JPH02255081A - Protease-producing bacterium, protease produced from the same bacterium and purification thereof - Google Patents

Abstract

PURPOSE:To provide the subject protease-producing bacterium having heat resistance and an optimum pH of the alkaline side and capable of producing a protease which can be readily separated and purified in a high yield. CONSTITUTION:A protease-producing bacterium belonging to Nocardiopsis genus, having cell walls of meso-form by the 2,6-diaminopimelic acid analysis and of III/C type and containing PIII type phospholipid. The above-mentioned bacterium is separated from the soil in Kyoto Prefecture, named Nocardiopsis sp. OPC-210 strain and committed as FERM P10508 to Bikoken. By preparing an acetone powder from the resultant OPC-210 strain, dissolving the prepared acetone powder in a weakly acidic solution, dialyzing the solution and purifying the dialyzed solution using the column chromatography, the objective protease of high purity can be obtained in a high yield.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to alkaliphilic actinomycetes, and in particular to protease-producing bacteria, protease produced from the bacteria, and a method for purifying the same.

(Prior art and problems to be solved by the invention) Actinomycetes are a group of bacteria that are practically important because they produce a wide variety of secondary metabolites. However, there are limited reports on the isolation and utilization of alkaliphilic actinomycetes compared to other microorganisms that grow in abnormal environments.

The purpose of the present invention is to isolate alkaliphilic actinomycetes, purify alkaline protease produced by alkaliphilic actinomycetes, and elucidate its properties, with the aim of searching for and utilizing microorganisms that grow in abnormal environments.

(Means for Solving the Problems) The present invention provides a microorganism belonging to the genus Nocardiopsis (alkaliphilic Actinobacteria, Nocard 1ops i ssp
．． OPC-210 strain, Microtechnical Research Institute No. 10508 (FE
RM P-10508)) and the protease produced from this bacterial cell.

In addition, the method for purifying protease produced from protease-producing bacteria of the present invention involves obtaining acetone powder from the above-mentioned OPC7210 strain, dissolving it in a weakly acidic solution (preferably an aqueous ammonium acetate solution), dialysis, and then using column chromatography. Characterized by refining. At this time, purification by column chromatography was carried out by passing it through a DEAR-Sephadex A-50 column equilibrated with the weakly acidic solution, and then CM-Sepharose CL-
Preferably, this is carried out by 6B column chromatography.

The microorganisms used in the present invention were newly isolated from soil in Kyoto Prefecture in November 1985. The mycological properties of the isolated strain (hereinafter referred to as OPC-210 strain) are as follows.

By the way. OPC-210 strain was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, as part of the Microbiological Research Institute No. 10508 (FERMP-
10508).

1) Morphological properties Medium hyphae are moderately branched and do not divide under normal conditions. Aerial hyphae grow abundantly on yeast/malt agar medium (ISP medium), and sporulation is good. Aerial hyphae are simply branched. Sporangium, sclerotia, and fecal hair spores are Not observed.

At the beginning of sporulation, the aerial mycelium appears somewhat zigzag-shaped;
It then fragments into long fragments, which in turn fragment into unevenly sized, smooth-surfaced spores. When observed using a scanning electron microscope, the spores are lemon-shaped and approximately IX1.7 in diameter.
It has a micron size and the spore surface is smooth.

Table 1 2) Growth status on various media As a result of culturing on the various media shown in Table 1 at a temperature of 27°C for 7 to 14 days, OPC, -210 strain was grown on yeast extract/malt extract agar medium (ISP medium 2). , Growth on starch/inorganic salt agar medium (ISP medium) is extremely good.Growth on tyrosine agar medium (ISP medium NcX7), glucose/asparagine medium is moderate, and almost no growth on starch synthetic agar medium. No. The observed growth conditions are shown in Table 1.

3) Physiological properties a) Growth temperature and pH Shows good growth at 27-32°C, and its optimum p) 1
was 8 to 10.3.

b) Decomposition of starch Positive C) Coagulation of milk Negative d) Production of melanin pigment Negative e) Utilization and degradability of carbon sources Prudham-Gottlieb agar (TSP medium 9)
*JIS Z
8721 compliant standard color table (Japanese Standards Association) was followed.

The assimilation properties were observed.

D-glucose Inositol is strongly utilized Lactose is not utilized D-mannitol is utilized D-mannose is utilized Slightly utilized but unclear D-raffinose L-rhamnose is not utilized Sucrose is not utilized D-xylose is not utilized strongly 4) Cell wall Composition The method of Lucivalier et al.
my ofactinomycete, In : ^
ctinomycete taxnomy (Dieiz
, A & D, H, Thatter ed,', 5
ociety forIndustrial Micro
obiology+ Virginia, pp 22
(5 (1980)) analyzed 2,6-diaminobimelic acid in the cells of OPC-210 strain, and found that it was meso-type and cell wall type III/C, and characteristic reducing sugars and mycolic acids were not observed. Ta.

The phospholipids were of pHl type.

In view of the above-mentioned properties, it was determined that the strain ○P (, -210 strain is a microorganism actinomycete belonging to the genus Nocardiopsis), and Nocardiopsis sp, OP C-21
It was named 0 stock.

