JPH0586184B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel Bacillus sp H014. 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. Additionally, Escherichia coli is generally used as a host for the production of genetically modified microorganisms, but in Escherichia coli, the peptides and proteins synthesized by recombinant genes remain within the cells and are not secreted into the culture medium. Therefore, its production volume was naturally 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, he selected Bacillus brevis (from about 1,200 strains) as a microorganism that secretes and produces large amounts of proteins. Five strains were isolated and identified: four strains of Bacillus brevis and one new strain of Bacillus proteiformans. [Agric.Biol.
Chem., 40(3), 523-528 (1976)] On the other hand, Bacillus subtilis was also used as a secretory host-vector, and various foreign proteins such as α-amylase and interferon were successfully accumulated in the culture medium. However, the production amount is limited or degraded by strong proteases inside and outside the bacterial body.
Good results have not been obtained. Previously, Udaka et al.
Plasmid containing the thermostable α-amylase gene of
When Bacillus brevis 47 carrying pBAM101 incorporated into pUB110 and Bacillus subtilis were cultured at 37°C for 48 hours, Bacillus brevis 47 produced approximately 15,000 U/ml,
In Bacillus subtilis, it was confirmed that approximately 3000 U/ml of α-amylase was produced and accumulated in each culture medium. (J.
Bacteriol., 164, (3), 1182-1187 (1985)]. Here, between Bacillus brevis 47 (described later) and Bacillus subtilis, which carry exactly the same plasmid, Bacillus brevis 47 is approximately 5 times more efficient in producing heat-stable α-amylase. It has been found that a heterologous gene product can be secreted and produced efficiently by using the protein secretion ability of the producing bacterium. However, five strains of Bacillus brevis 47, 144, 481, and 899, and Bacillus proteihomans 444, which were previously isolated and identified as bacteria that secrete and produce large amounts of protein outside their cells, all contained bovine serum albumin in the culture medium. It was confirmed that when grown with the addition of BSA (hereinafter referred to as BSA), BSA was degraded, and four strains, Bacillus brevis 144, 481, 899, and Bacillus proteihomans 444, also had casein-degrading activity. Ta. 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, the efficiently secreted and produced peptides and proteins are processed by proteolytic enzymes. It was thought that it would be decomposed. Therefore, the present inventors secreted a significant amount of protein,
Moreover, if we could find a strain that does not produce any proteolytic enzymes outside the cell, it would be an excellent host for genetic recombination, so we carried out extensive selection in search of such a strain.
Out of approximately 100,000 strains isolated from various samples, they succeeded in isolating a strain that produces a significant amount of protein outside the bacterial body, but does not produce proteolytic enzymes outside the bacterial body. When the species of the strain isolated here was identified, it was identified as belonging to Bacillus, leading to the completion of the present invention. The present invention is directed to Bacillus sp., which produces a significant amount of protein extracellularly but does not produce proteolytic enzymes.
It is H014. Conventionally, protein-producing strains of the genus Bacillus have been known, but all of the known strains produce proteolytic enzymes.
Bacillus sp H014, which produces a significant amount of protein extracellularly but does not produce proteolytic enzymes extracellularly, is completely unknown and is new. In the present invention, strains that secrete and produce 5 g of protein or more into the medium and do not degrade either BSA or casein proteins were selected and isolated. First, about 100,000 cells were isolated from soil and other samples.
strains on T2 agar plates (1% glucose,
The bacteria were inoculated into 1% peptone, 0.5% meat extract, 0.2% yeast extract, 1.5% 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 cultured with shaking (30℃, 48 hours) in a T2 liquid medium (150ml Erlenmeyer flask, medium volume 10ml), and the culture filtrate was
We obtained 80 strains that produced 1.2g/or more of protein. To measure extracellular proteins, add an equal volume of 0.2N NaOH to the culture solution, stir, centrifuge at 10,000 rpm for 5 minutes to remove the bacterial cells, and add an equal volume of 10% trichloroacetic acid to the supernatant. After 1 minute, centrifuge at 3000 rpm for 10 minutes to collect the precipitate and dissolve it in 1N NaOH.
It was quantified by the Lowry method [J. Biol. Chem. 193, 265 (1951)], and the protein amount was converted into bovine serum albumin. The medium shown in Table 1 was selected as a high protein production medium.

