JP4340382B2 - Alkaline cellulase gene - Google Patents

Description

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【図面の簡単な説明】
【図１】本発明の組換えアルカリセルラーゼ活性に及ぼすpHの影響を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a gene encoding an alkaline cellulase that can be blended in a laundry detergent or the like.
[0002]
[Prior art]
Cellulose is the most abundant biomass resource on earth. Since the 1973 oil shock, as part of the development of alternative petroleum resources, research on technology to convert biomass resources into renewable resources such as ethanol for fuel, industrial materials, and medical materials Is being conducted in a wide range of research institutions around the world. Cellulase, which uses cellulase to reduce environmental impact, was considered to be applied to laundry detergents. However, conventional cellulase is an enzyme that works only in the middle acidic range and works in alkaline clothing detergents. What was possible was not known. However, an alkaline cellulase derived from an alkalophilic Bacillus genus has been found by Minegoshi (Japanese Patent Publication No. 50-28515, Horikoshi & Akiba, Alkalophilic Microorganisms, Springer, Berlin, 1982), and cellulase has been considered difficult so far. Can be applied to heavy detergents for clothing, and alkaline cellulases (Japanese Patent Publication No. 60-23158, Japanese Patent Publication No. 6-030578, US4945053, etc.) actually produced by alkalophilic Bacillus bacteria have been developed. It came to be blended into detergents. The addition of alkaline cellulase to clothing detergents has not only improved detergency but also contributed to making detergents more compact and has become widely established.
[0003]
In general, it has been empirically recognized that, as a common feature of protease, amylase, or lipase, which exhibits an excellent effect as an enzyme for detergents, all have a high isoelectric point. The power is also considered high. Therefore, cellulase as a detergent enzyme is also required to have a high isoelectric point. Alkaline cellulase having a high isoelectric point has so far been derived from some fungi ( Fusariumu oxysporum ). Alone (WO95 / 24471), its isoelectric point is 9, and its detergency is not sufficient.
[0004]
[Problems to be solved by the invention]
Accordingly, the present inventors screened various cellulase-producing bacteria to obtain an alkaline cellulase having a high isoelectric point from the natural world, found a novel alkaline cellulase for detergent having an isoelectric point of about 9.3, and filed a patent application. (Japanese Patent Application No. 2000-50116).
An object of the present invention is to provide a transformant capable of obtaining a gene encoding an alkaline cellulase useful as an enzyme for detergents and producing a large amount of a single alkaline cellulase by genetic recombination.
[0005]
[Means for Solving the Problems]
The present inventor cloned a gene encoding alkaline cellulase from an alkaline cellulase-producing bacterium named as Bacillus sp. KSM-N257 strain and deposited as FERMP-17473, and is excellent in productivity of alkaline cellulase by gene recombination. It was found that transformants obtained were obtained.
[0006]
The present invention provides an alkaline cellulase gene encoding the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence in which one or several amino acids of the amino acid sequence have been deleted, substituted or added.
The present invention also provides a recombinant vector containing the above alkaline cellulase gene and a transformant containing the recombinant vector.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
