JPS5946593B2 - Method for producing alkaline protease - Google Patents

Description

[Detailed description of the invention] Background of the invention The present invention relates to a method for producing alkaline protease.

Note that protease is a general term for hydrolytic enzymes that act on proteins and intermediate products of their hydrolysis to promote the decomposition of peptide bonds, and among these, those that have an optimal pH on the alkaline side are called alkaline proteases. It is known for a wide variety of uses, including addition to pharmaceuticals, detergents, and use in the food and leather industries.

Microorganisms have traditionally been used as enzyme sources from which enzymes can be collected stably and in large quantities.
genus, Arthrobacterium
bacterium of the genus Aspergillus
lus ), the fungi Deuteromycota of the genus Fusarium and Streptomyces (Streptm.
Many products produced by actinomycetes of the genus S. yces have been reported.

However, there are very few microorganisms that produce proteases that exhibit activity over a wide pH range and are stable in highly alkaline regions, and it is particularly difficult to obtain highly active proteases in the case of Deuteromyces, so they have not yet been put to practical use. The current situation is that there is no vine.

(n) Summary of the Invention The present inventor investigated various microorganisms that efficiently produce proteases that are active in a wide pH range and are stable in a highly alkaline region, and as a result, the present inventor discovered that they have been found to be completely unknown as alkaline protease producing microorganisms until now. Acremonium (A)
Therefore, the method for producing alkaline protease of the present invention involves culturing alkaline protease-producing bacteria belonging to the genus Acremonium in a medium, and then culturing alkaline protease-producing bacteria belonging to the genus Acremonium in a medium. This method is characterized by producing protease and collecting it.

(In) Detailed description of the invention The present invention will be specifically explained below.

Microorganisms to be Used In the present invention, any alkaline protease-producing bacteria belonging to the genus Acremonium can be used.
No. 22], Acremonium versikinum CB8169
．． Strains such as S. 65 can be advantageously used.

The genus Aku*um is a member of the Deuteromycetes, Hyphomycetes (Hyp
homy ce tes ), Phialo-type conidial fungi group (
Phia 1csporae) and 7th grade
-6 It is closely related to the genus Res and Penicillium, but differs from these two genera in that it lacks a distinct conidiophore.

The mycological properties of the 12 Acremonium-mounted bacteria, which are one of the microorganisms used in the present invention, are shown below.

(1) Growth status in medium (i) Malt extract/yeast extract agar medium A large number of aerial mycelia are formed, and the bacterial flora is densely spread.

The color is initially white, but after two weeks of culturing at 25°C, some of the bacterial flora changes from yellowish-brown to yellowish-green.

(11) Czapek agar medium Shows the same growth conditions as malt extract/yeast extract agar medium.

(iii) Oatmeal agar medium The bacterial flora spreads after forming aerial hyphae.

The color changes to light brown after culturing at 25°C for 2 weeks.

On a malt extract/yeast extract agar medium, conidia begin to form after one week of culture and are colorless.

Conidiogenic cells grow early directly from vegetative hyphae as phialides, which are almost unbranched, colorless, thin, tapered, chained at the tip, and rarely irregularly atrophied, forming conidia. .

The formation of conidia is phialoid, and the conidia are colorless, thick membraned, obovate-elliptic, and consist of a single cell.

A microscopic photograph of the conidial structure on a malt extract/yeast extract agar medium is shown in FIG.

(2) Physiological and ecological properties (1+ Optimum growth conditions pH: 5-7 Temperature: 20-30°C (11) Growth range pH: 3-10 Temperature = 5-35°C The following characteristics of this strain G, C, Ainswort
h, F, K, Sparrow, A, S, Sus
sman edition'J TheFungilVAj (Aca
demic Prees, NewYorkand L
ondon (1973)) and W, Gam5 [C
ephalosporium-artige S
Ch immel-pi lze (Hyphomyc
etes) J (G, FischerVerlag,
It is clear that this microorganism belongs to the genus Acremonium, in accordance with the characteristics of the genus Acremonium described in Stuttgart (1971).

