JPH01235581A - 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-H501 (FERM P-9742). This strain is capable of secreting and producing ca. >=5g/l of protein in a culture medium.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel Pseudomonas bacterium.

Conventionally, when protein is produced by a microorganism in -m, the protein has been obtained by culturing the microorganism, grinding the microorganism cells, and then extracting and purifying the protein.

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, peptides and proteins can be extracted by cell trituration113
However, nine drawbacks have been identified, including that the operation becomes complicated.

Udaka previously researched microorganisms that secrete proteins outside of their cells as host bacteria for genetic recombination, and found that they were found to be microorganisms that secrete and produce large amounts of proteins.
Bacillus plevis (Bacillus plevis) was selected from 0 strains.
brevis) 4 strains, new strain Bacillus proteifor+m
Ans) A total of 5 strains, 1 strain, were isolated and identified. [
Agric, Biol.

Che+++, 40 (3), 523-528 (1
[976] On the other hand, Bacillus subtilis has also been used as a secretion host-vector, and various heterologous proteins such as α-amylase and interferon have been successfully accumulated in the culture medium. Good results have not been achieved due to limited amounts or decomposition.

Previously, Udaka et al.
Bacillus stearothermophil
us) Bacillus plevis 47 carrying pBAM10+ in which the thermostable α-amylase gene of DY-5 was incorporated into the plasmid pUBllo and Bacillus subtilis were incubated at 37°C and 48°C.
When cultured for an hour, Bacillus plevis 47 was about 15.
o00U/+++I, 3,000υ/III for Bacillus subtilis
MFa produces approximately I α-amylase in the culture medium.
I confirmed that it does. [J, Bacteriol,
, 164゜<31, 1132-1187 (1985)
]Here, Bacillus possessing exactly the same 1 lasmid.
Based on the fact that Bacillus plevis 47 is about 5 times more efficient in producing heat-stable α-amylase than Bacillus subtilis, it is possible to produce a heterologous gene product by using the protein secretion ability of protein-producing bacteria. It was found that secretion and production can be carried out efficiently.

Therefore, based on the idea that seedlings that secrete a large amount of protein are excellent host bacteria for gene loss, the inventors conducted a thorough search for such strains, and found that Out of approximately 100,000 strains isolated from various materials, we isolated a strain that produces a significant amount of protein outside the bacterial body.

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

The present inventors further performed ii selection and succeeded in creating a monolayer of strains other than those of the genus Bacillus that secrete and produce a significant amount of protein outside the bacterial cells. When the strain isolated here was identified as belonging to the genus Pseudomonas, the present invention was completed.

The present invention is a Pseudomonas vL bacterium that produces a significant amount of protein extracellularly.

Conventionally, strains of Bacillus that produce proteins have been known, but Pseudomonas strains produce protein at a rate of 5 g/
There are no known seedlings that produce these types of bacteria 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
The strains were grown on T2 agar plates (1% glucose, 1%^
Pton, 0.5% meat extract, 0.2% yeast extract, 1.
5% agar powder, pH 7.0) is inoculated, and the area around the colony becomes cloudy due to 5% perchloric acid on a plate medium! Next, the bacterial strain isolated here was transferred to T2 liquid medium (15
Culture in S shake culture (30°C, 48 hours) in a 0ml Erlenmeyer flask (medium volume 10ml), and add 1.2g/ml to the culture solution.
80 strains that produce at least one protein were obtained.For the measurement of extracellular proteins, the culture solution was mixed with equal amount of Q, 2N NaO.
After adding H and stirring 10. Centrifuge for 5 minutes at OOOrpm to remove bacterial cells, add an equal volume of 10% trichloroacetic acid to the supernatant, and after 10 minutes, centrifuge at 3,000 rpm + Xio to collect the precipitate, dissolve it with IN Mail, and then
owry method [J, Biol, Ches, 193.26
5 (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 1 High protein production medium The 80 strains obtained earlier were cultured with shaking in all of these five types of media, and strains that produced 5 g or more of extracellular protein in any of the media were cultured. 31 stocks were selected.

