JPH07274944A - New variant and method for producing protein hydrolysate - Google Patents

Abstract

PURPOSE:To obtain Aspergillus oryzae resistant against polyene antibiotics, enhanced in protease activity, acting on proteins to give protein hydrolysates having high nitrogen contents and high glutamic acid contents, and suitable for producing seasonings, e tc. CONSTITUTION:This Aspergillus aryzae resists against polyene antibiotics such as amphotercin B and nystatin, has an enhanced protease activity of 1.5-4.5 times that of the parent strain, and is deposited as FERM-P-14259. The Aspergillus oryzae is obtained by subjecting Aspergillus oryzae to a tautomeric treatment, and subsequently screening the polyene antibiotic-resistant strain. The culture product of the Aspergillus oryzae is preferably allowed to act on a protein for the production of the protein hydrolysate.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a highly active mutant of a protein hydrolase and a method for producing a protein hydrolyzate using a culture of the mutant. Specifically, as a method for efficiently obtaining a highly active mutant of a protein hydrolase, a method of selecting a polyene antibiotic resistant strain after mutation treatment of Aspergillus oryzae is adopted, and the resulting polyene antibiotic is obtained. Aspergillus oryza with substance resistance and high proteolytic enzyme activity
e ). In addition, a method for producing a protein hydrolyzate, which comprises allowing the culture of Aspergillus oryzae to act on a protein such as defatted soybean, that is, a protein hydrolyzate having a high nitrogen content, and a strong taste. The present invention provides a method for producing a protein hydrolyzate having a high content of amino acids such as glutamic acid. In addition, this protein hydrolyzate has a high utility value as a seasoning.

[0002]

2. Description of the Related Art Aspergillus oryzae, which is also known as Aspergillus oryzae, is a representative bacterium of Aspergillus oryzae in Japan, and is the most useful koji mold used for brewing miso, soy sauce, sake, mirin, amazake, etc. . The protein hydrolase produced by Aspergillus oryzae is used in the production of traditional Japanese fermented seasonings such as soy sauce and miso. In the production of these seasonings, proteases such as protease and peptidase are used. Increasing the activity of is very important in industrial production. Therefore, there have been many reports on the creation of highly active mutant strains by mutation breeding using X-ray irradiation, UV irradiation, and a mutagen (Nobuyoshi Iguchi: Agriculture, 2
9,73, (1955), Nobuyoshi Iguchi, Yoshishiro Yamamoto: Agriculture 29,394, (195
5), H. Sekine S. Nasuno and N. Iguchi: Agric . Biol . C
. hem, 33,1477, (1969) , S.Nasuno and T.Nakadai:. J Fe
rment . Technol ., 49,544, (1971), T. Nakadai and S. Nas
uno: J. Ferment Technol ., 55,273, (1977)).

In recent years, by mutating an extracellular enzyme-producing bacterium of the genus Bacillus and then adding a cell membrane synthesis inhibitor such as vancomycin or ristocetin, that is, an antibiotic,
Increase the production amount of extracellular enzymes such as protease and cellulase to 2
~ 4 times improvement method has been reported (S.Ito Y.Ohta
M.Shimooka M.Takaiwa K.Ozaki S.Adachi and K.Okamo
to: Agri c. Biol . Chem ., 55 (9), 2387, (1991), Japanese Patent Fair 1-
222771), high production of extracellular enzymes by vancomycin-resistant mutant strains is a commonly used method (H. Kawa.
hara et al .,: Biosci . Biochem ., (1993)).

However, it is reported that antibiotic resistant strains in eukaryotes can obtain excellent mycophenolic acid-producing mutants by selecting polyene antibiotic resistant strains of mycophenolic acid producing strains belonging to the genus Penicillium. (JP-A-53-115880), it has been reported that yeast strains belonging to the genus Saccharomyces are resistant to polyene antibiotics in freezing resistance (JP-A-59-203441), but the purpose is to increase enzyme activity. There is no report of antibiotic resistant strains.

[0005]

DISCLOSURE OF THE INVENTION The present inventors have made a culture of Aspergillus oryzae act on a protein so that the nitrogen content is high and the amino acid content is high, especially the content of glutamic acid having a taste. In order to obtain a protein hydrolyzate having a high activity, it was necessary to construct a highly active mutant strain of protein hydrolase.

[0006]

[Means for Solving the Problems] As a result of the inventors' earnest research on a method for efficiently obtaining a highly active mutant of a protein hydrolase (hereinafter referred to as a highly active mutant), which is the above-mentioned object, It was found that a highly active strain can be efficiently obtained by mutating Aspergillus oryzae and then selecting a polyene antibiotic resistant strain. Furthermore, when the culture of the highly active mutant strain was allowed to act on the protein,
It was found that a protein hydrolyzate having a high nitrogen content and a high amino acid content, particularly a glutamic acid content with a taste, was obtained, and the present invention was completed.

