JPH11313688A - Extreme thermostable chitinase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an extreme thermostable chitinase comprising an extreme thermostable chitinase (modified substance) containing a specific amino acid sequence, having a chitin hydrolyzing action, produced by a host cell using a gene isolated from an extreme thermophilic archaebacterium. SOLUTION: An extreme thermostable chitinase contains an amino acid sequence of the formula or a new extreme thermostable chitinase comprises its modified substance containing an amino acid sequence in which one or plural amino acids are deleted, substituted or added in the amino acid sequence of the formula and having chitinase activity, is a chitin hydrolyzing enzyme, has a controlling function of pathogenic microorganisms in a plant and can develop a plant having disease tolerance. The extreme thermostable chitinase is obtained by isolating a DNA encoding extreme thermostable chitinase from an extreme thermophilic archaebacterium KOD-1 strain, transferring the DNA to a host cell and culturing the DNA.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a hyperthermostable chitinase and a DNA encoding the same.

[0002]

2. Description of the Related Art Chitin is a kind of mucopolysaccharide that naturally exists in large amounts as a cell wall substance of arthropods, mollusks, crustaceans, insects, fungi, bacteria and the like.

[0003] Chitinase is an enzyme that hydrolyzes chitin, and includes snail gastric juice, insect molting fluid, fruit peel,
It is found in microorganisms and the like.

[0004] Chitinase can be industrially useful for the purpose of decomposing chitin, which is present in a large amount in nature as described above, into a form usable by microorganisms and the like. In addition, chitinase is considered to originally play a role in the defense mechanism against pathogenic bacteria in plants, and attempts have been made to develop disease-resistant plants by introducing a gene encoding this enzyme.

For the above-mentioned industrial use, a novel chitinase that is structurally more stable than conventionally known chitinases derived from normal-temperature organisms has been desired.

[0006] Because hyperthermophilic archaeons survive at high temperatures, the proteins (eg, enzymes) produced by the microorganism are generally highly thermostable (ie, structurally stable). In addition, the evolution of these organisms is evident from the fact that it has been proposed that the archaeon to which hyperthermophilic archaea belong is different from the conventionally known prokaryotes and eukaryotes. And different. Therefore, even though they have similar functions to known enzymes derived from prokaryotes and eukaryotes, enzymes derived from hyperthermophilic archaea are structurally and enzymatically different from conventional enzymes. Is often different. For example, the hyperthermophilic archaeon KOD-1 strain (Morikawa, M. et al., Appl. E
nviron. Microbiol. 60 (12), 4559-4566 (1994)) was isolated from Escherichia coli GroE.
It has the same function as L. However, GroEL itself forms a 14-mer, and GroES forms a further 7-mer.
In contrast, chaperonins from the KOD-1 strain function alone (Yan, Z et al., App.
l. Environ. Microbiol. 63 : 785-789).

[0007]

SUMMARY OF THE INVENTION The present invention provides a hyperthermostable chitinase and a DNA encoding the same.
It is an object of the present invention to provide a vector containing the DNA and a host cell containing the vector, and to provide a method for producing a hyperthermostable chitinase including a step of culturing the host cell.

[0008]

According to the present invention, a hyperthermostable chitinase comprising the amino acid sequence of SEQ ID NO: 2 or one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 2 A variant thereof comprising a modified amino acid and having chitinase activity is provided.

[0009] In one embodiment, the hyperthermostable chitinase or a variant thereof is derived from the hyperthermophilic archaeon strain KOD-1.

According to the present invention, there is provided a DNA encoding the hyperthermostable chitinase or a variant thereof.

In one embodiment, the DNA comprises the nucleotide sequence of positions 79 to 3723 of SEQ ID NO: 1.

[0012] In one embodiment, the DNA is derived from the hyperthermophilic archaeon strain KOD-1.

According to the present invention, there is provided a vector containing the above DNA.

According to the present invention, there is provided a recombinant host cell containing the above-mentioned vector.

According to the present invention, there is provided a method for producing a hyperthermostable chitinase or a variant thereof, comprising the step of culturing the host cell.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to a hyperthermophilic chitinase produced by a hyperthermophilic archaeon, by culturing a hyperthermophilic archaeon that originally produces the enzyme, or a DNA encoding the enzyme. Isolate, construct a vector containing this DNA,
The present invention relates to a method for producing a host cell containing the vector, and culturing the host cell to produce a hyperthermostable chitinase.

Chitinase is an enzyme that hydrolyzes the β-1,4 bond of chitin to produce N-acetylglucosamine, and has the system name poly (1,4-β- (2-acetamido-2-deoxy-D
-Glycoside)) having glycanohydrolase.

Chitin is a kind of mucopolysaccharide, β-poly-
It has the structure of N-acetylglucosamine. Chitin is naturally present in large amounts as cell wall materials such as arthropods, molluscs, crustaceans, insects, fungi, and bacteria.

The hyperthermostable chitinase of the present invention preferably has an optimum temperature of 60 ° C. or higher, more preferably 70 ° C. or higher, and most preferably about 80 ° C. The hyperthermostable chitinase of the present invention is preferably 3.0 to 7.0, more preferably 4.0 to 6.0,
Most preferably it has an optimum pH of about 5.0. Alternatively, a hyperthermostable chitinase of the invention may have an optimum pH of about 8.0.

The mode of degradation of the chitinase of the present invention may be endo-type or exo-type, but is preferably endo-type. Analysis of the mode of degradation can be predicted, for example, by comparing the reaction rates for disaccharide and trisaccharide derivatives (Robbins, PW, J. Bi.
ol. Chem., 263 (1), 443-447 (1988)). If the reaction rate for the disaccharide derivative is higher, the enzyme is predicted to be exo-type, while if the reaction rate for the trisaccharide derivative is higher, the enzyme is endo-type. is expected. Based on this description, the hyperthermostable chitinase of the present invention is determined to be endo-type.

The hyperthermophilic archaeon used in the present invention is defined as a microorganism that grows at 90 ° C. or higher. Preferably, the hyperthermophilic archaeon is a thermostable thiol protease-producing bacterium KOD-1 (Morikawa, M. et al., Appl. Environ. Micr), which produces a hyperthermostable chitinase.
obiol. 60 (12), 4559-4566 (1994)). The KOD-1 strain has been deposited at the National Institute of Bioscience and Human Technology,
Its accession number is FERM P-15007. This KOD-
One strain was initially classified as belonging to the genus Pyrococcus, as described in the above literature. But DNASIS
Comparison of 16S rRNA sequences using the registered data of GenBank R91.0 October, 1995 + DailyUpdate input to Hitachi Software Engineering Co., Ltd. showed that the KOD-1 strain was Thermococcu rather than Pyrococcus sp.
It has been suggested to be closely related to the s genus.

Culture of the hyperthermophilic archaeon producing the original hyperthermostable chitinase of the present invention can be performed, for example, by the method of Appl. Environ.
robiol. 60 (12), 4559-4566 (1994) (supra). The culture can be either static culture or aeration and agitation culture with nitrogen gas, and can be either continuous or batch.

The conditions for culturing the recombinant host cell are appropriately selected depending on the type of the host cell used. As the host cell, any host cell that can be used in recombinant DNA technology can be used. These include, for example, bacterial cells, yeast cells, animal cells, plant cells, insect cells, and the like. Preferred host cells are bacterial cells.

