JP4438479B2 - Endo-1,4-β-glucanase - Google Patents

Description

  The present invention relates to endo-1,4-β-glucanase useful as an industrial enzyme and a gene encoding them.

  Endo-1,4-β-glucanase is a general term for enzymes that hydrolyze polysaccharides in which glucose is β-1,4-linked, and can degrade plant-derived cellulose, lichenan, and the like. Cellulose is a main component of plant cell walls and is a representative biomass that is effectively used for clothing, paper, building materials, and the like. Rikenan is a storage polysaccharide also called lichen starch, which is a polysaccharide in which glucose present in lichens is β-1,3, β-1,4 linked. It is possible to convert oligosaccharides produced by the degradation of cellulose and lichenan into fuel substances and higher value-added metabolites via glucose. It is an essential enzyme when plant cells are converted to protoplasts. Therefore, endo-1,4-β glucanase is one of the enzymes that have been energetically studied in various countries around the world (see, for example, Patent Documents 1 and 2).

  Furthermore, in recent years, with the development of genetic engineering, production of industrial enzymes has been mass-produced by genetic recombination. Endo-1,4-β-glucanase is not an exception, and many genes have already been cloned and nucleotide sequences have been determined and used in actual production (see, for example, Patent Documents 3 and 4).

Accordingly, an object of the present invention is to find an endo-1,4-β-glucanase useful as an industrial enzyme, obtain a gene encoding it, and use a large amount of a single endo-1,4-β-glucanase using the gene. Is to establish a method of manufacturing.
Sawao Murao et al., Cellulase, Kodansha Scientific, 1987 Tomme et al., Advances in Microbial Physiology, 37, pp 1-75, 1995 International Publication No. 91/17243 Pamphlet Japanese Patent Laid-Open No. 10-313859

The present inventor screened endo-1,4-β-glucanase producing bacteria from the natural world. As a result, the present inventor found a microorganism producing an enzyme suitable for the above purpose, and further encoded endo-1,4-β-glucanase from the microorganism. The present invention has been completed by cloning the genes to be cloned.

That is, the present invention provides the following protein (a) or (b):
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 1,
(B) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of (a) and having endo-1,4-β-glucanase activity, and encodes the protein A gene, a recombinant vector containing the gene, and a transformant having the recombinant vector are provided.

  By using the endo-1,4-β glucanase of the present invention, it is possible to construct a system that decomposes polysaccharides such as cellulose and lichenan and converts them into fuel substances and higher-value-added metabolites. In addition, if the endo-1,4-β glucanase gene of the present invention is used, a useful enzyme can be produced in a single and large amount.

  The protein of the present invention (hereinafter also referred to as “endo-1,4-β-glucanase”) is (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, or (b) (1) in the amino acid sequence of (a). It is a protein having an amino acid sequence in which several amino acids are deleted, substituted or added, and having endo-1,4-β-glucanase activity, and the gene of the present invention is DNA encoding the protein.

Here, in the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence in which one or several amino acids are deleted, substituted or added means an amino acid sequence equivalent to the amino acid sequence of SEQ ID NO: 1, Is an amino acid sequence in which 1 to 10 amino acids are deleted, substituted or added, and still retains endo-1,4-β-glucanase activity. Includes the addition of one to several amino acids.
The base sequence encoding the equivalent amino acid sequence can be prepared by using a known method such as site-directed mutagenesis, in addition to being obtained from nature. For example, it can be prepared by introducing a mutation using a mutagenesis kit [Mutan-super Express Km kit (Takara)] using site-directed mutagenesis.

Comparing the amino acid sequence of endo-1,4-β glucanase (hereinafter referred to as Egl-546H) shown in SEQ ID NO: 1 of the present invention with the amino acid sequence of a conventionally known endoglucanase, the Bacillus lautas PL236 strain was compared. The identity with the produced endoglucanase B (Jorgensen & Hansen, Gene, 93, 55-60, 1990) was the highest, 64.4%. Next, 59% identity with an endoglucanase derived from Bacillus sp. CBS669.39 (European Patent No. 1275712A2), endo-1,4-β-glucanase produced by Bacillus halodurans (Takami et al., Nucleic Acids Res., 28 , 4317-433, 2000), which was 57.7%, and all showed moderate identity with the endoglucanase encoded by the gene of the present invention. This suggests that Egl-546H is a novel endo-1,4-β-glucanase and therefore endo-1 having up to 65% identity with the amino acid sequence shown in SEQ ID NO: 1. , 4-β glucanase is included in the present invention.

