JPH04190793A - Cellulase gene-containing recombinant plasmid and production of cellulase by recombinant bacterium - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a recombinant plasmid containing a cellulase gene, a recombinant microorganism containing this plasmid, and a method for producing cellulase using the recombinant microorganism.

[Prior Art] Research on cellulases in the past several decades has a history of being conducted from the viewpoint of the effective use of biomass resources and the saccharification of malt in the brewing industry. (A
s er 1llus), Acremonium (Acre
monium) genus, Humicola (I (ulicoll)
a) Filamentous fungi such as the genus Clostridium, Pseudomonas, Ruminococcus and Cellulomonas, etc. (Sawao Murao et al., "Cellulase," Kodansha (1987)). Recently, a new industrial use of cellulase related to laundry detergent compositions has been developed.
Genus Cellulomonas and Streptomyces
Alkaline cellulase produced by bacteria of the genus Toa + ces has been attracting attention (Japanese Patent Publication No. 50-28.515, Japanese Patent Application Laid-open No. 58-224686, Japanese Patent Application Laid-open No. 1987-61).
280276, JP 63-109771, JP 63-240785, etc.).

However, all of these microorganisms have poor cellulase productivity, and furthermore, cellulase inducers such as cellulose fibers are required for cellulase production, and they also produce a wide variety of similar enzyme groups. Therefore, there have been problems such as difficulty in separating and purifying the target cellulase component.

For this reason, there are very few examples of development of cellulases suitable for industrial use, and only one example is the production of alkalinease as a laundry detergent composition by Bacillus sp. KSM-635, a type of Bacillus bacterium. An example of this can only be found in JP-A-63-109771).

On the other hand, in recent years, in order to solve the above problems, cellulase genes derived from cellulase-producing microorganisms, specifically Trichoderma (Penttjla et al., Gene,
45, p. 253, (1986)), Clostridium (acteriol, from Beguin et al., J.
Vol. 162, p. 102 (1985), Joliff et al.
5, (1986) and Grepinet et al., Nucl.
eic Ac1ds master, vol. 14, p. 1791, (19
86)), Cellulomonas (Wong et al., Gene,
Volume 44, page 315, (1986), Nakamura
et al., J. Biotechnol, vol. 4, p. 247, (1986)), the genus Bacillus (Robson and Ch.
ambliss.

J, Bacte, Vol. 4169, p. 2017, (1
987), Fuku++ori et al., J. Bacteri
ol, vol. 168, p. 479, (1986), Fuk
umori et al., J. Gen. Microbiol.

Volume 132, page 2329, (1986) and JP-A-1-2
81090), Streptomyces genus (Naka
Kawai et al., Hirokugi Vol. 165, p. 229, (1988)) and Ruminococcus (Kawai et al., A ric, Bi
ol, chea+.

51, p. 59, (1987)) have been cloned using genetic manipulation techniques.

The aim of these efforts is to improve the productivity of cellulases through genetic engineering techniques, and it has been hoped that by cloning genes encoding only the desired cellulase, it will be possible to simplify the production and production of cellulases. . Furthermore, it is thought that it will be possible to improve the functions and properties of cellulases using protein engineering techniques, and this is an extremely significant endeavor.

However, since Escherichia coli has been used as the host bacterium for gene cloning in most cases, the productivity of cellulase by genetically modified microorganisms is low, and the cellulase produced is It is not secreted outside the body and is stored in the periplasmic space (periplasmic space).
Ht'B accumulates in the cells (asg+ic gap), making it difficult to separate Ht'B from intracellular proteins of E. coli.

Also, Bacillus subtilis
The neutral cellulase gene derived from the IF 0 3034 strain (Japanese Patent Application Laid-Open No. 61-280284) and the alkaline cellulase gene from the alkalophilic Bacillus sp. Although these recombinant Bacillus subtilis strains have been cloned, the production of cellulases is low, and so far no examples of economical industrial production of cellulases using recombinant microorganisms are known.

[Means for solving the problem] The present inventor obtained a recombinant microorganism that shunts a large amount of cellulase, and as a result of conducting extensive research in order to advantageously obtain cellulase using this, the present inventor found that a recombinant microorganism derived from Bacillus bacteria was obtained. The cellulase gene was transferred to a specific shuttle vector (5huttle v
It was discovered that a plasmid created by ligating with B. completed.

