JPH0371877B2 - - Google Patents

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 can produce powerful protease and glutaminase. In general, the protease production capacity of koji mold for soy sauce is 208
Unit/g koji, and glutaminase production ability is
It is known that 3.19 units/g of koji (Shoken)
Vol. 7, No. 4, 1981, p 166-172 "Differences in brewing characteristics depending on the classification of koji mold for soy sauce", p 169 "Table 4 (differences in enzymatic power of soy sauce koji)"). One method of obtaining superior koji molds is to increase enzyme production ability through artificial mutation, but the obtained mutant strain may have higher levels of protease or glutaminase than the parent strain, but conversely the other may be lower. In many cases,
In both cases, it was difficult to obtain strains that were 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. protoplast fusion,
When the fused cells were cultured in a hypertonic regeneration medium, it was found that Aspergillus oryzae having a high protease-producing ability and a high glutaminase-producing ability could be obtained therefrom, and based on this knowledge, the present invention was completed. That is, the present invention obtains protoplasts from cultured cells of a high protease producing strain of 210 units/g Koji or more and a high glutaminase producing strain of 3.2 units/g Koji or more belonging to the genus Aspergillus, and fuses these with protoplasts. The fused cells are cultured in a hypertonic regeneration medium, and the culture has a high protease production ability of 300 units/g or more of koji and
This is a method for producing koji mold, which is characterized by collecting koji mold having a high glutaminase production ability of 1.0 units/g koji or more. The present invention will be explained in detail below. First, the strain belonging to the genus Aspergillus used in the present invention and producing a high protease of 210 units/g or more of koji may be any strain as long as it belongs to the genus Aspergillus and has the above-mentioned protease production ability. It can be easily obtained by searching for strains with high protease production ability from among the brewing koji molds used in the production of miso, sake, etc. A specific example is Aspergillus
Examples include Sawyer ATCC 42249, ATCC 42250, and 42251, but variants or mutant strains of these can also be used. Also, 3.2 units/g belonging to the Aspergillus genus
As a strain producing glutaminase higher than that of koji, any strain belonging to the genus Aspergillus and having the above-mentioned glutaminase production ability may be used. can be obtained. Specific examples include Aspergillus sawyer 262 (FERM-P2128) and Aspergillus sawyer 2165 (FERM-P2128).
P-7280), 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, sutucarose, etc. are used as a carbon source, and ammonium sulfate, ammonium nitrate, etc. are used as a nitrogen source. A specific example is Czapek medium, which is prepared by adding 0.5% yeast extract and 0.2% casamino acids to a pH of 6.0 and then diluted 10 times with water (hereinafter abbreviated as Czapek modified medium). can be mentioned. Further, the composition of the natural medium includes cracked wheat, etc. as a carbon source, and skim milk, defatted soybean flour, etc. as a nitrogen source. In addition, appropriate inorganic salts such as phosphoric acid, potassium, magnesium, and calcium can be appropriately used in the above medium, and amino acids, vitamins, etc. necessary for the growth of bacteria can be added to the medium as necessary. can. As for the culture method, pure cultured conidia of Aspergillus oryzae are inoculated into the above-mentioned suitable medium, such as Czapetz modified medium, which has been heat sterilized by a conventional method. Leave it still, shake it for 5 to 15 hours, which is enough for the spores to grow to 5 to 15 times the outer diameter of the original spores (the sum of the length of the mycelium extending from the spores). Cultivate in an aerobic manner, such as by aerobic cultivation. In addition, when performing fixed culture, carbon sources such as wheat, nitrogen sources such as soybeans,