That is, the present invention provides an efficient method for producing a highly active mutant strain by mutating Aspergillus oryzase and then selecting a polyene antibiotic resistant strain. The present invention is also a highly active mutant strain obtained by the same method. Furthermore, the present invention provides a method for producing a protein hydrolyzate by allowing a culture of these highly active mutant strains to act on a protein, that is, a method for producing a protein hydrolyzate containing high nitrogen and high glutamic acid. The present invention is to be as (1) a polyene antibiotic resistant claimed, Aspergillus oryzae (Asperg the protein hydrolase high activity was increased
illus oryzae ), and a method for producing a protein hydrolyzate, characterized in that the culture of Aspergillus oryzes according to (2) (1) is allowed to act on a protein.

The polyene antibiotic used in the present invention is a substance that damages the membrane as a result of changing the fluidity of the membrane by binding to the cell membrane sterol of fungi or other eukaryotic cells, and its example is amphotericin. B, nystatin, pimalysin and the like.

The polyene antibiotic resistant strain of Aspergillus oryzae of the present invention (hereinafter abbreviated as resistant strain) is a nitrosoguanidine that has been conventionally used as a mutagenizing agent for Aspergillus oryzae producing various proteolytic enzymes. Then, these strains were cultured on an agar medium containing a high concentration of polyene antibiotics, and resistant strains growing there were obtained.

Regarding mutagenesis, in addition to the above-mentioned nitrosoguanidine as a mutagen, other commonly used chemical substances such as hydroxylamine and ethylmethylsulfonic acid, or irradiation with ultraviolet rays, radiation, X-rays, etc. Alternatively, it can be obtained by so-called spontaneous mutation obtained without mutation treatment.

Here, in the present specification, "tolerance" means
It means that a certain microorganism has acquired the property of being able to grow in an environment in which a polyene antibiotic is present at a concentration equal to or higher than the minimum growth inhibitory concentration of the parent strain. Therefore, in order to select a resistant strain from among mutant strains, the minimum growth inhibitory concentration of the parent strain is measured, and the mutant strain is cultured on an agar medium containing a polyene antibiotic above the concentration to form colonies. The collected strains should be collected. For example, when amphoterin B is used as a polyene antibiotic, the minimum inhibitory concentration of Aspergillus oryzae is about 20 μg / ml, and 20 to 200 μg / ml of amphotericin is used to select a resistant strain from among mutant strains. The mutant strain may be cultured on an agar medium containing B, and the colony-forming strain may be collected.

Further, regarding the method for obtaining a highly active mutant of a proteolytic enzyme from the resistant strain of the present invention, for example, a spore suspension of the resistant strain in an agar medium containing defatted soybean powder and potassium hydrogen phosphate. Add 1-10 μl to 4-8
After culturing for a day, a highly active mutant strain is selected based on the ratio of the radius of the part (decomposed halo) where the defatted soybean powder was decomposed into a transparent zone (decomposed halo) and the radius of mycelium. This makes it possible to remove poorly growing strains or strains with reduced proteolytic activity among the resistant strains, and to select highly active mutant strains efficiently.
As the protein contained in the medium, casein, wheat flour, gelatin or the like can be used in the same manner depending on the purpose.

As a representative example of the highly active mutant strains of the present invention thus obtained, Aspergillus oryzae AJ
Amphotericin B (hereinafter abbreviated as AMB) -resistant Aspergillus oryzae AJ117286, AJ117287, AJ11 derived from 177281 as a parent strain
7289 and AJ117290. The main mycelial properties of this highly active mutant strain are the same as those of the parent strain. However, it is significantly different in that it grows even in the presence of AMB, and the protease activity is 1.5 to 4 times that of the parent strain.

The culture of the highly active mutant strain of the present invention comprises a liquid culture or a solid culture, that is, koji. Usually, liquid culture is selected because it is easy to manage. Since these cultures contain various degrading enzymes, it is preferable to use the liquid culture or the solid culture as they are, but the required enzyme can be separated from the culture and purified before use. That is, bacterial cells are separated from a liquid culture by centrifugation or filtration, and proteins are precipitated from the bacterial cells and the culture solution by an ordinary separation means such as salting out method, isoelectric focusing method, and solvent precipitation method. Alternatively, the enzyme solution can be concentrated by the ultrafiltration method, and the purified product separated and collected by the usual purification method can be used alone or in combination. Also, the enzyme solution may be extracted from the solid culture and then similarly separated and purified, and the extract, the separated enzyme solution and the purified enzyme may be used alone or in combination.