【table】

[Table] The 80 strains previously obtained were cultured with shaking in all of these five types of media, and 31 strains that produced 5 g of extracellular protein or more were cultured in any of the media.
Selected stocks. Regarding the obtained 31 strains, the following measurements of BSA degradability and milk casein degradability were performed. (Measurement of degradability of BSA) Pour 10 ml of T2 medium into a 150 ml Erlenmeyer flask, sterilize it in an autoclave, and aseptically filter the BSA.
(SigmaA4503) solution was added to give a final concentration of 3.2 mg/ml, and after inoculating 0.2 ml of the strain that had been precultured overnight, culture was carried out at 37°C with shaking at 200 rpm. The culture filtrate sampled after 24 hours, 48 hours, and 72 hours of culture was centrifuged at 10,000 rpm for 5 minutes, and 625μ of the culture supernatant was mixed with 125μ of 0.5M Tris-Cl (PH6.8) for 10 minutes.
%SDS200μ, β-mercaptoethanol 50μ
0.05% after stirring and heat treatment in boiling water for 3 minutes.
0.0625M Tris− with BPB and 70% glycerol
0.1 ml of Cl (PH6.8) was added to prepare a sample for SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Slab SDS-PAGE was performed at a 10% acrylamide concentration. Protein detection was performed by staining with Coomassie brilliant blue. culture 24
A strain that did not degrade BSA for 48 hours or 72 hours was defined 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 ml of pure water, and a solution in which 1 g of agar was dissolved in 50 ml of pure water were sterilized separately in an autoclave, mixed together, and divided into portions. 5%, 2%, and 1% milk agar plates were prepared. 37℃ after inoculating the strain on the plate medium
After culturing for 3 days, it was observed whether the area around the colony became transparent. A strain that did not produce any transparent circles on all of the 5%, 2%, and 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 H014 was selected as a strain that does not produce proteolytic enzymes outside the bacterial cells, since it does not degrade both BSA and milk casein. H014 strain, Bergey's Manual Determinative
Bacteriology (8th edition) and The Prokaryote
(A Handbook on Habitats, Isolation and
Identification of Bacteria), this strain was first recognized to belong to the genus Bacillus in that it is aerobic, positive for Gram staining, and forms 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, it was found that it was different from any other bacterial species of the genus Bacillus. Furthermore, this bacterial species 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 H014. Bacillus sp. H014 has been deposited with the Microtech Institute as FERM P-8891. Next, the mycological properties of Bacillus sp H014 are shown.

【table】

【table】

【table】

【table】

[Table] Although the bacteria of the present invention produce a significant amount of protein outside the cells,
Bacillus that does not produce proteolytic enzymes outside the bacterial body
sp H014. Depending on the properties of the protein produced in large quantities by culturing the novel Bacillus sp H014 of the present invention,
It has very high industrial utility, such as its use as food processing materials such as food proteins, gelling agents, and leavening agents, or as industrial materials such as surface treatment of glass-like materials, paper, artificial leather, etc. Furthermore, when the novel sp H014 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 can be degraded. Therefore, it would be an excellent host bacterium for genetic recombination. This system can be 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. . 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 ml of the 5Y medium listed in Table 1 above was dispensed into a 2-volume jar fermenter, and heated to 121°C by a conventional method.
After sterilization for 20 minutes, it was cooled. Separately, a test tube containing 5 ml of 5Y medium was sterilized by autoclaving, and Bacillus
One platinum loop of spH014 was inoculated and cultured with shaking at 37°C for 14 hours. 5 ml of this preculture was inoculated into a jar fermenter and cultured at 37°C for 48 hours at an aeration rate of 0.5/min and a rotational speed of 400 rpm. After culturing, add an equal volume of 0.2N NaOH to the culture, stir, and centrifuge at 10,000 rpm for 5 minutes to remove bacterial cells. Add an equal volume of 10% trichloroacetic acid to 100 ml of supernatant, and after 110 minutes, centrifuge at 3,000 rpm.
The precipitate was collected by centrifugation for 10 minutes. Wash with 5% trichloroacetic acid and collect the precipitate by centrifugation to 1N
After dissolving with 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 5g/
It was hot.

Claims (1)

[Claims] 1. Bacillus sp., which produces a significant amount of protein extracellularly, but does not produce proteolytic enzymes extracellularly.
H014.