JPS62269692A - Cellulase gene - Google Patents

Abstract

PURPOSE:A DNA which hybridizes DNA sequence of N-4ACMCase gene and codes polypeptide having cellulase activity, obtained by a transforming host from a chromosome DNA fragment of Bacillus sp. No.N-4 strain. CONSTITUTION:Chromosome DNA of Bacillus sp. ATCC21833 strain is scissored with restriction enzyme Hind III, linked to vector pBR322 similarly scissored with the enzyme in the presence of ligase and transduced to Escherichia coli to give plasmid pNK2 containing Hind III fragment with 2.1kb and plasmid pNK2 containing Hind III fragment with 2.8kb from a transformant. Plasmid pNK1 to code cellulase is approximately equal to plasmid pNK2 except a fact that the plasmid pNK1 has 60 direct repeat amino acid sequence in the vicinity of C-terminal end. Cellulase obtained from the plasmids substantially no difference from cellulase produced by N-4 strain.

Description

DETAILED DESCRIPTION OF THE INVENTION (Technical Field) The present invention relates to a cellulase gene, and more specifically to Bacillus sp.
sρ, NaN-4)-derived cellulase gene.

(Background of the Invention) Cellulase is called 1,4-β-glucan glucanohydrolase (EC3,2,1,4) and has been found in many microorganisms. Most of these cellulases are enzyme systems consisting of multiple components, and are said to have activity in an acidic or neutral pH region. In recent years, regarding cellulases of bactericidal origin, Clostridium thermocellum (Clostridium thermocellum) has been studied.
and Cellulomonas fuimi (Cellulomon
as fimi) enzyme system consisting of multiple components derived from
Cloning of the structural gene of lulase in Escherichia coli has been reported [Cornet et al., FEMS Microbiology Letters.

FEMS Microbiology Letters
) 16, 137-141 (1983); Gilkes et al., Journal of Biological Chemistry.

Journal of Biological Che
mistry) 259, 10455-10459
(1984)].

In addition, the cellulase gene of Clostridium thermocellum and Bacillus subtilis
There is also a report that the nucleotide sequence of the β-glucanase gene of S. 5ubtilis has been determined [Begui et al.
net atlJontnal of Bacter
iology) 162.

102-105 (1985); Jurphy et al., Nucleic
Ac1ds Re5ear+: h) Yu, 309-321
(1984)].

On the other hand, some of the present inventors have studied the high pH region (pH 1'0
We have already isolated many alkaliphilic microorganisms that grow well in the alkaline region. et al., “Alkaliphilic Bacteria”, Springer-Verlag, New York (Horik
oshi, K. & T., Akiba, Alka.
-1ophilic microorganisms,
Springer-Verlag, New York)
(1982)].

Some of the present inventors previously reported that alkaline cellulase [alkaline carboxymethyl cellulase (
(hereinafter referred to as “N-4ACMCase”)] [Horikoshi et al., Canadian Journal of Microbiology]
, Canadian Journal of Mic
robiology) 30°Nα6.774-779
(1984)].

This enzyme is a specific cellulase that has weak activity against Avicel, but extremely strong activity against CMC (carboxymethylcellulose), and has an optimum pH of 6.7 and 1010.

(Object of the Invention) An object of the present invention is to provide a Bacillus sp. NαN-4-derived lulase gene.

Furthermore, the object of the present invention is a DNA sequence that hybridizes to the DNA sequence of the N-4A CMCase gene, which is obtained naturally or semi-synthetically, and which includes nucleotide substitutions and nucleotide changes to the above DNA sequence. The object of the present invention is to provide a DNA sequence encoding a polypeptide having cellulase activity as described above, which is related by deletions, insertions of nucleotides, inversions of the nucleotide sequence, and other mutations.

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

Furthermore, it is an object of the present invention to provide a recombinant DNA molecule that contains any of the above DNA sequences, and in which the DNA sequence is operably linked to an expression control sequence.