(Effects of the Invention) Actinomycetes, which produce a wide variety of secondary metabolites, are a group of bacteria that are practically important. According to the present invention, it is possible to purify alkaline protease produced by alkaliphilic actinomycetes. By efficiently isolating alkaline protease produced by alkaline-loving actinomycetes, the range of its use can be expanded.

Furthermore, enzymes that are active under extreme conditions have so far been mainly obtained from thermophilic bacteria.

However, the protease obtained in the present invention is heat resistant and has an optimum pn on the alkaline side. From this point of view, we can learn more about the structure and function of enzymes, as well as the effective use of enzymes, by comparing them with enzymes from normal organisms or by investigating the correlation between amino acid residue substitutions and activity using mutant strains. It is possible to obtain knowledge of

(Example) Hereinafter, the present invention will be specifically explained based on Examples.

(Example 1) Isolation of strain OPC-210: A strain of alkaliphilic actinomycetes obtained from soil in Kyoto Prefecture in November 1985 was isolated according to the method of Horikoshi et al.
ric, B to ol, Chem,, 35. 1407
(1971)).

Culture conditions: medium composition: 1% glucose, 1% by weight
casein, 0.5 wt% yeast extract, 0.1 wt% 2HPO4, 0.02 wt% MgSO4,-
7H20, pH 10, consisting of 1% by weight Na, CO3,
Seed culture for 2 days at a culture temperature of 27°C in a medium of
Subculturing was performed for 5 days with shaking at 120 rpm and an amplitude of 7 cm.

Purification and properties of alkaline protease 20pc-2
Acetone powder was obtained by adding 3 times the volume of cold acetone to the culture filtrate of the 10 strains in a conventional manner. This powder 30
．． 0g to 10mM CH3CO0NHn (pH 6,6)
DEAE-Sephadex A50 was dissolved in 20.0- and the supernatant was dialyzed overnight in the same buffer and then equilibrated in the same solution.
Chromatography was performed using a column (Sephadex is a trade name manufactured by Pharmacia). The fraction that passed through without being adsorbed was designated as Fr-1. Fr-1 is further 0. IMN
10mM Mc7) containing aCf CH3COONH4, (p
CM-Sepharose CL-6 equilibrated with H5,0)
Purification was performed by performing chromatography once using a B column (Sepharose is a trade name manufactured by Pharmacia, column dimensions: 1.9 x 50.0 cm) (protease I).

Table 2 shows each purified fraction.

Table 2 a) Protein was determined by the method of Lowry et al.

b) 1 unit of enzyme activity in enzyme solution 1.0 mi', 1
Defined as 1.0 μg of tyrosine per minute.

An electrophoretically homogeneous sample of this protease I was obtained. We also investigated various properties of this enzyme and obtained the following results.

) Molecular weight: approximately 21,000 (SDS electrophoresis method, K
.

Weber+ M, 0sborn, J, Biol
, Chem, 244+ 4406ii) Optimal pH:
10-12 (substrate casein) iii) Optimal temperature = 60
~70°C (substrate casein) iv) Thermal stability: pH 1
When treated at 0 for 30 minutes, it is stable up to 50℃, but at 70℃
Then it becomes completely inactive.

v) pH stability: At pH 84-8, there is a residual activity of 80% or more after treatment at 60° C. for 30 minutes, but at pH 10, the activity is almost lost.

vi) Stabilization: The residual activity is about 25% after treatment at pH 2, 50° C., for 150 minutes, but is stabilized to 50% in the presence of 1 mM Ca 2+.

vii) Effect of inhibitors: 2mM PCMB and 10
It was not inhibited by mM EDTA.

Strain identification: Mycological properties (culture properties and physiological biochemical properties) were determined using the ISP method (E, B, Shirlinga
nd D, Gottlieb, Int, J, 5
yst, Bacteriol,.

Kawa, 313 (1966)), and the chemical classification method (diaminopimelic acid, reducing sugars, mycolic acids, and phospholipids) was according to the method of Rechev Ariel et al. (M.

P, Lecbevalier and H, A, L
echevalier Int, J.

5yst, Bacteriol, 20.435
(1970)).

The results are shown in Tables 3 and 4.

Table 4 In addition, since protease I was completely inhibited by 5mM PMSF, it was confirmed that it is a serine protease.

Claims (6)

[Claims] (1) The cell wall is meso type and cell wall type III/C type as determined by 2,6-diaminopimelic acid analysis, and the phospholipid is P
Type III microorganisms belonging to the genus Nocardiopsis (alkaliphilic actinomycetes, Nocardiopsis sp. OP
Strain C-210, Microtechnical Research Institute No. 10508 (FERM
P-10508)) is a protease producing bacterium. (2) Purification of protease produced from protease-producing bacteria, characterized in that acetone powder is obtained from OPC-210 strain according to claim 1, dissolved in a weakly acidic solution, dialyzed, and purified by column chromatography. Method. (3) DEAE-Sephadex A-5 purified by column chromatography and equilibrated with the weakly acidic solution
0 column, then CM-Sepharose C
Claim 2 carried out by L-6B column chromatography
Method described. (4) The method according to claim 3, wherein the weakly acidic solution is an aqueous ammonium acetate solution. (5) A protease produced from the OPC-210 strain according to claim 1. (6) A protease obtained by the purification method according to claim 2.