After culturing, the hyperthermostable chitinase of the present invention can be purified from the resulting culture by a method known in the art. When the expression product is secreted extracellularly, for example, the culture is centrifuged or filtered to obtain a supernatant, which is directly purified or concentrated by a precipitation method or ultrafiltration and then purified. If the expression product accumulates in the cells, the cells are disrupted using cell wall lytic enzymes, changes in osmotic pressure, glass beads, homogenizer or sonication to obtain a cell extract, which is purified.
Purification can be performed by a combination of methods known in the art, such as ion exchange chromatography, gel filtration, affinity chromatography, and electrophoresis.

The DNA encoding the hyperthermostable chitinase is
For example, it can be obtained by isolating chromosomal DNA from hyperthermophilic archaeon, preparing a library containing the chromosomal DNA, and screening this library.

The chromosomal DNA of the hyperthermophilic archaebacterium is obtained by lysing cultured bacterial cells using a surfactant (eg, N-lauryl sarcosine) and subjecting the resulting lysate to an equilibrium density It can be obtained by fractionation by gradient ultracentrifugation or the like (for example, Imanaka et al.,
J. Bacteriol. 147 : 776-786 (1981)). The library is obtained by cleaving the obtained chromosomal DNA with various restriction enzymes, and then cutting the same restriction enzyme or a restriction enzyme giving a common cleavage end to a vector (such as a phage or plasmid), and then adding T4 DNA ligase or the like. And can be obtained by linking. Screening of the library can be performed by selecting a clone containing DNA encoding the desired hyperthermostable chitinase from the library. The selection can be performed using, for example, an oligonucleotide designed based on a predetermined partial amino acid sequence of the hyperthermostable chitinase, a cloned DNA presumed to have homology with the target DNA, or the like as a probe. . Alternatively, selection can be performed by expressing the enzyme of interest. For example, expression can be detected by detecting the activity of an expression product against a substrate added to a plate when the activity of the enzyme of interest can be easily detected, or when an antibody against the enzyme of interest is available. Can be carried out by utilizing the reactivity between the expression product and the antibody.

[0027] In one embodiment, selection can be performed through cloning of adjacent genes. In various organisms, genes having related and / or similar functions are located in adjacent regions on the chromosome. Therefore,
The gene encoding the target enzyme can be obtained by screening a chromosomal region adjacent to the gene encoding the enzyme having a related and / or similar function by performing chromosome walking or the like. For example,
The gene encoding chitinase, which hydrolyzes the mucopolysaccharide chitin, is an enzyme that acts on other sugars (eg,
(β-galactosidase or β-glycosidase).

Analysis of the resulting cloned DNA can be performed, for example, by isolating the selected DNA, creating a restriction map thereof, and determining the nucleotide sequence. Preparation of cloned DNA, restriction enzyme treatment,
Techniques such as subcloning and nucleotide sequence determination are well known in the art, and include, for example, “Molecular
Cloning: A Laboratory Manual Second Edition "(Sambrook, Fr
edited by itsch and Maniatis, Cold Spring Harbor Laborato
ry Press, 1989).

Next, the obtained cloned DNA is operably inserted into an expression vector compatible with the host cell to be used, and the host cell is transformed with the expression vector. By culturing, a hyperthermostable chitinase can be expressed.

Hereinafter, the present invention will be described in more detail by way of examples. However, the invention is not limited by these examples.

[0031]

EXAMPLES..... (Example 1: Isolation of DNA encoding the refractory chitinase) 1.1 PCR 1.1.1 Preparation KOD-1 strain chromosomal DNA of KOD-1 strain, Appl Environ Microbiol 60 (12), 4559-
4 × 2216 marine broth medium described in 4566 (1994) (221
6 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 (N
aCl 28.16 g / L, KCl 0.7 g / L, MgCl ₂・ 6H ₂ O 5.5 g / L, Mg
_{SO 4 · 7H 2 O 6.9 g} / L), distilled water 500 mL, was inoculated into pH 7.0) 1,000 ml, it was incubated with a 2-liter fermenter. During the culture, the inside of the fermenter was replaced with nitrogen gas, and the internal pressure was maintained at 0.1 kg / cm ² with the same gas. Culture is performed at a temperature of 85 ± 1 ° C for 1 hour.
The cells were cultured for 4 hours. The cultivation was performed by stationary cultivation, and aeration and agitation of nitrogen gas were not performed during the culture. After the completion of the culture, the culture was centrifuged at 10,000 rpm for 10 minutes to recover the cells.

1 g of the obtained cells was added to 10 ml of an A solution (50 mM Tr
is-HCl, 50 mM EDTA, pH 8.0) and centrifuged (8,00
After collecting cells by 0 rpm, 5 minutes, 4 ° C.), the cells were suspended in 3 ml of A solution containing 15% sucrose, incubated at 37 ° C. for 30 minutes, and then 1% N
3 ml of solution A containing lauryl sarcosine were added. To this solution, 5.4 g of cesium chloride and 300 μl of a 10 mg / ml ethidium bromide solution were added, and the mixture was added at 55,000 rpm for 16 hours.
Ultracentrifugation was performed at 8 ° C. 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 (pH 8.0),
(0.1 mM EDTA) overnight to obtain chromosomal DNA. 1.1.2. Two amino acid sequences highly conserved between the amino acid sequences of various enzymes belonging to PCR glycolysis family 1 were selected (Voorhorst, WGB et al., J. Bacteriol., 117 , 7).
105-7111 (1995); and Moracci, M. et al., Protein Engin.
eering, 9 , 1191-1195 (1996)).

[0033] Two primers were designed and synthesized based on the selected amino acids. These primers ce
The nucleotide sequences of l3 and cel4 are listed in the Sequence Listing as SEQ ID NO: 3.
And as 4.

The PCR was performed using 1 μg of the chromosomal DNA prepared above,
In a 50 μl reaction solution containing 20 pmol of each primer and KOD polymerase (Toyobo Co., Ltd.), at 55 ° C. for 60 seconds at 98 ° C.
1 cycle of 30 seconds at 74 ° C and 60 seconds at 74 ° C and 98 ° C
The reaction conditions were 15 seconds, 55 ° C. for 15 seconds and 74 ° C. for 60 seconds under 29 cycles of reaction conditions.

The resulting PCR product of about 500 bp was subcloned and its nucleotide sequence was determined.
A was found to encode polypeptides having amino acids homologous to the amino acid sequences of the various enzymes belonging to the known glycolytic family 1 (data not shown). This fragment of the PCR product was used as a probe in the following experiments. 1.2. Screening of chromosome library The chromosomal DNA prepared in 1.1 above was partially digested with a restriction enzyme EcoRI, and then ligated to a phage vector λEMBL4 (Stratagene) using T4 DNA ligase. This concatenated mixture is used as a Max Plax ^™ Packaging Extract (EPICENTER TEC).
HNOLOGIES).

The phage library has 1.1 × 10 ⁹ pfu
To Escherichia coli XL1-Blue MRA (P2) strain,
The resulting plaque was screened by a conventional method using the above PCR fragment as a probe (see “Molecu
lar Cloning: A Laboratory Manual Second Edition "Sambrook,
Fritsch and Maniatis eds, Cold Spring Harbor Labora
tory Press, 1989). About 15 kb in positive clone
Was analyzed by digestion with various restriction enzymes and Southern blotting using the above probe, thereby limiting the position of the gene encoding β-glycosidase. 1.3. Sequence Analysis Upon determining the nucleotide sequence of the 8 kb EcoRI fragment defined in 1.2 above, a 1452 bp open reading frame containing the sequence of the PCR clone was found (data not shown). The nucleotide sequence downstream of this open reading frame was determined, and an additional 3645 bp open reading frame was found. This nucleotide sequence is shown in SEQ ID NO: 1. FIG. 1 shows the positional relationship between the two open reading frames.

