JPH0665304B2 - Cellulase gene - Google Patents

Description

TECHNICAL FIELD The present invention relates to a cellulase gene, more specifically, Bacillus sp. No. 1139 (Bacillus sp. No. 113) having a specific alkaline cellulase producing ability.
9) related cellulase gene.

(Background of the Invention) Cellulase is referred to as 1,4-β-glucan glucanohydrolase (EC 3.2.1.4) and is found in many microorganisms. Most cellulases are enzyme systems composed of multiple components and are said to have activity in the acidic or neutral pH range. Recently, regarding cellulase of bacterial origin, Clostridium thermoceras (Clostridium
thermocellum and Cellulomonas
fimi) -derived multienzyme cellulase structural gene has been reported to be cloned by Escherichia coli [Corne et al., FE
MS Microbiology Letters (Cornet et al., F
EMS Microbiology Letters) 16 , 137-141 (1
983); Gilkes et al., Gilkes et al., (Journal of Biolgica).
Chemistry) 259 , 10455-10459 (198).
See 4)].

Also, the cellulase gene of the above Clostridium thermocellum and Bacillus subtilis.
It has also been reported that the nucleotide sequence of the β-glucanase gene of Escherichia coli was determined [Beguin et al., Journal of Bacteriolo.
gy) 162 , 102-105 (1985); Murphy et al., Murphy et al.,
Nucleic Acids Research) 9 , 309-321 (198)
See 4).

On the other hand, some of the present inventors have found that a high pH region (pH 10 to 1
We have already isolated many alkalophilic microorganisms that grow well in 1) [Horikoshi et al. (Horikoshi, K., & T. Akiba, Alkalophilic
microorganisms), Springer-Verlag, New York (198
2)]]. These microorganisms produce many extracellular enzymes having an active pH optimum in the alkaline region.

Some of the inventors of the present invention have previously reported that alkaline cellulase [carboxymethylcellulase (hereinafter referred to as “CM” has a specific action from Bacillus sp.
The name "Case" is isolated [Fukumori et al., Journal of General Microbiology (Fukumori et a
l., Jaurnal of General Microbiology) 131 , 333.
9-3345 (1985)].

As will be described later, this enzyme is a novel cellulase which hydrolyzes cellotriose or cellotetraose but does not hydrolyze cellobiose, has a molecular weight of about 92 K daltons, and has an optimum reaction pH of 9.

(Object of the Invention) An object of the present invention is to provide a cellulase (hereinafter referred to as “NO.1139CMCase”) gene derived from Bacillus sp.

Another object of the present invention is to use DN of NO.1139 CMCase gene.
A DNA sequence hybridizing to the A sequence, which is obtained by natural or semi-synthesis, and has the above-mentioned DNA.
Related to the sequence by nucleotide substitutions, nucleotide deletions, nucleotide insertions and nucleotide sequence inversions and other mutations, and
It is to provide a DNA sequence encoding a polypeptide having NO.1139 CMCase activity.

Further, it is an object of the present invention to provide a recombinant DNA plasmid containing any of the above DNA sequences.

Furthermore, it is an object of the present invention to provide a recombinant DNA plasmid containing any of the above DNA sequences, and the DNA sequence being operably linked to an expression control sequence.

(Constitution of the Invention) <Preparation of Chromosomal DNA> The mycological properties of Bacillus sp. NO. 1139 are as follows.

Form: bacillus Size: 0.6 to 0.8 μ × 3 to 4 μ Pigment formation in agar slant medium: None Motility: Yes Gram stainability: Yes Spores: Form but do not swell.

Catalase: Positive Oxidase: Positive Attitude toward oxygen: Aerobic Forges-Proscawell (VP) reaction: Negative Growth temperature: Growth up to 45 ° C Optimal growth temperature 30 to 37 ° C Optimal growth pH: 8.8 to 10.0 in the presence of NaCl Growth: 5% (w / v) Positive 7% (w / v) Negative Starch hydrolysis: Decomposition Nitrate reduction: Reduction Indole formation: No formation Medium: PY-CMC medium Carboxymethylcellulose 10 g, polypeptone 5 g Yeast extract 5g, NaCl 5g, KH ₂ PO ₄ 1g, MgSO ₄ · 7H ₂ O 0.2%, Na
₂ CO ₃ 0.5% (W / V), 1.5% (W / V) agar is dissolved in 1 distilled water. (pH10.3) The Bacillus sp. NO. 1139 was isolated from the soil in the Tama area of Tokyo.
The 39 strains were deposited at the Institute for Microbial Engineering, Institute of Industrial Science, on April 1, 1986, and the deposit number is Microindustrial Research Institute No. 8722.