(Structure of the Invention) Preparation of Chromosomal DNA> The N-4A CMCase-producing bacterium of the present invention is Bacillus sp. l
7 (The American Type Cult
Deposit number AT
It was deposited under CC21833 and is available to anyone (The American Type Cu1tu).
re Co11ection Catalog o
f Str, ains.

14th Edition 1980, p. 43).

The above N-4 strain is aerobically cultured at 37°C in an alkaline medium. After collecting bacterial cells in the early stage of logarithmic growth, pNA extraction was performed using the phenol method [Saito et al., Biochimica Biophysica Acta (Saito et al.

H., & Miura, K., Biochimica e.
t Biophysia Acta), 619-6
29 (1963)], the chromosomal DNA of the N-4 strain was extracted and purified.

<Insertion of DNA fragment into vector/plasmid and transformation> Vector/plasmid pBR322 can also be prepared by the method of Bolivar et al. [Bolivar et at Gene 2
, 95-113 (1977)], and commercially available products can also be used (e.g., Bethesda Research Laboratories).
manufacturers).

The above chromosomal DNA is cut with restriction enzyme mH+ndl [.

On the other hand, plasmid pBR322 was cut with HindI, and the cut chromosomal DNA was added to the DNA! A DNA strand binding reaction is performed using J gauze. The resulting binding mixture was prepared using conventional methods (e.g., Röderperg et al., Journal
Off-bacteriology (Lederberg, E.
, M., & Cohen, S.N., Journa.
l of Bacterio-1ogyH19,107
2-1074 (1974)) to obtain approximately 20,000 transformed strains per 1 μg of DNA (approximately 10% of the Ap' strain was TD''). Among convertible stocks, LB including CMC
Draw a shallow crater around the colony on the agar plate.
There were 8 stocks that formed. These strains are all Ap
', a transformed strain exhibiting cellulase activity.

This transformed strain was propagated, the plasmid was extracted and purified by a conventional method, and the 2.1 kb (kilobase) Hind m
Plasmids pNK1 and 2.8 containing the fragment b Hin
A plasmid pNK2 containing the d m fragment is obtained.

(Homology between inserted DNA fragment and chromosomal DNA) pBR
In order to analyze the origin of the DNA inserted into 322, plasmids pNKl and pNK2 labeled with 32p were immobilized on a nitrocellulose sheet, and N-4 strain and Escherichia coli HBIOI ( ε5cher 1ch-iacoli H
BIOI) chromosomal DNA was hybridized.

Radioactively labeled DNA fragments of plasmids pNK1 and pNK2 were hybridized with unlabeled fragments of plasmids pNK1 and pNK2, respectively.

In addition, the b fragments were also hybridized to 2.1 and 2.8 derived from the N-4 strain.

As a result, the respective fragments of plasmids pNKl and pNK2 were partially homologous, but
No NA sequence complementary to the plasmid pNK1 and pNK2 fragments was detected in the chromosomal DNA fragment of oli HBIOI.

(DNA sequence and amino acid sequence encoding cellulase) By sub-cloning, the cellulase of plasmid pNK2 is 1, 5 KbEcoRI-Hlnd m
At least the sites necessary for expression are present on the fragment. On the other hand, the cellulase of plasmid pNK1 is 2. IKb
It was found that at least a site necessary for expression exists on the HindI[I fragment. The strategy and restriction enzyme cleavage map for sea fencing of these fragments is shown in FIG. Sea fencing is based on the dideoxy chain termination method.
on method: Sanger et al, Proceedings of the National Academy of Sciences
・Sub-science (Proceedings of
the National Academy of 5c
iences), U, S, A,, 74.5463-54
67 (1967)]. That is, the HindIII fragment of plasmid pNK1 and the EcoRI-Hlndm fragment of pNK2 were combined with (α-”P)dC
It was cloned into a phage M13 vector for sea fencing using TP (Amersham) and M-13 sequencing kit (manufactured by Takara Shuzo).

