JPH0767392B2 - Gene that encodes a factor that increases the production of macromolecular degrading enzymes - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention relates to a DNA fragment encoding a thermophilic bacterium-derived polymeric substance degrading enzyme increasing factor, a recombinant plasmid incorporating the DNA fragment, and the recombinant plasmid. And a method for producing a polymer degrading enzyme using the transformant.

[Prior art]

Conventionally, for example, bacteria of the genus Bacillus, etc., have been used for industrial scale enzyme production because they secrete and produce various kinds of polymeric substance degrading enzymes such as protease and cellulase. Attempts have also been made to introduce a gene for a polymer-degrading enzyme into these bacteria by using a method to enhance the enzyme productivity.

However, satisfactory enzyme productivity has not always been obtained by the conventional method of merely introducing a gene for a polymer-degrading enzyme.

[Problems to be Solved by the Invention]

An object of the present invention is to develop new means for dramatically improving the productivity of high-molecular substance decomposing enzymes such as protease and cellulase by using gene manipulation techniques.

[Means for Solving the Problems]

As a result of earnest research on means for increasing the productivity of polymer-degrading enzymes, the present inventors have found a gene encoding a factor that causes an increase in the production of various types of polymer-degrading enzymes than thermophilic bacteria, and arrived at the present invention. It was done.

It is to be noted that the existence of a gene encoding a factor that causes an increase in the production of various types of macromolecular substance-degrading enzymes in thermophilic bacteria has not been known at all until now, and was first discovered in the present invention. .

That is, the present invention is: 1. A DNA fragment that is derived from a thermophilic bacterium and that contains a base sequence that encodes the following amino acid sequence and that encodes a factor for increasing the production of a polymeric substance-degrading enzyme.

2. The DNA fragment according to 1, containing the following nucleotide sequence.

A recombinant plasmid containing the DNA fragment according to any one of 3.1 to 2.

A transformant obtained by transforming a host microorganism with the recombinant plasmid described in 4.3.

A method for producing a polymeric substance degrading enzyme, which comprises culturing the transformant according to 5.4 in a medium and collecting the polymeric substance degrading enzyme.

It consists of

Examples of the polymer degrading enzyme produced in the present invention include, but are not limited to, alkaline protease, levansucrase, xylanase, cellulase, and amylase.

In the present invention, examples of thermophilic bacteria as a collection source of a DNA fragment encoding a factor that increases the production of a polymer degrading enzyme include, for example, Bacillus stearothermophilus (Ba
cillus stearothermophi-llus) is preferred,
The fragment is Bacillus stearothermophilus (Bacillus
stearothermo-phillus) chromosomal DNA. For example, Bacillus stearothermophilus (Baci
llus stearothermophillus) NCA1503 (J.Appl.Bacterio
l. 38 , 301-304, 1975) and a vector pTB53 with a chromosomal DNA digested with a restriction enzyme Pst I, and then mixed and ligated to obtain a recombinant plasmid such as Bacillus subtilis. A microorganism having the ability to produce a polymer-degrading enzyme is transformed, and a high-producing strain of a polymer-degrading enzyme such as alkaline protease or levansucrase is isolated. The plasmid pDT145 (22.3 kb) obtained from the high-producing strain has a chromosomal DNA fragment of about 5 kb, which was further miniaturized and reconstituted by treatment with various restriction enzymes and DNA polymerase I (Klenow fragment) to obtain plasmid pDT80. (12.3kb) is obtained. pDT80 is about
It has a chromosomal DNA fragment of 1.6 kb, and this DNA fragment encodes a polymeric substance degrading enzyme increase production factor. The nucleotide sequence of the above DNA fragment determined by the dideoxy method is shown in Fig. 2 and is 1116 bp (372 amino acids, molecular weight 41
There is an open reading frame consisting of 2,244). Furthermore, this DNA fragment has a promoter, terminator, and SD sequence necessary for the above-mentioned open reading frame to be actually translated as a protein, and a high molecular substance having the amino acid sequence shown in FIG. 1 as a gene product is decomposed. Enzyme production factor is produced.

