JP2544177B2 - DNA fragment containing the cellulase gene - Google Patents

Description

TECHNICAL FIELD The present invention relates to a cellulase gene, and in particular, it is derived from a so-called alkalophilic Bacillus bacterium that exhibits optimal growth at alkaline pH. The alkaline cellulase gene, a DNA molecule containing the gene, and a microorganism having the DNA molecule.

[Conventional technology and its problems]

Conventionally, cellulase is understood as a complex enzyme group that catalyzes an enzymatic reaction that decomposes cellulose into glucose, or cellobiose, or cellooligosaccharide, and by its action mechanism, C ₁ enzyme, C _X enzyme and β-glucosidase,
Alternatively, it is said to contain enzymes called by names such as exo-β-glucanase, endo-β-glucanase, and cellobiase. Cellulase research for the past few decades has focused on the effective utilization of biomass resources.
For example, molds such as Trichoderma, Aspergillus, Acremonium, and Humicola have been required to provide the source.

Further, as a novel industrial use of cellulase, there can be mentioned one related to a detergent composition for clothes. However, most of the cellulases produced by microorganisms including the above-mentioned molds are so-called neutral or acid cellulases having optimum and stable enzyme activity at neutral to acidic pH. That is, the highest activity in the alkaline pH region which is a necessary condition for a detergent composition for clothes, and stable, the presence of so-called alkaline cells is slightly, those derived from bacteria of the genus Bacillus and Streptomyces. Only known. Therefore, the present inventors have conducted a search for fine substances that produce alkaline cellulase, and found that Bacillus sp. KSM-635 (FERM BP-1485), which is a type of alkalophilic bacterium, is a detergent for clothing. It was found that a large amount of alkaline cellulase suitable as a composition was produced (Japanese Patent Application No. 61-257775).
issue).

On the other hand, in recent years, bacterial cellulase gene,
Specifically, genes of the genera Clostridium, Cellulomonas, Bacillus, Streptomyces and Luminococcus have been isolated using gene manipulation techniques.
Among these genes, Clostridium thermocellum (Beguin, P., Cornet, P.,
and Aubert, JP, J.Bacteriol., 162,102, (1985), Jol
iff, G ,, Beguin, P., and Anbert, JP, Nucleic Acids Be
s., 14,8605, (1986) and Grepinet, O., and Beguin, P., N.
Nucleic Acids Res., 14,1791, (1986), Cellulomonas fimi (Wong, WKR, Gerhard,
B., Guo, ZM, Kilbun, DG, Anthony R., Warren, J., and M
iller, RCJ, Gene, 44,315, (1986), Cellulmonas uda (Nakamura, K., Misawa, N., and
Kitamura, K., J. Biotechol., 4,247, (1986)), Bacillus subtilis (Murph
y, N., Mcconnell, DJ, and Cantwell, BA, Nucleic Acid
s Res., 12,5355, (1984), Bobson, LM, and Chamblis
s, GH, J.Baceriol., 169,2017, (1987), Maskay, RM,
Lo, A., Willick, G., Znker, M., Baird, S., Dove, M., Moranel
li, F., and Seligy, V., Nucleic AcidS Res., 14,9159, (1
986) Nakamura, A., Uozumi, T. and Beppu, T., Eur, J. Bioch
em., 164,317, (1987))) and alkalophilic Bacillus bacteria (Fukumori, F., Sashihara, N., Kudo, T. and Horiko)
shi, K., J. Bacteriol., 168,479, (1986) and Fukumori,
F., Kudo, T., Narahashi, Y.and Horikoshi, K., J.Gen.Micr
The nucleotide sequence of the gene derived from obiol.132, 2329, (1986) has already been determined, and the encoded amino acid sequence is also known.

By the way, an attempt to isolate a cellulase gene by using a genetic engineering technique and determine its nucleotide sequence has been carried out by breeding a producing strain and protein by a genetic approach.
This is extremely significant when considering the improvement of cellulase function and characteristics by the technique of engineering.
However, so far, there is almost no knowledge about the three-dimensional structure and active center of cellulases in general, and the functions and characteristics are different in order to achieve the above-mentioned purpose,
There is a need for nucleotide and amino acid sequence information for more cellulases.

[Means for solving the problem]

The present inventor has previously found that recombinant Escherichia (Esch.
erichia) 8 strains were prepared. About 4 strains containing the essential activity region of the alkaline cellulase gene
As a result of isolating and analyzing a 0 Kb DNK fragment, it was found to be a novel DNA fragment having a unique restriction map (Japanese Patent Application No. 61-259923). In addition, from another strain, the above DNA
Approximately 6.6 Kb including the fragment and the entire region of the gene
Was isolated. Therefore, the entire nucleotide sequence of the gene and the encoded amino acid sequence of alkaline cellulase were determined, and these were compared with the nucleotide sequences of other cellulase genes derived from Bacillus bacteria known so far and the encoded amino acid sequence. As a result, they have found that the DNA fragment of the present invention has a unique nucleotide sequence, and that the encoded amino acid sequence of alkaline cellulase is different from the others, and thus completed the present invention.