This open reading frame is 1215
It encodes a polypeptide consisting of amino acids (SEQ ID NO: 2) (estimated molecular weight 134,227). This size is considerably larger than the size of known chitinases (about 300-800 amino acids). The hydrophobicity of this amino acid sequence was analyzed.
As a result, the first 29 amino acids from the N-terminus were presumed to be a signal peptide for secretion.

Next, this deduced amino acid sequence was converted to DNASIS-M
The amino acid sequence of a known protein registered in the DB Express database using ac ver 3.6, or DDBJ / EM using the National Institute of Genetics open program BLAST
It was compared with the amino acid sequence of a known protein registered in BL / GenBank. As a result, regions having homology to the amino acid sequences of known proteins were found at four positions. The first region is located approximately at amino acids 234 to 301 of SEQ ID NO: 2, and is composed of Bacillus circulans, Trichoderma harz
ianum, Aphanocladium album, and Serratia marcesc
This region shows homology with the amino acid sequence of the ens-derived kinase (FIG. 2; referred to as "Bacillus circulans chitinase homology region"). Here, 289 and 291 of SEQ ID NO: 2
Asp of active site at position (reaction intermediate stabilization;
) And Glu (proton donor; indicated by 中 in the figure)
The residues were found to be conserved. The second and third regions comprise amino acids 625-715 of SEQ ID NO: 2 and
Almost in the 768th place to 858th place, butyrivibrio fib respectively
A region known as the cellulose binding domain in cellulases from risolvens (Berger, E. et al., Mol. Gen. Gen.
et., 219 , 193-198 (1989); and Gilkes, NR et al., Mic.
robiol. Rev., 55 , 303-315 (1991)) (FIG. 2; referred to as "cellulose binding domains (CBD) 1 and 2"). The fourth area is
Almost located at amino acids 975 to 1034 of SEQ ID NO: 2, Str
It is a region showing homology to the amino acid sequences of chitinase of eptomyces erythraeus and two Aeromonas sp. (FIG. 2; referred to as "Streptomyces erythraeus chitinase homologous region"). Here, 1022 and 1024 of SEQ ID NO: 2
Asp of active site at position (reaction intermediate stabilization;
) And Glu (proton donor; indicated by ◎ in the figure) residues were found to be conserved. Thus, the chitinase derived from the KOD-1 strain had a characteristic structure including a repeating structure of two active sites and a cellulose binding site.

Example 2 Expression of Chitinase Gene The chitinase gene obtained in Example 1 was replaced with Escherichia coli.
The following were performed for expression in E. coli.

Using the primers F1 (SEQ ID NO: 5) and R1 (SEQ ID NO: 6), an approximately 1.6 kb fragment containing the 5 'portion of the mature chitinase coding region was obtained using the 8 kb EcoRI fragment as a template. This fragment is
After digestion with SacI, a 288 bp fragment was obtained, which was inserted into plasmid pET25b (+) (Novagen) to construct pENS1. The 3.5 kb SacI fragment cut out from the EcoRI fragment was inserted into this plasmid to obtain an expression vector pEC1. Using this plasmid, Escherichi
a. BL21 (DE3) strain was transformed.

The resulting ampicillin-resistant transformant was transformed into an NZCYM medium (1% NZ) containing ampicillin (50 μg / ml).
Amine, 0.5% NaCl, 0.5% yeast extract, 0.1% casamino acids, was inoculated into _{0.2% MgSO 4 · 7H 2 O} (pH7)), O at 37 ° C.
Culture until _D660 reaches 0.3, then isopropyl-β-D
-Thiogalactopyranoside (IPTG, 0.1 mM)
The culture was further continued at 37 ° C. After completion of the culture, the cells were collected by centrifugation, crushed by sonication, and then subjected to centrifugation again to collect a soluble fraction.
Heat treatment was performed at 70 ° C. for 10 minutes. When a sample obtained by centrifuging the thermostable soluble fraction was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), an expected band of about 130 kDa was detected.

The heat-treated sample was subjected to ammonium sulfate precipitation (40%
(Saturation), anion exchange column (HiTrapQ), gel filtration column, and anion exchange column (MonoQ) until purification as a single band on SDS-PAGE (FIG. 3). (Example 3: Examination of properties of chitinase)
Colloidal chitin was used as a substrate according to the method described in "Chitin-Chitosan Experiment Manual" (edited by Chitin-Chitosan Research Group, published by Gihodo). 1μmo per minute
The amount of the enzyme that produces a reducing sugar corresponding to 1 N-acetylglucosamine was 1 U.

Colloidal chitin as a substrate was prepared by the following procedure. 10 g of chitin (Wako Pure Chemical Industries) was dissolved in 500 ml of 85% phosphoric acid and stirred at -4 ° C for 24 hours. This viscous liquid was added to a 10-fold volume of deionized water with stirring. A precipitate is obtained by centrifugation and this is deionized water.
Washing was repeated until the pH reached 5.0 or more. PH with NaOH
Adjusted to 7.0 and then washed once more with deionized water. This was dissolved in a small amount of water and autoclaved.

The optimum temperature of the hyperthermostable chitinase of the present invention was determined by determining the activity of the purified enzyme by using 50 mM sodium phosphate (pH
7.0) for 60 minutes at various temperatures. The reaction was stopped by ice cooling. FIG. 4 shows the results. The hyperthermostable chitinase of the present invention has been shown to have an optimum temperature of about 80 ° C.

The optimum pH of the hyperthermostable chitinase of the present invention is
The activity of the purified enzyme was determined by measuring at 80 ° C. for 60 minutes at various pH using the following buffer: 50
mM disodium hydrogen citrate-HCl (pH 2.5 to 4.0); 50 mM
Sodium acetate (pH 4.0 to 5.5); 50 mM MES-NaOH (pH 5.5
To 7.0); 50 mM Tris-HCl (pH 7.0 to 9.0); 50 mM glycine
-NaOH (pH 9.0 to 10.0). The reaction was stopped by ice cooling. FIG. 5 shows the results. The hyperthermostable chitinase of the present invention comprises about 4.
It was shown to have an optimal pH at 0. Further, a peak was observed around pH 8.0.

The effect of salts on the activity of the hyperthermostable chitinase of the present invention was determined by determining the activity of the above purified enzyme by adding 50 mM sodium phosphate (p.
H 7.0) for 120 minutes at 80 ° C. The reaction was stopped by ice cooling. FIG. 6 shows the results.
In FIG. 6, .largecircle. Indicates the results obtained using NaCl, and .quadrature. Indicates the results obtained using KCl. The activity of the hyperthermostable chitinase of the present invention increased with the addition of salt, and in particular, increased about twice by the addition of KCl.

The effect of the hyperthermostable chitinase of the present invention on oligosaccharides and colloidal chitin was examined. Oligosaccharides used were N-acetyl-D-glucosamine (G1), di-
N-acetyl-chitobiose (G2), tri-N-acetyl-chitotriose (G3), tetra-N-acetyl-chitotetraose (G4), penta-N-acetyl-chitopentaose (G
5), and hexa-N-acetyl-chitohexaose (G
6). 0.7 mg of each oligosaccharide, 70 mM sodium acetate buffer (pH 6.0), 200 mM KCl, and purified enzyme (G1-
0.9 μg for G3, 1.8 μg for G4 to G6)
50 μl of the reaction was incubated at 80 ° C. and sampled at 0, 5, 15, 30, 60, or 120 minutes. For colloidal chitin, 0.16 mg colloidal chitin, 50 mM sodium acetate buffer (pH 5.0), and
One ml of the reaction containing 0.6 μg of purified enzyme was incubated at 80 ° C. and sampled at 1.5, 3.0, and 4.5 hours and concentrated 20-fold by centrifugation. These were then subjected to TLC as follows. Kieselgel 60 sili
The sampled solution was spotted on a ca gel plate (Merck) and developed using a developing solution (n-butanol: methanol: 25% ammonia solution: water = 5: 4: 2: 1). After development, the plate was dried, and a color reagent (aniline 4 ml, diphenylamine 4 g, acetone 200 ml, 8
(Prepared by mixing 30 ml of 5% phosphoric acid), and this was heated at 180 ° C. for about 5 minutes to develop a color. The results for oligosaccharides are shown in FIGS. 7 and 8, and the results for colloidal chitin are shown in FIG.