That is, an object of the present invention is to provide a recombinant plasmid obtained by ligating a DNA fragment containing a cellulase gene derived from a Bacillus bacterium and a shuttle vector capable of replicating in E. m. and Bacillus subtilis.

Another object of the present invention is to provide a recombinant microorganism containing this M flexible plasmid.

Furthermore, another object of the present invention is to provide a method for producing cellulase by culturing the above-mentioned M-flexible microorganism.

The plasmid of the present invention is prepared by linking a cellulase gene derived from a bacterium belonging to the genus Bacillus to a shuttle vector capable of replicating in E. coli and Bacillus subtilis according to a known method.

The microorganism that serves as a donor for the DNA fragment containing the cellulase gene of the present invention is a microorganism of the genus Bacillus that has the ability to produce cellulase, and is preferably a so-called microorganism that has the ability to produce cellulase and exhibits good growth in an alkaline pH region. It is an alkalophilic bacterium belonging to the genus Bacillus. An example of an alkalophilic Bacillus bacterium that has the ability to produce cellulase is Bacillus sp., which produces -64 in alkaline cellulase.
s sp, ) K SM -64 (FERMP-1
0482), etc. This Bacillus sp. KSM-64 was isolated by the present inventors from the soil of Nasu District, Tochigi Prefecture as a microorganism that produces a significant amount of alkaline cellulase -64 outside the bacterial body. It has been deposited as No. 10482. Regarding the taxonomic characteristics of the KSM-64 strain,
Patent application specification dated May 2nd (Japanese Patent Application No. 1-140642)
The main points are as follows.

Since this strain is a type of so-called alkaliphilic bacteria that does not grow at pH below neutral and grows vigorously at alkaline pH, the culture medium used for taxonomic tests usually contains 1. 0% sodium carbonate (N a 2 CO 2 ) was added to a separate sterilized container and used.

(Taxonomy characteristics of Bacillus sp. KSM-64 strain) (a) Microscopic observation results ■Morphology: rod-shaped ■Size 2 0.5-1.2μmX 1.2-6.6
μm ■Spores: Quasi-terminal endospores, cylindrical (0.7-1.
0 μm x 1.5 to 2.5 μm (b) Growth status in various media ■ Broth agar plate medium: Shape of well-growing colonies; Round surface; Round clear edges; Smooth color tone; Milky luster; ■Beat juice liquid medium: Good growth ■Meat juice gelatin high-rise medium (puncture culture): Good growth, liquefies gelatin.

■Lidomus milk medium: No coagulation or peptonization of milk was observed. Discoloration of lidomas cannot be determined because the medium is alkaline (c) Physiological properties ■ Sulfate reduction: Positive ■ Denitrification reaction: Negative ■ MR test: Undeterminable (because the medium is alkaline) ■ VP test: Positive ■Indole raw f6. ．． : Negative■Generation of hydrogen sulfide: Negative■Hydrolysis of starch: Positive■Use of citric acid: Without burning ■Use of inorganic nitrogen source: Both nitrates and ammonium salts are used.

[Phase] Pigment generation: Produces yellow pigment.

■Urease double negative @ oxidase: positive ■Catalase: positive 0 Growth temperature range = 20-40″C0 optimum temperature is 3
0-37°C ■Growth pH range: pH 7-110 Optimum pH is 1
0,5 [Phase] Attitude towards acidity: Aerobic O-F test: Growth only under aerobic conditions, discoloration cannot be determined (because the medium is alkaline) [Phase] Positive for sugar utilization: L-arabinose , D-xylose, glucose, D-mannose, D-fructose, D-galactose, maltose, sucrose, lactose, trehalose, D-mannite, inosit, glycerin, starch negative: D-sorbit in 0 salt-containing medium Growth in: Good growth with 7% salt. No growth at 10%.

[Strain] Decomposition of casein: Positive @ G+C content: Approximately 37.7 mo1% The enzymatic properties of alkaline cellulase-64 produced by the above-mentioned microorganisms are also detailed in the above-mentioned patent application specification. The main points are listed below.

(Enzymological properties of alkaline cellulase-64) (1) Effect It acts on cellulose fibers and purifies reducing sugars.

