JPH01235582A - 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-H502 (FERM P-9743). 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 protein using microorganisms, it was obtained by culturing the microorganisms, grinding the microorganisms, 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 in the cannon and are not secreted into the culture medium. However, the amount of production 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 previously researched microorganisms that secrete proteins outside of their cells as host bacteria for genetic recombination, and found that approximately 1.200 microorganisms can secrete and produce large amounts of proteins.
Among the strains, Bacillus plebis
brevis) 4 strains, new strain Bacillus proteifors+a
A total of 5 strains, including 1 strain (ns), were isolated and identified. [Ag
ric. Biol.

Chem. , 40 (3>, 523-528 (197
6)] In addition, haystack has been used as a host-vector for R1 secretion, 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 stearother++ophi
lus) Bacillus plevis 47 and Bacillus subtilis carrying pRAM+01 in which the thermostable α-amylase gene of DY-5 was integrated into plasmid pυ811o and Bacillus subtilis at 37°C.
When cultured for 8 hours, Bacillus and Frehiss 47 te were approximately 15
．． It was confirmed that approximately 000U/me of α-amylase was produced and accumulated in the medium, and for Bacillus subtilis it was approximately 3.000U/me. [J, Bacteriol,, 164
<3>, 1182-1187 (1985)] Here, between Bacillus plevis 47 and Bacillus subtilis, which carry exactly the same plasmid, Bacillus plevis 47 is approximately 5 times more efficient in producing heat-stable α-amylase. Based on the fact that the production efficiency is high, it has been found that the foreign r4 gene product can be secreted and produced efficiently by using the protein secretion ability possessed by protein producing countries.

Based on the idea that bacteria that secrete significant amounts of protein would be excellent host bacteria for genetic recombination, the inventors conducted a diligent selection process in search of such strains, and found various materials. Among the approximately 100,000 strains isolated from the bacteria, strains that produced a significant amount of protein outside the bacterial cells were placed in a monolayer.

When the strains isolated here were identified by species, 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 relates to a Pseudomonas bacterium that produces a significant amount of protein extracellularly.

Strains of the genus Bacillus that produce proteins are known, but strains of the genus Pseudomonas that produce proteins are known.
No bacterial strain is known that produces significantly more than e.

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 in vitro, is completely new.

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

First, approximately 100,000
The strain was grown on T2 agar plate medium (1% glucose, 1% peptone, 0.5% yeast extract, 0.2% yeast extract, 15%
Agar powder, pH 7.0> was inoculated, and bacteria around the colony were selected to become cloudy with 5% perchloric acid on a plate medium.Next, the aaii strain isolated here was transferred to a liquid medium (150
Shake culture (3
0°C for 48 hours), and 80 strains producing 1.2g or more of protein were obtained in the culture solution.For the measurement of extracellular proteins, an equal amount of 0.2N Na0)1 was added to the culture solution. Add and stir? ! 10. Centrifuge at OOOrpm x 5 minutes to remove bacterial cells, add an equal volume of 10% trichloroacetic acid to the supernatant, and after 10 minutes, 3. OOOrpmx f&L was collected by centrifugation for 10 min and dissolved in IN NaOH.
owry method [J, Biol, Chem, 193.26
5 (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: High protein production medium 14 cultures of the 80 strains previously obtained were cultured in all of these 5 types of media, and which of the media produced 5 g or more of extracellular protein. We selected 31 stocks.

These 31 strains were analyzed using Bergey's Manual o
f Determi-native Bacteri
ology (8th edition), Bergey'Manua
l of 5ystea+atic Bacterio
ligy vol, 1 and TheProkaryot
e (A Handbook on)Iabitat
s.

l5olation and identification
ion of Bacteria), the six strains were initially aerobic, Durham stain negative, bacilli,
Among the strains recognized as belonging to the genus Pseudomonas in that they do not form spores, 8502 strains were compared with one previously known species of the genus Pseudomonas on other morphological properties, growth conditions in various media, and physiological properties. The results showed that it was different from any other bacterial species in the genus Pseudomonas.

