JPS6363374A - Novel bacillus sp h1402 - Google Patents

Abstract

PURPOSE:To produce a large amount of protein outside a cell without preparing a protease outside the cell, by cultivating Bacillus sp H1402 (FERM p-8892) of a novel strain. CONSTITUTION:Bacillus sp H1402 capable of producing >=5g/l has the following mycological properties. (A) Morphological properties: having 0.7 - 0.2 X 2.5 - 4mu cell size, being a bacillus free from polymorphism, having mobility by peripheric flagellum, containing a spherical - elliptic spore (0.5 X 0.5 - 1mu) at the central part and having a swollen shape of sporangia and positive Gram'stain free from acid-fastness. (B) Growth states in each medium as shown in the table. (C) Physiological properties: positive in reduction of nitrate, MR test, use of inorganic nitrogen source and catalase, negative in denitrification reaction, VP test, formation of indole, formation of H2S, hydrolysis of starch, use of citric acid, formation of dyestuff, urease and oxidase and not decomposing in O-F test.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel Bacillus sp H1402.

Conventionally, when proteins were produced by microorganisms, they were generally obtained by culturing the microorganisms, grinding the microbial cells, and then extracting and purifying the proteins.

In addition, Escherichia coli is generally used as a host for genetically modified microorganism production, but in Escherichia coli, the peptides and proteins synthesized by recombinant genes remain within the cells and are not secreted into the culture medium. Naturally, its production was limited.

However, it has been pointed out that extracting and purifying peptides and proteins by cell grinding has drawbacks such as complicated operations.

Udaka had previously researched microorganisms that secrete proteins outside of their cells as host bacteria for genetic recombination, and as a result, selected Bacillus plebis (Bacillus plebis) from among about 1,200 strains as microorganisms that secrete and produce large amounts of proteins. Bacillus b
revis) 4 strains, new strain Bacillus proteiformans (Bacillus proteiformans)
We were able to isolate and identify 5 strains of 1 strain. (Agric,
Biol.

Chew, 40(3), 523-528 (197
6)) On the other hand, Bacillus subtilis has also been used as a secretion host-vector and has been successful in accumulating various heterologous proteins such as α-amylase and interferon in the culture medium, but the production volume is limited due to strong proteases inside and outside the bacterial body. are limited or degraded, and good results have not been achieved.

Previously, Udaka et al.
Bacillus stearothermophil
us) When Bacillus plevis 47 and Bacillus subtilis carrying pBAMlol, in which the thermostable α-amylase gene of DY-5 was integrated into plasmid ptiB110, and Bacillus subtilis were cultured at 37°C for 48 hours, Bacillus plevis 47 exhibited approximately 15.0
OOU/mu, α of about 3.0000/mQ for Bacillus subtilis
- It was confirmed that amylase was produced and accumulated in the culture medium. (J. Bacteriol.

164, (3), 1182-1187 (1985))
.

Here, between Bacillus plevis 47 (described later) and Bacillus subtilis, which carry exactly the same plasmid, Bacillus plevis 47 is approximately 5 times more efficient in producing heat-stable α-amylase.
The fact that the production efficiency is twice as high indicates that a heterologous gene product can be efficiently secreted and produced by using the protein secretion ability of protein-producing bacteria.

However, Bacillus plevis 47.1, which was previously isolated and identified as a bacterium that secretes and produces large amounts of protein outside the bacterial body.
44.481.899 and Bacillus proteihomans 444, all of which contained bovine serum albumin (
Hereinafter referred to as BSA. ), it decomposes BSA and further increases Bacillus plebis 144゜481, 89.
It was confirmed that four strains of B. 9 and Bacillus proteihomans 444 also had casein-degrading activity. Therefore, when we secrete and produce heterologous gene products using recombinant genes using bacteria that secrete and produce large amounts of these proteins outside the bacterial body, we believe that the secreted and produced peptides and proteins will be efficiently degraded by proteolytic enzymes. It was done.