The fragments labeled by Sanger's method above were separated into 40 cm
The gel was dissolved in an 8% acrylamide gel with a size of 20 cm x 0.3 mm and a thickness of 0.3 mm. The buffer solution was 8M-urea, 8M-urea, 8M-urea,
9mM-) Liss buffer, 89mM-Borate buffer and 2
mM-EDTA (pH 8,3) was used. Also, M1
3 phage derivatives, mp8 and mp9 (Mess
ing, J. 1983°New M13 vect
or forcloning, 34methodsεn
zymo1. ), the host is Escherichia coli J.
M 101 (Messing, J, etal
l 981, A system for show
tgun DNA sequencing, Nucle
ic Ac1ds Re5earch 9:309-3
21) was used. The cellulase-encoding DNA and amino acid sequences of plasmid pNK1 thus obtained are shown in FIG. 2, and the cellulase-encoding DNA and amino acid sequences of plasmid pNK2 are shown in FIG.

From FIG. 2, 2. l kb ) f
The ind■ fragment encodes 488 amino acids.
It had an open reading frame of 64 bp (base pairs). On the other hand, from FIG. 3, 1.
The 5kb EcoRI-HindI fragment contained a 227bp open reading frame encoding 409 amino acids.

In both Figures 2 and 3, the nucleotides are A and 1.
Also, the amino acid is methionine (!J
Start counting with e t ) as (1).

(Comparison of non-cellulase-encoding regions) Liposome binding sites (S, D, sequences), i.e., Bacillus
s 5ubtilis l 5 S ribonormal RN
An AGGAGG sequence complementary to the 3'-end of A was found upstream of the translated sequence of the cellulase gene of plasmids pNKl and pNK2, 51]P.

3'- of the cellulase gene of plasmid pNKl
Downstream of the terminus, there is an inverted repeat sequence (parindromic
inverted repeat), followed by a prokaryotic ρ-dependent termination site ρ-dep present in the gene encoding the tyrosine t-RNA5us of E. coli.
en-dant terminator) (Kupp
er etal, Nature 272:423-4
28 (1978)] was bound to the TAACAAC sequence.

On the other hand, the 3'-
Downstream of the terminus, an inverted structure (parin-drome)
ic5structure), there is a 4T region and a transcription termination site (p-1ndependent t
(ranscriptiveterminator) was present. The calculated values of ΔGs (Gibbs free energy) of the RNA hairpin structure are as follows: plasmid pNK1,
pNK2 is -21 and 8 Kcal/mol, respectively.
-25.8 Kcal/mol.

Moreover, no characteristic homology was found between the two inverted structures.

(Similarity between cellulases encoded by plasmids pNKl and pNK2) Dot plot matrix of predicted amino acid sequences of cellulases
It was analyzed by The results are shown in FIG. As a result, the cellulase encoded by plasmid pNK1
The two cellulases were found to be nearly identical, except for having a 60 pure (direct repeat) amino acid sequence not present in the cellulase encoded by pNK2. FIG. 5 shows a comparison of the DNA and amino acid sequences of the two cellulase proteins. In Figure 5, 1 is plasmid p
It is a cellulase gene contained in NK1, and 2 is
This is a cellulase gene contained in plasmid pNK2, and - indicates that it is the same nucleotide as the cellulase gene encoded by pNKl. Also, different amino acids are indicated below or above the nucleotide sequence. Nucleotides are counted with the first A as 1. As a result, these sequences were found to have extremely strong homology, except that the base at the third position of the codon may differ. This suggests that the amino acid sequence retains enzymatic activity.

On the other hand, a region with little homology was found near (or within) the signal peptide region. However, the amino acids in the signal peptide region were similar to those found in secreted proteins (Beguin e
tal, Journal of Bacteriol
-Ogy, 162: 102-105 (1985)
;5tephens.