The gene of the present invention has a base sequence encoding the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence in which one or several amino acids of the amino acid sequence have been deleted, substituted or added. As long as the alkaline cellulase activity is not lost, the amino acid deletion, substitution or addition (hereinafter sometimes referred to as mutation) in the amino acid sequence is not particularly limited. In addition, one to several amino acids may be added, deleted, or substituted at the amino terminus in the amino acid sequence (amino acid numbers 1 to 407) of the mature enzyme shown in SEQ ID NO: 1.
[0008]
Comparing the amino acid sequence of alkaline cellulase (hereinafter referred to as N257 cellulase) shown in SEQ ID NO: 1 of the present invention with the amino acid sequence of a conventionally known cellulase, the cellulase produced by Bacillus circulans (Bueno et al., Nucleic Acids Research) 18, 4248, 1990), the highest homology with 75.9%, and cellulase derived from Bacillus sp. KSM-330 strain (Ozaki et al., J. Gen. Microbiol., 137, 41-48, 1991) And 44.4%, and cellulase derived from Clostridium josui (EMBL D16670, G391654) was 27.8%. The cellulase produced by Bacillus circulans that showed the highest homology (homology 75.9%) is a lichenase belonging to endo β-1,3-1,4 glucanase (EC 3.2.1.73), and the gene of the present invention N257 cellulase encoded by (EC 3.2.1.4) is an enzyme that is classified differently. In addition, cellulase (EC 3.2.1.4) derived from Bacillus sp. KSM-330 is a so-called acid cellulase. Therefore, the alkaline cellulase encoded from the gene of the present invention is a novel enzyme.
The homology search was performed using GENENTYX-CD biodata software [Software Development Co., Ltd., ver. 36] using the amino acid sequence homology search method.
[0009]
The alkaline cellulase gene of the present invention only needs to encode the amino acid sequence shown in SEQ ID NO: 1 or a variant thereof.
The alkaline cellulase gene of the present invention can be obtained, for example, by cloning from Bacillus sp. KSM-N257 strain (FERM P-17473) or the like using a known shotgun method, PCR method or the like.
[0010]
In addition, in order to produce a recombinant vector containing the alkaline cellulase gene of the present invention, it is possible to maintain replication in a host cell, to stably express the enzyme, and to a vector that can stably hold the gene. An alkaline cellulase gene may be incorporated. Examples of such vectors include pUC18, pBR322, pHY300PLK and the like when Escherichia coli is used as a host, and pUB110, pHSP64 (Sumitomo et al., Biosci. Biotechnol, Biocem., 59, 2172-2175, 1995) when Bacillus subtilis is used as a host. ) Or pHY300PLK.
[0011]
Transformation of a host bacterium using the recombinant vector thus obtained can be performed using a protoplast method, a competent cell method, an electroporation method, or the like. Host bacteria are not particularly limited, but Gram-positive bacteria such as Bacillus (Bacillus subtilis), Gram-negative bacteria such as Escherichia coli (Escherichia coli), Streptomyces (actinomycetes), Saccharomyces (yeast), Aspergillus (mold), etc. Of fungi.
[0012]
The obtained transformant may be cultured under suitable conditions using a medium containing an assimilating carbon source, nitrogen source, metal salt, vitamin and the like. From the culture solution thus obtained, the enzyme can be separated and purified by a general method, and the necessary enzyme form can be obtained by lyophilization, spray drying, and crystallization.
[0013]
【Example】