The NO.1139 strain is aerobically cultured in a PY-CMC medium at 37 ° C until the logarithmic growth phase. After culturing, the cells are collected, and then the DNA extraction method by the phenol method [Saito et al.
Bio Kimika Bio Physica Actor (Saito, H. & Miu
ra, K., Biochimica et Biophysica Acta) 72 , 619
-629 (1963)], the chromosomal DNA is extracted and purified by the DNA extraction method to obtain DNA.

<Insertion of DNA Fragment into Vector Plasmid and Transformation> Vector plasmid pBR322 can be prepared by the method of Bolivar et al., [Bolivar et al., Gene (Bolivar et al. Gene 2 , 95-113 ( 197
7)], commercially available products can also be used (for example, Bethesda Research Lab.
oratories) company).

The chromosomal DNA is cut with the restriction enzyme HindIII. On the other hand, the plasmid pBR322 was cleaved with HindIII, the cleaved chromosomal DNA was added, and the DNA was digested with DNA ligase.
Perform chain binding reaction. The resulting binding mixture was subjected to a conventional method (for example, Lederberg, EM & Cohen, SN Journal of Biology).
Bacteriology) 119 , 1072-1074 (197).
4) refer to 1) and transform E. coli into 1 μg
About 10,000 transformants of Ap ^r and Tc ^s are obtained per DNA (about 15% of Ap ^r transformants were Tc ^s ). Among these transformants, there were 8 strains that formed shallow craters around the colonies on LB agar plates containing CMC. All of these are transformants showing Ap ^r and cellulase activity, one of which was grown, and the plasmid was extracted and purified by a conventional method to obtain 4.6 kb (kilobase).
The plasmid pFK1 containing the HindIII fragment of

The plasmid pFK1 has a restriction enzyme cleavage map as shown in FIG. 1, has a HindIII fragment of 4.6 kb, and Hinc
It is also cleaved by II, Kpn I, Pvu II and Xho I.

In order to analyze the origin of the DNA inserted into pBR322, the plasmid pFK1 labeled with ³² P was analyzed.
Was digested with a restriction enzyme immobilized on a nitrocellulose sheet and strain No. 1139 and E. coli. Hybridized with chromosomal DNA of E. coli HB101. As a result, the plasmid pFK1 labeled with radioactive ³² P was identified as NO.11.
Although it hybridized with the 4.6 kb fragment of the 39 strain, coli D
In the NA fragment, the DNA to be added to the plasmid pFK1
The sequence was not detected.

<DNA sequence> By subcloning experiment, 2.9 kb Hinc II-Hind II
It was found that the I fragment was essential for cellulase production (in FIG. 1, the thick line part). 2.9kb Hinc I
The restriction enzyme digestion map of the I-Hind III fragment is shown in Fig. 2. The DNA sequence of this fragment was determined by the dideoxy chain termination method using phage M13.
method; Sanger et al., Proceeding of the National Academy of Science (Sanger et a
l.Proceeding of the National Academy of Science),
USA 74 , 5463-5467 (1977). The DNA sequence and amino acid sequence are shown in FIG. According to this, the 2.9 kb Hinc II-Hind III fragment described above encodes a polypeptide of 800 amino acids.
400kb open reading frame (open re
ading frame). Also the expected ribosome binding site (SD sequence), ie B. subtilis 16S AGGAGG complementing the 3'-end of ribosomal RNA
The sequence was found upstream of the open reading frame.

<Amino acid sequence> The amino acid composition of cellulase derived from the NO.
It was in very good agreement with that obtained from the DNA sequence.