M, ^, etal 1 bid and 8:369-372 (1
984); Yan, M., et al Nucle
ic Ac1ds Re5search 11+237
-249 (1983)). From this, it was predicted that these changes would not be very important for the function of this region.

(Need for a pure repeat sequence for expression of cellulase activity) A pure repeat sequence with a 60 amino acid sequence is found near the C-terminal end of the cellulase-encoding plasmid pNKl. Conventionally, a 23 amino acid region located near the C-terminus is isolated from Clostridium thermocellum (Clostri-dium thermocellum
It was predicted that this repeating unit would be involved in recognizing and cleaving the glucose bond of cellulose molecules [Begin et al. Journal of Bacteriology (Beguin, P.e.
tal, Journey of Bacteriolo
gY 162: 102-105 (1985)]
.

However, even though pNK2, which encodes cellulase, does not have a manipulated sequence, this protein expresses enzymatic activity (cellulase). Plasmid pN
Two Δ''I restriction sites are present in Kl near the 3'-end of each pure repeat sequence. Nco
One of the pure repeat sequences was removed using I. The resulting plasmid NK1-Δ (upper three) is a plasmid in which 261 base pairs (DNA fragments from 1186th to 1446th) have been removed. This plasmid pNK
The protein encoded by l-ΔNcoI still has cellulase activity.

As a result, it was predicted that pure repeat sequences are not necessarily required for enzyme activity.

Cellulases produced by Escherichia coli strain N-4 and Escherichia coli HBI 01 (pNKl) and HBIOI (pNK
2) Comparison of produced cellulases> When the pH stability, thermostability, and molecular weight of each cellulase was investigated,
No substantive differences were observed.

Example <1. Preparation of chromosomal DNA> The N-4 strain (ATCC21833) was cultured in an alkaline medium (
10g starch 5g yeast extract (pifco), 5g polypeptone 5K2HPO41g SMgSO4
・7H,00,2g 5Na2CO3 (separate sterilization) 10g
, if distilled water (pH 10,0)) at 37°C for 3 hours.
Culture was carried out aerobically, bacterial cells at the early stage of logarithmic growth were collected, and chromosomal DNA was extracted and purified by DNA extraction using the phenol method to obtain 5 mg of chromosomal DNA.

<2. Insertion of chromosomal DNA fragment into vector plasmid> Take 0 μg of the above chromosomal DNA and add restriction enzyme HindI.
I[ was added and reacted at 37°C for 14 hours to partially cleave. On the other hand, plasmid pBR322 (Bethesd
a Re5Earch Laboratories (
(manufactured in the United States) was added with HindIII, reacted for 1 hour, completely cut, and heat-treated at 65° C. for 5 minutes. after that,
Mix 1 Mg of cut vector plasmid DNA and 3 μg of cut chromosomal DNA, and
Add NA ligase and incubate at room temperature overnight.
A! 31 ligation reaction was performed, and after heat treatment at 65° C. for 5 minutes, 3 times the volume of ethanol was added to the reaction solution to precipitate and collect plasmid DNA incorporating chromosomal DNA.

<3. Transformation using plasmids〉Escherichia
Escherichia coli (Escherichia coli), a Vibrift strain with 12 strains of Escherichia coli and 8 strains of Escherichia coli.
ia coli) HB 101 strain (Gol-dfar
b et al, Proceedings of t
he National^cade+y+y of 5
Sciences of the United 5ta
tes of A+nerica 79, 5886 -
5890 (1982)] (Genetic traits: "2,
leu B, B, 1 embryo Y, dan dR, h seal x+.

ara 14. gal K2. xyl-5, m
tl-1,”E44. F-.

endor, recA, str') was used for transformation according to the method of Lederberg et al.

ε, M., & Cohen, S. N., 119. 1
072-1074 (see 1974).