From these results, the hyperthermostable chitinase of the present invention does not show a decomposing effect on substrates below disaccharide, and when chitin is used as substrate, it produces chitobiose, a disaccharide as a main product. It was shown that.

The effect of the hyperthermostable chitinase of the present invention on 4-methylumbelliferone (4-MU) oligosaccharide was examined. _{GlcNAc-4-MU, GlcNAc 2} -4-MU or GlcNAc ₃ -4-MU,
(0.01 mM) 10 μl, 100 mM acetate buffer (pH 5.0) 990 μl,
And 20 μl (18 ng) of purified enzyme were incubated at 80 ° C. 100 μl in 0, 5, 15, 30, 45, 60 or 180 minutes
Of the reaction solution, and add 900 μl of ice-cold 100 mM
The reaction was stopped by adding to glycine-NaOH (pH 11). The excitation light at 350 nm and the fluorescence at 440 nm of this sample were measured using a spectrofluorometer. The results are shown in FIG. In FIG. 10, ○ indicates the results when GlcNAc-4-MU was used as the substrate, ● indicates the results when GlcNAc ₂ -4-MU was used as the substrate, and □ indicates GlcNAc ₃ -4-MU as the substrate. The results when used are shown. From this graph, the reaction rate for each substrate was determined. Table 1 shows the results.

[0050]

[Table 1]

It has been reported that by comparing the rate of reaction with a derivative of a disaccharide and the rate of reaction with a derivative of a trisaccharide, it is possible to predict whether the mode of cleavage of the enzyme is endo or exo. (Robbins, PW,
J. Biol. Chem., 263 (1), 443-447 (1988)), where the reaction rate for the disaccharide derivative is higher, the enzyme is expected to be in exo form, while the trisaccharide If the rate of reaction on the derivative of is higher, the enzyme is predicted to be endo-type. Based on this description, the hyperthermostable chitinase of the present invention is determined to be endo-type.

The function of each domain of the hyperthermostable chitinase of the present invention was examined by preparing various deletion mutants. Primers F1, R2, F2 (SEQ ID NO: 7), R2 (SEQ ID NO: 8), F3 (SEQ ID NO: 9), and R3
(SEQ ID NO: 10) was converted to F1-R2, F2-R3, F1-R1, and F3
Using the combination of -R3 and the above 8 kb EcoRI fragment as a template, PCR was performed to obtain an amplification product. This product was digested with NcoI and HindIII, BamHI, NcoI and BglII, and BamHI, respectively, and digested with NcoI and HindIII, respectively, pET25b (+), NcoI, blunt-ended, and then digested with BamHI. PET-8
c (Novagen), pET-25b digested with NcoI and BamHI
(+), As well as digested with NcoI, blunt-ended, and then inserted into BamHI digested pET-8c. These plasmids were each deleted using the deletion mutant Pk-ChiAΔ1 (first Bacill
us circulans chitinase homology region and two cellulose binding domains), Pk-ChiAΔ2 (fourth Streptom
yces erythraeus chitinase homologous region and two cellulose binding domains), Pk-ChiAΔ3 (first Baci
llus circulans chitinase homologous region), and
Pk-ChiAΔ4 (containing a fourth Streptomyceserythraeus chitinase homology region) was used for expression.

A crude enzyme solution was obtained from a culture of an E. coli transformant carrying each plasmid by heat treatment at 70 ° C. for 10 minutes. The activity was examined by spotting the crude enzyme solution on a colloidal chitin plate (0.5% colloidal chitin, 1.5% agar) and incubating it (FIG. 11). The deletion mutant having only the first chitinase homologous region showed little activity, and the deletion mutant having only the fourth chitinase homologous region showed little activity. Deletion mutants having any of the chitinase homology regions and two cellulose binding domains showed high activity.

30 μl of the crude enzyme solution of the deletion mutants Pk-ChiAΔ2 and Pk-ChiAΔ4 was mixed with 30 μl of 1% colloidal chitin and incubated at 70 ° C. for 1 hour. Then
The reaction was centrifuged to obtain a supernatant and a precipitate containing colloidal chitin. For the precipitate, the precipitate was washed twice with 50 mM sodium phosphate (pH 7.0), and these were subjected to SDS-PAGE.
Was served. The results are shown in FIG. In FIG. 12, lane 1
And 5 are samples without colloidal chitin, lane 2
And 6 show the supernatant after the addition of colloidal chitin and lane 3
And 7 show precipitation after addition of colloidal chitin, and lanes 4 and 8 show molecular weight markers, lanes 1-3
Shows the results for Pk-ChiAΔ2, and lanes 5 to 7
The results for Pk-ChiAΔ4 are shown.

These results indicate that two cellulose binding domains are required for binding to chitin and chitinase activity.

[0056]

According to the present invention, a hyperthermostable chitinase and a DNA encoding the same are provided. The present invention also provides a vector containing the DNA and a host cell containing the vector. Further, the present invention provides a method for producing a hyperthermostable chitinase, comprising a step of culturing the host cell.

[0057]

