JPS6075280A - Production of aspergillus by protoplast fusion - Google Patents

Abstract

PURPOSE:To obtain an aspergillus strain having high activity of produce protease and glutaminase, by preparing a protoplast fusion cell of a strain having high activity to produce protease and belonging to Aspergillus genus and a strain having high activity to produce glutaminase, and culturing the obtained fusion cell in a hypertonic regeneration medium. CONSTITUTION:A protoplast is obtained from a strain having high activity to produce protease and belonging to Aspergillus genus (e.g. Aspergillus sojae ATCC 42249) and a strain having high activity to produce glutaminase [e.g. Aspergillus sojae 262 (FERM-P No.2128)] by the use of a cell wall lyase (e.g. a combination of beta-1,3-glucanase and chitinase). The fused cell obtained by the protoplast fusion is cultured in a hypertonic regeenration medium (e.g. a medium obtained by adding agar to the soup of malted rice containing sorbitol), and an aspergillus strain capable of highly producing both protease and glutaminase is separated from the cultured product.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing Aspergillus oryzae having a high protease-producing ability and a high glutaminase-producing ability by protoplast fusion.

Soy sauce is a seasoning whose flavor is mainly derived from amino acids, so when brewing soy sauce, great consideration is given to improving the nitrogen utilization rate and increasing the amount of glutamic acid produced. It is extremely important for the brewing industry to obtain koji molds that produce potent protease and glutaminase.

Conventionally, one method of obtaining such excellent koji molds is to increase the enzyme production ability by artificial mutation, but the obtained mutant strain has at least a higher level of protease or glutaminase than the parent strain. On the other hand, in many cases the values were low, and it was difficult to obtain strains with both values higher than the parent strain.

Therefore, as a result of various studies aimed at obtaining Aspergillus aspergillus having high protease-producing ability and high glutaminase-producing ability, the present inventors have developed a high-protease-producing strain and a high-glutaminase-producing strain belonging to the genus Aspergillus. When protoplasts were fused and the fused cells were cultured in a hypertonic regeneration medium, the inventors discovered that Aspergillus oryzae having a high protease-producing ability and a high glutaminase-producing ability could be obtained from the fused cells, and based on this knowledge, they completed the present invention. .

That is, the present invention obtains protoplasts from cultured cells of a high protease-producing strain and a high glutaminase-producing strain belonging to the genus Aspergillus, fuses these with protoplasts, cultures the fused cells in a hypertonic regeneration medium, and extracts hypertonic cells from the culture. This is a method for producing Aspergillus oryzae, which is characterized by collecting Aspergillus oryzae having protease production ability and high glutaminase production ability.

The present invention will be explained in detail below.

First, the high protease-producing strain belonging to the genus Aspergillus used in the present invention may be any strain as long as it belongs to the genus Aspergillus and has a high protease-producing ability. It can be easily obtained by searching for a strain of koji mold with high protease production ability. A specific example is Aspergillus sojae ATCC42249, ATCC4
2250, 42251, etc., but variants or mutant strains of these can also be used.

In addition, the high glutaminase producing strain belonging to the genus Aspergillus may be any strain as long as it belongs to the genus Aspergillus and has a high glutaminase producing ability. can be obtained. As a specific example, Aspergillus sawyer 262 (FE几M
-P2128), 2165 (FB'RM P-7Zl
rO) and the like, but variants or mutant strains of these can also be used.

Next, cultured cells of the above production strain can be obtained by culturing conidia of the production strain in a nutrient medium.