Therefore, the present inventors secreted a significant amount of protein.

In addition, if we can find a strain that does not produce any proteolytic enzymes outside the bacterial body, we believe that it would be an excellent host for genetic recombination, so we carried out intensive selection in search of such strains. As a result, approximately 1
Out of 00,000 shares.

They succeeded in isolating a strain that produces a significant amount of protein extracellularly, but does not produce proteolytic enzymes extracellularly.

When the strain isolated here was identified as belonging to Bacillus, the present invention was completed.

The present invention produces a significant amount of protein outside the bacterial cells.

Bacillus sp H140 that does not produce proteolytic enzymes
It is 2.

Conventionally, strains of Bacillus that produce protein have been known, but all of the known strains produce proteolytic enzymes, and the present invention, which produces a significant amount of protein outside the bacterial body, Bacillus sp H1402, which does not produce proteolytic enzymes outside its cells, is completely unknown and is new.

In the present invention, strains that secrete and produce 5 g or more of protein into the medium and do not degrade either BSA or casein proteins were selected and isolated.

First, approximately 100,000
strains were grown on T2 agar plates (1% glucose, 1% peptone, 0.5% meat extract, 0.2% yeast extract, 1.
The bacteria were inoculated onto 5% agar powder, pH 7.0), and bacteria whose periphery became cloudy with 5% perchloric acid were selected on a plate medium. Next, the bacterial strain isolated here was transferred to a T2 liquid medium (150 mQ
Culture with shaking (30°C,
48 hours), and 80 strains producing 1.2 g IQ or more of protein in the culture filtrate were obtained.

When measuring extracellular proteins, add an equal amount of 0.2
Add N NaOH, stir, and centrifuge at 10,000 rpm for 5 minutes to remove bacterial cells. Add an equal amount of 10% trichloroacetic acid to the supernatant, and after 10 minutes, centrifuge at 3,000 rpm for 5 minutes.
The precipitate was collected by centrifugation for 10 minutes, dissolved in IN NaOH, and then subjected to the Lowry method (J, Biol, Chem.
, 193.265 (1951) 3, and the protein amount was converted into bovine serum albumin. The medium shown in Table 1 was selected as a high protein production medium.

The 80 strains obtained above were cultured with shaking in all of these five types of media, and 31 strains that produced 5 g or more of extracellular protein in any of the media were selected.

Regarding the obtained 31 strains, the following measurements of BSA degradability and milk casein degradability were performed.

(Measurement of degradability of BSA) T2 medium was dispensed into a 150 mQ Erlenmeyer flask at 10++Ω, sterilized in an autoclave, and sterile-filtered BSA (S
igmaA4503) solution to a final concentration of 3.2 mg/mf.
The strain was pre-incubated overnight and the strain was added to a concentration of 0.21 l.
After inoculation, culture was carried out at 37°C with shaking at 20°rpm.

Culture filtrate sampled after 24 hours, 48 hours, and 72 hours of culture was collected in 10. OOOrpm 0.5M Tris-C1 (p
H6,8) 125μQ, 10% SDS 200μ2, β
- After adding 50 μQ of mercaptoethanol and stirring, heat treated in boiling water for 3 minutes, then 0.0625M Tris-CI (pue,
0.1 mQ of a) was added to prepare a sample for 5DS-polyacrylamide gel electrophoresis (SO3-PAGE).Slab 5O3-PAGE was performed at a 10% acrylamide concentration.

Protein detection was performed by staining with Coomassie brilliant blue. A strain that did not degrade BSA during all 24, 48, and 72 hours of culture was designated as a strain that does not have the ability to degrade BSA.