Inoculum is inoculated and mixed into a solid medium that has been sterilized by steaming or other sterilization using other organic or inorganic materials as raw materials, and the germinated spore length is 5 to 15 times the original spore outer diameter. It is preferable to culture aseptically for a sufficient period of time to ensure proper growth. In the present invention, it is important to use cultured cells in which the length of germinated spores has grown to 5 to 15 times the outer diameter of the original spores. That is, when the amount is less than 5 times, almost no protoplasts of Aspergillus oryzae can be formed,
On the other hand, if it exceeds 15 times, the formation rate of protoplasts of Aspergillus oryzae becomes low, which is not preferable. Next, to obtain protoplasts from the cultured cells thus obtained, a germinated spore suspension with a concentration of 10 ⁶ to ^{10 8} cells/ml (hypertonic solution) is prepared for each of the above-mentioned germinated spores, and After mixing equal amounts of the suspension and centrifuging to collect the bacteria, either add a cell wall lytic enzyme that has been previously sterilized and treat the suspension, or centrifuge each germinated spore suspension to collect the bacteria. Thereafter, this is first treated by adding a cell 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, quinatase, etc. may be used alone or in combination, or in general. Mixed enzymes used as cell wall lytic enzymes can be used. Experiments conducted by the present inventors revealed that among these enzymes, β
It has been found that the combination of -1,3-glucanase and chitinase significantly increases the protoplast formation rate of Aspergillus oryzae, so it is preferable to use the above two enzymes together. As the chitinase, an enzyme agent having chitinase activity is used, such as Chitinase No. 100466 manufactured by ICN, USA, Chitinase No. 100466, manufactured by Sigma Corporation, USA.
C-6137, Chitinase Fungal No. 100290 sold by Seikagaku Kogyo Co., Ltd., and the like. In addition, as β-1,3-glucanase, Zymolyase manufactured by Kirin Brewery Co., Ltd.
5000, 60000, and two crude enzymes prepared by the present inventors from the respective culture solutions of the microorganisms Pachirus circulans IAM1165 and Streptomyces 0143 (FERM P-7276). Enzymes are preferred. The crude enzyme prepared from Bacillus circulans IAM1165 and the crude enzyme prepared from Streptomyces strain 0143 were obtained as follows. [] Crude enzyme preparation method from Pacillus circulans IAM1165 Place 1 medium (PH7.2) consisting of 1% meat extract, 1% polypeptone, 2% wet bacterial cells of Aspergillus and water in a 5-volume Erlenmeyer flask and sterilize under high pressure. Add 10 ml of the inoculum solution of Pacillus circulans IAM1165, which was precultured (48 hours at 30°C) in the same medium as above, except that the wet cells of Aspergillus oryzae were not added.
was inoculated and incubated at 30°C for 48 hours with shaking (170 r.p.
m.) did. To obtain the crude enzyme from the culture solution obtained in this way, centrifuge it to separate and obtain the supernatant, add ammonium sulfate to this and salt out using a conventional method.
The produced precipitate fraction was collected. Dissolve this in 25ml of water, put it in a cellophane tube, and keep it at 5℃ overnight.
The resulting dialyzed fluid was lyophilized and purified with crude enzyme (hereinafter referred to as "Bacillus Bacillus").
circulans-derived crude enzyme) was obtained. [] Crude enzyme preparation method from Streptomyces 0143 Medium composition 1 KH ₂ PO ₄ 0.2% Yeast extract 0.05% Kc1 0.05% Glucose 0.25% MgSO ₄・7H ₂ O 0.1% Neopeptone 0.1% NaC1 0.05% NaNO ₃ 0.1% Aspergillus oryzae Wet bacterial cells 2.1% Distilled water 1.0 PH 5.0 Streptomyces precultured in medium 1 (PH5.0) with the above composition and the same composition as this.
0143 (FERM-P-7276) was inoculated with 10 ml of inoculum solution and kept at 30℃ until the pH of the culture solution rose to 7.0 (7.