That is, the present invention includes endo-1,4-β-glucanase having 65% or more identity when the corresponding sequence in the amino acid sequence shown in SEQ ID NO: 1 is appropriately aligned. The endo-1,4-β-glucanase of the present invention has an identity in the amino acid sequence shown in SEQ ID NO: 1, preferably 70% or more, more preferably 75% or more, still more preferably 85% or more, and most preferably 95%. The above is desirable.
The amino acid sequence identity described here is calculated by the Lipman-Pearson method (Science, 227, 1435, (1985)). Specifically, using a homology analysis (Search homology) program of genetic information processing software Genetyx-Win (Ver. 5.1.1; software development), Unit size to compare (ktup) is set to 1. Is calculated by

  The endo-1,4-β glucanase gene of the present invention may be any protein as long as it encodes a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 or a protein consisting of an amino acid sequence equivalent to the amino acid sequence, as described above. Hybridizes under stringent conditions to a gene having the base sequence shown in SEQ ID NO: 2 or a base sequence in which one or more bases of the base sequence are deleted, substituted or added, or DNA comprising the base sequence A gene containing a DNA encoding an alkaline protease is preferable.

  Here, the “stringent conditions” include, for example, conditions described in Molecular cloning-a Laboratory manual 2nd edition (Sambrook et al., 1989). For example, 6XSSC (composition of 1XSSC: 0.15M sodium chloride, 0.015M sodium citrate, pH 7.0), 0.5% SDS, 5X Denhart and 100 mg / mL herring sperm DNA together with the probe at 65 ° C. Examples include conditions of constant temperature for 8 to 16 hours for hybridization.

The endo-1,4-β-glucanase of the present invention is cloned from a microorganism belonging to the genus Bacillus , preferably Bacillus sp. KSM-N546 strain (FERM P-19729), etc. using a shotgun method or PCR method. In addition, it can be produced in large quantities using appropriate vectors and host bacteria.

In order to produce endo-1,4-β glucanase using the gene of the present invention, the above endo-1,4-β glucanase gene is added to any vector suitable for expressing the gene in the target host. Integration, transformation of the host using the recombinant vector, culturing the resulting transformant, and collecting endo-1,4-β-glucanase from the culture medium.
The culture may be carried out by inoculating a medium containing a carbon source, a nitrogen source and other essential nutrients that can assimilate microorganisms, and following a conventional method.

  Collection and purification of endo-1,4-β-glucanase from the thus obtained culture can be performed according to a general method. That is, the cells can be removed from the culture by centrifugation or filtration, and the target enzyme can be concentrated from the obtained culture supernatant by conventional means. The enzyme solution or dry powder thus obtained can be used as it is, but can be further crystallized or granulated by a known method.

  As characteristics of the endo-1,4-β glucanase of the present invention, the following properties are preferable. That is, it is desirable that the molecular weight is about 60 kDa (SDS-polyacrylamide gel electrophoresis), the optimum reaction pH is around 5 (acetate buffer), and the optimum temperature is around 55 ° C.

  The endo-1,4-β glucanase gene of the present invention can be cloned from, for example, the Bacillus sp. KSM-N546 strain having the following mycological properties.

(Mycological properties of Bacillus sp. KSM-N546 strain)
A: Morphological properties (a) Cell shape and size: Neisseria gonorrhoeae (0.3-0.6 × 1.5-4.3 μm)
(B) Polymorphism: None (c) Mobility: Existence (d) Spore shape, size, position, presence / absence of swelling: oval, 0.4-0.6 × 0.8-1.4 μm, Central near edge, no swelling (e) Gram staining: positive

B: physiological properties (a) nitrate reduction: positive (c) VP test: negative (d) indole production: negative (e) hydrogen sulfide production: negative (f) starch hydrolysis: positive (g) casein Hydrolysis: negative (h) gelatin liquefaction: positive (i) citric acid utilization: positive (j) propionic acid utilization: negative (k) tyrosine degradation: negative (l) phenylalanine degradation: negative (m) lecithinase reaction : Negative (n) Catalase: Positive (o) Oxidase: Positive (p) Temperature range of growth: 15-50 ° C
(Q) Effect of oxygen on growth: Grows under anaerobic conditions.
(R) Growth at pH 5.7: No growth (s) Growth at pH 6.8: Growth (t) Growth at pH 9.0: Growth (u) Gas production from glucose: Negative (v) Resistance to sodium chloride : Grows in the presence of 2% sodium chloride.
(W) Utilization of sugar: Acid is produced using glucose, arabinose, xylose and mannitol.