Example 1 Screening for Alkaline Cellulase-Producing Bacteria Suspension of soil in various places in Japan was heat-treated at 80 ° C. for 30 minutes and applied to an agar plate medium having the following composition [2.0 wt% %) Carboxymethylcellulose (A10MC), 1.0% meat extract (Oxoid), 1.0% bactopeptone (Difco), 1.0% sodium chloride, 0.1% potassium dihydrogen phosphate, 0 .5% sodium carbonate (separately sterilized)]. The culture was allowed to stand for 3 days in a 30 ° C. incubator, and after growth of the bacteria, a 0.1% (w / v) Congo red solution was poured and dyed. Thereafter, the cells were decolorized with a 1M sodium chloride solution, and those that were found to have dissolved spots associated with the degradation of carboxymethylcellulose around the grown bacteria were selected, and single colonization was repeated. These strains are 2.0% polypeptone S (Nippon Pharmaceutical Co., Ltd.), 1.0% fish meat extract (Wako Pure Chemical Industries), 0.15% potassium monohydrogen phosphate, 0.1% yeast extract (Difco), 0.07. Using a liquid medium composed of 7% magnesium sulfate heptahydrate, 0.1% carboxymethylcellulose and 0.5% sodium carbonate (separately sterilized), shaking culture was performed at 30 ° C. for 3 days. The culture supernatant was collected and subjected to isoelectric focusing using Phast-system (Pharmacia; IEF gel, pH 3.0-9.0). The electrophoresed gel was immersed in 10 mM phosphate buffer (pH 7.0), and then an agar plate containing carboxymethylcellulose [1.0% carboxymethylcellulose, 50 mM glycine-sodium hydroxide buffer (pH 9.0), 1.5 % Agar]. After leaving at 30 ° C. for 3 hours, the gel was removed and a 0.1% Congo red solution was poured. About 10 minutes later, the solution was decolorized with a 1M sodium chloride solution, and the one having a dissolution spot associated with the decomposition of carboxymethylcellulose was selected at a high isoelectric point to obtain Bacillus sp. KSM-N257 strain.
[0014]
The Bacillus sp. KSM-N257 strain had the following mycological properties.
A. Morphological properties (a) Cell shape and size: Neisseria gonorrhoeae (0.6-0.8 × 3.2-6.8 μm)
(B) Polymorphism: None (c) Mobility: Existence (d) Spore shape, size, position, presence / absence of swelling: elliptical, 0.8-1.0 × 1.0-1.6 μm, Quasi-end position, swelling.
(E) Gram staining: indefinite, but does not grow on crystal violet (CVT) agar.
(F) Antiacid: Negative
B. Physiological properties (a) Nitrate reduction: positive (b) Denitrification reaction: negative (c) MR test: positive (pH 5-5.5)
(D) VP test: negative (e) indole production: negative (f) hydrogen sulfide production: negative (g) starch hydrolysis: positive (h) casein hydrolysis: negative (i) gelatin liquefaction: negative (J) Use of citric acid: negative (k) catalase: positive (l) oxidase: positive (m) urease: negative (n) growth temperature range: 13-14 ° C
(O) Growth pH range: pH 6-10
(P) Effect of oxygen on growth: grow under anaerobic conditions.
(Q) Sodium chloride tolerance: Grows in the presence of 10% sodium chloride.
(R) Gas production from glucose: negative (s) Acid production from sugar: glucose, arabinose, xylose, mannitol, galactose, sucrose, mannose, maltose, lactose, trehalose, fructose, melibiose, ribose, salicin, glycerol, Acid production is observed from rhamnose and the like, and no acid production is observed from sorbitol and inositol.
[0016]
Example 2 Preparation of genomic DNA of Bacillus sp. KSM-N257 strain
Bacillus sp. N257 strain was cultured with 2% polypeptone S (Dainippon Pharmaceutical), 0.1% carboxycellulose (A10MC), 0.1% yeast extract (Difco), 1% fish meat extract, 0.15% phosphoric acid Shake (125 rpm) at 30 ° C. for 40 hours using a medium consisting of potassium monohydrogen, 0.07% magnesium sulfate heptahydrate, 0.5% sodium glutamate (separate sterilization) and 0.5% sodium carbonate (separate sterilization) I went there. The cells were collected from about 300 mL of the obtained culture solution by centrifugation (12000 × g, 15 minutes, 5 ° C.). Genomic DNA was prepared from the cells by the method of Saito and Miura.
[0017]