The nutrient medium used here may be any medium as long as it contains the nutrients required by Aspergillus oryzae, and a synthetic medium, a semi-synthetic medium, or a natural medium may be used. As a synthetic medium, glucose, sucrose, etc. can be used as a carbon source, and ammonium sulfate can be used as a nitrogen source.1. Ammonium nitrate etc. are used. As a specific example, 0.5% of yeast extract and 0.2% of casamino acids are added to Czapck medium, and P H
Examples include a medium prepared at a concentration of 6.0 and diluted 10 times with water (hereinafter abbreviated as Czapek's modified medium). In addition, the composition of the natural medium includes cracked wheat, wrinkles, etc. as a carbon source, skim milk, defatted soybean flour, etc. as a nitrogen source, and appropriate inorganic salts such as phosphoric acid, potassium, magnesium, and calcium are added to the medium. Can be used as appropriate, and if necessary, amino acids necessary for bacterial growth,
Vitamins and the like can be added to the medium.

As for the culture method, use the above-mentioned suitable medium, such as Czapek's modified medium, and heat sterilize it in a conventional manner. Cultivate aerobically by standing, shaking, aerating, etc. for 5 to 15 hours, which is sufficient to double the size. In addition, when performing solid culture, use carbon sources such as wrinkles, wheat, nitrogen sources such as soybeans, and other organic or inorganic materials as raw materials, inoculate and mix the inoculum into a solid medium that has been sterilized by steaming, etc. It is preferable to culture aseptically at ~38°C for a time sufficient to grow the germinated spores to a length of 5 to 15 times the original spore outer diameter.

In the present invention, the length of germinated spores and the outer diameter of 5
It is important to use cultured cells that have grown ~15 times. That is, when it is less than 5 times, it is hardly possible to form protoplasts of Aspergillus oryzae, and on the other hand, when it exceeds 15 times, the formation rate of protoplasts of Aspergillus oryzae becomes extremely low, which is not preferable.

Next, in order to obtain protoplasts from the cultured cells thus obtained, for each of the above germinated spores, 106 to 10
11 il of germinated spore suspension with a concentration of 8 spores/child (hypertonic solution)
After mixing equal amounts of each prepared suspension and collecting the bacteria by centrifugation, either add a cell wall lytic enzyme that has been sterilized in advance and treat it, or treat each germinated spore suspension. After collecting bacteria by centrifugation, this is first treated by adding a follicle wall lytic enzyme, and then equal amounts of each treatment solution are mixed.

The cell wall lytic enzyme used here may be any enzyme as long as it has the activity of lysing the cell wall of Aspergillus oryzae, and cellulase, β-1,3-glucanase, chitinase, etc. may be used alone or in combination, or in general. Mixed enzymes used as cell wall lytic enzymes can be used. As a result of experiments conducted by the present inventors, it was found that the combination of β-1,3-glucanase and chitinase among these enzymes significantly increases the protoplast formation rate of Aspergillus oryzae. is preferred.

As the chitinase, an enzyme agent having chitinase activity is used, such as Chitinase J and 100466. Chitinase 75C-6 manufactured by Sigma, USA
137, Chitinase Fanga sold by Seikagaku Corporation/'
(fun g aI ) IFh 100290 and the like.

In addition, as β-1,3-glucanase, Zymolyase 5000 manufactured by Kirin Brewery,
60,000 and the microorganisms Bacillus circulans IAM1165 and Streptomyces 0
143 (FEBMP-7, 27J), and the like, and the latter two crude enzymes are particularly preferred.

The crude enzyme prepared from Bacillus circulans IAM 1165 and the crude enzyme prepared from Streptomyces strain 0143 were obtained as follows.

[Engineering] Preparation of crude enzyme from Hachilas Circulans IAMII65 1% of meat extract, 1% of polypeptone, 2% of wet bacterial cells of Aspergillus oryzae and 1/2 of a medium (pH 7,2) of water was placed in a 51-volume Erlenmeyer flask and heated under high pressure. Pre-cultivate in the same medium as above except that the wet cells of Aspergillus oryzae are not added to the sterilized medium (48°C at 30°C).
Time) Hachilas Circulans IAM 116
5 was inoculated with 10 m of the inoculum solution, and cultured with shaking (170r, p, rll) at 30°C for 48 hours. To obtain the crude enzyme from the culture solution obtained in this way, centrifuge it and
The H solution was separated and obtained, ammonium sulfate was added thereto and salted out by a conventional method, and the resulting silt fraction was collected. This was dissolved in 25M water, placed in a cellophane Cheap, and dialyzed against distilled water overnight at 5°C. The original crude enzyme) was obtained.