DNA encoding the polymeric substance degrading enzyme production-increasing factor of the present invention
Whether the fragment has a part of the nucleotide sequence shown in FIG. 2 or has a part of the nucleotide sequence deleted, substituted or inserted, it causes an increase in the production of the polymer degrading enzyme. As long as it encodes a factor, it is within the scope of the invention.

The DNA fragment of the present invention is preferably expressed using a Bacillus bacterium host-vector system. As a host of the bacterium of the genus Bacillus, for example, Bacillus subtilis or Bacillus stearothermop
hillus) and the like. The vector must have an origin of replication in a bacterial host and a gene capable of complementing the auxotrophy of the host, such as a tetracycline resistance gene, a kanamycin resistance gene, an antibiotic resistance gene, or a leu gene, which serves as a selection marker. As such a vector, pTB53 (J. Bacteriol. 146: 1091-1097 (1981)),
pTB90 (J. Bacteriol. 149: 824-830 (1982)), pC194
(Proc.Natl.Acad.Sci.USA74: 1680-1682 (1977
)), PUB110 (Proc.Natl.Acad.Sci.USA75: 1428-1
432 (1978)) and the like. Further, if these vectors have a promoter capable of controlling the expression of the above DNA fragment, more effective action can be expected. Examples of such a promoter include a promoter of a penicillinase gene derived from Bacillus licheniformis, a promoter of various amino acid synthase genes, and the like.

Examples of the method for transforming a bacterial host with the plasmid obtained by inserting the DNA fragment of the present invention into the above vector include the competent cell method and the protoplast method. The competent cell method of Bacillus subtilis (Proc. Natl. Acad. Sci (Wash.) 44: 1072-1078 (19
58), the protoplast method was Molec.Gen.Genet.168: 11.
1-115 (1979), the Bacillus stearothermophillus protoplast method is described in J. Bacteriol. 149: 824-830 (1982), respectively. In addition, the transformed bacterial host produces the polymeric substance-degrading enzyme production-increasing factor of the present invention by culturing it using a general medium consisting of a nitrogen source, a carbon source, essential nutrients, trace metal salts, etc. Can be made.

Hereinafter, the present invention will be described in more detail with reference to Examples.
The present invention is not limited to this.

In the following examples, the gene encoding the polymeric substance degrading enzyme production-enhancing factor of the present invention is abbreviated as degT.

Example 1 Bacillus stearot
hermophillus) About 2 μg of chromosomal DNA of NCA1503 and vector
About 1 μg of pTB53 was treated with the restriction enzyme Pst I, mixed and ligated, and then Bacillus subtilis (Bacillus subtili
s) MT-2 (tysC2 leuC7 r M - m M - Npr -) was transformed by the competent cell method, Bacillus subtilis (Bacill
We isolated one strain with increased production of alkaline protease of us subti-lis). Plasmid pDT1 carried by this strain
45 (22.3 kb) contained a DNA fragment of about 5 kb derived from Bacillus stearothermophillus NCA1503 (Fig. 3). Also, Bacillus subtilils carrying pDT145
Found that, in addition to alkaline protease, it increased the production of macromolecule-degrading enzymes such as levansucrase, xylanase, and cellulase, and that the DAN fragment had a gene (degT) encoding a macromolecule-degrading enzyme production factor .