(2) Substrate specificity In addition to carboxymethyl cellulose (CMC), it acts well on lichenan and also acts on laminarin. In addition, filter paper,
It also has some enzymatic activity against p-nitrophenyl glucoside and p-nitrophenyl cellobioside.

(3) Working pH and optimum pn The effect pn range on CMC is from 3 to 13, and the optimum working pH is 8.5-10.

(4) pH stability Stable within the pH range of 5 to 10.5 (30°C21
time saving).

(5) Working temperature and optimum temperature It acts in the range of 10 to 70°C, and the optimum temperature is 50°C.

(6) Temperature stability Stable at temperatures below 50'C (pH 9, 10 minute treatment).

(7) Two types of molecular weights, approximately 180,000 and approximately 80,000, are provided (gel filtration method using Biogel A-0.5m).

(8) Effect of metal ions It is inhibited by Hg2+ and activated by C○2+ and Mn2+.

(9) Effect of surfactant Straight chain alkylbenzene sulfonate, alkyl sulfate,
Polyoxyethylene alkyl sulfonates, α-olefin sulfonates, 2-sulfo fatty acid salts, secondary alkanesulfonates, dodecyl sulfonates, soaps, and polyoxyethylene secondary non-alkyl ethers hardly inhibit the activity. I,).

(10) Effect of protease Resistant to proteases.

(11) Effect of chelating agents EDTA, EGTA, citric acid, sodium phosphate, and zeolite do not inhibit the activity.

A method for obtaining a DNA fragment containing a cellulase gene from a donor strain belonging to the genus Bacillus is, for example,
(1+1arvur) method (J, Mol.

Biol, Vol. 3, p. 208, (1961)) and Saito and Miura's method (Biochim, Bioph).
ys, Acta.

72, p. 619, (1963)), but other similar methods may also be used.

A DNA fragment containing the gene is prepared by cleaving the chromosomal DNA thus obtained with a restriction enzyme. Any type of restriction enzyme can be used as long as it does not disrupt the gene. can.

For example, Bacillus sp.
When using Hindm as a restriction enzyme, a fragment of approximately 4.4 kb having the restriction enzyme cleavage map shown in FIG. 3 can be obtained without disrupting the gene. Furthermore, even when using a restriction enzyme that cleaves the gene or the essential region for its activity, it is possible to produce tA DNA fragments containing the entire region of the gene by using partial cleavage conditions.

In addition, if a DNA fragment containing the cellulase gene has already been cloned using another vector, the DNA fragment can be cut with an appropriate restriction enzyme number.
NA fragments can be prepared. The present inventors obtained the following by cutting chromosomal DNA with the restriction enzyme Hindm.
Approximately 4.4 kb of DNA (Fig. 3) containing the gene was cloned into the plasmid vector pBR322 for E. coli, and then cut with the restriction enzyme Hindm to create DNA containing the 164 gene in alkaline cellulase. Fragments were prepared.

The 164 genes for alkaline cellulase in the present invention are:
Approximately 4.4kb with the restriction enzyme cleavage map shown in Figure 3
The structural gene is contained in a DNA fragment of approximately 2.5 kb indicated by an arrow at the top of the map. Furthermore, the vicinity of the 164 gene for alkaline cellulase is
It has the base sequence shown in FIG. In this array, A
Translation starts from the TG codon (base number 1), and an open reading frame (open reading frame) that encodes a sequence consisting of 822 amino acid residues shown below the base sequence.
adding frame, 0RF). ○R
10 bases upstream of F is the 3'-end sequence of Bacillus subtilis 168 ribosomal RNA (Mc-Laughlin et al., J.
Biol, Chew, vol. 256, p. 11283, (1
There is an AGGAGGT sequence that is highly complementary to 981)), and further upstream is the common sequence of the σ43 type promoter (
Gitt et al., J. Biol, Chew.

260, p. 7178, (1985), etc.), there are three sequences with high homology (base numbers -123 to -94, -246).
-213 and -403 to -373, the sidelined portion at the top of the base sequence). Furthermore, downstream of the translation stop codon TAA at base number 2467, there is an inverted repeat base sequence that is thought to be a transcription terminator.
eat 5 sequence) exists (base number 24
87-2528, the sidelined part at the bottom of the base sequence).

On the other hand, examples of shuttle vectors that can be replicated in E. coli and Bacillus subtilis used in the present invention include plasmid vector pHY300PLK (manufactured by Yakult Honsha Co., Ltd.).