Therefore, the present invention provides an alkaline cellulase gene, and further provides a DNA molecule containing the gene. The present invention also provides a microorganism containing an alkaline cellulase gene.

In the present invention, as a microorganism serving as a donor of the alkaline cellulase gene, for example, Bacillus sp. KSM-635, which is a kind of alkalophilic Bacillus bacterium, can be used. The present strain was isolated by the present inventors from the soil of Haga-gun, Tochigi Prefecture as a strain producing a large amount of alkaline cellulase K outside the bacterial cell, and has been deposited as Micro Engineering Research Article No. 1485. . The classification property of KSM-635 is the patent application No. 1 of 1986 filed on October 28, 1986 by the present inventors.
No. 257775.

As a method for obtaining chromosomal DNA from a donor strain, for example, the Marmer method (Marmur J., Mol. Biol., 3,208, (19
61)) and Saito and Miura's method (Saito, H.and Miura, KI, B
iochim, Biophys, Acta, 7d, 619, (1961)) and the like are mentioned, but other similar methods can be used. A DNA fragment containing an alkaline cellulase gene is prepared by cleaving the chromosomal DNA thus obtained with a restriction enzyme. Any kind of restriction enzyme can be used as long as it does not divide the gene. But you can use it.
Examples of such restriction enzymes include EcoR I, EcoR V or
Examples include restriction enzymes such as BamHI. Further, a DNA fragment containing a part of the gene can be prepared if the restriction enzyme used does not divide the region essential for the enzyme activity expression by the gene. Furthermore, even when a restriction enzyme that cuts the gene or the essential activity region is used, the purpose can be achieved by using the conditions for partial cutting. For example, restriction enzymes
When Hid III is used, a complete cleavage condition can be used to prepare a DNA fragment of about 4.0 Kb containing the essential activity region of the gene, and in the case of partial cleavage, it contains the entire region of the gene. , It is possible to prepare a DNA fragment of about 6.6 Kb.

On the other hand, the host / pecter system used is one in which the host strain can express the alkaline cellulase gene, and the recombinant DNA molecule is replicable in the host bacterium and can stably retain the incorporated gene. Anything can be used if it exists. For example, using Escherichia coli K-12 strain as a host
EK and Bacillus subtilis Mar
The BM system using the burg strain as a host may be mentioned, but preferably, the EK system, which has been most studied genetically and has a wide variety of vectors, gives good results. Specific examples of host strains include HB101 strain, C600 strain, JM109 strain and the like for EK strains, and BD170 strain, MI112 strain and the like for BM strains. As the vector, in addition to the above description, it is convenient to combine with a chromosomal DNA fragment if a plasmid vector that can be cleaved at only one site by a restriction enzyme cleaving the chromosomal DNA is used. Specifically, chromosomal DNA is cut with Hind III and EK
Vectors such as pBR32, pUC12 and pUC18 can be used in the system, and vectors such as pC194 and pBD8 can be used in the BM system. Also, when the vector used by the restriction enzyme that cuts the donor chromosomal DNA is not cut, a method using a synthetic linker or a homopolymer binding method (Nelson, T. and Brutlag, D.,
Mathods in Enzymology, 68,41, Academic Press, New Yor
k (1980)) and the like.

The above-mentioned chromosomal DNA fragment and a vector DNA molecule cleaved by a restriction enzyme are ligated to produce a recombinant DNA molecule. Examples of the ligation method include a method using DNA ligase and a homopolymer ligation method. Can be used.

The method for transforming the host strain with the recombinant DNA molecule is not particularly limited, but for example, in the case of the EK host strain, the calcium chloride method (Mandel, M. and Higa, A., J. Mol. Biol., 53,
159, (1970)) and the ribidium chloride method (Bolivar, F. and Ba
ckman, K., Method in Enzymology, 68,253, Academic Pres
s (1979)), and in the case of BM host strains, the content cell method (Contente, S. and Dabuan, D, Mol.Gen.
Genet., 177, 459, (1979)) and the protoplast method (Ghan
g, S. and Cohen, SN, Mol.Gen.Genet., 168,111, (197
8)) etc. can be used. Among recombinant substances such as antibiotic resistance coded on vector DNA molecules, first of all, recombinant microorganisms are selected by using vector-derived DNA as an indicator of a trait that is not inactivated by insertion of foreign chromosomal DNA fragment.
Those transformed with the DNA molecule containing the fragment are primarily selected. Specifically, for example, as a vector, EK-based pB
Using R322, the Hind III cleavage site
When the III fragment is inserted, the tetracycline resistance gene is inactivated, so primary selection may be performed using ampicillin resistance, which has no Hind III cleavage site in the gene, as an index.