[I'l Crude enzyme preparation method from Streptomyces o143 carcasses Medium composition 1 i<l-l2P 040.2% Yeast extract o, o59g KO,l O,05% Glucose 0.25% Mg504j 7 l-1200,1 %neopeptone
0.1% NaC10.05% NaN0. , 0.1% Aspergillus wet cells 2.1% Distilled water 1.0! PH5,0 Streptomyces 0143 (F
10 copies of the inoculum solution of E (MP-727A) were inoculated and cultured with shaking (170 r, p, nl) at 30°G until the P II of the culture solution rose to 7.0 (7 days). Hereinafter, the crude enzyme (crude enzyme) was obtained from the culture solution in exactly the same manner as the above-mentioned method for obtaining crude enzyme from the culture solution of Bacillus circulans.
Hereinafter referred to as "crude enzyme of Streptomyces origin") was obtained.

The two crude enzymes obtained above were tested for β-1,3-glucanase activity using β-1,3-glucan as a substrate by a conventional method, and it was confirmed that they strongly degraded the substrate. It was found that it could be used as β-1,3-glucanase.

Both enzymes mentioned above still act on the agar plate medium containing the washed and homogenized wet cells of Aspergillus oryzae and form a transparent halo, so both of the crude enzymes dissolve the cell wall of the Aspergillus cells. This was also confirmed.

Next, conditions for treating germinated spores of Aspergillus oryzae with a cell wall lytic enzyme will be explained. In other words, it is preferable that the cell wall lytic enzyme used be at a concentration sufficient to dissolve the cell wall of the germinating spores of Aspergillus oryzae and obtain a complete protoplast; for example, in the case of a crude enzyme derived from Bacillus circulans. , 10
~50 mg/ml, preferably 25-35+++
g/d. If chitinase is also used, the concentration used is 1-10 mg/m, preferably 3-5 mg/m. The enzyme treatment time must be sufficient to dissolve the cell wall of the koji mold and obtain protoplasts, and is usually preferably 30 minutes to 6 hours. Also, the pH is 6.0-7.
0, especially 6.5 is preferred.

Next, after the enzyme treatment is completed, a washing solution for retaining bacterial cells, such as 0.7-1.5M sorbitol or 0.5-1.5M sorbitol, is added.
The bacterial cells are washed with a 1.0 M potassium chloride solution or the like to obtain protoplasts in which the cell wall of Aspergillus oryzae has been dissolved and removed.

Next, the fusion treatment of the protoplasts obtained here and the regeneration of the fused protoplasts are performed, for example, by the method of Annp et al. (J, Gen, Microbi.

1. 92, 413 (1976)'].

That is, 0.7-1.5M sorbitol, 5-10Q
15-33% polyethylene glycol 6000 (P
H7, 5), add and mix the washed protoplasts of the high protease producing strain and the high glutaminase producing strain obtained above, and process at 20 to 30°C for 15 to 45 minutes to obtain protoplast fused cells. I can do it.

Next, the protoplast-fused cells obtained in this way are regenerated using a hypertonic regeneration medium that can regenerate the cell wall while preserving the protoplasts, such as rice malt juice (or malt) containing 0.7 to 1.5 M sorbitol. Hypertonic regenerated agar medium (pH 6.5) containing 2% Sokadai (soup) and 0.7~
1.5M sorbitol in Czapek liquid medium with 2% agar
The protoplast fused cells obtained above were transferred to a hypertonic minimal regeneration agar medium (P)I6.5) containing
This is carried out by culturing at ~35°C for 2 to 10 days.

In this way, the protocylast fused cells regenerate a cell wall around them and grow on the medium to form a regenerated colony.