This shuttle vector is an approximately 4.9 kb shuttle vector encoding an ampicillin resistance gene and a tetracycline resistance gene (Ishiwa and 5hibah
ara, Jpn, J, Genet,, 60 volumes,
p. 235, (1985)). This vector is stably maintained and replicated in both E. coli and Bacillus subtilis, and the tetracycline resistance gene is expressed in both host bacteria, so resistance to this antibiotic can be obtained during transformation. It can be used as an indicator. In addition, a polylinker is linked to the vector, and Hind present on this polylinker
I[I, XbaI, BglII, Sal I, BamH
The restriction enzyme cutting points of I, SmaI and EcoR are as follows:
Since it is not present in other positions on the vector, the vector DNA is made into a linear molecule by cleaving the vector with one of these restriction enzymes, and the cellulase gene, for example, alkaline cellulase, is
It can be used for cloning 4 genes. Furthermore, if the same type of restriction enzyme used for linearization is used to prepare the DNA fragment containing the cellulase gene, for example, the 164 gene for alkaline cellulase, the subsequent It is convenient for joining both DNAs.

A mixture of M-replaced DNA molecules can be prepared by mixing the cellulase gene fragment thus obtained and a linear vector DNA molecule and adding DNA ligase to perform a ligation reaction. Next, a host microorganism is transformed with the mixture of recombinant DNA molecules obtained in this way, the host bacteria containing the desired recombinant plasmid is isolated, and the desired recombinant plasmid is isolated from the desired host strain. Plasmids can be extracted and purified. At this time, it is also possible to directly transform Bacillus subtilis with a mixture of recombinant DNA molecules; Therefore, the WI half that allows the desired recombinant plasmid to be obtained is low, and in some cases, it may not be possible to obtain the desired plasmid. For this reason, it is better to first transform E. coli with the ligation reaction product, amplify the desired recombinant plasmid in E. coli, extract and purify it, and introduce it into Bacillus subtilis. You may get

As for the type of host bacteria, any strain can be used as long as it can stably retain and replicate the desired recombinant plasmid and can produce cellulase. For example, in the case of E. coli host, H8101
strain, 0600 strain or JMI09 strain, and in the case of Bacillus subtilis host, BD170 strain, 168 strain, or l5W strain.
1214 stocks etc. can be mentioned.

The method for transforming a host strain with a recombinant DNA molecule is not particularly limited, but for example, in the case of a large mN host strain, Mandel and Figa &
(J, Mo1. Biol, vol. 53, p. 159 (
1970) ) and Hanahan's method (
J, Mo1. Biol, vol. 166, 55
7 (1983)), and in the case of Bacillus subtilis host strains, competent cells.
) method (Contente and Dabnau, Mo1.
Gen, Genet, vol. 177, p. 459 (19
79) ) and protoplasts
) method (Chang and Cohen, Mo1. Gen.

Genet, vol. 168, p. 111 (1978)'
) etc. can be used.

The method for screening recombinant microorganisms is not particularly limited, but may be performed as follows, for example. That is, first, using the tetracycline resistance trait encoded on the vector DNA molecule as an indicator,
Those transformed with a DNA molecule containing a vector-derived DNA fragment are primarily selected. For example, the transformed bacterial suspension was treated with tetracycline (15 μg/
ml), culture this at an appropriate temperature, and isolate the colonies that appear as transformant microorganisms. Next, this was transplanted onto an appropriate agar plate by a replica method or the like, and a colony was made to appear by culturing. Agar (45°C to 50°C) whose pH has been adjusted to around 9 with an appropriate buffer is overlaid, and after solidification, the culture is continued. After this, for example Congo Red (Congo Red)
go red) method (7eather and Wood, Ap
pl.

Environ, Microbiol, vol. 43,
777 pages (1982) ') and other commercials around the village.
Colonies in which decomposition of C was detected are isolated as target recombinant microorganisms.

Recombinant DNA held by the recombinant microorganism thus obtained
The molecule is amplified by growing recombinant microorganisms in a suitable medium, followed by the conventional plasmid preparation M1 method (Ma
Niatis et al., Molecular Cloni
ng, Co1d Spring HarborL
laboratory, New York, (1982
) etc.). Furthermore, cleavage patterns by various restriction enzymes were analyzed by electrophoresis (Meyers, J.

