JPH01174378A - Novel microorganism of genus pseudomonas - Google Patents

Abstract

NEW MATERIAL:A microorganism capable of extracellularly producing a remarkable amount of proteins. EXAMPLE:Pseudomonas sp. H53 (FERM P-9741). USE:A microorganism capable of producing proteins useful as medicines, food proteins, etc., or host microorganism in genetic recombination without producing a proteolytic enzyme. PREPARATION:A microorganism separated from soil, etc., is initially inoculated into a T2 agar plate culture medium to select a microorganism in which the periphery of a colony is clouded with 5% perchloric acid. The resultant microorganism is then cultivated in a T2 liquid culture medium by a shaking culture method to provide a strain capable of producing >=1.2g/l amount of produced proteins in a cultivation filtrate. The obtained strain is subsequently cultivated in a culture medium for highly producing proteins to select the aimed microorganism.

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.

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.

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, selected Bacillus plebis (Bacillus plebis) from among about 1,200 strains as microorganisms that secrete and produce large amounts of proteins. Bacillus b
Bacillus revis) 4 strains, new strain Bacillus proteiformans
A total of 5 strains (1 strain) were isolated and identified. (Agric
, Biol.

Chem, 40 (3), 523-528 (197
6)) On the other hand, Bacillus subtilis has also been used as a secretory 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 PBAMIOI, in which the thermostable α-amylase gene of DY-5 was incorporated into plasmid pUB110, and Bacillus subtilis were cultured at 37°C for 48 hours, Bacillus plevis 47 had a concentration of about 15%,
0000/mQ, 3,000U/mfl for Bacillus subtilis
It was confirmed that a certain amount of α-amylase was produced and accumulated in each medium. [J, Bacterj,ol,,1
6C(3), 1182-11.8'7 (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.
44.48], 899, and Bacillus proteihomans 444, when grown in the medium with bovine serum albumin (hereinafter referred to as BSA), BSA
Bacillus plebis 144, 481
．． It was confirmed that four strains, B. 899 and Bacillus proteihomans 444, also had casein-degrading activity. Therefore, when a heterologous gene product is secreted and produced by a recombinant gene using a bacterium that secretes and produces many of these proteins outside the bacterial body, the secreted and produced peptides and proteins are efficiently degraded by proteolytic enzymes. it was thought.

Therefore, the present inventors came up with the idea that if a strain could be found that secretes a significant amount of protein and does not produce any proteolytic enzymes outside the cell, it would be an excellent host strain for genetic recombination. As a result of intensive selection in search of such strains, approximately 1.00,000 strains were isolated from various samples.
Among the 000 strains, they succeeded in isolating a strain that produces a significant amount of protein extracellularly, but does not produce proteolytic enzymes extracellularly.

When the strains isolated here were identified, most of them were identified as belonging to the genus Bacillus.

After further intensive selection, the present inventors succeeded in isolating a strain other than the genus Bacillus that secretes and produces a significant amount of protein outside the bacterial cell.

When the species of the strain isolated here was identified, it was identified as belonging to the genus Pseudomonas, leading to the completion of the present invention.

The present invention is a 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 strains of the genus Pseudomonas that produce a significant amount of protein outside their cells have 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. A 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, 5 g/Ω or more of protein is added to the medium in -ζ
- Bacterial strains that secrete and produce proteins and do not degrade either BSA or casein proteins were selected and isolated.

First, approximately 100,000 bacterial strains isolated from samples such as soil were grown on a T2 agar plate medium (1% glucose, 1% peptone, 0.5% meat extract, 0.2% yeast extract, 1.5%
% agar powder, pH 7.0), and bacteria around the colony that became cloudy with 5% perchloric acid were selected on a plate culture medium. Next, the bacterial strain isolated here was cultured with shaking (30°C,
1.48 hours) in the culture filtrate. ．． We obtained 80 strains that produced 2 g/f1 or more of protein.

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, 3. OOOrpm X 1
The precipitate was collected by centrifugation for 0 min, dissolved in IN NaOH, and then subjected to the Lowry method (J, Biol, Chem,
193.265 (1951)), and the protein amount was converted into bovine serum albumin.

The media shown in Table 1 were selected as high protein production media.

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

Regarding the obtained 31 strains, the following measurements of BSA degradability and milk casein degradability were performed. Autoclaved and sterile filtered BSA (Sig
maA4503) solution was added to give a final concentration of 3.2 mg/mfl, and the strain was pre-cultured overnight. After inoculation with 0.2 mρ, the strain was cultured with shaking at 37° C. and 20 Orpm.