Next, add this to 2% of carpoxymethyl cellulose (CM
C) is transferred to a suitable agar medium by the replica method and cultured, and after colonies appear, for example, the Congo red method (Teather, RMand Wood, PJ, Appl. Env.
Iron.Microbiol., 43,777, (1982)) and the like can be selected as the target recombinant microorganism, which is a strain in which CMC around the colony has been degraded.

Recombinant DNA retained by the recombinant microorganism thus obtained
Molecules were prepared by conventional plasmid preparation or phage DNA preparation (Maniatis, T., Fritach EF, and Sambrook, J., Molec).
ular Cloning, Cold Spring Harbor Laboratory, New Yor
k (1982) etc.) and analyzed the cleavage patterns by various restriction enzymes by electrophoresis etc., whereby the recombinant DNA molecule bound to the vector DNA molecule and the DNA fragment containing the alkaline cellulase gene. You can confirm that it is a thing.

The alkaline cellulase gene according to the present invention is contained in a DNA fragment of about 6.6 Kb having the restriction map shown in FIG. 3, and is particularly present in the portion of about 3.5 Kb indicated by the thick line. . In addition, of the two DNA fragments that make up a DNA fragment of approximately 6.6 Kb and whose both ends are Hind III cleavage points,
One of the about 4.0 Kb fragments contains the essential activity region of the gene, which can be further truncated to about 2.4 Kb indicated by the thin line. DN of these about 4.0Kb and about 2.4Kb
The fragment A has the restriction maps shown in FIGS. 4 and 5.

Next, the nucleotide sequence of a portion of about 3.5 Kb containing the alkaline cellulase gene was transferred to the M13 phage vector (Messi
ng, J., Methods in Enzymol., 101, 20, (1983)) using the Sanger method (Sanger, F., Nicklen, S. and Coulson, A.
B., Puoc. Natl. Acad. Sei. USA, 74, 5463, (1977)), it was revealed that it had the sequence shown in FIG. This sequence shows the sequence from the right side to the left side of the about 3.5 Kb portion shown in Fig. 3 in the direction of 5'to 3 ', and the sequences of nucleotide numbers 1 to 7 are subcloned. It is derived from the synthetic linker (manufactured by Pharmacia) used in the above. Therefore, the nucleotide sequence of 3498b excluding this is derived from the chromosomal DNA of the KSM-635 strain, and translation is initiated from the ATG of nucleotide number 612 in this sequence, and amino acid 941 shown in FIG. An open rating frame encoding a sequence of residues was found. Open reading
Bacillus subtilis (Bacillus subti) upstream of frame 5b (base)
lis) 16S liposomal RMA sequence <3 '(McLaughli
n, JR, Murray, CLand Rabinowitz, JC, J.Biol.Che
m., 256,11283, (1981) ) and there is a high GGGAGG sequence complementarity, further upstream, the consensus sequence of 6 ^43-inch promoter near the cutting point by restriction enzyme Sap I (Gitt, M., Wang , JF
and Doi, RH, J. Bio1, Chem., 260, 7178, (1985)), and a highly homologous TAGACG-17b-TATTAT sequence was observed. In addition, the translation termination codon TA at nucleotide number 3435
Inverted repeat sequence that appears to be a transcription terminator downstream of A (Fig. 6) Existed).

The alkaline cellulase gene of the present invention has been
When compared with the cellulase gene derived from the bacterium, the sequence and the amino acid sequence of the encoding cellulase were determined, this gene has a unique DNA sequence, and the encoded amino acid sequence is not the same as that of the above cellulase. It was different and new.

Recombinant DNA containing the entire region of alkaline cellulase gene
A preferable example of the molecule is plasmid pBC100 (Fig. 7) and the like. This plasmid is the vector plasmid pBR3.
It is derived from the KSM-635 strain at the Hind III cleavage site of 22 and has a DNA fragment of about 6.6 Kb shown in FIG. 3 inserted therein. still,
On the chromosomal DNA of KSM-635 strain, this DNA of about 6.6 Kb
The fact that the two Hind III fragments constituting the fragment are adjacent to each other means that Southern
Confirmed using the hybridization method. Further, as a preferred example of a recombinant DNA molecule containing the active essential region of the gene, recombinant plasmids pBC101, pBC102,
pBC111, pBC112, and the like. Each of these plasmids was obtained by inserting a DNA fragment derived from the KSM-635 strain into the Hind III cleavage site of the vector plasmid pBR322, and each of the inserted DNA fragments was about 4.0 shown in FIG. 4 in the case of pBC10 and pBC102. Kb, and in pBC111 and pBC112, it is a DNA fragment of about 2.5 Kb shown in FIG. 2.4 Kb D
In the case of NA fragments, there is no Hind III cleavage site at their one end, so synthetic Hind
III linkers were added. As shown in FIGS. 8, 9, 10, and 11, respectively, pBC101, pBC102, and pBC1
In 1 and pBC112, the direction of the inserted DNA fragment is opposite to that of the vector plasmid pBR322. In either case, the gene can be expressed in the host strain.