That is, the 88101 strain was cultured in LB medium [Maniatis et al., Molecular Cloning, A Laboratory Maniatis/l/ (Maniatis et al., ) 4
o1ec-ular cloning, a 1abo
ration manual), pp 68-69 (
1981)] : Purified water 11 per cent, Trizo) 7 (
Dirco) 10 g, yeast extract 5 g, glucose 1 g, NaCj! A solution containing 10 g was adjusted to pH 7 and inoculated at >10 mJ2, cultured with shaking at 37°C, grown to late logarithmic growth, and then harvested.

This was cooled with water at a final concentration of 0.03M CaCI12.
The cells were sequentially suspended in a solution of □ to prepare competent cells.

The plasmid DNA solution obtained in 2 above was added to this cell suspension, and the mixture was allowed to react for 60 minutes under water cooling, followed by heat shock at 42° C. for 1 to 2 minutes to incorporate the plasmid into the cells. In this way, approximately 20,000 transformed strains were obtained per 1 Mg of DNA (approximately 10
% was Tc'>. Among these transformed strains,
On the LB agar plate containing CMC, there were 8 strains that formed shallow craters around the colony. All of these strains were transformed strains exhibiting Ap' and cellulase activities. This transformed strain was grown, and the cell suspension was separately inoculated into the LB medium, cultured with shaking at 37°C for 3 to 5 hours, harvested, washed, and transformed with plasmids pNK1 and pNK2, respectively. Converted Escherichia coli HB101 (pNKl) and Escherichia coli HB101 (pNK2), respectively.

This bacterial cell was treated as follows to obtain a MW plasmid.

Bacterial cells 50 mj2 Polypropylene centrifugal tube lysis. Gently mix, centrifuge on water bath for 15 to 30 minutes. 17.000
r, pom, 4℃, 40 minutes supernatant liquid ↓ 250+mj! Glass bottle centrifugation 650 (]r, ρ1m, 4℃, 15 minutes Upper layer 1 equivalent of phenol; Chloroform centrifugation 650 Qr, I), m, 4℃, 15 minutes ↓ Top "Centrifugation 6500r, p, m, -20℃, 60 minutes ↓ DNA pellet, dry excess liquid and mix A-50 column (2 x 35 cm, 1 fraction DNA
Fraction (A26° peak) [DNA pellet CsC1 gradient centrifugation, 36000 r, p, m
.

Collect the DNA in the lower band bit by bit.

[Dowex 50W-X8 column (detected by UV) [(maintained in bright state) Dialysis (10mM) Squirrel, lmM E
DTA, pH830ITll Cortex centrifuge tube centrifugation 6500r, p, n+, -20℃, 60
min [DNA precipitation 1-21Tlβ TEN buffer purified plasmid DNA (1+g/ml culture 1-20~
Cellulase production using transformed strain stored at -70°C> (Culture method) Each of Escherichia coli HBLOl (pNKl) and Escherichia coli HBI 01 (pNK2) was inoculated into the above LB medium, and 500 mj! After culturing aerobically for 24 hours at 37°C in a large-sized flask, the bacteria were harvested using the osmotic shock method [Kato et al., European Journal of Applied Microbiology and Biotechnology (Osmotic 5hock)].
Methods: Niato etal Eur, J.

Appll Microbiol, Biotechnol
) (1983) 18:339-343] into extracellular, veliplasmic space, and extracellular fractions.

(Enzyme activity measurement) CMC (2.0%) 1 ml, Glyci7-NaC1-N
0.5 mL of enzyme solution per 1 ml of aOH buffer (pH 9,0)
mj! and react at 40°C for 20 minutes.

After completion of the reaction, 3,5-dinitro-salicylic acid method [3.
5-ro1nitro-salicylic acid
(DNS) method) to quantify reducing sugars. That is, 11 Tl of DNS reagent was added to 0.25 ml of reaction solution.
1, heated at 100℃ for 5 minutes to develop color, cooled, and heated at 100℃ for 5 minutes.
1TII! Dilute by adding distilled water. Wavelength 50
Colorimetrically determined at 0 mμ.

