JPH01269492A - Production of thermostable protease - Google Patents

Abstract

PURPOSE:To eliminate dispersion in productivity under the same culture conditions and efficiently obtain the subject enzyme by cultivating nonthermophilic bacteria containing a gene capable of coding a thermophilic bacteria-delived thermostable protease. CONSTITUTION:A gene capable of coding a thermophilic bacteria-derived thermostable protease [preferably thermostable protease gene of Bacillus.stearothermophilus TELNE (FERM-P 9439)] is initially integrated into a chromosomic DNA of nonthermophilic bacteria (preferably Bacillus.subtilis and the above-mentioned bacteria are cultivated to collect the aimed enzyme from the afore-mentioned culture.

Description

[Detailed Description of the Invention] (Industrial Application Field) Thermostable proteases can be used in food production, detergent production, peptide synthesis, etc., and the development of an effective production method is extremely significant. .

(Prior Art) A method for producing a thermostable protease generally involves culturing a thermostable protease-producing bacterium, and isolating and purifying the secreted thermostable protease from the culture supernatant. However, since the productivity of protease is not sufficient, in recent years a method for producing heat-stable protease has been developed using a transformant strain that has been transformed with a recombinant plasmid containing a gene necessary for the production of heat-stable protease. .

For example, the Journal of Hacteriology (Jou
RNA of Bacteriology) No. 154
Volume 831 (1983) describes Bacillus stearothermophilus.
Bacillus subtilis (Bacillus s.
A method for producing a thermostable protease using MT-2 (pNP22) has been reported.

(Problems to be Solved by the Invention) Although the method for producing protease using a microorganism transformed with a recombinant plasmid containing a thermostable protease gene can be used industrially in terms of productivity, it does not require long-term use. (100 to 150 hours), the recombinant plasmid is often missing from the microorganism during the culture period, or the heat-stable protease gene is deleted from the plasmid, so protease productivity may vary. Therefore, a more stable manufacturing method was desired.

(Technical means to solve the problem) As a method for solving the problem described in 1, the present invention provides a method for solving the problem described in 1. This method uses bacteria to produce heat-stable protease. Furthermore, a thermostable protease is produced using Bacillus subtilis, which has a gene encoding a thermostable protease derived from a thermophilic bacterium incorporated into its chromosomal DNA.

The thermophilic bacteria of the present invention include Bacillus stearothermophilus.
There is moph-.

The thermostable protease derived from a thermophilic bacterium of the present invention is a protease obtained from the above-mentioned bacterial strain, for example, a protease derived from a strain of the genus Bacillus stearothermophilus, which retains 70% or more even when heated at 80°C for 60 minutes. Neutral protease exhibiting residual activity (Patent Application No. 193409/1982),
Examples include alkaline protease produced by Bacillus stearothermophilus NCA 1503.

Genes encoding thermostable proteases derived from thermophilic bacteria of the present invention include genes encoding thermostable proteases derived from thermophilic bacteria, such as Bacillus stearothermophilus (B.
acillus stearothermopbilu
s) Gene encoding a thermostable protease produced by TIELNETERM-P No. 9439 (patent application 1986)
2-184323) and the neutral flotease gene of Bacillus stearothermophilus C112].

Upstream of the gene encoding the thermostable protease, gene sequences necessary for expression of the thermostable protease gene, such as a promoter and SD sequence, are included.

The chromosomal DNA in the present invention is chromosome 11 NI'l of a non-thermophilic bacterium. Preferably it is Bacillus subtilis chromosomal DNA.

Integration of the above gene into chromosomal DNA takes place within the body of a non-thermophilic bacterium.

Bacillus subtilis has a thermostable protease gene integrated into its chromosomal DNA.
iris) will be described in order below.

(8) gene DN8 encoding a thermostable protease;
Genetic DNA encoding an enzyme that inactivates antibiotics such as kanamycin and tetracycline and the host Bacillus subtilis
After preparing fragment DNA by cutting the chromosomal DNA of s) into 1 to 10 kb bases by enzymatic or mechanical treatment according to a conventional method, it is mixed in an aqueous solution and treated with an enzyme ("F4D").
ligate using NA ligase).

(B) Using the DNA ligated in (8) above, Bacillus subtilis
) is transformed according to a conventional method, and transformants exhibiting antibiotic resistance are selected.

(C) The transformant obtained in (B) above was inoculated into a medium for protease production, and after culturing for 10 to 150 hours, the protease activity of the supernatant was measured, and the thermostable protease gene was detected in the chromosome. Bacillus subtilis is integrated into DNA and produces thermostable protease.
1 us 5 ubtilis).