Bacteriol, vol. 127, p. 1529 (19
76)) etc., the recombinant D
The NA molecule is a shuttle vector, e.g. pHY300PL
It can be confirmed that the DNA fragment containing the K molecule and the cellulase gene, such as alkaline cellulase, and the 164 gene are combined.

Shuttle vector 1) Plasmid pHC is a suitable example of a recombinant plasmid containing the entire region of one 64 genes for alkaline cellulase prepared using HY300PLK.
L64 (Figure 1) and pHCL64R (Figure 2). This plasmid is a vector plasmid pHY3
Bacillus sp.K at the Hindm cleavage site of 00PLK
It is derived from the SM-64 (FERN P-10482) strain and has the approximately 4.4 Kb DNA fragment shown in FIG. 3 inserted therein. The fact that this approximately 4.4 Kb Hindm fragment was derived from the KSM-64 (FERM P-10482) strain was confirmed by Southern hybridization analysis using the DNA fragment isolated from the plasmid as a probe. , can be confirmed. A suitable example of a recombinant Bacillus subtilis containing the recombinant plasmid includes Bacillus subtilis I SW 1214 (pHcL64) strain.

This strain has a recombinant plasmid pHCL64 of Bacillus subtilis l5W.
1214 strains (Ishiwa and 5hibahara, J
pn, J. Genet, vol. 60, p. 235 (19
85)) using the protoplast transformation method, and the tetracycline resistance gene encoded by the shuttle vector pHY300PLK and Bacillus sp. K S M-64 (FERM P-10482
) to the alkaline cellulase derived from the chromosomal DNA of the strain
Both genes are expressed and are resistant to tetracycline;
In addition, it has an ability to produce -64 alkaline cellulase.

When producing cellulase using the recombinant Bacillus subtilis of the present invention, the recombinant Bacillus subtilis is cultured in an appropriate medium, and the enzyme is recovered from the culture supernatant from which bacterial cells have been removed by means such as centrifugation. Good. The type of medium used is
It is not particularly limited as long as the recombinant Bacillus subtilis can grow and produce the enzyme. For example, as a carbon source, monosaccharides such as glucose and fructose,
Disaccharides such as sucrose, maltose or cellobiose, or C
Examples include polysaccharides such as MC and soluble starch, and nitrogen sources include ammonium sulfate, ammonium chloride salts, nitrates such as sodium nitrate, polypeptone, yeast extract,
Examples include meat extract, Pharmamedia, soy flour, casein, corn gluten meal, and amino acids such as glutamic acid. These nitrogen sources and carbon sources are appropriately combined at appropriate concentrations, and inorganic salts at appropriate concentrations, such as
Sodium chloride, sodium soluble acid, sodium sulfate, etc. may be added. Further, the pH of the medium is not particularly limited as long as it is within the range in which the recombinant Bacillus subtilis used can grow, but it is preferably adjusted to pH 6.5 to 7.5. Furthermore, regarding the culture conditions, 15°C to 50°C
℃, preferably 25° C. to 35° C. for 1 to 4 days with shaking or aeration with stirring.

[Effects of the Invention] By integrating a novel recombinant plasmid encoding the cellulase gene of the present invention into a host microorganism, a recombinant microorganism that produces alkaline cellulase in significant amounts can be obtained.

Furthermore, by culturing the recombinant microorganism of the present invention in an appropriate medium, a significant amount of cellulase can be produced in the medium.

[Example] Hereinafter, the present invention will be specifically explained with reference to Examples.

Note that cellulase (CMCase) activity was measured as follows. That is, 0.4 ml of 2.5% CMC (Sunrose AOIMC manufactured by Sanyo Kokusaku Valve Co., Ltd.), 0.2 ml of 0.5 M glycine buffer (pH 9,0), and 0.2 ml of deionized water.
Add 0.1 ml of enzyme solution to 3 ml of substrate solution and repel at 40°C for 20 minutes.
nitrosalicylic acid (DNS)
(Miller et al., Anal, Biochem.

2, p. 127 (1960)). The enzyme titer was 1.5% per minute under the above conditions. The amount of enzyme that produces reducing sugar equivalent to amol of glucose was defined as 1 unit. Protein quantification was performed using a Bio-Rado protein assay kit (manufactured by Bio-Rad), and calculations were made using bovine plasma albumin as the standard protein.