[Sequence List] SEQUENCE LISTING <110> Imanaka, Tadayuki <120> Highly heat-resistant chitinase <130> J1-98432443 <150> JP 10-39285 <151> 1998-02-20 <160> 10 <170> PatentIn Ver 2.0 <210> 1 <211> 3780 <212> DNA <213> KOD-1 <220> <221> CDS <222> (79) .. (3723) <400> 1 aaaacattta taagcatgga agttcatgta aaatatgcaa catctcccaa aacattcaaa 60 gctcaagggg tgttgtgt gtg aag aag att tgg act tca ata gtt ttg acc 111 Val Lys Lys Ile Trp Thr Ser Ile Val Leu Thr 1 5 10 gca gtg ctc ctg ctg tcc ctg gtc cag gtg ggc act ttc ccc tgg gct 159 Ala Val Leu Leu Leu Val Gln Val Gly Thr Phe Pro Trp Ala 15 20 25 tca gcg gcg gag agc gta agc ctg agc ggg agc gct ata gca tgg gac 207 Ser Ala Ala Glu Ser Val Ser Leu Ser Gly Ser Ala Ile Ala Trp Asp 30 35 40 gtt gtg aac ctg act tgg agc ccg tac agc agc gcc aag gcg tac gag 255 Val Val Asn Leu Thr Trp Ser Pro Tyr Ser Ser Ala Lys Ala Tyr Glu 45 50 55 gtg tac agg agc acg gat ccc tcc aac ctg ttc tca ccg ac ctg 303 Val Tyr Arg Ser Thr Asp Pro Ser Asn Leu Phe Ser Pro Asn Asn Leu 60 65 70 75 ctg gtg gtc gtt aac tgg agc agc tat ccg aaa tat gag cca ggg aag 351 Leu Val Val Val Asn Trp Ser Ser Tyr Pro Lys Tyr Glu Pro Gly Lys 80 85 90 aca tac aac cag ggc gat gtc gtt gag tac aac ggg aag ata tgg cgc 399 Thr Tyr Asn Gln Gly Asp Val Val Glu Tyr Asn Gly Lys Ile Trp Arg 95 100 105 gtt aag tac tgg acc cag agc gtt cca gga agc gac gac tcc tgg gag 447 Val Lys Tyr Trp Thr Gln Ser Val Pro Gly Ser Asp Asp Ser Trp Glu 110 115 120 ctc gta ggg gat gtt gtc cca acc aca agc tac ctc gac cag tat cac 495 Leu Val Gly Asp Val Val Pro Thr Thr Ser Tyr Leu Asp Gln Tyr His 125 130 135 ctg aag gca aac aca acc tac tac tac ggg gtc gtc ccg gtt ctc gcc 543 Leu Lys Ala Asn Thr Thr Tyr Tyr Tyr Gly Val Val Pro Val Leu Ala 140 145 150 155 gat gga agc agg gga agt ccc tcg aac gtc ctc gcc acg act ccc 591 Asp Gly Ser Arg Gly Ser Pro Ser Asn Val Leu Ala Ile Thr Thr Pro 160 165 170 ctc gaa cct tac agg gtc ata gtc tac tac atc tcc tgg gga agg tac 639 Leu Glu Pro Tyr Arg Val Ile Val Tyr Tyr Ile Ser Trp Gly Arg Tyr 175 180 185 gcg agg aag ttc tac gtg agc gac atc ccc tgg gag aag gtt acc cac 687 Ala Arg Lys Phe Tyr Val Ser Asp Ile Pro Trp Glu Lys Val Thr His 190 195 200 gtc aac tac gcc ttc ctc gac ctc aag gag gat gga acc gtt gcc ttc 735 Val Asn Tyr Ala Phe Leu Asp Leu Lys Glu Asp Gly Thr Val Ala Phe 205 210 215 tac gat acc tac gct gat cct ctc aac ctc gag gcc atg aag gag tac 783 Tyr Asp Thr Tyr Ala Asp Pro Leu Asn Leu Glu Ala Met Lys Glu Tyr 220 225 230 235 aaa agg aag tat cca gcc gtc aag gtt ctc atc tca gtc ggc gga tgg 831 Lys Arg Lys Tyr Pro Ala Val Lys Val Leu Ile Ser Val Gly Gly Trp 240 245 250 act ctc agc aag tac ttc tcg gtg gtc gcc gca gac ccg gcc aag aga 879 Thr Leu Ser Lys Tyr Phe Ser Val Val Ala Ala Asp Pro Ala Lys Arg 255 260 265 cag cgc ttc gcc gag act gcc ata gag atc ctc aag tac aac ctc 927 Gln Arg Phe Ala Glu Thr Ala Ile Glu Ile Leu Arg Lys Tyr Asn Leu 270 275 280 gac gga att gat atc gac tgg gag tac ccg ggc ggc gga ggt atg gcg 975 Asp Gly Ile Asp Ile Asp Gyr AslePro Gly Gly Gly Gly Met Ala 285 290 295 ggc aac tac gag agt ccc gac gac ggt aag aac ttc gtt ctt ctc ctc 1023 Gly Asn Tyr Glu Ser Pro Asp Asp Gly Lys Asn Phe Val Leu Leu Leu 300 305 310 315 aag gat agg gag gcc ctc gac aag gcc gca aaa gag gac cac aag 1071 Lys Asp Leu Arg Glu Ala Leu Asp Lys Ala Ala Lys Glu Asp His Lys 320 325 330 gac tac ctc cta acg gcc gca acg ccg gcc gat ccg gtt aag gct Asp Tyr Leu Leu Thr Ala Ala Thr Pro Ala Asp Pro Val Lys Ala Gly 335 340 345 agg ata gat tgg gtg gag gcg agc aag tac ctc gat tca atc aac atc 1167 Arg Ile Asp Trp Val Glu Ala Ser Lys Tyr Leu Asp Ser Ile Asn Ile 350 355 360 atg acc tac gac tac cac ggc gcc tgg gag acc ata acc ggc cac ctt 1215 Met Thr Tyr Asp Tyr His Gly Ala Trp Glu Thr Ile Thr Gly His Leu 365 370 375 gcc ccg ctc tac tgc gat cca aac gcg cca tac acc gat gag aac gtc 1263 Ala Pro Leu Tyr Cys Asp Pro Asn Ala Pro Tyr Thr Asp Glu Asn Val 380 385 390 395 aag tac cac ttc tgc gtc aac tac acc gtc cag tgg tac atc cag cac 1311 Lys Tyr His P he Cys Val Asn Tyr Thr Val Gln Trp Tyr Ile Gln His 400 405 410 gtt ccc gat aag acc aag ata acc gtc ggc ctg ccc ttc tac agc agg 1359 Val Pro Asp Lys Thr Lys Ile Thr Val Val Gly Leu Pro Phe Tyr Ser Arg 415 420 425 agc ttt gcc aac gtc ccg ccc gag aac aac ggc ctc tac cag ccc ttc 1407 Ser Phe Ala Asn Val Pro Pro Glu Asn Asn Gly Leu Tyr Gln Pro Phe 430 435 440 agc ggc acc cca gct gga acc tcc ggaccg gag acc tac gga 1455 Ser Gly Thr Pro Ala Gly Thr Trp Gly Pro Ala Tyr Glu Thr Tyr Gly 445 450 455 gtt atg gac tac tgg gac gtt gcc gag aag aac cag agc agc gag tac 1503 Val Met Asp Tyr Trp Asp Val Ala Glu Lys Asn Gln Ser Ser Glu Tyr 460 465 470 475 gag tac cac tgg gat ccg ata gcc cag gtg gcc tgg ctc tac tcc ccg 1551 Glu Tyr His Trp Asp Pro Ile Ala Gln Val Ala Trp Leu Tyr Ser Pro 480 485 490 490 agc aag aag ata ttc ata acc ttc gac gat ccc agg gcg atc ggg ata 1599 Ser Lys Arg Ile Phe Ile Thr Phe Asp Asp Pro Arg Ala Ile Gly Ile 495 500 505 aag gtt gac tac atg ctg aag aac ggc ctc ggc gga ggg atg 1647 Lys Val Asp Tyr Met Leu Lys Asn Gly Leu Gly Gly Val Met Ile Trp 510 515 520 gag atc aca gct gac agg aag ccc ggg acc aac gac cac ccg ctt ctc 1695 Glu Ile Thr Ala Asp Arg Lys Pro Gly Thr Asn Asp His Pro Leu Leu 525 530 535 gat act gtt ctc cag cac ctc ggc gag aag ccg ccg gcg tgg att ccg 1743 Asp Thr Val Leu Gln His Leu Gly Glu Lys Pro Pro Ala Trp Ile Pro 540 545 550 555 gac acc tac tac atc ggc aac atc ccg agc aac ata acc gtt cca 1791 Asp Thr Tyr Tyr Ile Gly Ser Asn Ile Pro Ser Asn Ile Thr Val Pro 560 565 570 gag ccg acg cca ctg ccg ccg agc aac gag aca acc cct gag gac aat 1839 Glu Pro Thr Pro Leu Pro Pro Ser Asn Glu Thr Thr Pro Glu Asp Asn 575 580 585 cag acc aat cca aac cca tca cag gga aat gaa acc aat cca aac ccg 1887 Gln Thr Asn Pro Asn Pro Ser Gln Gly Asn Glu Thr Asn Pro Asn Pro 590 595 600 tca cct gga aac gag acc aca ccc tcg gac aac cag acg act cca tcc 1935 Ser Pro Gly Asn Glu Thr Thr Pro Ser Asp Asn Gln Thr Thr Pro Ser 605 610 615 acc ggg gat ttt gtc aag ccg ggt tct ttg agc gtc aag gta acc gac 1983 Thr Gly Asp Phe Val Lys Pro Gly Ser Leu Ser