JPH10210967A - Highly active variant and production of protein hydrolyzate using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject hydrolyzate strong in flavoring, useful for seasoning, high in nitrogen content and glutamic acid content, by treating a culture product of a specific variant belonging to Aspergillus oryzae with a protein in the absence of salt or under conditions of low salt content. SOLUTION: A variant [e.g. Aspergillus oryzae AJ11731 strain (FERM P-15956)] excellent in culture properties, high in glutaminase activity, obtained by a method for treating Aspergillus oryzae with a nitrosoguanidine as a mutation agent is subjected to liquid culture. A protein such as defatted soybeans, soybean protein or wheat gluten is treated with the obtained culture product to give a protein hydrolyzate high in nitrogen content and glutamic acid.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a highly active mutant having high glutaminase activity and protease activity in liquid culture, and a method for producing a protein hydrolyzate having a high nitrogen content and a high glutamic acid content using the mutant. .
More particularly, Aspergillus oryzae (Aspergillu
s oryzae ), a highly active mutant having both high glutaminase activity and high protease activity in liquid culture, and a culture of the mutant is reacted with proteins such as defatted soybean, soybean protein, and wheat gluten to contain nitrogen. And a method for producing a protein hydrolyzate having a high glutamic acid content. This protein hydrolyzate has a strong taste,
It is highly useful as a seasoning.

[0002]

2. Description of the Related Art Since soy sauce, miso and the like contain a high concentration of sodium chloride, microorganisms having a high salt tolerance and a high halophilicity are used as microorganisms for producing them.
For example, Zygosaccharomy
Other ce s rouxii) yeast, such as, Aspergillus oryzae is a koji mold, Aspergillus sojae (Aspergillu s s
oja e) etc. are used. These microorganisms produce enzymes such as proteolytic enzymes, amylase enzymes and glutaminase in the production of soy sauce, miso and the like. Proteolytic enzymes, ie, proteases and peptidases, are enzymes necessary to break down proteins into amino acids. Glutaminase is an enzyme that converts glutamine released during proteolysis into glutamic acid having a strong taste. When glutaminase activity is insufficient in the production of soy sauce, miso, and the like, glutamine is non-enzymatically changed to pyroglutamic acid having no umami. Therefore, in order to obtain a protein degradation product having a strong taste from a protein as a raw material, it is necessary that these proteases, peptidases and glutaminases are sufficiently present.

[0003] For the purpose of improving the activity of these enzymes required for the production of soy sauce and miso, a microorganism having these enzyme activities has been mutated by UV irradiation, treatment with a mutagen or the like for a long time. Active mutants have been created (Nobuyoshi Iguchi: Agricultural Science, 29, 73)
(1955), Nobuyoshi Iguchi, Yoshiro Yamamoto: Agriculture, 29, 394 (195
5), H. Sekine, S. Nasuno and N. Iguchi: Agric. Biol.
Chem., 33, 1477 (1969), S. Nasuno and T. Nakadai: J.
Ferment. Technol., 49, 544 (1971), S. Yamamoto am
d H. Hirooka: J. Ferment. Technol., 52, 564 (197
4), T. Nakadai and S. Nasuno: J. Ferment. Technol., 5
5, 273 (1977)).

It is said that it is difficult to obtain a mutant having both high protease activity and high glutaminase activity in solid culture. In recent years, high activity has been achieved by cell fusion between a high protease activity and a high glutaminase activity. Strains have also been created (S. Ushijima, T. Nakada
i: Agric. Biol. Chem., 51, (4), 1051 (1987), S. Us
hijima, T. Nakadai, K. Uchida: Agric. Biol. Chem.,
51, (10), 2781 (1987), S. Ushijima, T. Nakadai, K.
Uchida: Shoken, 17, (3), 89 (1991), Tokuhei 3-73271
Gazette, Japanese Patent Publication No. 3-68672).