The unit of enzyme titer is 1 unit (L) when producing reducing sugar equivalent to 1 μmol of glucose per minute under the above conditions.
J).

When we investigated the distribution of cellulase produced outside the cell, in the Beriplasmi gamma space, and within the cell, we obtained the results shown in the following table.

Next, cellulase produced by anti-4 strain, cellulase produced by Escherichia coli HBI 01 (pNKl) (pNK1 cellulase), and cellulase produced by Escherichia coli HBI 01 (pNK2) (pNK2NK2 cellulase at pH Activity (pH
stability), thermal stability and molecular weight were investigated and compared.

(pH stability) pH 4, 4 and 5.0 are acetate buffer, pH 7, 5 and 8.
0 is Tris-HCl buffer, pH 9, 2, 10, 1, 10
No. 69, 11, 7, 12, and 8 used glycine buffer.

In addition, lomlI was used for each cellulase.
After reacting the j buffer solution and the enzyme mixture at 60° C. for 10 minutes, cellulase activity was measured in the same manner as the measurement method described above. In addition, the enzyme activity is the activity at pH 10.1
It was set as 00%. 1 The results are shown in Figure 6.

From Figure 6, all cellulases are stable at pH 5 to 11, and the optimal pH for enzyme action is pH 5.0.
It can be seen that it is between 10.9 and 10.9.

(Thermal Stability) In the activity measurement method described above, the reaction was carried out for 10 periods while changing the temperature, and the residual potato activity at pH 5, 5 and 10.0 was investigated. As a result, both cellulases b were stable up to 175°C.

(Molecular weight) P taken by gel filtration method using Sephacryl S-200
The molecular weights of NK1 cellulase and pNK2 cellulase are
They were approximately 58,000 and approximately 50,000, respectively.

Thus, it was confirmed that the respective cellulases are almost identical in pH stability, stability, and molecular weight.

[Brief explanation of the drawing]

Figure 1 is a diagram showing a restriction enzyme cleavage map of the region in which the cellulase gene derived from the N-4 strain is troubled and the strategy for Sequence Engine R. The arrow (-) indicates the direction and range of DNA sequence analysis. The concave line indicates the coding region of the cellulase gene, A is the pNK1 cellulase region, B is the pN
K2 supracellular region is shown. Figure 2 shows 2. of plasmid pNK1. lkb Hind
Figure 3 shows the nucleotide sequence of the cellulase gene encoded in the plasmid p
1.6kb of NK2 'EcoRI-HlndI[I
The nucleotide sequence of the cellulase gene encoded in the fragment is shown. FIG. 4 shows a computer survey of homologous regions of the amino acid sequences of pNK1 cellulase versus 1) NK2 cellulase. FIG. 5 is a diagram showing a comparison of the base sequences and amino acid sequences of pNK1 cellulase and pNK2NK2 cellulase. Figure 6 is derived from N-4 strain, E. coli HB 10
1 (pNKl) and Snake, coli HBI 01
(pNK2) is a diagram showing the pH stability of cellulase produced by pNK2. Figure 4 PNKl Cellulase Amino Shun Sequence ←→1 to PN h---h 2 H to PN

Claims (3)

[Claims] (1) A DNA sequence encoding a cellulase having the following amino acid sequence. [There is a gene sequence] (2) A DNA sequence that hybridizes to the DNA sequence set forth in claim (1), which is obtained naturally, synthetically, or semi-synthetically; A DNA sequence that is related to the sequence by nucleotide substitutions, nucleotide deletions, nucleotide insertions, nucleotide sequence inversions, and other mutations and that encodes a polypeptide that has cellulase activity. (3) The DNA sequence described in claim (2) is
A DNA sequence that is the following DNA sequence. [There is a gene sequence]