On the other hand, we reduced the size of pDT145 and limited the degT included in pDT145 (Fig. 3). First, pDT145 was treated with the restriction enzyme BamHI, and after ligation, Bacillus subtilis (Baci
llus subtilis) MT-2 was transformed, and the plasmid pDT99 (15.2 kb) was obtained from the transformant. Next, pDT99 was simultaneously treated with restriction enzymes Sal I and BamH I to obtain Sal I-B of about 4.2 kb.
The amHI fragment was isolated. This and Esh.
erichiacoli) vector pBR322 (Gene2: 95-113 (1977
)) Was simultaneously treated with Sal I and BamH I, mixed and ligated, then Esherichiacol (Esherichiacol
i) JM103 (ΔClac-pro) supE thi strA sbcB15endA hs
pR4F ′: traD36 proAB lacI ^q Z ΔM15) was transformed by the competent cell method (J. Bacteriol. 119: 1073-1074 (1974)), and the plasmid pBR-BS (8.3k) was transformed from the transformant.
b) got. This pBR-BS was treated with restriction enzymes Dra I and Sal I at the same time to separate a Sal I-Dra I fragment of about 2.7 kb. This and pBR322 treated with restriction enzymes EcoRV and Sal I were mixed and ligated, and then Escherichia coli (Escher
ichia coli) JM103 was transformed, and a plasmid pBR-DS (6.6 kb) was obtained from the transformant. pBR-DS and pDT99 were treated with restriction enzymes Hind III and Sal I, mixed and ligated, and then Bacillus subtilis MT-
2 was transformed with the plasmid pDT77 (11.
8 kb) was obtained. On the other hand, pBR-BS was treated with the restriction enzyme Acc I, and then DAN polymerase I (Klenow fragment)
After treatment with, a fragment of about 1 kb was separated. This fragment and pBR32
Escherichia coli (Eshe-richia coli) JM103 was transformed with the mixture of the restriction enzyme 2 treated with EcoRV and ligated, and the plasmid pBR-Ac3 (5.4 kb) was obtained from the transformant. Finally, pBR-Ac3 and pDT99 were treated with the restriction enzyme Sph I, and after mixed ligation, Bacillus subtilis (Baci
-Llus subtilis) MT-2 was transformed, and a plasmid pDT80 (12.3 kb) was obtained from the transformant. Bacillus subtilis MT-2 (p containing pDT77
DT77) has been deposited with the Institute of Microbiology as Microbiology Research Institute No. 10826. pDT99 is approximately 4.3 kb, pDT77 is approximately 3.2 kb, pDT80 is approximately 1.
6 kb Bacillus stearothermophilus
earothermophillus) NCA1503-derived DNA fragment containing degT.

The nucleotide sequence of the DNA fragment containing about 1.6 kb of degT was determined by the dideoxy method (Meth) using a Sequenase kit (manufactured by Toyobo).
ods Enzymol. 101: 20-78 (1983)). The result is shown in FIG. 2 (in addition, the amino acid sequence deduced therefrom) is shown.

The various restriction enzyme treatments, ligations, and DNA polymerase I (Klenow fragment) treatments were performed according to their respective protocols.

Example 2 (Effect of degT on alkaline protease) The protease activity was determined by the method of Hagihara et al. (J. Biocchem. (T
45: 185-194 (1958)).

That is, the degT-introduced transformant of the present invention obtained in Example 1, Bacillus subtilis (Bacillus subti)
lis) MT-2 (pDT80) and degT-free plasmid pTB5
3 transformed Bacillus subtilis (Bacillus su
btilis) MT-2 (pTB53) was cultivated in each, and 1 ml of each enzyme solution (bacterial culture supernatant) and substrate solution (50 mM Tris-HCl, 5 m) obtained
M CaCl ₂ , 2% casein (Hamarsten method), pH 7.5) 1 ml
Were mixed and reacted at 37 ° C. for 20 minutes. Then, 2 ml of TCA solution (0.1 M TCA, 0.33 M acetic acid, 0.22 M sodium acetate) was added to stop the reaction, and the mixture was filtered through Whatmar filter paper No. 1 φ7.0 cm to measure the absorbance at 275 nm.

The results are shown in Table 1.

Bacillus subtilis (Baci-llus s) introduced with degT
ubtilis) MT-2 had about 3-fold increased activity.