[0005] Soy sauce and miso are produced by solid cultivation, but since the cultivation is an open system, it is difficult to control the growth of Aspergillus or to control contaminating microorganisms other than Aspergillus. On the other hand, liquid culture can be performed in a closed system using an ordinary fermenter, so that the culture medium and culture conditions can be kept constant and pure culture can be performed. For liquid culture, there are reports and related patents on protease production by Aspergillus oryzae and Aspergillus soya (S. Ueno, M.
Miyama, Y. Ohashi, M. Izumiya, and I. Kusaka: App
l. Microbiol. Biotechnol., 26, 273 (1987);
2-248485, JP-B-63-248390, JP-B 3-2
No. 77280, Japanese Unexamined Patent Publication No. Hei 3-277289, and Japanese Patent Publication No. 6-7975) have no patents on the production of glutaminase derived from Aspergillus oryzae and Aspergillus soya, and there are few reports (S. Yamamoto and H. Hirooka). :
J. Ferment. Technol., 52, (8), 564 (1974), T. Yano,
S. Ashida, T. Tachiki, H. Kumagai, and T. Tochikur
a: Agric. Biol. Chem., 55 (2), 379 (1991), Tatsuo Yamazaki, Kazuo Inamori, Kazuo Uchida: Shoken, 22, (1), 13 (1996)).

Glutaminases derived from Aspergillus oryzae and Aspergillus soya have low heat stability and salt tolerance. Therefore, in order to compensate for the glutaminase deficient during protein hydrolysis, it is necessary to add a glutaminase activity derived from a Bacillus genus or a protease preparation containing glutaminase having high glutaminase activity and excellent stability, yeast of the genus Cryptococcus, etc. (Y. shimizu, A. Ueyama, K. Goto: J. Brew. Soc.
Japan. 86, (6), 441 (1991), F. Harayama: Joukyo, 8
7, (7), 503 (1992), JP-B-60-173214, JP-B-6
No. 4-10957, Japanese Patent Application Laid-Open No. 1-108955, Japanese Patent Publication No. 1-10895
No. 5, Japanese Patent Publication No. 1-16465).

[0007]

SUMMARY OF THE INVENTION In order to obtain a protein hydrolyzate having a high nitrogen content and an amino acid content, particularly a high glutamic acid content and a strong taste, by allowing a culture of Aspergillus oryzae to act on a protein. Requires the production of a mutant strain of Aspergillus oryzae having both high protease activity and high glutaminase activity. Accordingly, an object of the present invention is to construct the mutant strain and to provide a method for producing a protein hydrolyzate using the mutant strain.

[0008]

The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, Aspergillus
Olyse was subjected to a mutation treatment to obtain a mutant showing high glutaminase activity even in liquid culture (hereinafter sometimes abbreviated as a high-activity mutant). This highly active mutant strain not only has a high glutaminase activity in liquid culture, but also retains a protease activity and a peptidase activity necessary for production of a protein hydrolyzate, and good culture properties. When the culture of this highly active mutant strain was allowed to act on a protein, it was found that a protein hydrolyzate having a high nitrogen content and amino acid content, particularly having a high glutamic acid content, having a tasty taste, was obtained, and the present invention was completed. did.

[0009] That is, the present invention relates to a highly active mutant belonging to Aspergillus oryzae, which has both high glutaminase activity and protease activity in liquid culture, and a culture of the high active mutant, which is subjected to salt-free or low-salt conditions. The present invention provides a method for producing a protein hydrolyzate having a high nitrogen content and a high glutamic acid content, wherein the protein hydrolyzate is made to act on a protein by using the same.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The highly active mutant strain of Aspergillus oryzae of the present invention can be obtained, for example, by allowing nitrosoguanidine, which has been conventionally used as a mutagen, to act on Aspergillus oryzae which produces various proteases. It can be obtained from a mutant strain having good culture properties, high glutaminase activity and high protease activity. However, it is not limited to this acquisition method, and other commonly used chemical substances such as hydroxylamine and ethyl methyl sulfonic acid as a mutagen, or irradiation with ultraviolet rays, radiation, X-rays, or no mutagenesis treatment It is also possible to obtain a mutant strain having the above properties by so-called spontaneous mutation or the like obtained in (1).

Specific examples of the highly active mutant of the present invention which can be obtained by the above method include Aspergillus oryzae AJ
There is a highly active mutant G (AJ117331) derived from 117290 as a parent strain. This bacterium was deposited at the National Institute of Bioscience and Human-Technology, National Institute of Advanced Industrial Science and Technology, and its accession number was F
ERM P-15956. The main bacteriological properties of this highly active mutant are not different from the parent strain. However, the glutaminase activity of the high activity mutant is about 6 times that of the parent strain. In addition, the protease activity of this highly active mutant is as high as that of the parent strain.