Val Lys Val Thr Asp 620 625 630 635 tgg ggc aac act gaa tac gat gtc acc ctc aac ctc ggt gga acc tat 2031 Trp Gly Asn Thr Glu Tyr Asp Val Thr Leu Asn Leu Gly Gly Thr Tyr 640 645 650 gac tgg gtc gtc aag gtc aaa ctc aag gac ggt tca agc gta tcg agc 2079 Asp Trp Val Val Lys Val Lys Leu Lys Asp Gly Ser Ser Val Ser Ser 655 660 665 ttc tgg agc gcg aac aag gcc gag gaa ggc ggt tac gtc gtc ttc acg 2127 Phe Trp Ser Ala Asn Lys Ala Glu Glu Gly Gly Tyr Val Val Phe Thr 670 675 680 ccg gtg agc tgg aac agg ggg ccg acg gca acg 2175 Pro Val Ser Trp Asn Arg Gly Pro Thr Ala Thr Phe Gly Phe Ile Ala 685 690 695 act gga agc gag tcc gtt gaa gcg att tac ctc tac gta gac ggc cag 2223 Thr Gly Ser Glu Ser Val Glu Ala Ile Tyr Leu Tyr Val Asp Gly Gln 700 705 710 715 ctc tgg gat gcc tgg ccc tca aac acc caa caa ccg gag gaa aac cag 2271 Leu Trp Asp Ala Trp Pro Ser Asn Thr Gln Gln Pro Glu Glu Asn Gln 720 725 730 aca gtc cca agc ccg t ca cct ggc aac gag acg act ccc aca ccc tcc 2319 Thr Val Pro Ser Pro Ser Pro Gly Asn Glu Thr Thr Pro Thr Pro Ser 735 740 745 ccc gga aac gag acc gca ccc tca gag aac cag aca act ccg tcc acg 2367 Pro Gly Asn Glu Thr Ala Pro Ser Glu Asn Gln Thr Thr Pro Ser Thr 750 755 760 gga gat cta gtc aag ccg gat gca ttc agc gtt aaa atc cag gac tgg 2415 Gly Asp Leu Val Lys Pro Asp Ala Phe Ser Val Lys Ile Gln Asp Trp 765 770 775 gga agc acg gag tac gat gta acc ctg aac ctc ggt gga act tac gac 2463 Gly Ser Thr Glu Tyr Asp Val Thr Leu Asn Leu Gly Gly Thr Tyr Asp 780 785 790 795 tgg gtc gtt aaa gtc aag gctc aag gac tca gcc gtc tca agc gtc 2511 Trp Val Val Lys Val Lys Leu Lys Asp Gly Ser Ala Val Ser Ser Val 800 805 810 tgg agc gct aac aag gcc gag gaa ggt ggt tac gta gtc ttc act ccc 2559 Trp Ser Ala Asn Lys Ala Glu Glu Gly Gly Tyr Val Val Phe Thr Pro 815 820 825 gta agc tgg aac aag gga ccg acg gca acc ttc ggc ttc atc gcc act 2607 Val Ser Trp Asn Lys Gly Pro Thral Ala Thr Phe Gly Phe Ile Ala Thr 830 835 840 gg c agc gag ccc gtt gag gcc atg tac ctc tac gta aac gat cag ctc 2655 Gly Ser Glu Pro Val Glu Ala Met Tyr Leu Tyr Val Asn Asp Gln Leu 845 850 855 tgg gac gtc tgg cca gaa acc gca tca gcc ccg ggc agt act 2703 Trp Asp Val Trp Pro Glu Thr Ala Ser Ala Pro Gly Asn Glu Ser Thr 860 865 870 875 cca tcc gac aac cag acc aat ccg aat cca tca ccg ggc aac gaa acc 2751 Pro Ser Asp Asn Gln Thr Asn Pro Asn Pro Ser Pro Gly Asn Glu Thr 880 885 890 aat cca aat ccc agc cca gga aac gag acg ggc ccg tac gtt cca gca 2799 Asn Pro Asn Pro Ser Pro Gly Asn Glu Thr Gly Pro Tyr Val Pro Ala 895 900 905 ggc cca ggt ctt ccc gag cac ttc ttc gcc cca tac atc gac atg agc 2847 Gly Pro Gly Leu Pro Glu His Phe Phe Ala Pro Tyr Ile Asp Met Ser 910 915 920 ctc ggc ata cac aag ccg ctc gtt gag tac tac aac ctc Lec gag aca Ile His Lys Pro Leu Val Glu Tyr Tyr Asn Leu Thr Gly Thr 925 930 935 ccg tac ttc aca ctg gcc ttc gtc ctc tac agt tca gtc tac aac ggg 2943 Pro Tyr Phe Thr Leu Ala Phe Val Leu Tyr Ser Ser Val Tyr Asn Gly 940 945 950 955 ccg gcc tgg gct gga agc att ccc ttg gat gcc ttc gtt gat gag gta 2991 Pro Ala Trp Ala Gly Ser Ile Pro Leu Asp Ala Phe Val Asp Glu Val 960 965 970 aaa ggg ctc agg gag ggg ggg gtt att ata gcc ttc ggg ggc 3039 Lys Gly Leu Arg Glu Ala Gly Gly Asp Val Ile Ile Ala Phe Gly Gly 975 980 985 gca gtt ggc ccc tac ctc tgc cag cag gca aaa act cca gaa cag ctc 3087 Ala Val Gly Cys Gln Gln Ala Lys Thr Pro Glu Gln Leu 990 995 1000 gcc cag tgg tac att cag gtc ata gac acc tac aac gcc aca tac ctc 3135 Ala Gln Trp Tyr Ile Gln Val Ile Asp Thr Tyr Asn Ala Thr Tyr Leu 1005 1010 1015 gat ttt gac atc gaa tct ggt gta gac gcc gac aaa ctt gcg gat gct 3183 Asp Phe Asp Ile Glu Ser Gly Val Asp Ala Asp Lys Leu Ala Asp Ala 1020 1025 1030 1035 ctc ctg atc gtc cag agg gag agg ccg aacgt ttc aca 3231 Leu Leu Ile Val Gln Arg Glu Arg Pro Asn Val Arg Phe Ser Phe Thr 1040 1045 1050 ctc ccg agc gac ccg gga ata ggt ctc gcg ggc gga tac gga ata ata 3279 Leu Pro Ser Asp Pro Gly I le Gly Leu Ala Gly Gly Tyr Gly Ile Ile 1055 1060 1065 gag acc atg gca aag aag ggg gtc att gtc gac agg gtc aat ccg atg 3327 Glu Thr Met Ala Lys Lys Gly Val Ile Val Asp Arg Val Asn Pro Met 1070 1075 1080 acg atg gac tac tac tgg acg cca gca aac gcc gac aac gcc ata agc 3375 Thr Met Asp Tyr Tyr Trp Thr Pro Ala Asn Ala Asp Asn Ala Ile Ser 1085 1090 1095 gtg gcc gaa cac gtc ttt aac cag ctc aag cag atc tat aag 3423 Val Ala Glu His Val Phe Asn Gln Leu Lys Gln Ile Tyr Pro Asp Lys 1100 1105 1110 1115 agc gac gac gaa ata tgg ggc atg atc ggc ctg act ccg atg ata gga 3471 Ser Asp Asp Glu Ile Trp Gly Met Ile Gly Thr Pro Met Ile Gly 1120 1125 1130 acc aac gac gac aag agc gtc ttc tcg ctc cag gat gcc gaa aag ctc 3519 Thr Asn Asp Asp Lys Ser Val Phe Ser Leu Gln Asp Ala Glu Lys Leu 1135 1140 1145 gtc gac tgg atca cac aag ata cgc tcc ctt gcc ttc tgg agc 3567 Val Asp Trp Ala Ile Gln His Lys Ile Arg Ser Leu Ala Phe Trp Ser 1150 1155 1160 gtt gac aga gat cac cca ggg ccg aca gga gag gtc tca c ca atc cac 3615 Val Asp Arg Asp His Pro Gly Pro Thr Gly Glu Val Ser Pro Ile His 1165 1170 1175 agg gga acc agc gac ccc gat tgg gcc ttc agc cac gcc ttc ctg agg 3663 Arg Gly Thr Ser Asp Pro Asp Trp Ala Phe Ser His Ala Phe Leu Arg 1180 1185 1190 1195 ttc atg aag gca ttc cag ccg gtt gcc agc acg gct cag gtg gca gtt 3711 Phe Met Lys Ala Phe Gln Pro Val Ala Ser Thr Ala Gln Val Ala Val 1200 1205 1210 gca gtt cca gtt tgacccctct cttctcctct tttgtccata atgaaattgg 3763 Ala Val Pro Val 1215 agaaacagct aaggaag 3780 <210> 2 <211> 1215 <212> PRT <213> KOD-1 <400> 2 Val Lys Lys Ile Trp Thr Ser Ile Val Leu Thr Ala Val Leu Leu Leu 1 5 10 15 Ser Leu Val Gln Val Gly Thr Phe Pro Trp Ala Ser Ala Ala Glu Ser 20 25 30 Val Ser Leu Ser Gly Ser Ala Ile Ala Trp Asp Val Val Asn Leu Thr 35 40 45 Trp Ser Pro Tyr Ser Ser Ala Lys Ala Tyr Glu Val Tyr Arg Ser Thr 50 55 60 Asp Pro Ser Asn Leu Phe Ser Pro Asn Asn Leu Leu Val Val Val Asn 65 70 75 80 Trp Ser Ser Tyr Pro Lys Tyr Glu Pro Gly Lys Thr Tyr Asn Gln Gly 85 90 95 Asp Val Val Glu Tyr Asn Gly Lys Ile Trp Arg Val Lys Tyr Trp Thr 100 105 110 Gln Ser Val Pro Gly Ser Asp Asp Ser Trp Glu Leu Val Gly Asp Val 115 120 125 Val Pro Thr Thr Ser Tyr Leu