JPH01235585A - Novel pseudomonas bacteria - Google Patents

Abstract

PURPOSE:To provide novel Pseudomonas bacteria capable of producing a significant amount of protein out of the bacteria. CONSTITUTION:The objective novel Pseudomonas bacteria capable of producing a significant amount of protein out of the bacteria, have been isolated and was named as Pseudomonas SP-H022 (FERM P-9740). This strain is capable of secreting and producing ca. >=5g/l of protein in a culture medium.

Description

[Detailed description of the invention] It is something.

Conventionally, when producing proteins by microorganisms, the microorganisms were cultured, the microbial cells were ground, and the proteins were extracted and purified.

Additionally, Escherichia coli is generally used as a host for the production of microorganisms with genetic defects; ilm
Since the peptides and proteins synthesized by these genes remain within the cell and are not secreted into the culture medium, their production is naturally limited.

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

Udaka previously conducted research to find microorganisms that secrete proteins outside of their cells as host bacteria for gene loss, and found that approximately 1.200 microorganisms can secrete and produce large amounts of proteins.
Among the strains, Bacillus brevis
brevis) 4 strains, new strain Bacillus alloteihomans (Bacillus proteifor+ma)
A total of 5 strains were isolated and identified, with ns > 1 strain. [^gr
ic. Biol.

Chem. , 40 (3), 523-528 (197
6) 3 On the other hand, bamboo shoots have also been used as secretory host vectors, and various heterologous proteins such as α-amylase and interferon have been successfully accumulated in the culture medium, but due to strong proteases inside and outside the bacterial body. Good results have not been achieved due to limited production or decomposition.

Previously, Udaka et al.
Bacillus stearothermophil
us) The thermostable α-amylase gene of DY-5 was incorporated into the 1 rasmid pUBIIO.I) Bacillus brevis 47 carrying BAMIOI and Bacillus subtilis were incubated at 37°C for 4 hours.
When cultured for 8 hours, Bacillus and Frehiss 47 te were approximately 15
．． 00ri υ/me, dried grass iii teha 3.000U/
It was confirmed that approximately +wl of α-amylase was produced and accumulated in each medium. [J, Bacteriol, 1
64゜(3), +182-1187 (1985)] Here, between Bacillus plevis 47 and Bacillus subtilis, which carry exactly the same plasmid, Bacillus plevis 47 is about 5 times more efficient in producing heat-stable α-amylase. The fact that the production efficiency is high also indicates that it is possible to efficiently secrete and produce heterovolatile gene products by using the protein secretion ability of protein-producing bacteria.

Based on the idea that bacteria that secrete large amounts of protein would be excellent as host bacteria for genetic recombination, the inventors conducted a thorough search to find such strains, and found that they found a variety of strains. Among the approximately 100,000 strains isolated from the materials, we isolated a strain that produced a significant amount of protein outside the bacterial body.

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

The present inventors made further efforts to differentiate between the two, and succeeded in isolating a strain other than the genus Bacillus that secretes and produces proteins to a remarkable extent outside of the bacterial cell. When the strain isolated #L was identified as belonging to the genus Pseudomonas, the present invention was completed.

The present invention relates to a Pseudomonas bacterium that produces a significant amount of protein extracellularly.

Although strains of the genus Bacillus that produce protein are known, there are no known strains of the genus Pseudomonas that produce proteins of 58 or more lengths outside the bacterial body.

The new 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 outside the bacterial body, is completely new.

In the present invention, strains that secrete and produce 5 g or more of protein into the medium were selected and isolated.

First, approximately 100,000
strain on T2 agar plate medium (1% glucose, 1% peptone, 0.5% meat extract, 0.2% yeast extract, 15%
% agar powder, p) 17.0), and bacteria that became cloudy around the colony with 5% perchloric acid were selected on a plate medium.Next, the bacterial strains isolated here were transferred to a T2 liquid medium (150%
Culture with shaking (30°C, 48 hours) in an O+E Erlenmeyer flask, medium volume 10++U, and add 1.2g to the culture solution.
For in vitro protein measurement, an equal amount of 0.2N Na was added to the culture solution.
Add OH and stir at 1110.000 rpm x 5 minutes centrifugation M! I! After removing the bacterial cells, add an equal volume of 10% trichloroacetic acid to the supernatant. 13. QOQrpmx
The precipitate was collected by centrifugation for 10 minutes, dissolved in IN Na0)1, and then subjected to the Lowry method [J, Biol, Che+m,
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.