The culture of the highly active mutant strain of the present invention is a liquid culture of koji and contains not only glutaminase activity but also various degrading enzymes. Therefore, it is preferable to use the liquid culture as it is. Can be used by separating and purifying the required enzyme. That is, cells are separated from the liquid culture by centrifugation or filtration, etc.
The enzyme, which is a protein, can be precipitated from the cell lysate and culture solution by ordinary means, for example, salting out, isoelectric precipitation, or solvent, or concentrated by ultrafiltration to form an enzyme solution. it can. Further, purified products separated and collected by a usual purification method can be used alone or in combination.

The medium used in the liquid culture of the highly active mutant strain of the present invention may be any medium as long as the high active mutant strain can sufficiently grow, and is composed of a carbon source, a nitrogen source, an inorganic salt, a cofactor and the like. Normal medium may be used. As the carbon source, for example, soluble starch, wheat bran or wheat bran extract,
Glucose, sucrose, fructose, maltose, mannitol, erythritol, lactose, sorbose, galactose, linoleic acid, oleic acid, myristic acid, palmitic acid, and the like. Casein, peptone and the like; Further, as inorganic salts, potassium chloride, magnesium sulfate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, etc., and as auxiliary factors, oils such as corn steep liquor, rice oil, corn oil, etc .; Activators and the like.

The culture may be carried out by inoculating a highly active mutant strain into a medium and performing aeration culture according to a conventional method. Regarding the culture conditions, the pH is 4.0 to 9.0, preferably the pH is 5.0 to 8.0.
0, the temperature is suitably 20 to 40 ° C, preferably 27 to 33 ° C. The culture time is 10 to 96 hours, preferably 48 to 72 hours. A commercially available enzyme preparation, for example, an enzyme solution containing a protease, a cell wall degrading enzyme, glutaminase, or the like, or a purified enzyme preparation may be added to the thus obtained culture of the highly active mutant strain according to the purpose.

Next, 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, and further subjected to processing such as defatted soybean or puffing. Various proteins obtained, or proteins separated from these various protein raw materials may be used. The conditions under which the culture of the highly active mutant strain is allowed to act on the protein are as follows. For example, 0.2 to 50%, preferably 1 to 20%, of the raw material of the protein such as defatted soybean and isolated soybean protein can be highly active. Mix the strain cultures and mix 5-60
C., preferably at 30-50 C. for 6 hours to 15 days,
The reaction is preferably performed for 24 hours to 10 days. During the reaction, ethanol or salt may be added for preservative purposes. The protein hydrolysis reaction is carried out under salt-free or low-salt conditions, but is preferably carried out in the absence of salt. Note that the low salt condition means a condition in which the salt concentration is 5 (weight / volume)% or less. After completion of the reaction, unreacted insoluble substances such as raw material proteins and bacterial cells may be removed by a conventional separation method such as centrifugation or filtration. Glutamic acid release rate and amino acid release rate of each generated protein hydrolyzate can be determined by glutamic acid determination by enzymatic method, determination of free amino acid amount by amino acid analysis,
It can be obtained from the quantification of the total nitrogen amount. The protein hydrolyzate obtained by the present invention has a high nitrogen content and a high content of amino acids such as glutamic acid, and has a strong taste.

[0016]