Example 3 (Effect of degT on levansucrase) The recombinant plasmid pDT80 containing degT of the present invention obtained in Example 1 and the plasmid pTB53 not containing degT were used to express Bacillus subtilis MI113 (arg-1).
^{5 trpC2 r M - m M -} ) respectively transformed to obtain transformants.

Next, 300 μl of each enzyme solution (bacterial culture supernatant) and substrate solution (50 mM K-Pi, 16% suc) obtained by culturing each of them were obtained.
rose, pH6.0) 300μ, mix and perform at 37 ℃ for 20 minutes.
The reaction was stopped by heating at ℃ for 5 minutes, and levansucrase activity was measured. The levansucrase activity was measured by quantifying the amount of glucose produced by the above enzymatic reaction using "Glucose Test B-Wako (Wako Pure Chemical Industries)". The results are shown in Table 2.

Bacillus subtilis (Baci-llus s) introduced with degT
ubtilis) MI113 (arg-15 trpC2 r M - m M -) is about 13 times the increase in production was observed. Inducibility by sucrose was not lost.

Example 4 (Effect on xylanase and cellulase) The xylanase and cellulase productivity was assayed by a plate test using each transformant obtained in Example 3 above.

(A) Assay for cellulase productivity After growing the bacteria on a plate containing 0.5% CM-cellulose, the cells were overlaid with 0.6% agar, a 1.5% Congo red solution was poured, and the mixture was allowed to stand for 15 minutes. The solution was removed, the plate was washed several times with a 1 M NaCl solution, and assayed with a halo around the colony.

(B) Assay for xylanase productivity 0.4% xylan, 2% (NH ₄ ) ₂ SO ₄ , 0.01% casamino acid, 5 μg
/ ml required amino _{acids, 60mM K 2 HPO 4 44mM K} 2 HPO 4, 3mM Trisodi
_{um-citrate, 2mM MgSO 4,} 0.01mM MnCl 2, 0.5mM CaCl 2, 10m
After growing the bacteria on a plate composed of g / ferric ammonium citrate, the bacteria were overlaid with 0.6% agar. After that, the same operation as in the cellulase production assay was performed for the assay. The results are shown in FIG.

Bacillus subtilis (Baci-llus s) introduced with degT
ubtilis) MI113, increased production of these enzymes was observed.

Thus, it was shown that the strain into which degT was introduced increased the production of various types of polymer degrading enzymes.

〔The invention's effect〕

As is clear from Examples 2 to 4, the transformants transformed with the recombinant plasmid containing the DNA fragment encoding the macromolecule-degrading enzyme production factor of the present invention are various macromolecule-degrading enzymes. In the production of the above, it showed an extremely excellent effect of increasing production, and by the present invention, it became possible to efficiently produce various polymeric substance degrading enzymes in good yield.

[Brief description of drawings]

FIG. 1 shows the amino acid sequence of the polymer-degrading enzyme production-increasing factor of the present invention. FIG. 2 shows the nucleotide sequence of a DNA fragment encoding a polymeric substance degrading enzyme production-enhancing factor. FIG. 3 shows the outline of plasmid construction. Figure 4 shows Bacillus subtilis MI113 (pDT80) and Bacillus subtilis MI113 (pTB53).
It is a figure showing the comparison of the cellulase and xylanase productivity of.

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Claims (5)

[Claims] 1. A DNA fragment which is derived from a thermophilic bacterium and which contains a base sequence encoding the following amino acid sequence and which encodes a factor for increasing the production of a polymer degrading enzyme. 2. The DNA according to claim 1, which contains the following nucleotide sequence:
fragment. 3. A recombinant plasmid containing the DNA fragment according to claim 1. 4. A transformant obtained by transforming a host microorganism with the recombinant plasmid according to claim 3. 5. A method for producing a polymeric substance degrading enzyme, which comprises culturing the transformant according to claim 4 in a medium and collecting the polymeric substance degrading enzyme.