Actual 1M example 1 Bacillus sp. KS M-64 (FERNP-10482), which is a -64 producer of alkaline cellulase
Polypeptone (Inugo Nutrition Co., Ltd. 0.5%, Yeast Extract (manufactured by Difco) 0.5%, KH2P○40.1%, M
gSO4.7H200.02% and Na2CO3
The cells were cultured in 100 ml of a medium containing 0.5% with shaking at 30"C for 12 hours. The bacterial cells were collected by centrifugation, and the cells were collected using the method of Saito and Miura (Biochim).

Biophys, Acta, volume 72, page 619 (1
Approximately 50 μg of purified chromosomal DNA was obtained according to 963).

10μg of the obtained chromosomal DNA was added using a conventional method (!4ani
atfs et al., Molecular Cloning,
ColdSpring Harbor Laborat
(1982) et al.) using the restriction enzyme HindI.
After cutting with [I (Boehringer Mannheim), it was mixed with 1 μg of the vector plasmid pER322 (Boehringer Mannheim) which had also been cut with HindIII, and then ligated with T, DNA ligase (Boehringer Mannheim). The binding reaction was performed using

Transformation of E. coli with the ligation reaction was performed using E. coli.
The treatment was carried out using HB101 competent cells (manufactured by Takara Shuzo Co., Ltd.), and the bacterial suspension after treatment was transferred to an LB agar plate medium (1.0% tryptone (manufactured by Difco)) containing 50 μg/m1 of ampicillin (manufactured by Sigma). , yeast extract (manufactured by Difco Co., Ltd.) 0.5%, NaC 11.0%, Bacto agar (Difco Co., Ltd. tJ) 1.5%) and 37°
The cells were cultured for 12 hours at C.

The colonies of transformants that appeared were transplanted onto two LB agar plates (containing ampicillin) and cultured at 37° C. for 24 hours in an alternating manner. After culturing, 1°0% agar containing 0.5% CMC and 1 mg/ml of Wazozyme (manufactured by Sigma) and adjusted to pH 9 with 50 mM glycine buffer was overlaid and culture was continued. As a result, CMC around the village
The strain decomposed by the Congo red method (Tether
and Wood, Appl, Envfron, Mic
ro-bios, vol. 43, p. 777 (1982>
), and five recombinant E. coli strains were isolated.

Fruit #j, Example 2 The 5 recombinant E. coli strains obtained in Example 1 were treated with ampyridine (
50μg/ml) containing 5ml QLB medium (tryptone 1.0%, yeast extract 0.5%, NaC 11.0%)
) and cultured overnight at 37°C. 50 of this
0ml M9CA medium (Na2HP0,0.6%, KH
2P0.0.3%, NaC1O, 05%, NH, C10
, 1%, Casamino acid (manufactured by Difco) 0.2%, Mg8
0.2mM, CaCl20.1mM, glucose 0.
2% ampyridine 50 AIg/ml).
After further shaking culture at 37°C for about 5 hours, 170 mg of chloramphenicol (manufactured by Sigma) was added, and shaking culture was carried out at 37°C for 15 hours.

Bacterial cells were collected from the obtained culture solution by centrifugation, and the alkaline lysis method (Birnboim and Doly) was used.

Nucleic Ac1ds Res, Volume 7, 15
13 (1979) and Maniatis et al., Mole
cular Cloning, ColdSpri
ng Harbor Laboratory, (198
Approximately 500 μg of each recombinant plasmid was prepared according to 2)). Agarose gel electrophoresis (Meyers et al.
J, Bacteriol, Vol. 127, p. 1529 (1976)), the #J restriction enzyme cleavage point of the five types of recombinant plasmids obtained was analyzed. As a result, each plasmid contained approximately 4.4K as shown in Figure 3.
It was found that the HindlIl fragment of b was contained, and in addition to this fragment, 2 to 4 Hindm fragments were contained.

Therefore, by cutting the recombinant plasmid with the restriction enzyme HindIII and performing a religation reaction with T, DNA ligase, approximately 4.4 Kb of HindDII was obtained.
When attempting to create a recombinant plasmid containing only the fragment, a recombinant E. coli containing the desired recombinant plasmid was obtained. Since this strain produced cellulase, it was confirmed that the cellulase gene was present in the approximately 4.4 Kb fragment shown in FIG. The obtained recombinant plasmid (8.8 Kb) was named pCL64, and the E. coli H8101 strain containing pCL64 was named HB101 (pCL64) strain.