The culture filtrate sampled after 24 hours, 48 hours, and 72 hours of culture was centrifuged at 10.00 rpm for 5 minutes. 0.5M Tris-C1 (
p)16.8) 125μΩ, 10%SDS 200μ
Q 0.062'5M Tris-C1 containing 0.05% BPB and 70% glycerol after stirring and heat treatment in boiling water for 3 minutes after adding 50 μQ of β-mercaptoethanol (
pH 6,8) 0. In+R was added to prepare a sample for 5DS-polyacrylamide gel electrophoresis (SDS-P, AGE). 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 5g, 2g, and 1g of skim milk were each suspended in 50mM pure water, and a solution in which 1g agar was dissolved in 50+d pure water were sterilized separately in an autoclave, then mixed and placed in a petri dish. The mixture was divided into 5%, 2%, and 1% milk agar plates. After seedlings of the bacterial strain were planted on a plate medium, they were cultured at 37°C for 3 days, and it was observed whether the area around the colony 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 853 was selected as a strain that does not produce proteolytic enzymes outside the bacterial cells because it does not degrade both BSA and milk casein.

) 153 stocks to Bergey's annual of
Determinative Bacteriolog
y (8th edition), Bergey's Manual, o
f Systematic Bacteriology
vol.1 and The Prokaryote (
A Handbook on Habitats, l
5olation and identity
As a result of identification using the method (n of Bacteria), this strain was first recognized to belong to the genus Pseudomonas in that it is aerobic, negative for Durham staining, does not form bacilli, and does 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 other bacterial species of the genus Pseudomonas. In addition, this bacterial species also contains casein,
It also lacked the ability to break down BSA.

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

Thus, this strain was named Pseudomonas sp. 853. Pseudomonas 8p, H53 is FORM P
-9741 and has been deposited with the Institute of Fine Technology.

Next, the mycological properties of Pseudomonas sp. H53 will be shown.

(A) Morphological properties (B) Growth status in each medium (C) Physiological properties = (E) Requirement for carbon source Acetate +5uccina
te +Fumarate
-L-Malate
+β-Hydroxybutylate
+Lactate +C1tr
ate +a - Ketoglut
arate-Grycerol
+L-Alanine
+L-Apart
-L-Glutamate 10L-Arginine -L-
Proline +LT
yrosine +D-Fuc
osa ±Maltos
e ±Ce1lobi
ose ±Lactose
±D-Arabino
se ±Arabitol
+5tarch
±Inulin
+0xalate
-Etylene glycol
±L-Threonine
-D-Riboge
±Mannitol
±D-Xylose ±
L-Arabinose +L
-Rhamnose +G
lucose +D-
Mannose ±D-G
alactose +L-)
1ydroxyproline +In
osine +Bet
aine-D″Fr
actose ±5ucro
se + Treharo
se ±Gluconate
-Propionate
-Malonate
-D(-)-Tartrate
+5orbito1 Propylene glycol +E
thanol +n-B
utanol +0xala
te +m-Hydrox
ybenzoate-p-Hydrox
ybenzoate-Phenol
-Glycine
+L-5erine 11-16ucine ±L-
Isoleucine fL-V
aline-L-Ly
sine +L-Ornith
ine ±L-Histidin
e +L-Phenylala
nine +L-Tryptophan
±N-^cety1glucos
amine ±Raff1nose
±Creatine
±Deoxycholate
+D-Glucronate
-Free ±
The present invention produces a significant amount of protein outside the bacterial cells.

Pseudomonas bacteria that do not produce proteolytic enzymes outside the bacterial body, especially Pseudomonas sp. H53.

Depending on the properties of the protein produced in large quantities by culturing Pseudomonas sp.
It has great industrial utility, such as use as an industrial material for surface treatment of glass-like materials, paper, artificial leather, etc.

Furthermore, when Pseudomonas sp. 853 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, food processing materials such as high-quality food proteins, gelling agents, and leavening agents, and industrial materials for surface treatment such as glass exploration 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 The 5YKC medium 500-ml listed in Table 1 above was dispensed into 2N jar fermenters, sterilized at 121°C for 20 minutes by a conventional method, and then cooled.

Separately, a test tube containing 5d of 5'/KC medium was sterilized by autoclaving, and Pseudomonas sp.
, H53 was inoculated into one platinum loop, and cultured with shaking at 37°C for 14 hours. 5mM of this preculture was inoculated into a jar fermenter at 37°C for 48 hours with no aeration rate. Nail/min rotation speed 40O
Cultured at rpm+. After incubation, add an equal amount of 0.
After adding 2N NaOH and stirring, 110,000 rpm x 5
After centrifuging to remove bacterial cells, add an equal amount of 10% trichloroacetic acid to 100ml of supernatant, and after 10 minutes, spin at 3000 rpm.
A+X Centrifugation was performed for 10 minutes to collect the precipitate. Wash with 5% trichloroacetic acid and collect the precipitate by centrifugation.
After dissolving with aOH, it was quantified by the LOvry method. Proteins are expressed in terms of bovine serum albumin. the result,
The amount of protein produced outside the bacterial cells was 1 g/fl.

Agent Patent Attorney 1) Parent Male Procedural amendment February 19, 1988

Claims (1)

[Claims] A new Pseudomonas bacterium that produces a significant amount of protein outside the bacterial body.