Asp Gln Tyr His Leu Lys Ala Asn Thr 130 135 140 Thr Tyr Tyr Tyr Gly Val Val Pro Val Leu Ala Asp Gly Ser Arg Gly 145 150 155 160 Ser Pro Ser Asn Val Leu Ala Ile Thr Thr Pro Leu Glu Pro Tyr Arg 165 170 175 Val Ile Val Tyr Tyr Ile Ser Trp Gly Arg Tyr Ala Arg Lys Phe Tyr 180 185 190 Val Ser Asp Ile Pro Trp Glu Lys Val Thr His Val Asn Tyr Ala Phe 195 200 205 Leu Asp Leu Lys Glu Asp Gly Thr Val Ala Phe Tyr Asp Thr Tyr Ala 210 215 220 Asp Pro Leu Asn Leu Glu Ala Met Lys Glu Tyr Lys Arg Lys Tyr Pro 225 230 235 240 Ala Val Lys Val Leu Ile Ser Val Gly Gly Tly Thr Trp Thr Leu Ser Lys Tyr 245 250 255 Phe Ser Val Val Ala Ala Asp Pro Ala Lys Arg Gln Arg Phe Ala Glu 260 265 270 270 Thr Ala Ile Glu Ile Leu Arg Lys Tyr Asn Leu Asp Gly Ile Asp Ile 275 280 285 Asp Trp Glu Tyr Pro Gly Gly Gly Gly Met Ala Gly Asn Tyr Glu Ser 290 295 300 Pro Asp Asp Gly Lys Asn Phe Val Leu Leu Leu Lys Asp Leu Arg Glu 305 310 315 320 Ala Leu Asp Lys Ala Ala Lys Glu Asp His Lys Asp Tyr Leu Leu Thr 325 330 335 Ala Ala Thr Pro Ala Asp Pro Val Lys Ala Gly Arg Ile Asp Trp Val 340 345 350 Glu Ala Ser Lys Tyr Leu Asp Ser Ile Asn Ile Met Thr Tyr Asp Tyr 355 360 365 His Gly Ala Trp Glu Thr Ile Thr Gly His Leu Ala Pro Leu Tyr Cys 370 375 380 Asp Pro Asn Ala Pro Tyr Thr Asp Glu Asn Val Lys Tyr His Phe Cys 385 390 395 400 Val Asn Tyr Thr Val Gln Trp Tyr Ile Gln His Val Pro Asp Lys Thr 405 410 415 Lys Ile Thr Val Gly Leu Pro Phe Tyr Ser Arg Ser Phe Ala Asn Val 420 425 430 Pro Pro Glu Asn Asn Gly Leu Tyr Gln Pro Phe Ser Gly Thr Pro Ala 435 440 445 Gly Thr Trp Gly Pro Ala Tyr Glu Thr Tyr Gly Val Met Asp Tyr Trp 450 455 460 Asp Val Ala Glu Lys Asn Gln Ser Ser Glu Tyr Glu Tyr His Trp Asp 465 470 475 480 480 Pro Ile Ala Gln Val Ala Trp Leu Tyr Ser Pro Ser Lys Arg Ile Phe 485 490 495 Ile Thr Phe Asp Asp Pro Arg Ala Ile Gly Ile Lys Val Asp Tyr Met 500 505 510 Leu Lys Asn Gly Leu Gly Gly Val Met Ile Trp Glu Ile Thr Ala Asp 515 520 525 Arg Lys Pro Gly Thr Asn Asp His Pro Leu Leu Asp Thr Val Leu Gln 530 535 540 540 His Leu Gly Glu Lys Pro Pro Ala Trp Ile Pro Asp Thr Tyr Tyr Ile 545 550 555 560 Gly Ser Asn Ile Pro Ser Asn Ile Thr Val Pro Glu Pro Thr Pro Leu 565 570 575 Pro Pro Ser Asn Glu Thr Thr Pro Glu Asp Asn Gln Thr Asn Pro Asn 580 585 585 590 Pro Ser Gln Gly Asn Glu Thr Asn Pro Asn Pro Ser Pro Gly Asn Glu 595 600 605 Thr Thr Pro Ser Asp Asn Gln Thr Thr Pro Ser Thr Gly Asp Phe Val 610 615 620 lys Pro Gly Ser Leu Ser Val Lys Val Thr Asp Trp Gly Asn Thr Glu 625 630 635 640 Tyr Asp Val Thr Leu Asn Leu Gly Gly Thr Tyr Asp Trp Val Val Lys 645 650 655 Val Lys Leu Lys Asp Gly Ser Ser Val Ser Ser Phe Trp Ser Ala Asn 660 665 670 Lys Ala Glu Glu Gly Gly Tly Val Val Phe Thr Pro Val Ser Trp Asn 675 680 685 Arg Gly Pro Thr Ala Thr Phe Gly Phe Ile Ala Thr Gly Ser Glu Ser 690 695 700 Val Glu Ala Ile Tyr Leu Tyr Val Asp Gly Gln Leu Trp Asp Ala Trp 705 710 715 715 720 Pro Ser Asn Thr Gln Gln Pro Glu Glu Asn Gln Thr Val Pro Ser Pro 725 730 735 Ser Pro Gly Asn Glu Thr Thr Pro Thr Pro Ser Pro Gly Asn Glu Thr 740 745 750 Ala Pro Ser Glu Asn Gln Thr Thr Pro Ser Thr Gly Asp Leu Val Lys 755 760 765 Pro Asp Ala Phe Ser Val Lys Ile Gln Asp Trp Gly Ser Thr Glu Tyr 770 775 780 Asp Val Thr Leu Asn Leu Gly Gly Thr Tyr Asp Trp Val Val Lys Val 785 790 795 800 Lys Leu Lys Asp Gly Ser Ala Val Ser Ser Val Trp Ser Ala Asn Lys 805 810 815 Ala Glu Glu Gly Gly Tyr Val Val Phe Thr Pro Val Ser Trp Asn Lys 820 825 830 Gly Pro Thr Ala Thr Phe Gly Phe Ile Ala Thr Gly Ser Glu Pro Val 835 840 845 Glu Ala Met Tyr Leu Tyr Val Asn Asp Gln Leu Trp Asp Val Trp Pro 850 855 860 Glu Thr Ala Ser Ala Pro Gly Asn Glu Ser Thr Pro Ser Asp Asn Gln 865 870 875 880 Thr Asn Pro Asn Pro Ser Pro Gly Asn Glu Thr Asn Pro Asn Pro Ser 885 890 895 Pro Gly Asn Glu Thr Gly Pro Tyr Val Pro Ala Gly Pro Gly Leu Pro 900 905 910 Glu His Phe Phe Ala Pro Tyr Ile Asp Met Ser Leu Gly Ile His Lys 915 920 925 Pro Leu Val Glu Tyr Tyr Asn Leu Thr Gly Thr Pro Tyr Phe Thr Leu 930 935 940 Ala Phe Val Leu Tyr Ser Ser Val Tyr Asn Gly Pro Ala Trp Ala Gly 945 950 955 960 Ser Ile Pro Leu Asp Ala Phe Val Asp Glu Val Lys Gly Leu Arg Glu 965 970 975 Ala Gly Gly Asp Val Ile Ile Ala Phe Gly Gly Ala Val Gly Pro Tyr 980 985 990 Leu Cys Gln Gln Ala Lys Thr Pro Glu Gln Leu Ala Gln Trp Tyr Ile 995 1000 1005 Gln Val Ile Asp Thr Tyr Asn Ala Thr Tyr Leu Asp Phe Asp Ile Glu 1010 1015 1020 Ser Gly Val Asp Ala Asp Lys Leu Ala Asp Ala Leu Leu Ile Val Gln 025 1030 1035 1040 Arg Glu Arg Pro Asn Val Arg Phe Ser Phe Thr Leu Pro Ser Asp Pro 1045 1050 1055 Gly Ile Gly Leu Ala Gly Gly Tyr Gly Ile Ile Glu Thr Met Ala Lys 1060 1065 1070 Lys Gly Val Ile Val Asp Arg Val Asn Pro Met Thr Met Asp Tyr Tyr 1075 1080 1085 Trp Thr Pro Ala Asn Ala Asp Asn Ala Ile Ser Val Ala Glu His Val 1090 1095 1100 Phe Asn Gln Leu Lys Gln Ile Tyr Pro Asp Lys Ser Asp Asp Glu Ile 105 1110 1115 1120 Trp Gly Met Ile Gly Leu Thr Pro Met Ile Gly Thr Asn Asp Asp Lys 1125 1130 1135 Ser Val Phe Ser Leu Gln Asp Ala Glu Lys Leu Val Asp Trp Ala Ile 1140 1145 1150 Gln His Lys Ile Arg Ser Leu Ala Phe Trp Ser Val Asp Arg Asp His 1155 1160 1165 Pro Gly Pro Thr Gly Glu Val Ser Pro Ile His Arg Gly Thr Ser Asp 1170 1175 1180 Pro Asp Trp Ala Phe Ser His Ala Phe Leu Arg Phe Met Lys Ala Phe 185 1190 1195 1200 Gln Pro Val Ala Ser Thr Ala Gln Val Ala Val Ala Val Pro Val 1205 1210 1215 <210 > 3 <211> 23 <212> DNA <213> Artificial Sequence <400> 3 ACAATGAAYG ARCCAAAYGT AGT 23 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <400> 4 TARTARTTTA CGCCGATCCA RTC 23 <210 > 5 <211> 30 <212> DNA <213> Artificial Sequence <400> 5 TTCCATGGCG GAGAGCGTAA GCCTGAGCGG 30 <210> 6 <211> 34 <212> DNA <213> Artificial Sequence <400> 6 GCAGATCTCA GCCGAGGTGC TGGAGAACAG TATC 34 < 210> 7 <211> 21 <212> DNA <213> Artificial Sequence <400> 7 CCGCCGGCGT GGATTCCGGA C 21 <210> 8 <211> 34 <212> DNA <213> Artificial Sequence <400> 8 GGAAGCTTCA ACCTGGGCCT GCTGGAACGT ACGG 34 <210> 9 <211> 23 <212 > DNA <213> Artificial Sequence <400> 9 CTTCCGGAGC ACTTCTTCGC CCC 23 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <400> 10 TGGGATCCAG CTGAAGAACT GGCTG 25