EXAMPLES Hereinafter, the method for isolating a highly active mutant of the present invention and the production of a protein hydrolyzate will be described in detail with reference to examples, but the present invention is not limited thereto. The methods for measuring glutaminase activity and protease activity in the examples are as follows. Glutaminase activity is determined by a modified method of Hartman et al. (Hartman, SC: J. Biol. Chem., 2) for quantifying γ-glutamyl hydroxamic acid produced by an enzyme reaction in the presence of hydroxylamine.
43, 853-863 (1968)). Specifically, reagent A (0.4 M trisaminomethane, 0.2 M hydroxylamine hydrochloride (pH 7.0)) and reagent B (100
200 μl of the culture solution of the highly active mutant is added to 1 ml of an equal volume mixture of mM L-glutamine and 20 mM reduced glutathione (pH 7.0) and incubated at 37 ° C. for 1 hour. Next, (1) 3
N hydrochloric acid, (2) 12% trichloroacetic acid and (3) 5
% Ferric chloride hexahydrate in a 0.1 N hydrochloric acid solution, 1 ml of an equal mixture of each was added, and the mixture was stirred.
The enzyme activity was determined by measuring the absorbance at 525 nm of the reaction solution supernatant. Under the above conditions, 1 μm per minute
The enzymatic activity for producing mol γ-glutamyl hydroxamic acid was defined as 1 unit (U).

The measurement of protease activity is carried out by the usual Anson-Hagihara modified method (B. Hagihara et al .:
J. Biochem., 45, 185 (1958)). Specifically, 400 μl of a 0.75% casein solution and 0.24%
M Disodium hydrogen phosphate solution (pH 7.5) 100
μl was preincubated for 5 minutes at 37 ° C., and 10-100 μl of an enzyme solution (culture supernatant, etc.) was added thereto, and
Incubate at 7 ° C for 10 minutes. Next, (1) 0.1 M trichloroacetic acid, (2) 0.2 to stop the reaction.
A mixture of equal volumes of 2M sodium acetate and (3) 0.33M acetic acid was added, and 200 μl of the supernatant of this reaction solution was added to 500 μl of 0.55M sodium carbonate solution, and a commercially available phenol reagent diluted twice was added thereto. 100 μl was added, the mixture was immediately stirred, incubated at 30 ° C. for 30 minutes, and the absorbance at 660 nm of the reaction solution was measured to determine the enzyme activity. Under the above conditions, one minute
The activity of producing a non-protein substance exhibiting Folin coloration equivalent to μg tyrosine was defined as 1 unit (U).

Example 1 Cultured parent strain, Aspergillus oryzae AJ1172
A 2 mg / ml nitrosoguanidine solution was mixed with a spore suspension obtained from a PDA slant medium (commercially available potato-dextrose medium) of 90 strains (FERM P-14259) in an equal amount with the spore suspension, and the mixture was stirred at 30 ° C. Incubated for 30 minutes. This spore suspension is applied to a PDA medium,
After culturing at 30 ° C. for 4 to 7 days, colonies that grew on the surface of the medium were collected, and cultured at 30 ° C. for 4 to 7 days on a PDA slant medium. The 500 mutant strains thus obtained were each cultured with shaking at 30 ° C. for 64 hours in a liquid medium A shown in Table 1, and the glutaminase activity of the culture and the protease activity of the culture supernatant were measured. From these mutants, a mutant G showing stable high enzyme activity even after subculture was selected. Glutaminase activity and protease activity of the mutant strain G were compared with those of a parent strain and a commercially available seed koji, a seed koji isolate B (Aspergillus oryzae var.
Efusus ( A. oryzae var. Effusus ) AJ1173
32 strains (FERM P-15957), and A. sojae Ben-1 (FERM P-752), which was further sold by the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology.
2) and PFA-118 (FERM P-7524). These two strains are highly active strains obtained by haploid transformation of a diploid strain by cell fusion, and have high protease activity and glutaminase activity in soy sauce koji (Japanese Patent Publication No. 3-73271). Gazette). Table 2 shows the results.

As is evident from Table 2, the mutant strain G did not change the protease activity as compared to the parent strain, and the glutaminase activity was increased 6 times. Seed koji isolate B is a isolate from commercial koji and has the highest glutaminase activity in liquid culture, but has significantly lower protease activity. The two highly active strains of soy sauce koji both had a protease activity and a glutaminase activity that were clearly lower than the mutant G. The mutant G
Is named Aspergillus oryzae AJ117331 strain, and has been deposited with the National Institute of Advanced Industrial Science and Technology, as described above. The accession number is FERMP-
15956.