Next, from the regenerated colonies obtained in this way, protoplast fusion cells are selected in advance by washing the protoplasts of the parent strain, the high protease producing strain, and the parent strain, the high glutaminase producing strain, in the same manner as for the fused cells. By keeping track of the morphological properties (hue, color, brightness, light σ, shape, size, etc.) and physiological properties of the resulting regenerated colonies, It is possible to select and collect +Ij live colonies that have characteristics different from those of the above.

In addition, the conidia of the parent strains of the high protease producing strain and the high glutaminase producing strain were subjected to artificial mutation treatment (irradiation with X-rays, ultraviolet rays, etc., chemical mutation treatment with nitrosoguanidine, ethylmethylsulfite, etc.). , the characteristic color and various nutritional requirements of the conidia of the parent strain, such as amino acid requirements such as alanine and methionine, biotin,
A mutant strain with requirements for nicotinic acid, para-aminobenzoic acid + 'i2, etc. is obtained, and this mutant strain is used as a parent strain and subjected to enzyme treatment and protoplast fusion treatment in the same manner as above, and then regenerated to form a regenerated colony. Certain mutant strains have a characteristic color, and protoplast fusion cells that do not have this color can be clearly distinguished and collected. Protoplasts, which are still auxotrophic mutants, cannot grow unless a specific nutrient source is present during regeneration, so protoplast fusion cells that can grow even in the absence of a specific nutrient source can be easily distinguished and collected. . By attaching markers such as color and auxotrophy to the biparental method in this way, the separation operation of the desired fused cells becomes extremely easy.

Mutant strain 22-9 of the high protease producing strain obtained above
1 (FERP-7λ77), and as a mutant strain of a high glutaminase producing strain, for example, Aspergillus sojae M12-2-61 (FE )LM P-72za/), etc.

Next, the protoplast fusion strain thus regenerated in a hypertonic regeneration medium is often a heterokaryon that easily reverts back to the parent strain and is therefore unstable. Therefore, we used the method of Koide et al. (Agric, Biol, C.
By applying ultraviolet irradiation (distance: 39 m, 1 to 5 minutes) and assaying according to the method described in Hem. .

Example t C. Aspergillus sojae D-78 is a stable diploid strain with high protease-producing ability and high glutaminase-producing ability obtained in this way, and is affiliated with the National Institute of Microbiology, Agency of Industrial Science and Technology. It has been deposited as Tsuta No. 72.

The advantages of the invention are as follows. Isolation of mutant strains by mutation treatments such as ultraviolet irradiation, which have been commonly used in the past, is highly random and requires a huge amount of effort for screening, making it extremely inefficient, and it is still difficult to obtain mutant strains that happen to have excellent enzyme productivity. The biggest drawback of this method for obtaining mutant strains is that even if the obtained mutant strain has a higher level of protease or glutaminase than the parent strain, the other is often lower than the parent strain; It was difficult to do so.

In contrast, according to the present invention, Aspergillus oryzae having a high protease-producing ability and a high glutaminase-producing ability can be efficiently collected and produced.

In addition, when the koji mold obtained in the present invention is inoculated and cultured in soy sauce koji raw material to make koji, and this koji is used to prepare and brew soy sauce, the soy sauce raw material is well decomposed and the nitrogen utilization rate is about 3~3.
Not only is it improved by 10%, but the glutamic acid content is also significantly increased, resulting in a soy sauce with extremely strong flavor, which is an industrially groundbreaking effect. (Refer to the results in Experimental Example 1 and Table 1), the protoplast formation rate of Aspergillus aspergillus is low.
This has been a major obstacle in research using blast fusion technology, but by using germinated spores that are 5 to 15 times longer than the original spore outer diameter as cultured cells, we can significantly increase the protoplast formation rate of Aspergillus oryzae. According to the method of the present invention, the desired koji mold cells can be efficiently obtained. (Refer to the results in Table 2 of Experimental Example 2) The effects of the present invention will be described below with reference to experimental examples.