Preferable examples of the recombinant microorganism containing the recombinant DNA molecule include Escherichia coli HB101 (pBC100) and HB101.
(PBC101), HB101 (pBC102), HB101 (pBC111), and
Each strain of HB101 (pBC112) can be mentioned. These strains were obtained by inserting the above-mentioned recombinant plasmid into the Escherichia coli HB101 strain by a usual transformation method, and by culturing in a medium usually used for culturing bacteria of the genus Escherichia, alkaline cellulase was introduced into the cells. Produce.
The optimum reaction pH of the produced enzyme is 9-10, and Bacillus espi-KSM-635 (FERM
The value of alkaline cellulase K produced by P-8872) was in good agreement, and in addition, the immunological identities of the enzymes produced by both were confirmed by an immunodiffusion test.

As a method for isolating a DNA fragment containing the entire region of the alkaline cellulase gene or the essential activity region from the above recombinant plasmid, the recombinant plasmid is used as a restriction enzyme Hind III.
It can be carried out by separating the DNA fragment by agarose gel electrophoresis, and then extracting and purifying from the gel after the partial or complete digestion with DNA. DNA from gel
As a method for extracting and purifying fragments, electroelution (McDo
nnell, MW, Simon, MNand Studier, FW, J.Mol.Biol.,
110, 119, (1977) and a method using a low melting point agarose gel (Weislander, L., Anal. Bioshem., 98, 305, (1979)) and the like.

〔Example〕

Hereinafter, the present invention will be specifically described with reference to Examples. The alkaline cellulase (CMCase) activity was measured as follows. That is, 0.1 ml of the enzyme solution was added to a substrate solution consisting of 0.2 ml of CMC (2.5%), 0.1 ml of 0.5M glycine buffer (pH 9.0) and 0.1 ml of deionized water, and the reaction mixture was reacted at 40 ° C to produce the reducing sugar. To 3.5-dinitrosalicylic acid (3.5-dinitro salicyli
c acid (DNS) method (Summer, JR and Somers, GF, Labo
ratory Experiments in Biological Chemistry, Academi
c Press, New York, pp.34, (1944)). The enzyme titer was defined as 1 unit of the amount of enzyme that produced a reducing sugar corresponding to 1 μmol of glucose per minute under the above conditions. In addition, protein quantification was performed using a Pio-Rad Protein Assay Kit (manufactured by Bio-Rad), and bovine plasma albumin was calculated as a standard protein.

Example 1 Alkaline Bacillus sp KSM-635 (FERM BP-1485), which produces alkaline cellulase K, was mixed with 5 ml of MYG.
Medium (meat extract (Rab Remco powder Oxoid) 1.0%, yeast extract (Daifuco) 0.5%, NeCl1.
0%, KH ₂ PO ₄ 0.1%, Na ₂ CO ₂ 1.0% (separated bacteria)), shake culture at 30 ° C for 2 days, and then inoculate 500 ml of the same medium. The culture was carried out at 30 ° C. for shaking. After this, the cells were collected by centrifugation and the method of Saito and Miura (Saito, H. and Miura, KI, Biochim. Biophys.Acta, 72, 6
19, (1963)), about 300 μg of purified chromosomal DNA was obtained.

Example 2 10 μg of the chromosomal DNA obtained in Example 1 was added to a restriction enzyme reaction solution (10 mM Tris-HCl buffer (pH 7.5), 10 mM MgCl ₂ , 5).
10 mM NaCl, 1 mM dithiothreitol) and the restriction enzyme Hind III (Boehringer Mannheim) 10
The unit was added and incubated at 37 ° C. for 2 hours to cut the chromosome DN. Vector plasmid pBR322 (Boehringer Mannheim) 1 μm, which was also digested with Hind III after removing restriction enzymes by phenol treatment,
g was added and ethanol precipitation was carried out. The resulting DNA precipitate was treated with a ligase reaction solution (20 mM Tris-HCl buffer (pH 7.
5), 10mM MgCl ₂ , 10mM dithiothreitol, 1mM ATP)
Dissolved in 50μ. To this, 2 units of T ₄ DNA ligase (manufactured by Boehringer Mannheim) was added, and the reaction was carried out at 16 ° C. for 12 hours to ligate the chromosomal DNA fragment and the vector plasmid to prepare a recombinant plasmid.

