JPH0588106B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel Pseudomonas bacterium. 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. Furthermore, 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. A total of five strains, four strains of Bacillus brevis and one new strain of Bacillus proteiformans, were isolated and identified. [Agric.Biol.
Chem. 40(3), 523-528 (1976)] 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. However, the production amount is limited or degraded by strong proteases inside and outside the bacterial body.
Good results have not been obtained. First, Udaka et al. Bacillus stearothermophilus DY-5
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, Bacillus brevis 47, 144, 481, 899 and Bacillus proteihomans 444, which were previously isolated and identified as bacteria that secrete and produce large amounts of protein outside the bacterial body, all contained bovine serum albumin in the culture medium. Bacillus brevis 144, 481, 899, and Bacillus proteihomans 444 have also been shown to have casein-degrading activity when grown with the addition of BSA (hereinafter referred to as BSA). confirmed. Therefore, when a foreign gene product is secreted and produced by a recombinant gene using a bacterium that secretes and produces large amounts of these proteins outside the bacterial body, the secreted and produced peptides and proteins are efficiently digested 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. Upon species identification of the strains isolated here, most were identified as belonging to the genus Bacillus. The inventors conducted further thorough selection and found that
In addition to the genus Bacillus, they succeeded in isolating a bacterial cell that secretes and produces a significant amount of protein outside the bacterial cell. When the strain isolated here was identified as belonging to the genus Pseudomonas, the present invention was completed. The present invention is a novel Pseudomonas bacterium that produces a significant amount of protein extracellularly, but does not produce proteolytic enzymes. Bacterial strains of the genus Bacillus that produce protein have been known, but no strain of the genus Pseudomonas that produces protein in significant amounts of 5 g or more extracellularly has been known. Furthermore, although the novel Pseudomonas bacterium of the present invention does not secrete proteolytic enzymes outside the bacterial body, nothing is known about such Pseudomonas bacterium. The new bacterial strain isolated for the first time in the present invention, ie, the new strain of the genus Pseudomonas that produces a significant amount of protein extracellularly and does not secrete proteolytic enzymes extracellularly, is completely 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, 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 that, the precipitate was collected by centrifugation at 3000 rpm for 10 minutes, dissolved in 1N NaOH, and then quantified by the Lowry method [J. Biol. Chem. 193, 265 (1951)]. The protein amount was determined as bovine serum albumin. Converted. 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 or more of extracellular protein 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 BSA degradability) Pour 10 ml of T2 medium into a 150 ml Erlenmeyer flask, sterilize it in an autoclave, and aseptically filter BSA (Sigma
A4503) solution was added to a final concentration of 3.2 mg/ml, and 0.2 ml of the strain was pre-cultured overnight at 37°C.
Shaking culture was performed at 200 rpm. The culture filtrate sampled after 24, 48, 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-HCl (PH6.8).
10% SDS 200μ 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 HCl (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, then mixed and separated into sieves. 5%, 2%, and 1% milk agar plates were prepared. After inoculating the bacterial strain onto a plate medium, it was cultured at 37°C for 3 days, and 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 H53 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. H53 strain, Bergey's Manual of
Determinative Bacteriology (8th edition),
Bergey's Manual of Sys tematic Bacteriology
vol.1 and The Prokaryote (A Handbook on
Habitats, Isolation and Identification of
Bacteria), this strain was identified as
First, it was recognized that it belonged to the genus Pseudomonas in that it was aerobic, Gram stain negative, bacillus, and did not form 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 Pseudomonas, it was found that it was different from any species of the genus Pseudomonas. 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 Pseudomonas. Therefore, this bacterial strain was named Pseudomonas sp.H53. Pseudomonas sp.H53 is FERM P-
It has been deposited with the Institute of Fine Technology as 9741. Next, the mycological properties of Pseudomonas sp.H53 are shown.

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【table】

【table】

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【table】

[Table] (E) Assimilation of carbon sources H53 Acetate + Succinate + Fumarate - L-Malate + β-Hydroxybutylate + Lactate + Citrate + α-Ketoglutarate - Glycerol + L-Alanine + L-Aspartate - L-Glutamate + L -Arginine - L-Proline + L-Tyrosine + D-Fucose - Maltose - Cellobiose - Lactose - D-Arabinose - Arabitol + Starch - Inulin + Oxalate - Ethylene glycol - L-Threonine - D-Ribose - Mannitol - D-Xylose - L-Arabinose + L-Rhamnose + Glucose + D-Mannose - D-Galactose + L-Hydroxyproline + Inosine + Betaine - D-Fructose - Sucrose + Treharose - Gluconate - Propionate - Malonate - D(-)-Tartrate + Sorbitol - Propylene glycol + Ethanol + n-Butanol + Benzoate + m-Hydroxybenzoate − p-Hydroxybenzoate − Phenol − Glycine + L-Serine + L-Leucine − L-Isoleucine − L-Valine − L-Lysine + L-Ornithine − L-Histidine + L-Phenylalamine + L-Tryptophan - N-Acetylglucosamine - Raffinose - Creatine - Deoxycholate + D-Glucuronate - The present invention uses Pseudomonas, which produces a significant amount of protein extracellularly but does not produce proteolytic enzymes extracellularly. For example, Pseudomonas sp.H53
(FERM P-9741) is exemplified. Depending on the properties of the protein produced in large quantities by culturing Pseudomonas sp. It has very high industrial utility, such as its use as an industrial material for surface finishing. Furthermore, when the Pseudomonas sp.H53 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. This makes it an excellent host for genetic recombination. This system can be expected to be used as a production means for pharmaceuticals, high-quality food proteins, food processing materials such as gelling agents and swelling agents, and industrial materials for surface treatment such as 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 5YKC medium listed in Table 1 above was dispensed into a 2-volume jar fermenter, and 121
After sterilization at ℃ for 20 minutes, it was cooled. Separately, a test tube into which 5 ml of 5YKC medium was dispensed was sterilized by autoclaving, one platinum loop of Pseudomonas sp. 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 amount of 0.2N NaOH to the culture and stir at 10,000 rpm.
Centrifuge for 5 minutes to remove bacterial cells, add an equal volume of 10% trichloroacetic acid to 100ml of supernatant, and after 10 minutes, spin at 3000 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.
Proteins are expressed in terms of bovine serum albumin. As a result, the amount of protein produced outside the bacterial body was 7g/
It was hot.

Claims (1)

[Claims] 1 Pseudomonas sp.H53 produces a significant amount of protein outside the bacterial body.