Experimental Example 1 The Aspergillus aspergillus D-78 (FEI (MP-7279)), which has a high protease production ability and a high glutaminase production ability obtained in Example 2 below, was roasted and steamed with 300 g of defatted soybeans. A soy sauce raw material consisting of 300 g of crushed wheat was inoculated, and koji was made in a koji pot according to the current soy sauce koji manufacturing method.The resulting koji was poured into 1200 m of 25% salt water, and fermentation was controlled using conventional methods. was fermented and aged at 30°C for 3 months.As a control method, aged soy sauce moromi was obtained in the same manner using both methods.Soyamoto (abbreviated as 'rN) , glutamic acid (Q)
u), Qlu/TN and nitrogen utilization were measured. The requests are shown in Table 1.

Table 1 Experimental Example 2 Example 1 was performed except that in Example 1 described later, germinated spores with various lengths of germinated spores were used as cultured cells.
In exactly the same manner as above, glotosolists of Aspergillus oryzae were obtained and their formation rate was investigated.

The results are shown in Table 2.

Table 2 Note 1 = Results used Aspergillus sawjae 2165 bacteria Note 2: Protoplast formation rate indicates the ratio of the number of protoplasts to the number of germinated spores.

Note 3: The number of germinated spores and prodobocysts was determined using a phase-contrast optical microscope (manufactured by Kon Co., Ltd.).

Example 1 High protease producing strain (Aspergillus sojae AT
CC42251) and high glutaminase producing strain [Aspergillus sojae 2165 (FERM P-72♂
Conidia were collected from rice malt juice slant medium that had been cultured for 66 hours at 30°C.
01% sorbitan fatty acid ester solution [Sorgen TW-
60 (manufactured by Daiichi Kogyo Seiyaku Co., Ltd.)" to prepare a spore suspension of 1 to 5 x 10 spores/d.

Next, this spore suspension 1d was added to Czapek modified medium 3Q.
ml and inoculated at 30°C in a 150 ml Erlenmeyer flask.
The cells were cultured with shaking for 12 hours to obtain a suspension of germinated spores (cultured cells) whose length and outer diameter were 10 times the original spore outer diameter.

The germinated spore suspension was then centrifuged (4500 g, 20
Collect the germinated spores, wash thoroughly with water, drain the water, mix with 1 ml of cell wall lytic enzyme, and shake gently (50 to 60 times per minute) at 27°C for 2 hours. After enzyme treatment, Aspergillus oryzae noprotoplasts were obtained.

The cell wall lytic enzyme used here was obtained by dissolving a crude enzyme originating from Bacillus circilians and a commercially available chitinase (manufactured by ICN, USA) at a ratio of 30 mg and 1 g of 5II to 1 g of solution, respectively.

Next, the protoplasts thus obtained were separated using a G-3 standard glass filter, washed several times with hypertonic solution (0.8M sorbitol solution), and the washed protoplasts obtained were transferred to hypertonic solution 1d. , suspend.

Next, the hypertonic solutions in which protoplasts were suspended were mixed and centrifuged at 20°C (700g, 15 minutes).
A pellet of mixed protoplasts was collected. This is 0.
8M sorbitol, 10mM Ca1l, and 50mM
20% polyethylene glycol 6000 containing glycine
(PH7.5) solution was mixed with 1 ml of the solution, and a protoplast fusion reaction was performed at 25°C for 30 minutes to obtain protoplast fused cells.

Next, the protoplast fused cells are regenerated by diluting the fused cells with a hypertonic solution to an appropriate cell concentration, pouring this into a petri dish in advance, and hardening it on a hypertonic regeneration agar medium (containing 0.8 M sorbitol at a sugar concentration of 8 degrees Celsius). rice malt juice agar medium (agar 2%
)], and then pour the same hypertonic regeneration agar medium as above except for 0.5% agar over it, solidify it, and incubate at 30°C.
The cells were cultured for 2 to 5 days, and regenerated colonies of protoplast fused cells were obtained.