Example 3 Transformation of E. coli with the recombinant plasmid prepared in Example 2 was carried out by the calcium chloride method (Mandel, M. and Higa, A.,
J. Mol. Biol., 53, 159, (1970)). As a host strain, Escheriohia coli HB101 strain (le
n, pro, thi, lacy, ara14, galK2, xy15, mtl1, strA, recA, sup
E44, hsdR, hsdM, endI) were used. Ampicillin (sodium salt, manufactured by Sigma) is used for the bacterial suspension that has been transformed.
LB agar medium containing 50 μg / ml (tryptone (manufactured by Difco) 1.0%, yeast extract (manufactured by Difco) 0.5%, NaCl1.
0%, Bact agar (Daifuco 1.5%) smeared at 37 ℃
The cells were cultured for 24 hours. About 10,000 transformant colonies that appeared were transferred to the LB agar medium containing 50 μg / ml of ampicillin and 2% of CMC by the replica method, and further cultured at 37 ° C. for 48 hours. Congo red method (Teather, RMand Wood, PJ, Appl. Env.
iron.Microbiol., 43,777, (1982)),
Eight strains of the target recombinant microorganism were isolated.

Example 4 The 8 strains of recombinant microorganisms obtained in Example 3 were treated with 10 ml of LB medium containing ampicillin (50 μg / ml) (tryptone (manufactured by Difco) 1.0%, yeast extract (manufactured by Difco) 0.5).
%, NaCl 1.0%) and allowed to stand overnight at 37 ℃, 500 ml of M9CA medium (Na ₂ HPO ₄ 0.6%, KH ₂
PO ₄ 0.3%, NaCl 0.05%, NH ₄ Cl 0.1%, Casamino acid (manufactured by Difco) 0.2%, MgCO ₄ 2mM (separate sterilization), CaCl ₂ 0.1mM
(Separate sterilization), glucose 0.2% (separate sterilization), ampicillin 50 μg / ml (disinfection)) and transplanted to 37 ° C for 4 to 5 hours with shaking. To this, 170 mg of chloramphenicol was added,
Further, the cells were cultivated with shaking at 37 ° C for 15 hours. The cells were collected from this culture solution by centrifugation and subjected to an alkaline lysis method (Birnboin, H.
C. and Doly, J., Nucleic Acids Res., 7,1513, (1979))
And cesium chloride-ethidium bromide (ce
sium chloride-ethidium bromide) Density gradient centrifugation (R
adloff, R., Bauer, W.and Vinograd, J., Proc.Natl.Acad.S
ci., 57,1514, (1967)) combined method (Maniati
s, T., Fritsch, EF, and Sambrook, J., Molecular Clonin
g, Cold Spring Harbor Laboratory New York (1982)
Recombinant plasmid was prepared according to. The 8 recombinant plasmids obtained were subjected to the conventional method (Maniatis, T., Fritsc
h, EF, and Sambrook, J., Molecular Cloning, Cold Spri
According to ng Hardor Laboratory New York (1982),
When a restriction enzyme cleavage map was prepared, it was revealed that each plasmid contained the Hind III fragment of about 4.0 Kb shown in FIG. Of these, two plasmids were those in which only this approximately 4.0 Kb NDA fragment was bound to the vector plasmid pBR322. As shown in Figs. The direction was right. The resulting recombinant plasmids were designated as pBC101 and pBC102, respectively. Also, pBC101
Alternatively, Escherichia coli HB101 strain transformed with pBC102 can be used as HB101 (pBC101) or HB101 (pBC102).
I named it. As shown in FIG. 7, another plasmid contains Hind III of about 2.6 Kb in addition to the fragment of about 4.0 Kb, which was named pBC100. The transformed HB101 strain was designated as HB101 (pBC100).