These 31 strains are listed in Bergey's Manual
of Detern+1-native Bacte
riology (8th edition), Bergey'Ma
natural of 5yste+natic bacte
rioligy vol.l and TheProkar
yote (A Handbook on)Iabi
tats.

l5olation and identification
ion of Bacteria), the six strains were initially aerobic, Durham stain negative, bacilli,
Strain 1501, which was recognized as belonging to the genus Pseudomonas in that it does not form spores, was compared with previously known bacterial species of the genus Pseudomonas in terms of other morphological properties, growth conditions in various media, and physiological properties. As a result of examination, it was found that it was different from any other bacterial species in the genus Pseudomonas.

Therefore, this bacterial species was identified as a new 1i111 of the genus Pseudomonas.

Thus, this bacterial species was named Pseudomonas sp, H3O1. Pseudomonas sp, H3O1 is FERM P
-9742 and has been deposited with the Institute of Fine Technology.

Next, we will show the national characteristics of Pseudomonas sp.) 1501.

(B) Growth of T-shaped deafness in each culture medium (D> Production of acid and gas (E) Assimilation of carbon source Acetate +5ucc
1nate +Fumarate
-L-Malate
+β-Hydroxybutylate
+Lactate
+C1trate +Set-Ke
toglutarate-Glycer
ol +shi Alanine
+L-Apartate
-L-Glutasate
+Arginine -L-Pr
oline 10L-Tyrosine +D-Fu
cose-Ma
ltose-
Cellobiosc
-Lactose
-O-8 rabinose
-＾rabitol + 5tarch
-Inulin
-Ethyle++ glycol
-L-Threonine
-D-Ribose
+Mannitol +D-X
ylose tre^rabinose 10L-Rha
mnose-Gluc
ose 10D-Mannose + 1l-Galactose
+ Shiichi 11ydroxyprol ine
) Inosi++e
+Betaine
-D-Fructose 15ucrose Treharose -I
C1uconate
--1'ropionatc MalnnaLe
-D(-)-Tartrate- 3orbitol
-Propylene glycol
-Ethanol + n-Butanol +B
enzoate-m
-Hydroxybenzoate
-p-11ydroxybenzoate
-Pheno I
-Glycine
-L-3erine
+shi-Lcucine
) L-1 soleucine
+L-Valine
+L-Lysine
-L-Ornithine
-L-Histidine
+L-Phenylalanine
+Raff1nose
-Creatine
+Deoxycholate
-D-Glucuronat
e - The present invention relates to Pseudomonas bacteria that produce a significant amount of protein extracellularly, particularly Pseudomonas sp.

It is H5O1.

Depending on the properties of the protein produced in large quantities by culturing Pseudomonas sp, H3O1 of the present invention, it can be used as a food protein, a food processing material such as a gelling agent, a swelling agent, or a glass-like material, paper, artificial leather, etc. It has very high industrial utility, such as its use as an industrial material for surface finishing.

Furthermore, when Neudomonas sp, H3O1 of the present invention is used as a host bacterium for genetic recombination, the product of genetic recombination can be efficiently secreted outside the bacterial body.
It would be an extremely good host bacterium 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 The 5PC medium 5ooII11 described in Table 1 above was dispensed into a 2Q volume jar fermentor and heated to 121°C by a conventional method.
After sterilization for 20 minutes, it was cooled.

Separately, a test tube containing 51 aliquots of 5PC medium was sterilized by autoclaving, and Pseudomonas sp.
One platinum loopful of H3O1 was seeded and cultured with shaking at 37°C for 14 hours. 5 ml of this preculture was inoculated into a jar fermenter at 37°C for 48 hours at an aeration rate of 0.51/rpm and a rotation speed of 400.
Cultured in rp+s. After incubation, add an equal volume of 0.
Add 2N NaOH and stir 1k 1010000 rpm
Centrifuge for 5 minutes to remove bacterial cells, and remove ifloOm.
Add proprietary 101 trichloroacetic acid to l and after 10 minutes 30
00rp+++8 Centrifugation was performed for 10 minutes and the precipitate was collected.

Wash with 5% trichloroacetic acid and collect the precipitate by centrifugation.
It was quantified by the 1&Lowry method dissolved in N Na011. Proteins are expressed in terms of bovine serum albumin.

As a result, the amount of protein produced outside the bacterial cells was 9 g/I.

Claims (1)

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