Example 3 Cloning of N257 Cellulase Gene Fragment (a) Genomic DNA restriction enzyme treatment N257 cellulase gene was obtained using LA PCR in vitro Clonig Kit (manufactured by Takara). First, Bacillus sp. KSM-N257 strain genomic DNA was partially decomposed by Eco Rl treatment according to the following procedure. Specifically, genomic DNA 1.5 μL (1 μg), 10-fold buffer 10 μL, deionized water 86.5 μL, and Eco Rl 2 μL (20 units) were mixed, treated at 37 ° C. for 3 hours, and after completion of the reaction, GFX PCR DNA and Gel Band Purification Kit (Pharmacia) and purified (25 μL).
[0018]
(B) Ligation reaction To 5 μL of the purified genomic DNA Eco Rl-degraded fragment solution, 2.5 μL (50 ng DNA) of Eco Rl cassette of LA PCR in vitro Clonig Kit, 15 μL of ligation solution I and 7.5 μL of ligation solution II were added, and 16 ° C. After 30 minutes of reaction, DNA was purified and recovered by GFX PCR DNA and Gel Band Purification Kit.
[0019]
(C) N257 cellulase gene amplification ligation purified by PCR 14.5 μL of deionized water was added to 20 μL of the purified solution, followed by heat treatment at 94 ° C. for 10 minutes, and then the first PCR was performed as follows. The culture supernatant of 34.5 μL of heat-treated solution, 5 μL of 10-fold concentration buffer in LA PCR in vitro Clonig Kit, 0.5 μL of LA Taq polymerase, 8 μL of dNTP mix, 1 μL (10 pmol) of primer C1 and Bacillus sp. KSM-N257 strain 1 μL (10 pmol) of composite primer 1 (SEQ ID NO: 3) estimated from the N-terminal sequence of the more purified alkaline cellulase was mixed. The PCR reaction conditions were 94 ° C, heat denaturation for 1 minute, and then 94 ° C, 30 seconds, 55 ° C, 2 minutes, 72 ° C, 1 minute (30 cycles). The obtained PCR reaction solution was diluted 100 times with deionized water, and a second PCR reaction was performed using 1 μL. 1 μL of a 100-fold diluted solution of 5 μL of 10-fold concentration buffer in LA PCR in vitro Clonig Kit, 0.5 μL of LA Taq polymerase, 8 μL of dNTP mix, 1 μL (10 pmol) of primer C2 and purified enzyme from Bacillus sp. KSM-N257 strain 1 μL (10 pmol) of the composite primer 2 (SEQ ID NO: 4) estimated from the N-terminal sequence was mixed, and the DNA was amplified with a thermal cycler 480 (Perkin Elmer). PCR reaction conditions were 94 ° C for 1 minute after heat denaturation, followed by 94 ° C, 30 seconds, 55 ° C, 2 minutes, 72 ° C, 1 minute (30 cycles). Using the thus obtained N257 cellulase gene partially amplified fragment (about 0.7 kb), the nucleotide sequence was determined. The base sequence of the obtained DNA fragment was determined using a DNA Sequencing Kit (Applied Biosystem) and a 377 DNA sequencer (Perkin Elmer Biosystem).
[0020]
Example 4 Total nucleotide sequencing (a) Inverse PCR ( Eco Rl)
The nucleotide sequence of the partially amplified fragment (about 0.7 kb) of the N257 cellulase gene was analyzed using primer C2 (3.2 pmol) and primer 2 (SEQ ID NO: 4, 3.2 pmol) as a sequence primer. Inverse PCR was performed based on the determined base sequence of about 0.5 kb. Bacillus sp. KSM-N257 strain genomic DNA solution 1.5 μL (1 μg), 10-fold concentration buffer 10 μL, deionized water 86.5 μL and Eco Rl 2 μL (20 units) were mixed, followed by restriction enzyme treatment at 37 ° C. for 3 hours. did. The resulting genomic DNA degradation product was purified with GFX PCR DNA and Gel Band Purification Kit (Pharmacia) (20 μL), and 5 μL was used for Ligation Kit Ver. 2 (manufactured by Takara) was used for self-ring closure (16 ° C., 1 hour). PCR was performed using 1 μL of the obtained ligation mixture as template DNA. Antisense primer 3 (SEQ ID NO: 5) and sense primer 4 (SEQ ID NO: 6) were used. The PCR reaction consists of 1 μL of a self-closing solution, 20 μL of each primer (1 μM), 10 μL of 10-fold concentration buffer, 8 μL of dNTP mix (2.5 mM), Pyrobest DNA polymerase (manufactured by Takara) 0.5 μL (2.5 units), and desorption. After mixing 40 μL of ionized water, the DNA was amplified with a thermal