Next, from among the regenerated colonies obtained here, the colonies formed by the protoplasts of the biparental method were divided into 4 forms y4.
A regenerated colo possessing this was selected, and a protoplast-fused regenerated strain was collected from it.Then, the collected protoplast-fused regenerated strain was inoculated into rice shunjitsu slant medium and incubated at 30°C for 4 hours.
Conidia were obtained by culturing for 1 day, and this spore suspension was irradiated with ultraviolet rays for 1 minute from a distance of 39 G. The obtained spores were applied to a rice malt juice plate medium and cultured at 30°C for 4 days. Among the colonies, Aspergillus sojae D-24 (FEB, M P-7 7t
)] was collected.

The enzyme production ability of the obtained Invention 1 bacterial cells and their parent methods was investigated.

The results are shown in Table 43.

Table 3 Enzyme production ability of Aspergillus Aspergillus Example 2 Russ Sawyer W2-91 (FERM P-7,277)
Aspergillus sawya M12-2-61 (FEBM P-721), which has yellow conidia and a nicotinic acid requirement, is used as a b1 high glutaminase producing strain.
7), protoplast fusion regeneration samples were collected in exactly the same manner as in Example 1, except that hypertonic minimal regeneration agar medium (Zapek agar medium containing 0.8 M sorbitol) was used as the hypertonic regeneration medium for glotoblast fusion cells. .

Yoneji then inoculated the regenerated strain obtained here into a koji juice slant medium and cultured it at 30°C for 4 days to obtain a heterokaryon strain with green, yellow, and white spores. The spores were irradiated with ultraviolet rays for 1 minute from a distance of 39 bacteria, and the obtained spores were again applied to the minimal agar plate medium.
After culturing at 0° C. for 4 days, Aspergillus aspergillus cells forming stable colonies consisting of only green spores were obtained. Among these koji mold cells, one strain, Aspergillus sojae D-, has a high grotease production ability and a high glutaminase production ability.
After obtaining 78 (FERM P-7, 27F), the enzyme production ability of the Aspergillus aspergillus of the present invention thus obtained and its parent methods was investigated.

The results are shown in Table 4.

Table 4 Enzyme production capacity of N bacteria Note 1): The protease activity in Table 4 was determined by using defatted soybeans 301 and roasted cracked wheat 30f'jt as raw materials in a petri dish with 15 scratches in diameter, and using normal soy sauce koji. Koji was made in the same manner as in the production method, and the obtained koji was extracted for 2 hours using 10 times the amount of water, and the activity of producing 1 μmol of tyrosine in 9 hours was expressed as 1 unit. The glutaminase activity was determined by reacting the solution obtained by homogenizing the extracted residual blue gold with L-glutamine by shaking at -30° C. for 1 hour, and after filtration, measuring the amount of glutamic acid in solution F. Glutaminase activity is
The activity of producing 1 μmol of glutamic acid per minute per 12 pieces of koji was expressed as one unit. 'Table 1 and 2
From the results in the table, it is found that strains with high protease have low glutaminase activity, and conversely, strains with high glutaminase activity have low protease activity, which is a characteristic of Aspergillus aspergillus. It can be seen that Aspergillus Aspergillus M having high glutaminase activity can be obtained.

Patent applicant: Kikkoman Corporation

Claims (2)

[Claims] (1) Obtain grotoplasts from cultured cells of a high protease-producing strain and a high glutaminase-producing strain belonging to the genus Aspergillus, fuse these with protoplasts, culture the fused cells in a hypertonic regeneration medium, and produce high protease from the culture. A method for producing Aspergillus oryzae, characterized by collecting Aspergillus oryzae having the ability to produce high glutaminase, (2) The production method according to claim 1, wherein the cultured cells are germinated spores whose length is 5 to 15 times the original spore outer diameter.