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

Thus, this bacterial species was named Pseudomonas sp, H3O2. Pseudomonas sp, 8502 is FERM
It has been deposited with the Institute of Fine Technology as P-9743.

Next, we will show the surname-like properties of Pseudomonas sp, )+502.

(A) Morphological properties (B) Growth status in each medium (C) Physiological properties (Continued) (D> Production of acid and gas (E) Assimilation of carbon source Acetate +5ucci
nate +Fu&1rate
-L-Malate
+β-Hydroxybutylate
+actate
+C1trate + *-Ketoglutarate -G
lycerol +L-Alan
ine +L-Aparta
te -L-Gluta+*ate
+L-Arginine
-L-Proline
+ Tyrosine -D
-Fucose-
MalLose
-Cellobiose
-Lactose
-D-Arabinose
+5 rbitol 15 tarch Inulin
-0xalate
-Ethylen glycol
-L-Threonine
-D-Ribose
+Mannitol
+D-Xylose
-L-Arabinose
-L-Rhamnose
-Glucose + D-Mannose
-D-Galactose
-Re1 lydroxyprol ine
)Inosine
+Betaine
-D-Fructose
-3ucrose 10Treharose
-Gluconate
-Propionate
-Malonate D(-)-Tartrate
+5orbitol
-Propylene glycol +n
-Butanol +
Benzoate
-5-11ydroxybenzoate
-p-■ydroxybenzoate
-Phenol
-Glycine + L-3erine
-L-Leucine
+L-1 soleucine
-L-Valine
-L-Lysine' +
L-Ornithine + L-Histidine
+L-Phenylanine 10L-Tryptophan +
N-Acety1glucosamine
+Raff1nose
+Create
+Deoxycholate
+D-Glucuronate
- The present invention relates to Pseudomonas bacteria that produce a significant amount of protein extracellularly, particularly Pseudomonas sp.

It is H3O2.

Depending on the properties of the protein produced in large amounts 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.H3O2 of the present invention is used as a host bacterium for genetic recombination, it is possible to efficiently secrete products resulting from gene damage to the outside of the bacterium, making it an excellent host bacterium for genetic recombination. It will become something new.

This system is expected to be used as a means of production for medical products, food processing materials such as high-quality food proteins, gelling agents, and leavening agents, and industrial materials for surface treatment such as glass-like materials, paper, and artificial leather. can.

As described above, the usefulness of the present invention is extremely significant in industry.

The present invention will be explained in more detail below by giving examples.

Example 1 The IP medium 500+++I described in Table 1 above was dispensed into a 2Q volume jar fermenter, and heated to 121°C2 by a conventional method.
After sterilization for 0 minutes, it was cooled.

Separately, the test tube containing the IP medium + 1!5 s + e was sterilized by autoclaving, and Pseudomonas s.
One platinum loop of p.p., H3O2 was inoculated and cultured with shaking at 37°C for 14 hours. Last time cultivated! Jarfur Mentor I thing 51
and cultured at 37° C. for 48 hours at an aeration rate of 0.51/) and a rotation speed of 400 rp+++. After culturing, cultivate! ! Add an equal amount of 2N NaOH to the product and stir 1k 1010
Centrifuge at 000 rpm for 5 minutes to remove bacterial cells.
10z trichloroacetic acid from Kasadai was added to 00ml, and after 10 minutes, the mixture was centrifuged at 3000 rpm for 10 minutes to collect the precipitate. The precipitate was washed with 5% trichloroacetic acid, collected by centrifugation, and electrolyzed by the fiLowry method, which was dissolved in IN Na011. Proteins are expressed in terms of bovine serum albumin. As a result, the amount of protein produced in vitro was 78/min.

Claims (1)

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