  As described above, the KSM-N546 strain is an alkali-resistant bacterium and is a gram-positive and catalase-positive spore-forming gonococcus, and thus was determined to be a Bacillus bacterium. Therefore, as a result of comparative studies on the morphology and physiological properties of this strain in accordance with Bergey's manual of Systematic Bacteriology (9th edition), it was considered that this strain is a close relative to Bacillus coagulans. However, since its properties do not completely match those of the known Bacillus coagulans and do not match those of other Bacillus species, this strain is regarded as a novel Bacillus genus and is a patented organism of the National Institute of Advanced Industrial Science and Technology. Deposited at the depository center as Bacillus SP KSM-N546 strain (FERM P-19729).

  As a method for cloning the endo-1,4-β-glucanase gene from the above-mentioned KSM-N546 strain, it can be carried out using a known means such as a shotgun method or a PCR method.

  Moreover, in order to produce a recombinant vector containing the endo-1,4-β-glucanase gene of the present invention, replication can be maintained in the host cell, the enzyme can be stably expressed, The endo-1,4-β glucanase gene may be incorporated into a vector that can be stably maintained. 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.

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.

  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 collected and purified by a general method, and the necessary enzyme form can be obtained by freeze-drying, spray-drying, and crystallization.

Example 1 Screening of endo-1,4-β-glucanase producing bacteria A small amount of soil samples from various parts of Japan were suspended in 10 mL of sterilized water and heat-treated at 80 ° C. for 15 minutes. After cooling at room temperature, 0.1 mL of the supernatant was applied to an agar plate medium having the following composition [3.0% (w / v) trypticase soy broth (BBL), 2.0% phosphoric acid swelling Cellulose (prepared from cellulose powder KC Flock W-100G, Nippon Paper Industries) (separate sterilization), 1.5% agar, 0.005% trypan blue (Merck, Inc .: separate sterilization), 0.01% sodium carbonate (separate sterilization) ]. After culturing at 30 ° C. for 7 to 10 days, a halo formed around the colony was selected as an alkaline cellulase-producing strain.

Example 2 Preparation of genomic DNA of Bacillus sp. KSM-N546 strain Bacillus sp. KSM-N546 strain was cultured in 2.0% (w / v) polypeptone S (Dainippon Pharmaceutical), 0.1% carboxycellulose (A10MC). , 0.1% yeast extract (Difco), 1.0% fish meat extract (Wako Pure Chemicals), 0.15% monopotassium phosphate, 0.035% magnesium sulfate heptahydrate, 0.3% sodium carbonate (other (Sterilization) was used, and the mixture was shaken (125 rpm) at 30 ° C. for 40 hours. 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 (Biochim. Biophys. Acta, 72, 619-629, 1963).