That is, the NH ₂ -terminal sequence of the cellulase of NO.1139 strain was analyzed by Edman method (Edman, P. & Henschen, A. Protein sequence det using the Beckman sequencer model 890M.
er-mination, 2'nd de., Springer-Verlag, Berlin, pp2
32-279 (1975)), Glu-Gly-Asn-Thr-Arg-Glu-Asp-Asn-Phe-L.
ys, which was in agreement with the sequence from Glu at the 31st residue. Therefore, it seems that the region from the first Met to Ala at the 30th residue is a signal peptide that is cleaved during secretion.

<Identity of cellulase> Cellulase derived from NO.1139 strain and plasmid pFK
E. coli transformed with E. When cellulase produced by Escherichia coli HB101 was compared, the identities of both were confirmed. That is, FIG. 4 shows the relative activities of cellulases at each pH, and it can be seen that they are in very good agreement. Moreover, in the measurement of the molecular weight by SDS-polyacrylamide gel electrophoresis, the former was 92,000, while the latter was 94,000, but the difference in this degree is due to the protein purification step, and the difference is substantial. Was not recognized. Also, immunologically, they are considered to be the same since they undergo a precipitation reaction.

Example <1. Preparation of Chromosomal DNA> The NO.1139 strain (FERM P-) was added to PY-CMC.
3 at 37 ° C in medium (the above medium minus agar)
After shaking culture aerobically for a period of time, the cells at the early stage of logarithmic growth were collected, and then chromosomal DNA was extracted and purified by the DNA extraction method by the phenol method to obtain 6 mg of chromosomal DNA.

<2. Insertion of Chromosomal DNA Fragment into Vector / Plasmid> 10 μg of the above chromosomal DNA was taken, restriction enzyme Hind III was added, and the mixture was allowed to react at 37 ° C. for 14 hours to partially cut it. On the other hand, plasmid pBR322 (Bethesda Research
Hind III was added to Loboratories (USA), and the mixture was reacted for 1 hour to be completely cut, and heat-treated at 65 ° C. for 5 minutes. After that, cleaved vector / plasmid DNA 1μ
g and 3 μg of the cleaved chromosomal DNA are mixed, DNA ligase derived from T4 phage is added and reacted overnight at 15 ° C to ligate the DNA chains. After heat treatment at 65 ° C for 5 minutes, 3 times volume of ethanol is added to the reaction solution. In addition, the plasmid DNA incorporating the chromosomal DNA was precipitated and collected.

<3. Transformation with plasmid> Escherichia coli K-12 strain and Escherichia coli B
Escheri coli, which is a hybrid strain of strains
chia coli) HB101 strain [Goldforb et al, Proceedings
of the National Academy of Sciences of the United
Stated of America 79 , 5886-5890 (198
2)] (Genetic traits: pro, leuB1, lac Y, hsd
R, hsd M, ara 14, gal K2, xyl-5, mtl
-1, sup E44, F, endoIrec A Str ^r ) was used for transformation according to the method of Lederberg et al. (Lederberg, EM, & Cohen, SN (Journal of Bacteriology 119 , 1072-1074 ( 1
974)).

That is, the HB101 strain was transformed into LB medium [Maniatis et al., Molecular Cloning, A Laboratory.
Manual (Maniatis et al., Molecular cloning, a lab
Oratory manual), pp68-69 (1981): 10 g of tryptone (Difco), 5 g of yeast extract per 1 pure
Glucose 1g, NaCl 10g adjusted to pH 7.0]
10 ml was inoculated, shake-cultured at 37 ° C., grown to the latter stage of logarithmic growth, and then collected. This was sequentially suspended in a solution of 0.03M CaCl ₂ at a final concentration under ice cooling to obtain competent cells. The solution of the plasmid DNA obtained in 2 above was added to this cell suspension and reacted for 60 minutes under ice cooling, and heat shock was applied at 42 ° C. for 1 to 2 minutes to allow the above plasmid to enter the cells.

Thus, about 10,000 strains of A per μg of DNA are obtained.
A transformant of p ^r and Tc ^s was obtained. Among these transformants, 8 strains were obtained which formed shallow craters around the colonies on a plate of LB agar medium containing carboxymethyl cellulose. These are all transformants showing Ap ^r and cellulase activities, one of them was grown, and the cell suspension thereof was separately inoculated into the LB medium and shake-cultured at 37 ° C. for 3 to 5 hours. After that, the cells were collected and washed to obtain Escherichia coli HB101 (pFK1) transformed with the plasmid pFK1.