(Measurement of degradability of milk casein) A solution in which 5 g, 2 g, and 1 g of skim milk were each suspended in 50 mQ pure water, and a solution in which 1 g agar was dissolved in 50 mfl pure water were mixed separately in an autoclave and sterilized, then separated into petri dishes. 5%, 2%, and 1% milk agar plates were prepared. After planting the bacterial strain on a plate medium, the seedlings were cultured at 37° C. for 3 days, and the surrounding area of the colony was examined to see if it became transparent. 5%, 2%
A strain that did not produce any transparent circles on any of the 1% milk agar plates was designated as a strain that did not have the ability to decompose milk casein.

As a result of the above measurements, strain 81402 was selected as a strain that does not produce proteolytic enzymes outside the cells, since it does not degrade both BSA and milk casein.

81402 strain, Bergey's Manual D
eterminative Bacteriology
(8th edition) and The Prokaryote (A
Handbook on Habitats, l5ol
ation and identification o
When the strain was identified by f Bacteria), it was first recognized that it belonged to the genus Bacillus in that it was aerobic, positive for Durham staining, and formed bacilli and spores.

In addition, as a result of comparing other morphological properties, growth conditions in various media, and physiological properties with conventionally known bacterial species of the genus Bacillus, they were found to be different from any other bacterial species of Bacillus. Furthermore, this bacterial strain did not have the ability to degrade casein and BSA.

Therefore, this bacterial species was identified as a new bacterial species of the genus Bacillus.

Thus, this bacterial strain was named Bacillus sp H1402.

Bacillus 5pH1402 has been deposited with the Microtech Institute as FERM P-8892.

Next, the mycological properties of Bacillus sp. H1402 will be shown.

(A) Morphological properties (B) Growth status in each culture medium The strain of the present invention that produces a significant amount of protein extracellularly but does not produce proteolytic enzymes extracellularly is Bacillus SPP4.
There is O10.

Depending on the properties of the protein produced in large quantities by culturing the novel Bacillus sp H1402 of the present invention, it can be used as food protein itself, food processing materials such as gelling agents and swelling agents, glass-like materials, paper, artificial leather, etc. It has very high industrial utility, such as its use as an industrial material for surface finishing.

Furthermore, when the novel sp H1402 of the present invention is used as a host bacterium for genetic recombination, the genetically recombinant product can be efficiently secreted outside the bacterial body, and the genetically recombinant product cannot be degraded. Therefore, it would be an excellent host bacterium for genetic recombination.

This system is expected to be used as a means of production for pharmaceuticals, food processing materials such as high-quality food proteins, gelling agents, and leavening agents, and industrial materials such as surface processing of glass-like materials, paper, and artificial leather. I can do it.

As described above, the usefulness of the present invention is extremely significant industrially.

The present invention will be explained in more detail below by giving Examples.

Example 1 500 mM of the 5Y medium listed in Table 1 above was dispensed into a 2Q jar fermentor, sterilized at 121° C. for 20 minutes by a conventional method, and then cooled.

Separately, a test tube containing 5mR of 5Y medium was sterilized by autoclaving, and Bacillus 5pH140 was added to the test tube.
2 was inoculated into one platinum loop, and cultured with shaking at 37°C for 14 hours.

5 mQ of this preculture was inoculated into a jar fermenter.
37℃, 48 hours, ventilation rate 0.5 fl/min, rotation speed 40
Culture was performed at 0 rpm. After incubation, add an equal amount of 0.
Add 2N NaOH and stir at 10,000 rpm + i
Centrifuge for 5 minutes to remove bacterial cells, and supernatant 100+++1
Add an equal amount of 10% trichloroacetic acid to 2 and use a lO fractionator 00.
The precipitate was collected by centrifugation at 0 rpm for 10 minutes. 5
% trichloroacetic acid and collect the precipitate by centrifugation.
After dissolving with N NaOH, it was quantified by the Lowry method. The amount of protein was expressed in terms of bovine serum albumin. As a result, the amount of protein produced outside the bacterial cells was 5 g/I2.

Claims (1)

[Claims] Bacillus spH1402 produces a significant amount of protein extracellularly, but does not produce proteolytic enzymes extracellularly.