37 ° C. EXAMPLE 3 Egl-546h gene fragment genomic DNA Cloning N546 strain (2 [mu] g) with restriction enzymes Hin dIII, after completely decomposed overnight, ethanol precipitated, and the resulting precipitate of 2.5μL deionized Dissolved in water. Also, previously treated with Hin dIII, pUC18 was dephosphorylated ethanol precipitated (Takara Bio) 50 ng, the precipitate was dissolved in deionized water 2.5 [mu] L. Each solution was mixed (5 μL) and Ligation Kit Ver. 2 (Takara Bio) solution I (5 μL) was added, and the DNA ligation reaction was carried out at 16 ° C. for 30 minutes. E. coli HB101 strain is transformed using the reaction solution, and LB agar medium containing 100 μg / mL ampicillin, 0.5% (w / v) carboxymethylcellulose (Kanto Chemical), 0.005% trypan blue (Merck) Smeared on. Plasmid DNA was extracted from transformants that formed halos with cellulase production using the High Pure Plasmid Purification kit (Boehringer Mannheim). Using the resulting plasmid DNA as a template, the base sequence was determined with a 377 DNA sequencer (PE Applied Biosystems) using M13 Primer M2 and RV (Takara Bio) complementary to the vicinity of the multicloning site of pUC18. As a result of analyzing the base sequence, it was found that the upstream was missing from the middle of the signal sequence. Therefore, genomic DNA was treated with the restriction enzyme Bam H I, and inverse PCR was performed. Specifically, genomic DNA (500 ng) of N546 strain was completely decomposed with restriction enzyme Bam H I, ethanol was added, and the resulting precipitate was dissolved in 10 μL of deionized water. Ligation kit Ver. 40 μL of 1 (Takara Bio) solution A and 10 μL of solution B were added to perform self-ring closure, ethanol was added, and PCR was carried out using the resulting precipitate dissolved in 5 μL of deionized water as a template. . As primers, upstream primer 1 (SEQ ID NO: 3) and downstream primer 2 (SEQ ID NO: 4) constructed using the already determined base sequence were used. PCR was performed according to the instructions of LA Taq polymerase (Takara Bio) using 0.5 μL of a self-closing solution and 2 pmol of each primer. The reaction conditions were heat denaturation at 94 ° C. for 2 minutes, followed by 16 cycles of 94 ° C. for 20 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 4 minutes. Next, for the purpose of gradually extending the extension reaction time, the extension reaction (72 ° C. 4 minutes) is programmed to increase by 20 seconds per cycle in a cycle of 94 ° C. for 20 seconds, 60 ° C. for 30 seconds, 72 ° C. for 4 minutes. The mixture was allowed to react for 12 cycles and then allowed to stand at 72 ° C. for 10 minutes. When 1.5% (w / v) agarose gel electrophoresis was performed using the obtained PCR product, a band was confirmed at a position of about 1.6 kb. The PCR product was purified using the High Pure PCR Product Purification kit (Boehringer Mannheim), and a direct sequencing was performed using a portion of the PCR product.

Example 5 Production of Egl-546H by transformed Bacillus subtilis The obtained gene encoding the mature region of Egl-546H was designated as an alkaline cellulase (Egl-S237) gene derived from Bacillus sp. KSM-S237 strain (Japanese Patent Application No. 11-013049). Between the upstream expression region and the downstream expression region. That is, the structural gene of Egl-S237 is removed from the plasmid pHYS237 in which the region containing the terminator from the upstream expression region of the Egl-S237 gene is linked to pHY300PLK (Takara Bio) previously treated with Sma I, and Egl- A plasmid was prepared by inserting the structural gene of 546H.

First, using PCRYS237 (about 100 ng) as a template, using upstream primer 3 (SEQ ID NO: 5), downstream primer 4 (SEQ ID NO: 6) 20 pmoL each and Pyrobest polymerase (Takara Bio), PCR (reaction conditions: 94 ° C., 20 seconds, 55 30 cycles of 30 ° C. for 30 seconds and 72 ° C. for 3 minutes). The obtained PCR product was purified using a High Pure PCR Purification kit (Boehringer Mannheim), and blunting of the ends and phosphorylation of the 5 ′ end were performed using a BKL kit (Takara Bio). Thereafter, it was treated with the restriction enzyme XbaI and purified with the Hight Pure PCR Purification kit.