(days) and shaking culture (170 r.pm). Hereinafter, a crude enzyme (hereinafter referred to as "crude enzyme originating from Streptomyces") was obtained from the culture solution in exactly the same manner as the method for obtaining the crude enzyme from the culture solution of Pacillus circulans. The two crude enzymes obtained above are β-1,
Using 3-glucan as a substrate, β-1,
As a result of examining the presence or absence of 3-glucanase activity, it was confirmed that it strongly degrades substrates, and β-1,3-
It was found that it can be used as a glucanase. In addition, since both of the above crude enzymes act on the agar plate medium containing the washed and homogenized wet cells of Aspergillus oryzae and form a transparent halo, both of the crude enzymes dissolve the cell wall of the Aspergillus oryzae cells. This was also confirmed. Next, conditions for treating germinated spores of Aspergillus oryzae with a cell wall lytic enzyme will be explained. That is, the concentration of the cell wall lytic enzyme used is preferably 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, the concentration is 10 to 50 mg/ ml, preferably 25-35 mg/ml. When chitinase is used in combination, the concentration used is 1 to 10 mg/ml, preferably 3 to 5 mg/ml. The enzyme treatment time needs to be long enough to dissolve the cell wall of the koji mold and obtain protoplasts, and is usually 30 minutes to 6 minutes.
time is preferable. Further, the pH is preferably 6.0 to 7.0, particularly 6.5. Next, after the enzyme treatment is completed, wash with a washing solution that protects the bacterial cells, such as 0.7-1.5M sorbitol or
The cells are washed with a 0.5-1.0M potassium chloride solution, etc., to obtain protoplasts in which the cell wall of Aspergillus aspergillus is dissolved and removed. Next, the fusion treatment of the protoplasts obtained here and the regeneration of the fused protoplasts are carried out by, for example,
The method of Ann′e et al. [J.Gen.Microbiol., 92 , 413
(1976)]. i.e. 0.7-1.5M. Sorbitol, 5-100mM.
The washed protoplasts of the high protease producing strain and the high glutaminase producing strain obtained above were added and mixed in 15 to 33% polyethylene glycol 6000 (PH7.5) containing CaC ₂ and 30 to 80 mM glycine, and then mixed for 20 to 30 minutes. Protoplast fusion cells can be obtained by treatment at 30°C for 15 to 45 minutes. The protoplast-fused cells thus obtained are then regenerated using a hypertonic regeneration medium capable of regenerating the cell wall while protecting the protoplasts, e.g.
A hypertonic regenerated agar medium (PH6.5) containing 2% agar in rice koji juice (or wort) containing 0.7 to 1.5M sorbitol, and 2% agar in a Czapek liquid medium containing 0.7 to 1.5M sorbitol. The protoplast fusion cells obtained above are transferred to a hypertonic minimally regenerating agar medium (PH6.5) containing the culture medium and cultured at 25° C. to 35° C. for 2 to 10 days. In this way, the protoplast fused cell regenerates a cell wall around it, grows on the medium, and forms 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. to play,
By understanding the morphological properties (hue, color, brightness, gloss, shape, size, etc.) and physiological properties of the obtained regenerated colonies, we can identify the characteristics that are different from those. As a regenerating colony,
Can be selectively collected. 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, ethyl methyl sulfate, etc.). , we obtained mutant strains that gave the conidia of the parent strain a characteristic color and various nutritional requirements, such as amino acid requirements such as alanine and methionine, and requirements for biotin, nicotinic acid, and para-aminobenzoic acid. When the parent strain is subjected to enzyme treatment and protoplast fusion treatment in the same manner as above, and then regenerated to form a regenerated colony, the mutant strain that is the parent strain has a characteristic color, and protoplast fusion cells