Example 3 HB101 (pCL64) statically cultured overnight in LB medium
The culture solution of the strain was mixed with LB medium (ampicillin 50 μg/m
1) and cultured with shaking at 37°C for 24 hours. After culturing, the culture solution was centrifuged, and the resulting bacterial cells were suspended in 1.0 ml of phosphate buffer (pH 7.0) and disrupted by ultrasonication. Insoluble matter is removed as a precipitate by centrifugation, and the pH at which the CMCase activity of the supernatant obtained is
When we determined the range and optimal pH for action, we found that this enzyme has a pH of 3.
It works in the range of pH 8.5 to 13, with the optimum effect at pH 8.5 to 10.
Bacillus sp. KSM-64 (FER14P-1048
2) The properties of -64 closely match the alkaline cellulase produced by the strain.

After cutting pCL64 with restriction enzyme HindlII,
Agarose gel electrophoresis was performed and electroelution from the gel (McDonnell et al., J.).

Mo1. Biol, vol. 110, p. 119 (197
7) ), an approximately 4.4 Kb Hindm fragment was isolated.

This Hindm fragment was subjected to DNA labeling and detection kit (manufactured by Boehringer Mannheim).
Probe DNA was prepared by labeling with.

On the other hand, after agarose gel electrophoresis of chromosomal DNA derived from Bacillus sp.
The bands were transferred to a Zeta Probe membrane (Bio-Rad) using an electroplotting instrument N (Bio-Rad), and hybridization with probe DNA was performed using a DNA labeling and detection kit. I did it. Chromosomal DNA derived from KSM-64
The HindIII cut product contains the probe DNA used.
It was confirmed that there is only a DNA fragment of about 4.4 Kb that hybridizes with the chromosomal DNA of Bacillus sp. KSM-64 (FERM P-10482). The origin was confirmed.

Example 4 Approx.
4Kb Hindm fragment and linear molecular shuttle vector pHY300P cut with restriction enzyme HindIII
LK was mixed and ligated with T, DNA ligase. E. coli strain HB101 was transformed using this bound DNA. Tetracycline (15 μg/m 1
Transformants were screened using resistance (manufactured by Sigma, Inc.) and cellulase productivity as indicators.
Recombinant plasmid pHCL64 (first
) and pHCL64R (Figure 2) were obtained. In both recombinant plasmids, the direction of insertion of the approximately 4.4 Kb fragment into the vector was opposite to each other. pHCL64 and p
The recombinant E. coli HB101 strain containing HCL64R was transformed into HB101 (pHCL64) strain and )I strain, respectively.
The strain was named B101 (pHCL64R). Both recombinant E. coli strains produced about 1000 U of -64 alkaline cellulase per 11 culture fluids.

Example 5 Recombinant plasmid pHCL64 was extracted and purified from the recombinant Escherichia coli HB101 (pHCL64) strain in the same manner as in Example 2. Using these recombinant plasmids,
Bacillus subtilis strain 15W1214 (leuA8. MetB5.
hsrMl) was transformed using the protoplast method (Cha
ng and Choen, Mo1. Gen, Genet
, Vol. 168, p. 111 (1978)), and protoplast regeneration medium containing tetracycline (DM3; sodium succinate (pH 7,3) 0.5
M, Casamino #10.5%, yeast extract 0.5%, K
Transformants were selected using 40.35% 2HP○, 0.15% KH2PO, 0.5% glucose, 220 mM MgCl, and 0.01% bovine serum albumin (manufactured by Sigma).

As a result, a recombinant haystack SWI 214 (pHCL64) strain containing the recombinant plasmid pHCL64 was obtained.

Example 6 Recombinant Bacillus subtilis l5WI 214 (pHCL64) strain was mixed with 2% polypeptone, 0.1% yeast extract, and KH2P0.
．． 0.1%, NaC10.5%, Tetracycline
3 in a medium (PY medium) consisting of 15 μg/m 1
After shaking culture at 0°C for 24 hours, various nitrogen sources shown in Table 1, 1% maltose, 0.5% fish extract (manufactured by Wako Pure Chemical Industries), 0.05% yeast extract, and KH2P0 were added. ．．
0.1%, Mg'SO.