The liquid culture of the highly active mutant strain in the present invention is a normal medium comprising a carbon source, a nitrogen source, inorganic salts, cofactors and the like, and aeration culture may be carried out according to a conventional method. As a carbon source, for example, soluble starch, wheat bran or wheat bran extract, glucose, mannose,
Examples of the nitrogen source include fructose, galactose, sucrose and the like, and examples thereof include soybean powder, defatted soybean, casein, yeast extract, peptone, ammonium sulfate and the like. The inorganic salts include potassium chloride, magnesium sulfate, disodium hydrogen phosphate, potassium dihydrogen phosphate, and the like, and cofactors such as corn steep liquor, oils such as rice oil, and surfactants such as sugar esters. Can be mentioned. Regarding culture conditions, pH is 4.0 to 8.
0, preferably pH 5.0-6.0, temperature 20-40
C., preferably between 27 and 33.degree. C. is suitable. A culture time of 24 to 72 hours, preferably 36 to 60 hours is suitable.

Further, solid culture, that is, koji making may be carried out under normal koji making conditions. As a raw material of koji, for example, defatted soybean,
Puffed defatted soybean, wheat, wheat bran, rice and the like can be mentioned.

Here, commercially available enzyme preparations were added to these cultures,
For example, an enzyme solution containing a protein hydrolase, a cell wall degrading enzyme, glutaminase, or the like or a purified enzyme preparation may be added depending on the purpose.

Next, the proteins that act on the culture of the highly active mutant strain of the present invention include, for example, soybean, wheat, cornmeal, milk casein, fishmeal and the like, which are further processed by degreasing or puffing. Proteins or proteins separated from these raw materials may be used.

The conditions for allowing the culture of the highly active mutant strain to act on the protein are as follows. For example, defatted soybean, soybean protein as a raw material, and the like are mixed with the culture of the highly active mutant strain, and the mixture is preferably heated at 5 to 60 ° C. For 6 hours at 30-50 ° C
The reaction may be performed for 15 days, preferably 24 hours to 10 days. During the reaction, salt or ethanol may be added for the purpose of preserving.

After the completion of the reaction, unreacted starting protein, insoluble matter such as bacterial cells may be removed by a conventional separation method such as centrifugation or filtration. The glutamic acid release rate and amino acid release rate of each of the produced proteolytic solutions may be determined from the determination of glutamic acid by the enzymatic method, the free amino acid content by amino acid analysis, and the total nitrogen content.

[0021]

INDUSTRIAL APPLICABILITY According to the present invention, a highly active mutant strain can be efficiently obtained. The highly active mutant strain of the present invention is 1.5 to 1.5 times that of the parent strain.
A protein hydrolyzate having a protease activity of 4.5 times and obtained by acting a culture of a highly active mutant strain of the present invention on a protein has a high nitrogen content, a free amino acid content,
In particular, it has a large amount of glutamic acid liberated, and is advantageously used as a seasoning having a strong taste.

[0022]

[Examples] Hereinafter, the method for isolating the resistant strain, the highly active mutant strain, the method for producing a protein hydrolyzate, and the like of the present invention will be described in detail with reference to Examples and Reference Examples. In addition, the measuring method of protease activity in an Example is as follows. The protease activity was measured according to the usual Anson-Hagihara modified method (B. Hagihara et al .,: J. Biochem., 45, 185, 1958). Specifically, 0.75% casein solution 4
00 μl and 0.24 M disodium hydrogen phosphate solution (p
H7.5) 100 μl was preincubated at 37 ° C. for 5 minutes, and the enzyme solution (culture supernatant, etc.) 10-100
μl was added and incubated at 37 ° C. for 10 minutes. To stop the reaction, an equal volume mixture of 0.1M trichloroacetic acid, 0.22M sodium acetate and 0.33M acetic acid was added. Next, 200 μl of the supernatant of this reaction solution was added to 500 μl of 0.55 M sodium carbonate solution, and 100 μl of a commercially available phenol reagent diluted 2-fold was added thereto, and immediately after stirring, 30
Incubated at 0 ° C for 30 minutes. 660n of this solution
The absorbance at m was measured to determine the enzyme activity. 1un
It is equivalent to 1 μg tyrosine per minute under the above conditions
It is defined as an activity of producing a non-protein substance showing Folin coloration.

(Reference example) Aspergillus oryzae AJ
A spore suspension of 117281 was used as a potato dextrose agar medium containing each concentration of AMB (hereinafter, abbreviated as PDA medium).
And the minimum growth inhibitory concentration of AMB was determined. That is, a PDA medium containing AMB applied with the spore suspension of Aspergillus oryzae AJ117281 was cultured at 30 ° C. for 5 days, and the growth of hyphae on the medium surface was visually observed. The results are shown in Table 1.