Example 5 11 μg of recombinant plasmid pBC10 was added to 50 μm of 10 mM MgCl ₂
Was dissolved in 10 mM Tris-HCl buffer (pH 7.5) containing 1 mM dithiothreitol, and the restriction enzyme Kpn I2 unit was added thereto, and the mixture was reacted at 37 ° C. for 2 hours to linearize pBC101. Kpn I was removed by a phenol treatment, ethanol precipitation was carried out, and the precipitate was dissolved in 50 μm of 50 mM Tris-HCl buffer containing 10 mM MgCl ₂ and 0.1 mM dithiothreitol. To this, 1.25 units of nuclease Bal31 was added and reacted at 22 ° C. for 15 minutes, the reaction was stopped by treatment with phenol, and ethanol precipitation was further carried out. Precipitate 50 mM NaCl, 10 mM MgCl ₂
Dissolve in 0 mM Tris-HCl buffer (pH 7.5) containing 1 mM dithiothreitol, add Hind III restriction enzyme unit, and
After reacting at 7 ℃ for 2 hours, agross gel electrophoresis was performed and the gel was electroeluted (McDonnell, MW, Simon, MNa).
nd Studier, FW, J. Mol. Biol., 110, 119, (1977)), and the DNA fragment of about 24 Kb shown in FIG. 5 was isolated. This D
0.2 μg NA fragment, pBR322 0.2 cleaved with Hind III
μg and synthetic Hind III linker 0.02 OD ₂₆₀ units of ligase reaction solution (20 mM Tris-HCl buffer (pH 7.5), 10 mM Mg
Cl ₂ , 10 mM dithiothreitol, 1 mM ATP) 20 μl. To this, 2 units of T ₄ DNA ligase (manufactured by Berger Mannheim) was added, a binding reaction was carried out at 16 ° C. for 12 hours, and then the Escherichia coli HB101 strain was transformed by the method of Example 3 to obtain. The presence or absence of production of alkaline cellulase in the transformant was examined. In addition, a brasmid was extracted from the transformant strain in the same manner as in Example 4, and it was confirmed that the target DNA fragment was inserted into the vector plasmid pBR322. As a result, cellulase productivity was confirmed in the recombinant microorganism having the recombinant plasmid in which the DNA fragment of about 2.4 Kb shown in FIG. After adding a synthetic Hind III linker to a DNA fragment of about 2. Kb, the recombinant plasmids inserted into the Hind III cleavage site of the vector plasmid pBR322 were named pBC111 and pBC112 (Figs. 10 and 11).
Figure). In addition, Escherichia coli HB101 strain transformed with each recombinant plasmid was transformed into HB101 (pBC1
11) and HB101 (pBC112), respectively.

Example 6 1 ml of the culture solution of each strain of HB101 (pBC100) and HB101 (pBC102), which had been statically cultured overnight in 10 ml of LB medium, was inoculated into 100 ml of LB medium (containing 50 μg / ml of ambicillin), and at 37 ° C., 24 Culture was performed with shaking for an hour. After culturing, the culture broth was centrifuged, and the precipitated bacterial cells were suspended in 10 ml of a phosphate buffer (pH 7.0), followed by ultrasonic disruption. Once again, insoluble matter was removed as a precipitate by centrifugation, and the working pH range and optimum working pH of the CMCase activity of the resulting supernatant were determined (Fig. 12). Works over a range, optimal for pH 9-10
It became clear that it has a pH value, and the Bacillus sp. KSM-635 (FERM
It was in good agreement with the properties of alkaline cellulase K produced by the BP-1485 strain.

In addition, double immunodiffusion (Ouchterlony, O., Hendbook of
Experimental Immunology, Blackwell Sci. Publ., Oxfor
d, (1967)), the alkaline cellulase of the HB101 (pBC100) strain forms a sedimentation line between the antiserum of CMCase I purified from the culture solution of KSM-635 (see Reference Example), and
It was completely fused with the sedimentation line of CMCase 1, indicating the immunological identity of both enzymes (Fig. 13).

Example 7 About 5 μg of pBC100 was cleaved with restriction enzyme Hind III, and then electrophoresed on agarose gel. In the same manner as in Example 5, two Hind III fragments of about 2.6 Kb and about 4.0 Kb about 0.5 μm.
Each g was isolated. The two Hind III fragments were combined with [α- ³² P] dCTR (manufactured by Amersham) and a nickel transfection kit (manufactured by Takara Shuzo Co., Ltd.) for the nickel transduction method (Rigby, PWJ, Dieckmanm, M. and Ber).
g, P., J. Mol. Biol., 113, 237, (1977)) was used to prepare a probe DNA of about 2.0 × 10 ⁸ cpm / μg. On the other hand, plasmids pBC100 (0.5 μg each) and KSM-635, which were individually cleaved with Xba I and Pvu II, respectively.
Chromosomal DNA (3 μg each) derived from the strain was subjected to agarose gel electrophoresis, and the DNA band was analyzed by Southern method (Southern, EM, JM).
ol.Biol., 98,503, (1975)) and transferred to a Zeta probe membrane (manufactured by Bio-Rad) to hybridize with the above-mentioned probe DNA.

As a result, as shown in FIG. 14, about 2.6 Kb after the Xba I digestion product and the Pvu II digestion of the chromosomal DNA derived from the KSM-635 strain.
It was revealed that there are DNA fragments of about 1.1 Kb and about 0.7 Kb, which hybridize with both of the two probe DNAs of about 4.0 Kb and about 4.0 Kb, respectively. X in the insert DNA fragment of pBC100
baI1.1Kb and PvuII0.7Kb exist in such a manner that they scissor the HindIII cleavage site, which is the contact point between the two HindIII fragments, and the two HindIII fragments do not hybridize with each other.
From the above, it was revealed that the two HindIII fragments constituting the approximately 6.6 Kb insert DNA fragment of pBC100 exist adjacently on the chromosomal DNA of the KSM-635 strain.

