arg Ala Leu Phe Gly Val Glu Leu Asn Leu Ala Val His Ser 565 570 575 Val Met Glu Glu Pro Ala Glu Phe Glu Ala Glu Lys P he Glu Val Asn 580 585 590 Asp Pro Tyr Gly Ile Gly Lys Val Glu Ile Glu Leu Asp Arg Arg Ala 595 600 605 Lys Val Trp Lys Tyr Pro Ile Lys Thr Leu Ser Gln Ser Glu Ser Gly 610 615 620 Trp Asp Phe Ile Gln Gln Gly Val Ser Tyr Thr Val Leu Phe Pro Val 625 630 635 640 Glu Gly Glu Leu Arg Phe Arg Leu Arg Phe Arg Glu Leu * 645 650

[0090]

[SEQ ID NO: 3] Sequence length: 20 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence GAYGAYGGNG ARAARTTYGG 20

[0091]

[SEQ ID NO: 4] Sequence length: 20 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence CCRTGCCART ANGCRTCRTT 20

[0092]

[SEQ ID NO: 5] Sequence length: 20 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence CCAAGCTTGC ATGCCTGCAG 20

[0093]

[SEQ ID NO: 6] Sequence length: 26 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence CGAAGCTTCC TGTGTGAAAT TGTTAT 2
6

[Brief description of the drawings]

FIG. 1: Amino acid sequence of 4-α-glucanotransferase, α-amylase of P. furiosus and D. thermophi
FIG. 7 is a diagram comparing the amino acid sequences of α-amylase of lum.

FIG. 2 is a photograph of electrophoresis showing purification of 4-α-glucanotransferase.

FIG. 3. 4-α-glucanotransferase, B. subtil
1 is a graph showing the blue value and reducing power when α-amylase of is and CGTase of B. macerans were reacted with soluble starch for various times.

FIG. 4 is a photograph showing thin-layer chromatography of a product obtained by reacting various substrates with 4-α-glucanotransferase.

FIG. 5 is a photograph showing thin-layer chromatography of a reaction product obtained by reacting starch with various sugars (acceptors) in the presence of 4-α-glucanotransferase.

FIG. 6 is a graph showing the relative activity of 4-α-glucanotransferase at each pH.

FIG. 7 (A) is a graph showing the relative activity at each temperature at pH 7.5. (B) is a graph showing the residual activity after being kept at each temperature for 30 minutes.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12R 1:01) (C12N 1/21 C12R 1:19) (C12N 9/10 C12R 1:19) (72) Inventor Yuji Suzuki 52, Nagase, Fukuchiyama Plant, Fukuchiyama City, Kyoto Prefecture, Japan Fukuchiyama Plant (72) Inventor Iwao Kojima, 52, Nagase, Fukuchiyama City, Fukuchiyama City, Kyoto Prefecture Fukuchiyama Plant, 72 (72) Inventor, Uzu Kensaku Lu 52-1, Nagatano-cho, Fukuchiyama-shi, Kyoto

Claims (10)

[Claims] 1. An α-glucanotransferase having the following physicochemical properties: (1) the optimum pH is around 6.0 to 8.0; (2) the optimum temperature at pH 7.5 is around 100 ° C. (3) after heat treatment at pH 7.5, 100 ° C. for 30 minutes, at least 90%;
% Or more activity remains; and (4) transferring one or several glucose units in α-1,4-glucan to α-1,4-glucan via α-1,4-glucosidic bond. 2. D-glucose, D-xylose, isomaltose, D-cellobiose, sucrose or D-glucose
The 4-α-glucanotransferase according to claim 1, which transfers one or several glucose units in α-1,4-glucan using fucose as an acceptor. 3. The 4-α-glucanotransferase according to claim 1, which is obtained from a hyperthermophilic strain KOD-1. 4. It has an amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence containing one or more deletions, additions, or substitutions of amino acids in the sequence, and has 4-α-glucanotransferase activity. 4-α-glucanotransferase. 5. An amino acid sequence having the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence containing one or more deletions, additions, or substitutions of amino acids in the sequence, and having the following physicochemical properties: α-glucanotransferase: (1) optimum pH is around 6.0 to 8.0; (2) optimum temperature at pH 7.5 is around 100 ° C .; (3) pH 7.5 at 100 ° C. for 30 minutes After heat treatment, at least 90
% Or more activity remains; and (4) transferring one or several glucose units in α-1,4-glucan to α-1,4-glucan via α-1,4-glucosidic bond. 6. D-glucose, D-xylose, isomaltose, D-cellobiose, sucrose, or D-glucose
The 4-α-glucanotransferase according to claim 5, which transfers one or several glucose units in α-1,4-glucan using fucose as an acceptor. 7. The 4-α- according to any one of claims 1 to 6,
DNA fragment encoding glucanotransferase. 8. An expression vector comprising the DNA fragment according to claim 7. 9. A host cell transformed with the expression vector according to claim 8. 10. A method for producing 4-α-glucanotransferase, comprising a step of culturing the transformed host cell according to claim 9.