that do not have this color can be clearly identified. Can be distinguished and collected. Additionally, protoplasts of 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 parent strains in this way, it becomes extremely easy to separate the desired fused cells. Examples of mutant strains of the high protease producing strain obtained above include Aspergillus sawjae W2-91 (FERM P-7277), which has white conidia and is endowed with a para-aminobenzoic acid requirement; Examples of mutant strains of the production strain include Aspergillus sojae M12-2-61, which has yellow conidia and is endowed with nicotinic acid auxotrophs.
(FERM P-7281), etc. Next, protoplast fusion strains regenerated in this hypertonic regeneration medium are often heterokaryons that easily return to the parent strain and are therefore unstable. Therefore, conidia of these heterokaryon strains were treated according to the method of Oda et al. [Agric.Biol.Chem., 27 , 758 (1963)].
By applying ultraviolet irradiation (distance 39 cm, 1 to 5 minutes) and assaying, stable diploid, ie, the desired koji mold cells can be obtained. For example, Aspergillus sojae D-78 is a stable diploid strain that has a high protease-producing ability and a high glutaminase-producing ability, and was submitted to the Institute of Microbiological Technology, Agency of Industrial Science and Technology. Deposited as No. 7279. 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, requires a huge amount of labor for screening, and is extremely inefficient.
Moreover, even if mutant strains with excellent enzyme productivity were obtained by chance, many of them immediately underwent reverse mutation and could not be put to practical use. The biggest drawback of the method of obtaining mutant strains through mutation treatment is that even though the obtained mutant strain has a higher level of protease or glutaminase than the parent strain,
On the other hand, it was often low, and it was difficult to obtain strains that were higher than the parent strain in both cases. In contrast, according to the present invention, Aspergillus oryzae having high protease-producing ability and 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 into 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 approximately 3 to 10%. Not only does this improve the glutaric acid content, but it also significantly increases the amount of glutaric acid, producing a soy sauce with an extremely strong flavor, which is an industrially groundbreaking effect. (Refer to the results in Experimental Example 1 and Table 1) In addition, several breeding methods for soy sauce koji mold and brewing koji mold using protoplast fusion have been known, but the protoplast formation rate of koji mold is low, and the protoplast fusion technology is not suitable. However, by using germinated spores with a length 5 to 15 times the original spore outer diameter as cultured cells, the rate of protoplast formation of Aspergillus aspergillus can be significantly increased. According to the method of the present invention, the desired koji mold cells can be obtained efficiently. (See the results in Table 2 of Experimental Example 2) Hereinafter, the effects of the present invention will be explained with reference to experimental examples. Experimental Example 1 Koji mold Aspergillus sojae D-78 (FERM P-7279) having high protease production ability and high gluminase production ability obtained in Example 2 below
was inoculated into a soy sauce raw material consisting of 300 g of defatted soybeans and 300 g of roasted cracked wheat, and made into koji using a koji lid according to the usual soy sauce koji manufacturing method.
The moromi fermentation is controlled by the usual method, and the fermentation temperature is 30°C.
Fermented and aged for a month. In addition, as a symmetrical method, aged soy sauce moromi was similarly obtained using both parent strains. Total nitrogen (abbreviated as TN), glutamic acid (Glu), Glu/TN, and nitrogen utilization rate were measured for the moromi soup thus obtained. The results are shown in Table 1.