The A12 bacterium is similar to Acremonium versikinum in terms of its conidial and phialide forms, but it differs from Versikinum in terms of flora, such as remarkable development of aerial hyphae and rapid growth rate.

The culture medium may be one commonly used for the growth of Acremonium bacteria, and there are no particular limitations. Examples include starch, dextrin, sucrose, glucose, etc. as a carbon source, peptone, meat extract, yeast, etc. as a nitrogen source. Contains organic and inorganic nitrogen-containing substances such as extracts, corn staple liquor, ammonia salts, and urea, other inorganic salts such as potassium salts, magnesium salts, calcium salts, and phosphates, vitamins, growth-promoting factors, etc. .

Note that either a solid medium or a liquid medium may be used.

Culture Cultivation is carried out at a pH of 4 to 9, especially 5, and a temperature of usually 20 to 30.
It is carried out at ℃.

The culture period is usually 4 to 20 days for solid culture and 3 to 20 days for liquid culture.
~10 days.

In liquid culture, culture can be performed by an appropriate culture method such as static culture, shaking culture, or aeration culture.

Since protease is produced and accumulated in the medium as the culture progresses, protease is collected from this culture in the next collection step.

Collection Production The accumulated protease can be collected by any conventional collection means.For example, in the case of solid culture, the enzyme solution is extracted with water or an aqueous solvent, and in the case of liquid culture, the enzyme solution is extracted from the culture solution by filtration. , sterilize by centrifugation, etc. to obtain an enzyme solution.

The enzyme solution obtained in this way is directly or concentrated, and an organic solvent such as ethanol or acetone is added thereto to precipitate the protease active fraction, followed by dehydration and drying to obtain the target crude alkaline protease. can get.

Properties of the alkaline protease produced (1) Action It decomposes proteins into peptides and amino acids, but relatively little release of single amino acids, which have a strong ability to liquefy proteins.

When Hammar-5ten was used as a substrate, results were obtained in which it was degraded in an endo-type decomposition mode.

(2) Substrate specificity Various proteins such as casein (Hamma
rs ten ), hemoglobin, and seratin.

The resolution of bovine serum albumin and wheat gluten was in the following order: casein > wheat gluten > hemoglobin > bovine serum albumin.

(3) Optimum 1) H and stable pH range When casein (Hammersten) is used as a substrate, the pH for high activity is 6 to 11, and the optimal pH is about 10.
．． 5, and the stable pH is 4-10.

A graph showing the influence of pH on protease activity is shown in FIG. 2, and a graph showing pH stability is shown in FIG. 3.

(4) Activity measurement method Enzyme solution (extract of solid culture, sterilized culture solution, or a solution obtained by dissolving a precipitate produced by adding an organic solvent after sterilization in water) 1.0 ml and 0.6% Hammersten casein solution (p
Mix 5.0 ml of H10,0) and react at 30°C for 10 minutes.

Immediately after the completion of the reaction, 5.0 g of 0.44M) IJ dichloric acid was added to stop the reaction, and the mixture was left at 30°C for 30 minutes.
Strain the reaction solution.

This P liquid 2. 5. 0.55M sodium carbonate solution in OM.
0rILlO and 2x diluted Folin's reagent 1.01
After adding nl and leaving it at 30℃ for 30 minutes, the coloring solution 66
Measure the absorbance at 0 nm.

66, which was colored by adding trichloroacetic acid to the enzyme solution and then adding casein solution using the same procedure.
Measure the absorbance at 0 nm and subtract this as a blind test.

The amount of enzyme that produces 1 μg of 1,000 rosin per minute under these conditions is defined as 1 unit.

(5) Range of suitable temperature for action The optimum temperature is around 40°C.

A graph showing the influence of temperature on protease activity is shown in FIG. 4, and a graph showing temperature stability is shown in FIG. 5.

(6) Deactivation conditions by pH 1 temperature, etc. At pH 12 or higher and pH 3 or higher, it is almost completely deactivated in 30 minutes at 30°C.