・7H200.02% and tetracycline 15μg
/ m 1 and cultured with shaking for 14 days at 30"C. When the alkaline cellulase activity of the resulting culture solution was measured, the recombinant Bacillus subtilis was found to be effective when using either nitrogen source. -6 to a significant amount of alkaline cellulase
4 was secreted into the medium (Table 2).

Table 1 Nitrogen source □(%) □(%) (NH4)isOa 21.21. lNaN0. 1
6.51.5 NH, C126,20,9 Polypeptone (Five Nutrients for Dogs) 13.5
1.8 Polypeptone S (macropore nutrition) 8
．． 5 2.8 Peptone-BMY (Pehringer) 13.5 1.8 Meat Extract (Oxoid) 12.4 1.9 Glutami Skin Sodium 7.5 3.2 (Left below) 2nd Table of Contents Alkaline Cellulase (U /
1) (NH,)2SO,190O NaNOs 220ONI
(, C11400 Polypeptone (Daigoeigaku) 7400 Polypeptone S (Ooana Nutrition) 6400 Peptone-BMY (Pellinger') 5
300 Meat Extract (Oxoid) 79
00 Glutamine Sodium 3500
Example 7 A culture solution of Bacillus Bacillus Bacillus SWI 214 (pHCL64), which had been cultured with shaking at 30° C. for 24 FR in a PY medium, was inoculated into each PY medium containing various carbon sources and cultured with shaking. As a result, as shown in Table 3, a significant amount of alkaline cellulase was produced no matter which carbon source was used.

Table 3 Carbon source Concentration (%) Alkaline cellulase (U/
l) Grape M 1.0 9100

[Brief explanation of the drawing]

Figures 1 and 2 show the recombinant plasmid pH.
It is a drawing showing restriction enzyme cleavage maps of CL64 and pHCL64R. In the figure, the bold line is Bacillus sp.
-64 (FIJM P-10482) strain, and the thin line is the shuttle vector pI (Y30O
This is a DNA fragment derived from PLK. Figure 3 shows Bacillus sp. KSM-64 (FER?I).
P-10482) strain derived from alkaline cellulase.
This is a restriction enzyme cleavage map of an approximately 4.4 Kb DNA fragment containing the gene. In the figure, the black area contains 164 alkaline cellulase genes. FIG. 4 is a drawing showing the base sequence near the 164 gene of alkaline cellulase. In each of the above figures, the abbreviations for restriction enzyme cleavage points are as follows. Ba; BamHI, Bg; Bgll. Ec; EcoRI; Hi; HindIn. Ps; PstI, Pv; Pbull. Sa; 5alI, Sc; 5caI. Sm; SmaI, Xb; XbaI. Xh; XhoI Above Figure 1: ll+m sa xb Hi Xh Hi
Pv Bg Hio 1.0 2.0
3.0 4.04.4 kb (012N lb/
)b C12R1:07) February 17, 1992

Claims (9)

[Claims] (1) D containing cellulase gene derived from Bacillus bacteria
A recombinant plasmid obtained by ligating an NA fragment and a shuttle vector that can be replicated in E. coli and Bacillus subtilis. (2) The recombinant plasmid according to claim 1, which has the restriction enzyme cleavage map shown in FIG. 1 or 2. (3) A recombinant plasmid in which a part of the recombinant plasmid according to claim 1 or 2 has a base deletion, substitution, or insertion. (4) The recombinant plasmid according to any one of claims 1 to 3, wherein the shuttle vector is pHY300PLK. (5) The recombinant plasmid according to any one of claims 1 to 4, wherein the cellulase gene derived from a bacterium belonging to the genus Bacillus is a gene encoding alkaline cellulase K-64. (6) A recombinant microorganism containing the recombinant plasmid according to any one of claims 1 to 5. (7) The recombinant microorganism according to claim 6, wherein the host microorganism is E. coli or Bacillus subtilis. (8) The recombinant microorganism according to claim 6 or 7, wherein the recombinant microorganism is Escherichia coli HB101. (9) A method for producing cellulase, which comprises culturing any of the recombinant microorganisms described in claims 6 to 8.