[Brief description of the drawings]

FIG. 1 shows the position of the cloned β-glycosidase and chitinase open reading frames.

FIG. 2 shows the positions of the B. circulans chitinase homologous region, the cellulose binding domain, and the S. erythraeus chitinase homologous region, and the alignment in the B. circulans chitinase homologous region and the S. erythraeus chitinase homologous region.

FIG. 3 is an electrophoresis photograph of an enzyme expressed and purified in E. coli.

FIG. 4 is a graph showing the optimal temperature of the chitinase of the present invention.

FIG. 5 is a graph showing the optimum pH of the chitinase of the present invention.

FIG. 6 is a graph showing the effect of salts on the chitinase of the present invention.

FIG. 7 is a photograph of TLC showing the action of the chitinase of the present invention on various oligosaccharides.

FIG. 8 is a photograph of TLC showing the effect of the chitinase of the present invention on various oligosaccharides.

FIG. 9 is a TLC photograph showing the effect of the chitinase of the present invention on colloidal chitin.

FIG. 10 is a graph showing the effect of the chitinase of the present invention on 4-MU.

FIG. 11 shows the chitinase activities of various deletion mutants.

FIG. 12 is an electrophoretic photograph showing the chitinase activity of various deletion mutants.

──────────────────────────────────────────────────続 き Continued on front page (51) Int.Cl. ⁶ Identification code FI (C12N 9/42 C12R 1:19)

Claims (8)

[Claims] 1. A hyperthermostable chitinase comprising the amino acid sequence of SEQ ID NO: 2, or a chitinase having one or more amino acids deleted, substituted or added in the amino acid sequence of SEQ ID NO: 2 and having chitinase activity Variant. 2. The hyperthermostable chitinase or its variant according to claim 1, which is derived from the hyperthermophilic archaeon KOD-1 strain. 3. A DNA encoding the hyperthermostable chitinase according to claim 1 or a variant thereof. 4. The DNA according to claim 3, comprising the nucleotide sequence of positions 79 to 3723 of SEQ ID NO: 1. 5. The DNA according to claim 3, which is derived from the hyperthermophilic archaeon KOD-1 strain. 6. A vector comprising the DNA according to claim 3. 7. A recombinant host cell comprising the vector according to claim 6. 8. A method for producing a hyperthermostable chitinase or a variant thereof, comprising a step of culturing the host cell according to claim 7.