cycler. The reaction conditions were 94 ° C, 2 minutes after heat denaturation, 94 ° C, 30 seconds, 57 ° C, 1 minute, 72 ° C, 2 minutes (30 cycles) and 72 ° C, 2 minutes. The obtained PCR product (about 0.7 kb) was purified using GFX PCR DNA and Gel Band Purification Kit (Pharmacia), and then antisense primer 5 (SEQ ID NO: 7) and sense primer 6 (SEQ ID NO: 8) were used. Then, sequencing was performed, and a base sequence of about 1.1 kb was determined between the Eco Rl site (base number 1) in the promoter region of the N257 cellulase gene and the Eco Rl site (base number 1090) in the downstream structural gene.
[0021]
(Ii) Inverse PCR ( Pvu II)
In order to determine the undetermined base sequence downstream of the Eco Rl site in the structural gene, inverse PCR was performed again using Pvu II. Bacillus sp.KSM-N257 strain genomic DNA solution 1.5μL (1μg), 10-fold concentrated buffer solution 10 [mu] L, after mixing deionized water 86.5μL and Pvu II 2μL (20units), 3 hours digested with 37 ° C. did. The obtained genomic DNA degradation product was purified by GFX PCR DNA and Gel Band Purification Kit (Pharmacia) (20 μL), and 5 μL was used for Ligation Kit Ver. 2 (manufactured by Takara) was self-closing (16 ° C., 1 hour). Using 1 μL of the obtained ligation mixture as template DNA, PCR was performed using antisense primer 7 (SEQ ID NO: 9) and sense primer 4 (SEQ ID NO: 6). The PCR reaction consists of 1 μL of a self-closing solution, 20 μL of each primer (1 μM), 10 μL buffer solution 10 μL, dNTP misk 8 μL (2.5 mM), Pyrobest DNA polymerase (Takara) 0.5 μL (2.5 units), and desorption After mixing 40 μL of ionized water, the DNA was amplified with a thermal cycler. The reaction conditions were 94 ° C., heat denaturation for 2 minutes, 94 ° C., 30 seconds, 57 ° C., 1 minute, 72 ° C., 2 minutes (30 cycles) and 72 ° C., 2 minutes. The obtained PCR product (about 1.5 kb) was purified using GFX PCR DNA and Gel Band Purification Kit (Pharmacia), and then sense primer 6 (SEQ ID NO: 8) and sense primer 8 (SEQ ID NO: 10) were used. Then, an undecided nucleotide sequence downstream of the Eco Rl site in the structural gene was determined. As described above, the entire nucleotide sequence of the N257 cellulase gene including the promoter region and the terminator region was determined.
[0022]
Sal l / Bam Hl and ligated into the site, ISW1214 strain Bacillus subtilis recombinant plasmids constructed plasmid (pHSP64) gene to the terminator downstream from the amino terminus of the production N257 cellulase N257 cellulase according to Example 5 Transformation Bacillus subtilis And transformed. The transformed strain was 3.0% polypeptone S, 3.0% maltose, 0.5% fish meat extract, 0.1% yeast extract, 0.1% potassium dihydrogen phosphate, 0.02% magnesium sulfate heptahydrate. And cultured in a medium (PM medium, pH 6.8) consisting of tetracycline (7.5 μg / mL) at 30 ° C. for 48 hours, and obtained by centrifugation (8000 × g, 20 minutes, 4 ° C.). The activity of cellulase in the culture supernatant was about 10,000 U / L.
[0023]
Example 6 Purification of Recombinant N257 Alkaline Cellulase Recombinant N257 culture supernatant was subjected to 85% saturated ammonium sulfate salting out, followed by centrifugation (12000 × g, 15 minutes, 5 ° C.) to collect a precipitate. This precipitate was suspended in an appropriate amount of 10 mM Tris-HCl buffer (pH 7.5) containing 2 mM calcium chloride, and dialyzed against a sufficient amount of the same buffer at 5 ° C. for 15 hours. Centrifugation (12000 × g, 15 minutes, 5 ° C.) of the dialyzed solution, and the obtained supernatant was previously equilibrated with 10 mM Tris-HCl buffer (pH 7.5) containing 2 mM calcium chloride. ) After being attached to a column (2.5 cm × 15 cm), the column was washed with the same buffer, and then the protein was eluted with a linear concentration gradient of 0 to 0.2 M sodium chloride in the same buffer. The target N257 alkaline cellulase was electrophoretically eluted as an almost single component at a sodium chloride concentration of around 0.02M. The specific activity was 14.8 U / mg.