[0024]

[Table 1]

As is clear from these results, the minimum inhibitory concentration for Aspergillus oryzae AJ117281 was about 20 μg / ml.

(Example 1) Aspergillus oryzae A
An example of a method for selecting an AMB resistant strain using J117281 as a parent strain is shown below. To the spore suspension obtained from the PDA slant culture medium of the cultured parent strain, 0 to 4 mg / ml of nitrosoguanidine solution was added in an amount equal to that of the spore suspension, and the temperature was kept at 30 ° C for 3
Incubated for 0 minutes. This spore suspension is
P containing 50 μg / ml at a concentration higher than the minimum growth inhibitory concentration of P
Apply to DA medium, culture at 30 ° C for 5-8 days, collect colonies growing on the medium surface, and use PDA medium at 30 ° C for 4-8
Cultured for 7 days. About 200 AMB resistant strains were obtained by the above operation.

(Example 2) An example of selection of a highly active mutant of a protein hydrolase from an AMB resistant strain is shown below. 2 μl of the spore suspension of the AMB-resistant strain obtained in Example 1 was added to medium A containing defatted soybean powder shown in Table 2 at 30 ° C. for 5
After culturing for 7 days, the ratio of the radius of the clear zone (halo) due to the decomposition of defatted soybeans around the mycelial colony to the radius of the mycelial colony (hereinafter, abbreviated as the decomposed halo ratio) was measured for 230 strains and the parent strain was degraded A strain was selected that was 5% or more larger than the halo ratio. The results are shown in Table 3.

[0028]

[Table 2]

[0029]

[Table 3]

As is clear from this table, the rate of increase in the decomposition halo ratio of the AMB-resistant strain was higher than that of the mutant strain treated with the mutation alone. The AMB-resistant strains with an increased decomposition halo ratio were tested in liquid medium B shown in Table 4 at 30 ° C. for 48 hours.
After shaking culture for a period of time, the protease activity of the supernatant was measured. The results are shown in Table 5.

[0031]

[Table 4]

[0032]

[Table 5]

As is clear from this table, the number of highly active mutants in which the protease activity of the liquid culture supernatant of the AMB-resistant strain was higher than that of the parent strain was obtained compared to the mutant strains only subjected to the mutation treatment. The maximum value of its activity reached 4.5 times that of the parent strain, which was 3 times that of the mutant strain only subjected to the mutation treatment.

This highly active mutant strain No. A2801 showing a protease activity of 450 u / ml was named Aspergillus oryzae 117290, and was deposited with the Accession No. FERM P-14259 at the Institute for Microbial Industrial Technology, Institute of Industrial Science and Technology.

(Example 3) Next, an example of a method for producing a proteolytic product obtained by acting a culture of a highly active mutant strain on a protein will be described below. The following operation was performed aseptically. 20 ml of a liquid culture of the highly active mutant strain selected by the decomposition halo of Example 2 was added to 5 g of defatted soybean flakes and 7.5 m of purified water.
1 and added to the autoclaved (121 ° C, 20 minutes) treated, mixed with 0.6 ml of ethanol, and added to 30
The reaction was carried out at 5 ° C for 5 days, then at 50 ° C for 5 days. The reaction product was centrifuged and the total nitrogen, amino acid release amount by amino acid analysis, and yield were measured and compared with the parent strain. Table 6 shows the results of the highly active mutant strains in which the glutamic acid release rate, the total free amino acid content, and the nitrogen utilization rate were clearly increased.
8 shows.

[0036]

[Table 6]

[0037]

[Table 7]

[0038]

[Table 8]

Table 9 shows the amino acid release rate (amino acid release amount (g) / total nitrogen amount (g)) of each amino acid determined from the amino acid analysis of these defatted soybean decomposition solutions.

[0040]

[Table 9]

The AMB-resistant highly active mutants Nos. B310, No. B1005 and No. B109 shown in Tables 6 to 8 were respectively treated with Aspergillus.
Olise AJ117289, AJ117287, AJ1
It was named 17286 and was deposited with the Institute of Microbial Technology, Institute of Industrial Technology. The deposit numbers are FERM P-1
4258, P-14257, and P-14256.

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Claims (2)

[Claims] 1. A is a polyene antibiotic resistance, protein hydrolase high activity Aspergillus oryzae elevated (Aspergillus oryzae). 2. A method for producing a protein hydrolyzate, which comprises allowing the culture of the Aspergillus oryzae according to claim 1 to act on a protein.