  Next, the Egl-546H gene fragment was obtained by PCR using primers 5 (SEQ ID NO: 7) and primer 6 (SEQ ID NO: 8) (reaction conditions: 94 ° C. for 20 seconds, 60 ° C. for 30 seconds, 72 ° C. for 2 minutes) 30 cycles). The obtained PCR product was blunted at the end and phosphorylated at the 5 'end with the BKL kit. Then, it processed with the restriction enzyme XbaI and refine | purified. These amplified gene fragments were ligated using the Ligation Solution attached to the BKL kit. This ligation solution was purified, dried, dissolved in 3 μL of sterilized deionized water, and the entire amount was used to transform the Bacillus subtilis ISW1214 strain by the protoplast method. DM3 regeneration medium [0.8% (w / v) agar (Japanese) Mitsui), 0.3M disodium succinate hexahydrate, 0.5% casamino acid technical (Difco), 0.5% yeast extract, 0.35% monopotassium phosphate, 0.15% phosphoric acid 2 Potassium, 0.5% glucose, 0.4% magnesium chloride hexahydrate. A colony showing a halo on 0.01% bovine serum albumin (Sigma), 0.5% CMC (Kanto Chemical) 0.005% trypan blue (Merck)] was selected as a transformant containing the target PCR product. The obtained transformant was treated with 3.0% (w / v) polypeptone S, 3.0% maltose, 0.5% fish meat extract, 0.1% potassium monophosphate, 0.02% magnesium sulfate 7 water. The culture was carried out with shaking at 30 ° C. for 72 hours in a medium consisting of salt and tetracycline (7.5 μg / mL). Cellulase activity in the culture supernatant obtained by centrifugation is 1.2 U / mL, and the molecular weight of the major protein band detected by SDS electrophoresis is about 60 kDa, which is deduced from the amino acid sequence. The molecular weight (61.3 kDa) almost coincided.

Example 6 Purification of Egl-546H After 150-fold dilution of the culture solution obtained in Example 5 with deionized water, SuperQ Toyopearl previously equilibrated with 50 mM Tris-HCl buffer (pH 7.5) It was attached to a column (2.5 × 8.5 cm). After washing with the same buffer, the adsorbed protein was eluted by a linear concentration gradient method up to 0.2M potassium chloride concentration. Further, the adsorbed protein was eluted with 0.5 M potassium chloride. Egl activity was eluted in this fraction, and concentrated and desalted with an ultrafiltration membrane. The obtained enzyme solution was added to DEAE-Toyopearl 650M (2.5 × 4.5 cm) which had been equilibrated in advance with 10 mM phosphate buffer (pH 6). After washing and elution, the adsorbed protein was eluted by a linear concentration gradient method up to 0.3M potassium chloride concentration. Egl activity was eluted around 0.2M potassium chloride concentration, and this fraction was collected, concentrated with an ultrafiltration membrane, and used for examining various properties.

[Enzyme activity measurement method]
A reaction solution consisting of 0.1 mL of 0.5 M acetate buffer (pH 5.0), 0.4 mL of 2.5% (w / v) carboxymethyl cellulose (A01MC; Nippon Paper Industries), and 0.4 mL of deionized water After adding 0.1 mL of appropriately diluted enzyme solution and reacting at 40 ° C. for 20 minutes, 1 mL of dinitrosalicylic acid reagent (0.5% dinitrosalicylic acid, 30% Rochelle salt, 1.6% sodium hydroxide aqueous solution) was added. The mixture was added and incubated for 5 minutes in boiling water. 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.

Reference Example 1 Optimal reaction pH and temperature of Egl-546H Quen buffer (pH 4-7), Phosphorus buffer (pH 6-8), Tris-HCl buffer (pH 7-9), Glycine-sodium hydroxide buffer (pH 8) To 11), and the optimum reaction pH was examined using each buffer solution (50 mM) of phosphate-sodium hydroxide buffer solution (pH 12 to 12.5). As a result, N546H cellulase was most in the citrate buffer solution at pH 5.3. High reaction rate was shown. In the acetate buffer (pH 5.0), the optimum reaction temperature for CMC degradation was found around 40 ° C.

Reference Example 2 Substrate specificity of Egl-546H When the degradation rate for CMC in acetate buffer (pH 5.0) of Egl-546H is 100%, 251% and 177% for lichenan, glucomannan and pectin, respectively. A relative speed of 38%. On the other hand, it had no effect on laminarin and curdlan.

Claims (6)

The following protein (a) or (b):
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 1,
(B) A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of (a) and having endo-1,4-β glucanase activity.   A gene encoding the protein according to claim 1. Endo-1,4-β glucanase gene comprising the following DNA (a) or (b):
(A) DNA represented by the base sequence shown in SEQ ID NO: 2,
(B) A DNA that hybridizes with a DNA comprising the base sequence of (a) under a stringent condition and encodes a protein having endo-1,4-β-glucanase activity.   A recombinant vector containing the gene according to claim 2 or 3.   A transformant comprising the recombinant vector according to claim 4.   The transformant according to claim 5, wherein the host is a microorganism.