Furthermore, when heat treated at 50° C. for 10 minutes, most of the activity is lost.

(7) Inhibition, activation and stabilization 40% with 2X10-3 moles of Cu++ and the same amount of Mn'',
It is activated by 10 to 20% with Ni''+ and about twice as much with 2×10 −2 moles of Cu++.

Ma7: 90% or more, 30%, and 10% inhibition by 2Xl□-a mol of Hg++, Ag", and Fe", respectively.

The effects of inhibitors or metal ions are summarized in the table below (an example of alkaline protease obtained from Acremonium genus/'1612 bacteria).

(8) Purification method The enzyme powder obtained from the above-mentioned T-collecting step is dissolved in water and subjected to gel filtration through a Sephadex G-50 column stabilized with water.

Collect polymer fractions with protease activity, M, /2
Dialyze in 0'J acid buffer (pH, 6°0) or
After lyophilization, the mafon was dissolved in the same buffer, equilibrated with the same buffer, and applied to a CM-Sephadex C-50 column.

By this operation, the protease active fraction is specifically adsorbed, and the unnecessary fraction is not adsorbed and passes through.

The active fraction is eluted by changing pHK from 6 to 8 in M/20 phosphate buffer to obtain purified enzyme.

(9) Molecular weight From the gel Pa, the molecular weight is estimated to be approximately 30,000 (!).

The present invention will be explained in more detail below with reference to Examples.

Example 1 Glucose 5%, Peftone 0.25%, Yeast Extract 0,
25%, KH2PO40.1%, MgSO4・7f,
,L200.05%+CaCl2・2H200,05
% and pH with trace amounts of iron, copper, zinc and manganese salts
Sterilize the liquid medium of 5 and add Acremonium%12 to it.
Inoculate the bacteria (Feikoken Bacteria No. 4222) and culture with shaking at 7.28°C for 7 days.

The propase titer of the supernatant obtained by sterilizing the culture solution by centrifugation or filtration was 55.3 units per ml.

Three times the volume of ethanol was added to this supernatant liquid 11 to obtain a precipitate, this precipitate was dissolved in water, gel filtered over a Sephadex G50 column, and ion exchange chromatography was performed using CM-Sephadex.I) The enzyme was isolated from the eluate of H8 with phosphate buffer by dialysis, and the purified enzyme was obtained by lyophilization.

67.3 units of enzyme per 1 mg of this dry matter was added to 40
5m1? I got one.

Example 2 Glucose 5%, dry yeast 0.5%, KH2PO40.1
%, MgSO,・7l-(200.05%
, CaCl2 2F (2() p containing 0.05% and trace amounts of iron, copper, zinc, manganese salts) (,5 sterilized liquid medium, Acremonium versikinum CB
516965 and culture with shaking at 30°C1
child.

After 7 days of culture, the bacterial cells were removed and the enzyme activity of the fermentation solution was measured and found to be 13.5 units/ml.

This furnace liquid 1. Add ethanol to i to produce 1 precipitate 2
．． When the enzyme activity of Og was measured, it was 5.4 units/4 and 1
child.

[Brief explanation of drawings]

Figure 1 is a micrograph of the conidial structure on the culture medium of Acremonium gallo 12 bacteria used in the present invention (NIFE accession number 4222), and Figure 2 is FIG. 3 is a graph showing the relationship between the activity of the protease obtained by the present invention and pH; FIG. 4 is a graph showing the relationship between the activity of the protease and temperature; FIG. Figure 5 is a graph showing the activity and humidity stability of the propase.

Claims (1)

[Scope of Claims] 1. A method for producing alkaline protease, which comprises culturing alkaline protease-producing bacteria belonging to the genus Acremonium in a medium to produce alkaline protease, and collecting the same. 2 Alkaline protease-producing bacteria belonging to the genus Acremonium are genus Acremonium A.
12. The production method according to item 1 above. 3 Acremonium versichinum (A-Pers) is an alkaline protease-producing bacterium belonging to the genus Acremonium.
icinum), the manufacturing method according to item 1 above.