[0024]
Example 7 Optimal reaction pH of recombinant N257 alkaline cellulase
The optimal reaction pH was examined using a recombinant N257 alkaline cellulase purified sample prepared as in Example 6. Acetate buffer (pH 5.1-6.6), phosphate buffer (pH 6.2-7.9), Tris-hydrochloric acid buffer (pH 7.6-9.1), glycine-sodium hydroxide buffer ( pH 8.5-10.7), carbonate buffer (pH 10.8-11.5), and potassium chloride-sodium hydroxide buffer (pH 10.5-11.4), each buffer solution (50 mM) is optimal. As a result of examining the reaction pH, this enzyme showed the highest reaction rate in a glycine-sodium hydroxide buffer solution at pH 8.5. Moreover, it showed an activity of 70% or more of the maximum activity between pH 7 and 9, and had an activity of 10% or more of the maximum activity even in a wide range of pH 5 to 10 (FIG. 1). Recombinant N257 alkaline cellulase adsorbs to the cation exchanger with a molecular weight of about 42 kDa and pH 7.5, and is eluted with sodium chloride in the same manner as the wild-type enzyme, so it has a high isoelectric point and an optimum reaction pH of pH 8. 5-20 N-terminal amino acid sequence Ala-Asn-Lys-Pro-Phe-Pro-Gln-His-Thr-Ser-Tyr-Thr-Ser-Gly-Ser-Ile-Lys-Pro-Asn-Asn is Bacillus Since it completely matches the N-terminal amino acid of the enzyme derived from sp. KSM-N257 strain, it is an enzyme having the same properties as N257 alkaline cellulase derived from the wild strain.
[0025]
[Enzyme activity measurement method]
From 0.2 mL of 0.5 M glycine-sodium hydroxide buffer (pH 9.0), 0.4 mL of 2.5% (w / v) carboxymethylcellulose (A01MC; Nippon Paper Industries), 0.3 mL of deionized water After adding 0.1 mL of an appropriately diluted enzyme solution to the reaction solution and reacting for 20 minutes, 1 mL of dinitrosalicylic acid reagent (0.5% dinitrosalicylic acid, 30% Rochelle salt, 1.6% sodium hydroxide aqueous solution) And coloring of reducing sugar was performed in boiling water for 5 minutes. After quenching in ice water, 4 mL of deionized water was added and the absorbance at 535 nm was measured to determine the amount of reducing sugar produced. In addition, after adding a dinitrosalicylic acid reagent to the reaction liquid processed without adding an enzyme liquid, the blank added the enzyme liquid and prepared the same color. One unit (1 U) of enzyme was defined as an amount that produces a reducing sugar equivalent to 1 μmol of glucose per minute under the above reaction conditions.
[0026]
【The invention's effect】
The alkaline cellulase gene of the present invention can be used to produce a single alkaline cellulase for detergents in large quantities.
[0027]
[Sequence Listing]

[0028]

[0029]

[0030]

[0031]

[0032]

[0033]

[0034]

[0035]

[0036]

[Brief description of the drawings]
FIG. 1 is a graph showing the effect of pH on the recombinant alkaline cellulase activity of the present invention.

Claims (5)

An alkaline cellulase gene encoding the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence in which one or several amino acids of the amino acid sequence are deleted, substituted or added. The alkaline cellulase gene according to claim 1, wherein the alkaline cellulase gene has a base sequence shown in SEQ ID NO: 2 or a base acid sequence in which one or several bases of the base sequence are deleted, substituted or added. A recombinant vector containing the gene according to claim 1 or 2. A transformant comprising the recombinant vector according to claim 3. The transformant according to claim 4, wherein the host is a microorganism.

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