Table 1: Culture medium for producing 1 g of protein The 80 strains obtained previously were cultured with shaking in all of these 5 types of media, and the strains that produced 5 g/S or more of extracellular protein in any of the media 31 stocks were selected.

These 31 strains f, Bergey's Manual
of Determi-native Bacter
iology (8th edition), Bergey'Manu
al of 5yste+natic Bacteri
origy vol.1 and TheProkaryo
te (A)Iandbook on Habita
ts.

l5olation and identification
ion of Bacteria>, the six strains were initially aerobic, Durham stain negative, bacilli,
The HO22 strain, which is recognized as belonging to the genus Pseudomonas in that it does not form spores, was compared with conventionally known seedlings of the genus Pseudomonas in terms of other morphological properties, growth status in each medium, and physiological properties. As a result of the 1st test, it was different from any species of Pseudomonas.

Therefore, this fungal species was identified as a new species of the genus Neudomonas.

Therefore, this bacterial species was named Pseudomonas sp. HO22. Pseudomonas Sp, 1lQ22 is FERM
It has been deposited with the Institute of Fine Technology as P-9740.

Next, the mycological properties of Neudomonas sp.) 1022 are shown.

(B) Growth-like muscle in each culture medium (C) Physiological properties continued CD) Production of acid and gas (E) Assimilation of carbon source Acetate +5ucci
nate -1-Fu+aara
te -shi-Malate
-β-tlydr
oxybutylate +actate
+C1
treat ＋＊
-Ketoglutarate -Gl
ycerol +L-Alani
ne- L-Apartate -shi-G
lutamate
-Le Arginine -L-Pr
ol ine -L-Tyros
ine -D-Fucose
-Maltose
--Cellobio
se -LacLose
-D-Ara
binose -^ra
bitol + 5tarch
-Inulin
+Ethylen glycol
-L-Threonine
-D-Ribose
-Mann i to l
-D-Xylose L-Arabinose
-shi-1'! han+nose
-GIucose
+D-Mannose
-D-Galactose
-L-11ydroxypr
oline +1nosine
-Betaine
-D-Fru
ctose-5uc
rose-T
reharose-
GluconaLe
-Propionate
+Malonate
-D(-)-Tartrate
+5orbitol
-Propylene glycol
-Ethanol
-n-Butanol
+Benzoate
-m-Hydroxyb
enzoate-p-11ydro
xybenzoate-pheno
l-Glyc
ine-L-Ser
ine -L-L
eucine +1nosi
ne -Le Lysin
e +L-Ornithine
-L→+1stidine
-L-Phenylal
anine-L-Tryptop
han'-N-Acety1
glucosa+m1ne-Raffin
ose −+Creatine
-Deoxychol
ate-D-Glucuro
-The present invention relates to a Pseudomonas bacterium that produces a significant amount of protein extracellularly, particularly Pseudomonas 5pH022.

Depending on the properties of the protein produced in large quantities by culturing Pseudomonas SP, 1 (Q22) of the present invention, it may itself be food protein, a food processing material such as a gelling agent, a swelling agent, a glass-like material, paper 1, artificial leather, etc. It has very high industrial utility, such as its use as an industrial material for surface finishing, etc.

In addition, when used as a host bacterium for the Pseudomonas sp, HO22 & Dodenshi recombination of the present invention, it is possible to efficiently secrete the products of genetic recombination outside the image, making it an excellent host bacterium for genetic recombination. It will become something new.

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 The 5PC medium 500+++9 described in Table 1 above was dispensed into a 29-volume jar fermentor and heated to 121°C by a conventional method.
After simmering for 20 minutes, it was cooled.

Separately, 5++ u of 5PC medium was dispensed into test tubes and sterilized by autoclaving, and Pseudomonas sp.
, 1 platinum loop of HO22 was inoculated, and cultured at 37° C. for 14 hours with shaking. Inoculate this previous item 1 into a jar fermenter with 51 scoops, and set the aeration rate to 0.51/min for 48 hours at 37°C at a rotation speed of 40.
Culture was performed at 0 rpm. After incubation, add an equal amount of 0.
After adding 2N Na0)1 and stirring 10000 rpm+x
Centrifuge for 5 minutes to remove bacterial cells, add an equal amount of 10x trichloroacetic acid to the supernatant 10011, and after 10 minutes add 3000
The precipitate was collected by centrifugation at rpmx for 10 minutes. Wash with 5% trichloroacetic acid and collect the precipitate by centrifugation.
It was quantified by the Lowry method using barley dissolved in a011. Protein was expressed as Ha calculated from bovine serum albumin. As a result, the amount of protein ii produced outside the bacterial cells was 8 g/I.

Claims (1)

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