The cells were treated as follows to obtain the purified plasmid pFK
Got 1.

<Cellulase Production by Transformant> (Culture Method) Escherichia coli HB101 (pFK1) was added to the above LB
After inoculating the medium and aerobically culturing in a 500 ml flask at 37 ° C for 24 hours, the cells were collected and osmotic shock method [Kato et al., European Journal of Applied Microbiology and Biotechnology (Osmotic shock Methods: Kato et al., Eur, J, Appl.
Microbiol. Biotechnol) (1983) 18: 339-3.
43], the cells were fractionated outside the cells, periplasmic space and outer surface of the cells.

(Enzyme activity measurement) CMC (2.0%) 1 ml, glycine-NaCl-NaOH buffer (pH
9.0) Add 0.5 ml of enzyme solution to 1 ml and react at 40 ° C for 20 minutes.

After completion of the reaction, the 3.5-dinitro-salicylic acid method [3.5
-Dinitro-salicylic acid (DNS) method] is used to quantify reducing sugars. That is, 1 ml of the DNS reagent was added to 0.25 ml of the reaction solution, heated at 100 ° C. for 5 minutes to develop color, and after cooling, 4 ml of distilled water was added to dilute. This is colorimetrically determined at a wavelength of 500 μm.

The unit of the enzyme titer is 1 unit (U) when reducing sugar corresponding to 1 μmol of glucose is produced per minute under the above-mentioned conditions.

As a result, when the distribution of cellulase outside the cells, periplasmic space and intracellular production was examined, it was found to be 66
(11.3%), 402 (68.8%) and 116 (19.9%) m
It was U / ml.

Next, cellulase and HB10 produced by NO.1139 strain
In order to check the identity of the cellulase produced by the 1 (pFK1) strain, (1) activity by pH and (2) molecular weight were examined.

(1) pH 6-8 is 0.05M sodium phosphate, pH 8.5 10.5 is
0.05M glycine-NaCl-NaOH, pH 11-13 is 0.05M
As a result of investigating the influence of pH by the above-mentioned measuring method using KCl-NaOH buffer, the identity of both enzymes was confirmed as shown in FIG.

(2) The molecular weight was determined by SDS-PAGE method using an immunoassay kit manufactured by Bio-Rad (Laemmli, UK, Nature,
Lando 227 , 680-685 (1970)), the cellulase of the NO.1139 strain was about 92,000, and that of HB101 (pFK1) was about 94,000. Was due to the difference in the protein purification process, and no substantial difference was observed.

[Brief description of drawings]

FIG. 1 shows a restriction enzyme digestion map of the plasmid pFK1. The lines indicated by medium and thick lines represent the fragments cloned from the NO.1139 strain, and the lines indicated by thick line contained the cellulase gene. It is a figure which shows the fragment subcloned. FIG. 2 is a view showing a policy for sequencing the cellulase gene of the present invention, and details of the Hinc II-Hind III fragment encoding the cellulase gene of the present invention and the site used for DNA sequencing. Shows a restriction map of various restriction enzymes, and an arrow under the line is a drawing showing the direction and range of the sequencing method by the dideoxy chain end method. FIG. 3 shows the nucleotide sequence of the cellulase gene of the present invention, and the DNA sequence of the Hinc II-Hind III fragment is
Shown in the 5'to 3'direction, the numbers above the sequence are from the predicted start site, nucleotide 1 and the predicted amino acid sequence is shown below the DNA sequence. FIG. 4 shows cellulase derived from the NO.1139 strain and E. coli transformed with the cellulase gene of the present invention. coli
It is the figure which compared the relative activity in each pH of the lulase which HB101 produces.

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Claims (2)

[Claims] 1. A DNA sequence encoding a cellulase having the following amino acid sequence: 2. A recombinant DNA plasmid in which a DNA sequence encoding a cellulase having the following amino acid sequence is operably linked to an expression control sequence.