[0020]

[Table 1]

[0021]

[Table 2]

Example 2 The following operation was performed aseptically. Autoclave treatment (1
(21 ° C., 20 minutes)
1, the parent strain of Example 1 (FERM P-14259),
Mutant G (FERM P-15956), seed koji isolate B
(FERM P-15957) and 5 ml of each culture of the highly active strains FERM P-7522 and FERMP-7524 in soy sauce koji were added and mixed.
Allowed to react for days. Parent strain (FERM P-14259),
Mutant G (FERM P-15956), seed koji isolate B
(FERM P-15957) and FERM P-75
22, for each culture of FERM P-7524,
Further, a commercially available enzyme preparation "glutaminase Daiwa" (manufactured by Daiwa Kasei Co., Ltd.) was added at 0.01%, which is a necessary and sufficient amount of glutaminase, and the mixture was reacted in the same manner as a comparative control. For the supernatant of these reaction products, the amount of released amino acids was measured by total nitrogen and amino acid analysis. Table 3 shows the glutamate release rate (glutamic acid release amount (g / dl) / total nitrogen (g / dl)) determined from the amount of glutamic acid released and total nitrogen. Table 3 shows a system without the addition of "glutaminase Daiwa" which is a commercially available enzyme preparation. As shown in Table 3, the total nitrogen amount of the mutant G was the same as that of the parent strain, but the glutamate release rate of the mutant G was remarkably higher than that of the parent strain, the isolate koji isolate B and the highly active strain of soy sauce koji. It was a high value.

Next, the parent strain (FERM P-1425) was used.
9), mutant G (FERM P-15596), seed koji isolate B (FERM P-15957) and FERM P-7522, which are highly active strains in soy sauce koji, FERM
For each of P-7524, a commercially available enzyme preparation “Glutaminase Daiwa” was not added (−G) or added (+
G) glutamate release rate and the glutamate release rate ratio ((glutamic acid release rate (−
G) / Glutamate release rate (+ G)) × 100)
It is shown in the table. As shown in Table 4, highly active strains FERM P-7522, F in parent strain, seed koji isolate B and soy sauce koji
Glutamate release ratio of ERM P-7524 is low,
Glutaminase activity was insufficient. However, the glutamate release ratio of the mutant G was 100%, indicating that the glutaminase activity of the mutant G was sufficient even without the addition of the commercially available enzyme preparation “glutaminase Daiwa”. In addition, parent strain (FERM P-14259), mutant strain G (FERM P-15956), seed koji isolate B (F
Glutamic acid release rate (amino acid release amount (g / dl) / total nitrogen (g / dl)) determined from amino acid analysis of each decomposition solution of highly active strain FERM P-7522 in soy sauce koji and ERM P-15957. The results are shown in Table 5.
As shown in Table 5, the amino acid release rate of the lysate from the culture of the mutant strain G was glutamic acid and most amino acids other than glutamic acid were determined to be higher in the parent strain, the seed koji isolate B and the soy sauce koji. This was a higher value than that of 7522, and was a decomposition solution having a strong taste.

[0024]

[Table 3]

[0025]

[Table 4]

[0026]

[Table 5]

[0027]

EFFECTS OF THE INVENTION The highly active mutant of the present invention has a glutaminase activity about 6 times that of the parent strain in liquid culture and a high protease activity. A protein hydrolyzate obtained by allowing a culture of this highly active mutant strain to act on a protein has a high nitrogen content and an amino acid content, particularly a high glutamic acid content, and is advantageously used as a seasoning having a strong taste. Can be used.

──────────────────────────────────────────────────続 き Continued on front page (51) Int.Cl. ⁶ Identification symbol FI (C12N 1/15 C12R 1:69) (72) Inventor Jiro Kataoka 1-1 Suzukicho, Kawasaki-ku, Kawasaki-shi, Kanagawa Prefecture Ajinomoto Co., Inc. Inside the Food Research Institute

Claims (3)

[Claims] [Claim 1] Aspergillus oryzae (Aspergillu
soyzae ), a highly active mutant strain having both high glutaminase activity and high protease activity in liquid culture. 2. The mutant strain is Aspergillus oryzae A.
The highly active mutant strain according to claim 1, which is a J117331 strain (FERM P-15956). 3. A protein hydrolyzate having a high nitrogen content and a high glutamic acid content, wherein the culture of the highly active mutant strain according to claim 1 is allowed to act on a protein under salt-free or low-salt conditions. Manufacturing method.