Example 8 The nucleotide sequence of a portion of about 3.5 Kb indicated by a bold line in FIG. 3 was used as an M13 phage vector (Messing, J., Method).
Enzymol., 10120, (1983)) mp18 and mp19 (Boehringer Mannhi) (Sanger F., Nicklen, S. and Coulson, AR, Proc. Natl.A).
cad.Sci., USA, 74,5463, (1977)). As a host E. coli strain for M13 phage, JM109 strain (re
cA1, ΔIac−pro, endA1, gyrA96, thi−1, hsdR17, relA1,
F′traD36, proAB ⁺ , lacI ^g gZΔM15) and M13
Sequence Singing Kit (Takara Shuzo) and [α- ³²
P] dCTP (manufactured by Amersham) was used. As a result, the alkaline cellulase gene was present so as to span both of the two Hind III fragments constituting the insert DNA fragment of pBC100.
It was found to have the nucleotide sequence shown in the figure. When the determined nucleotide sequence was analyzed, it was 2823 bp.
And an oven reading frame encoding the amino acid sequence of 941 residues shown in FIG. 2 was recognized. On the other hand, plasmids pBC101, pBC102, pBC111 and pBC1
In the case of 12, although the transformant strain was confirmed to have alkaline cellulase productivity, the downstream region of the gene was approximately 2.6 Kb.
Since it does not have the Hind III fragment of the above, it is clear that the DNA fragments of about 4.0 Kb and about 2.4 Kb shown in FIGS. 4 and 5 include the essential lees of the gene. .
This essential region for activity encoded the amino acid sequence of 584 residues shown in FIG.

Reference Example 1 1 g of soil in Kai Town, Haga-gun, Tochigi Prefecture was suspended in 10 ml of sterile physiological saline and heat-treated at 80 ° C. for 30 minutes. This heat treatment solution is appropriately diluted to obtain a master plate (meat extract (Oxoid) 1%, Batatopeptone (Difco) 1%, NaCl)
1%, KH ₂ PO ₄ 0.1%, Na ₂ CO ₃ 0.5% (separate sterilization) Bact agar
1.5%) and smeared at 30 ° C. for 3 days to form a colony. By the Lebrika method, it was transplanted to a sterilized agar medium containing 2% CMC in a medium having the same composition as the master plate and cultured at 30 ° C. for 3 to 4 days to form a colony, followed by the Congo-Red method. Strains capable of degrading CMC around the settlement were detected. The relevant colonies were selected by a master plate to isolate CMCase-producing bacteria.

Bacillus SB KSM-635 (FE
RM BP-1485).

Reference Example 2 Bacillus SB KSM-635 with 1.5% meat extract, 0.5%
Aerobic culture was carried out at 34 ° C. for 2 days in a liquid medium consisting of yeast extract, 1% CMC, 0.1% KH ₂ PO ₄ and 0.75% Na ₂ CO ₂ (separate sterilization). 3 cold ethanol (-10 ° C) was gradually added to the culture supernatant liquid 1 to cause protein precipitation, and the resulting precipitate was dissolved in a minimum amount of sterile deionized water and neutralized with dilute acetic acid. Then, it was dialyzed against running water for 15 hours to obtain 9.6 g of alkali cellulase K as a freeze-dried powder.

Reference Example 3 Alkaline cellulase K produced by KSM-635 in a medium was purified as follows. That is, the supernatant obtained by removing the bacterial cells from the culture solution by centrifugation is treated with streptomycin,
After performing ammonium sulfate separation (30-75% saturated precipitation fraction), preparative high performance liquid chromatograph (SW3000G column, Toyo Soda) DEAE-Toyopearl (Toyo Soda) chromatograph, bidroxyapatite (Seikagaku Corporation) ) Chromatography and then DEAE-Toyopearl chromatography. By performing elution with a linear concentration gradient of NaCl (0.25M to 0.35M) during the second DEAE-Toyopearl chromatography, two types of CMCase (CMCase I
And CMCase II). Both enzymes are the Davis method (Davis, DJ, Ann.NYAcad.Sci., 121, 404, (196
Electrophoresis was performed according to 4)) and stained with Coomassie Primitive Blue to give a single band.

CMCase I antiserum was prepared by injecting rabbits with the above-described purified CMCase I (1 mg / dose) according to a conventional method.