The method of culturing the transformant is aeration culture at a temperature of 25-3.
The 7°C time is 100-150 hours. Thermostable protease can be collected from a culture by collecting the culture solution, removing bacterial cells by centrifugation or filtration, and using a combination of salting out, ion exchange resin, and other known methods from the supernatant. be exposed.

(Effect) Bacillus subtilis (Bacillus 5ubt) in which a thermostable protease gene has been integrated into the chromosomal DNA
By using Bacillus subtilis carrying a recombinant plasmid containing a thermostable protease gene,
is), the productivity of thermostable protease increases when cultured for a long time (100 to 150 hours),
Variations in productivity under the same culture conditions are also reduced.

Therefore, it is possible to produce thermostable protease more efficiently.

(Examples) Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to these in any way.

(1) Bacillus stearolymophilus (ll
ac-illus stearothermophi
lus) TIELNIj FIERM heat] N
o.

Recombinant plasmid psp124 containing the thermostable neutral protease gene of 9439 (base number 19.3 kb,
Kanamycin resistant) [Figure 1] is prepared according to the conventional method (
'Patent Application No. 1982-11E323). Restriction enzyme EcoRI
cleaved with thermostable neutral protease gene (95), b
) and the kanamycin inactivating enzyme gene (759b).
using a nucleic acid recovery device (ELICA-Vl, Waka-mor
(manufactured by i).

(2) Bacillus subtilis
MT-2 chromosomal DNA was prepared according to a conventional method, and after treatment with restriction enzymes 1ic, o Rl, fragment DNA of 1 to 1 okb was obtained using a nucleic acid recovery device.
0 mg was prepared.

(3) Mix the DNA obtained in (+) and (2) above and incubate at 4°C using IOU of T4 DNA ligase in the presence of 1mM ATP and 10mM dithiothreitol.
A 212 hour DNA ligation reaction was performed.

(4) Using the reaction solution of (3) above, Bacillus subtilis MT-
2 was transformed by the competent cell method and spread on a medium containing 1% casein, 0.0004% kanamycin and 1.5% agar. Colonies were formed at 37°C for 2 days, and producing bacteria were selected by the halo formed around the colonies due to secretion of heat-stable neutral protease.

(5) The heat-resistant neutral protease production 17S medium obtained in (4) above was mixed with protease production medium (1% casein, 11% glucose, 0.2% yeast extract, 0.02% magnesium sulfate, 1.0.02% Calcium chloride.

0.002% zinc sulfate, 0.0004% kanamycin.

The most productive strain (pH 7.0) was inoculated into a 20-capacity test tube and inoculated with one platinum loop, and after culturing at 30°C for 100 hours, the protease activity of the supernatant was measured. IK-25) was selected.

= 7- (6) I K -25 and MT-2 (psP124)
One platinum loopful was inoculated into 2 mQ of protease production 17S solution, which was dispensed into a 15 m2 test tube, and cultured with shaking at 30°C for 30 hours. Production ifH place] mj!
The cells were inoculated and cultured with shaking at 30'C for 100 hours.

The same culture was performed a total of 5 times, and IK-25 and MT-2 (p
The culture titers of SP124) were compared (Table 1).As a result, the IL25 titer was 1.2 to 1.7 times that of MT-2 (psPI24), and the The variation in titer was also smaller for IK-25.

Table 1 1st 11.3 9.11 2nd
10.9 6.48 3rd 12゜1
9.32 4th 11.8 7.9
0 5th 12.5 10.25 (Effects of the Invention) Using the present invention, it becomes possible to efficiently produce a thermostable protease.

[Brief explanation of the drawing]

Figure 1 is a restriction enzyme map of the recombinant plasmid l"psP+24. The part corresponding to the linear DNA used in the Examples is indicated by a black line. EcoRI is a restriction enzyme map derived from Eacherichia coli. The enzyme, BamHI, is derived from Bacillus amyloliquefaciens.
ns), Kmr is a kanamycin inactivating enzyme gene, and Npr is a thermostable enzyme. Each represents a neutral protease gene. Patent applicant: Toyobo Co., Ltd.

Claims (1)

[Claims] A method for producing a thermostable protease, which comprises culturing a non-thermophilic bacterium in which a gene encoding a thermostable protease derived from a thermophilic bacterium has been integrated into its chromosomal DNA, and collecting the thermostable protease from the culture.