[Table] Experimental Example 2 In Example 1 described later, protoplasts of Aspergillus oryzae were obtained in exactly the same manner as in Example 1, except that germinated spores with various lengths were used as cultured cells, and their formation rate was determined. Examined. The results are shown in Table 2.

【table】

[Table] Example 1 High protease producing strain (Aspergillus sojae ATCC42251) and high glutaminase producing strain [Aspergillus sojae 2165 (FERM P-
7280)] was cultured for 66 hours at 30°C, conidia were collected from rice malt juice slant medium, and each was added to a 0.01% sorbitan fatty acid ester solution "Sorgen TW-60 (manufactured by Daiichi Kogyo Seiyaku Co., Ltd.)". dispersed,
A spore suspension of 1 to 5×10 ⁷ cells/ml was prepared. Next, 1 ml of this spore suspension was inoculated into 30 ml of Czapek modified medium, and 30 ml of this spore suspension was inoculated in a 150 ml Erlenmeyer flask.
The cells were cultured with shaking at ℃ for 12 hours to obtain a suspension of germinated spores (cultured cells) in which the length of the germinated spores was 10 times the outer diameter of the original spores. Then, the germinated spore suspension was centrifuged (4500g,
20 minutes), collect the germinated spores, wash them thoroughly with water, drain the water, mix with 1 ml of cell wall lytic enzyme, and gently shake at 27°C for 2 hours (50 to 60 times per minute).
The cells were then treated with enzymes to obtain protoplasts of Aspergillus oryzae. The cell wall lytic enzyme used here was obtained by dissolving a crude enzyme originating from Bacillus circulans and a commercially available chitinase (manufactured by ICN, USA) at a ratio of 30 mg and 5 mg in 1 ml of solution, respectively. Next, the protoplasts obtained in this way were separated using a G-3 standard glass filter, and washed several times with a hypertonic solution (0.8M sorbitol solution).
Transfer to and 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 was mixed with 1 ml of 20% polyethylene glycol 6000 (PH 7.5) solution containing 0.8 M sorbitol, 10 mM CaCl ₂ C ₂ and 50 mM glycine and heated at 25°C for 30 min.
A minute protoplast fusion reaction was performed to obtain protoplast fused cells. Next, the protoplast fused cells are regenerated using a hypertonic regeneration agar medium [containing 0.8 M sorbitol and a sugar concentration of 8°C] that has been diluted to an appropriate cell concentration with a hypertonic solution and poured in advance into a shear dish. Rice malt juice agar medium (2% agar)] was plated with the same hypertonic regeneration agar medium as above except for 0.5% agar, solidified and overlaid, and cultured at 30°C for 2 to 5 days to induce protoplast fusion. A regenerated colony of cells was obtained. Next, from among the regenerated colonies obtained here,
A regenerated colony with traits different from those formed by the protoplasts of the parent strains was selected, and a protoplast-fused regenerated strain was collected from it. Next, the collected protoplast fusion regenerated strain was inoculated into rice malt juice slant medium and cultured at 30°C for 4 days to obtain conidia, and this spore suspension was irradiated with ultraviolet rays for 1 minute from a distance of 39 cm. The resulting spores were applied to a rice malt juice plate medium and cultured at 30°C for 4 days. Among the resulting colonies, Aspergillus aspergillus cells having high protease activity and high glutaminase activity, which is the object of the present invention, were selected. Aspergillus sawya D-24
(FERM P-7278)] was collected. The enzyme production ability of the obtained Aspergillus oryzae cells of the present invention and their parent strains was investigated. The results are shown in Table 3.

[Table] Example 2 Aspergillus sojae W2-91 (FERM P-7277), which has white conidia and is endowed with para-aminobenzoic acid requirement, as a high protease producing strain.
As a high glutaminase producing strain, Aspergillus sojae M12-2-61 (FERM P-) with yellow conidia and nicotinic acid requirement was used.
7281), and a protoplast fusion regenerated strain was collected in exactly the same manner as in Example 1, except that a hypertonic minimal regeneration agar medium [Tsapek agar medium containing 0.8 M sorbitol] was used as the hypertonic regeneration medium for the protoplast fused cells. Next, the regenerated strain obtained here was inoculated into a rice malt juice slant medium and cultured at 30°C for 4 days to obtain a telokaryon strain with green, yellow, and white spores. The spores were irradiated with ultraviolet light for 1 minute from a distance of 39 cm, the obtained spores were again applied to the minimal agar plate medium, and cultured at 30°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 protease production ability and a high glutaminase production ability.
-78 (FERM P-7279) was obtained. Next, the enzyme production ability of the Aspergillus oryzae cells of the present invention and their parent strains thus obtained was investigated. The results are shown in Table 4.

【table】

[Table] From the results in Tables 1 and 2, strains with high protease have low glutaminase activity, and conversely, strains with high glutaminase activity have low protease activity. According to the invented method, protease activity and glutaminase activity are respectively 300 units/g or more and 1.0 units/g.
It can be seen that a high level of koji mold can be obtained, which is higher than that of koji.

Claims (1)

[Scope of Claims] 1 Protoplasts are obtained from cultured cells of a high protease producing strain of 210 units/g koji or more and a high glutaminase producing strain of 3.2 units/g koji or more belonging to the genus Aspergillus, and these are fused with protoplasts. , the fused cells are cultured in a hypertonic regeneration medium, and the koji mold having a high protease production ability of 300 units/g or more and a high glutaminase production ability of 1.0 units/g or more of koji is collected from the culture. A method for producing koji mold. 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 external shape.