(Physicochemical Properties of Alkaline Cellulase K) The physicochemical properties of alkaline cellulase K produced by Bacillus sp. KSM-635, which is the donor of the alkaline cellulase gene in the present invention, are as follows.

(1) Action Hydrolysis of carboxymethyl cellulose (C _X enzyme activity)
In addition, it has weak C ₁ enzyme activity and β-glucosidase activity.

(2) Substrate specificity It acts on carboxymethyl cellulose, crystalline cellulose, avicel, cellobiose and p-nitrophenyl cellobioside.

(3) Working pH range and optimum working pH The working pH range is 5 to 12, and the optimum working pH is 9 to 10.

(4) Stable pH range The stable pH range is 6.0-11.0 when left at 5 ℃ for 16 hours.
Is.

(5) Working temperature range and optimum working temperature It works in a wide range of 15-60 ℃, but the optimum temperature is about 40
Appears in ° C.

(6) Effect of chelating agent EDTA, EGTA, NTA, STPP and zeolite do not inhibit the activity.

(7) Effect of surfactant Linear sodium alkylbenzenesulfonate (LA
S), sodium alkyl sulfate ester (AS), polyoxyethylene alkyl sulfate (ES), sodium α-olefin sulfonate (AUS), sodium α-sulfonate fatty acid ester (α-SEF), sodium alkyl sulfonate ( SAS), polyoxyethylene secondary alkyl ether, fatty acid salt (sodium salt), dimethyldialkylammonium chloride, and other surfactants hardly inhibit the activity.

(8) Effect of protease It has strong resistance to protease.

[Brief description of drawings]

1 and 2 are drawings showing the amino acid sequence of alkaline cellulase. Figure 3 shows about 6.6 Kb DNA containing the alkaline cellulase gene.
It is a restriction map of fragments. The thick line portion contains the entire region of the alkaline cellulase gene, and the thin line portion contains the essential activity region. FIG. 4 is a restriction map of a DNA fragment of about 4.0 Kb containing the essential activity region of the alkaline cellulase gene. FIG. 5 shows the DNA sequence of a DNA fragment of about 2.4 Kb containing the essential activity region of the alkaline cellulase gene. FIG. 6 is the nucleotide sequence of the portion of about 3.5 Kb indicated by the bold line in FIG. Figures 7, 8, 9, 10 and 11 show recombinant plasmids pBC100, pBC101, pBC102, pBC111 and
It is a restriction map to pBC112, in which the thin line portion shows the DNA fragment derived from the vector BR322 and the thick line portion shows the DNA fragment derived from Bacillus sp. KSM-635. FIG. 12 is a graph showing the action pH range and the optimum action pH of alkaline cellulase produced in the cells of HB101 (pBC100) and HB101 (pBC101) strains. ○ indicates HB101 (pBC100),
● indicates cellulase data derived from HB101 (pBC102) and ▲ indicates cellulase data derived from Bacillus sp. KSM-635. FIG. 13 shows a double immunodiffusion test of HB101 (pBC100) alkaline cellulase and purified CMCase I of Bacillus sp. KSM-635. 1 is antiserum of purified CMCase I, 2 is purified CMCase I, 3 is It is an alkaline cellulase produced by the HB101 (pBC100) strain. FIG. 14 shows the results of autoradiography after Southern hybridization using Hind III fragments of about 2.6 Kb (A) and about 4.0 Kb (B) as probes. 1 for each lane
Pvu II cleavage product of chromosomal DNA from KSM-635 strain, 2 is pBC10
0 Pvu II digest, 3 is Xba I digest of chromosomal DNA and 4
Is an Xba I digest of pBC100.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication (C12N 1/21 C12R 1:19) (C12N 9/54 C12R 1:19) (72) Inventor Okamoto Akihiko 1-229-8 Nanachiza Town, Koshigaya City, Saitama Prefecture

Claims (7)

(57) [Claims] 1. A DNA fragment encoding a cellulase having the amino acid sequence shown in FIG. 2. A DNA fragment encoding a cellulase having the amino acid sequence shown in FIG. 3. A DNA fragment encoding a cellulase having a sequence in which an amino acid or a polypeptide is added to the carboxy terminus of the amino acid sequence according to claim 1. 4. Amino acid sequence according to any one of claims 1, 2, and 3 which is characterized by amino acid substitution, deletion, inversion, insertion, etc. A natural, synthetic, or semi-synthetic DNA fragment that encodes a related protein having cellulase activity. 5. The DNA fragment according to any one of claims 1, 2, 3, and 4 contains a DNA sequence for regulating gene expression. Characteristic DNA
fragment. 6. Claims 1, 2, 3,
A DNA molecule comprising the DNA fragment according to any one of claims 5 and 4 in the molecule. 7. A microorganism containing the DNA fragment according to claim 5.