JPH0337913B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for obtaining koji mold that produces a significant amount of enzyme and has a good growth rate. In general, koji mold plays an extremely important role in soy sauce brewing, and various improvements have been made to the koji mold strains used. Conventionally, mutation treatment such as ultraviolet irradiation has been generally adopted as a method for modifying Aspergillus oryzae. However, isolation of mutant strains through mutation treatments such as ultraviolet irradiation is highly random and requires a huge amount of effort for screening, resulting in extremely low efficiency. Furthermore, even if a mutant strain with excellent enzyme productivity was obtained by chance, it would immediately undergo reverse mutation or its growth rate would be significantly slow, making it impractical. Furthermore, if a mutant strain obtained by general mutation treatment is subjected to mutation treatment two or three times, its inferior growth rate will further deteriorate, making it far from practical use. The present inventor conducted research to find a breeding method for Aspergillus aspergillus that has high enzyme productivity, fast growth rate, and can be separated reliably and efficiently.
We have learned that if we carry out mutation treatment using an intermediate means of creating ploidy, we can reliably obtain mutant strains that meet these objectives. The present invention was completed based on this knowledge, and involves constructing a diploid strain using at least one of the parent strains using a highly enzyme-producing Aspergillus oryzae strain obtained through mutation, and further using the resulting diploid strain. This is a method for breeding Aspergillus oryzae, which is characterized by mutating and obtaining a more highly productive Aspergillus strain. In the present invention, alkaline protease, neutral protease,
Acid protease, leucine aminopeptidase, acid carboxypeptidase, glutaminase, amylase, cellulase, and pectin transeliminase are the targets, but it is extremely difficult to increase the titer of all of these enzymes at the same time. It is common to increase the potency of at least one or two to six substances at the same time. In the present invention, first, a strain with good enzyme productivity and growth is isolated from a commercially available Aspergillus seed mold, and then subjected to mutation treatment such as ultraviolet irradiation to isolate a strain with better enzyme productivity. do. Here, when targeting alkaline protease, 1.2
The productivity has increased by ~3 times, which is typical. However, this mutant strain usually has a slower growth rate and is more prone to reverse mutations. In the present invention, a diploid is constructed using the mutant strain obtained here as at least one parent strain. The other parent strain may be a mutant strain of the same strain, the original strain, or a different strain of the same species, but it is preferable to select one with a good growth rate. When constructing diploids, it is not necessary to specifically label the parent strains with auxotrophy, color change, etc., but if they are not labeled, later separation of heterokaryons and diploids will be difficult. This is not preferable because it becomes difficult to operate. Labeling of mutant strains with good enzyme productivity is mainly done by the following procedure. 1 Addition of auxotrophy The strain to which auxotrophy is to be added is heated at 30°C to 7°C.
UV irradiation is performed on the spore suspension obtained by culturing for 10 days. (Survival rate: 10 ^-2 -10 ^-3 ) Dilute to about 50 colonies per plate and apply the irradiated suspension of spores to a complete medium (rice malt juice, sugar content 10). Add an appropriate amount of sodium deoxycholate to this complete medium to prevent colonies from growing. After spores have settled on the koji mold colonies that have grown by culturing at 30° C. for 2 to 3 days, this plate is used as a master plate and replicas are made on a minimal medium (Zapek medium) and a complete medium according to a conventional method. Bacteria that grow on complete media but not on minimal media are auxotrophs. The required substance can be determined by the oxanograph method. An auxotrophic strain can also be effectively obtained by treatment with NTG (nitrosoguanidine) as a mutagen. 2. Imparting color change tone. Add auxotrophic strain (spore color is green) to
When UV treatment is performed, color mutants with spore colors such as white, yellow, and brown can be obtained at a frequency of 10 ^-2 -10 ^-3 . After confirming that the two properties of this double mutant (auxotrophy and spore color) are sufficiently stable, proceed to the next step. One parent plant treated in this manner is prepared, and a color mutant different from the other parent plant is prepared. Both of these strains can undergo anastomosis and propagate the anastomoses to obtain heterokaryons, which can then be propagated to naturally separate diploids from heterokaolins.
At that time, if you use discolored parent strains (white, yellow, or brown), the green one is diploid (it becomes green when it combines with the other parent strain), so it should be separated immediately. becomes possible. To describe specifically the anastomosis, the production of heterokaolin, and the production of diploids, spores of two types of double mutants (auxotrophic, discolored spores) are placed in anastomosis medium (sugar content 1.0, rice malt juice medium PH5.5). ) and culture at 23℃ for 7 days. Extract the spores from the area where they have grown thinly and put them on Czapetsk medium.
When cultured at 30°C for 3 days, heterokaryons will proliferate. Due to the proliferation of heterokaryons, the diploid population is 10 ^-5
It can be naturally produced and isolated with a frequency of ~ ^10-7 . Here, only green colonies are 2
Because it is a diploid, it is easily distinguished from white, yellow, and mixed heterooricans. In order to increase the frequency of diploid production, if UV irradiation is applied to reduce the survival rate to 5% or less, diploids can be obtained at a frequency of about 10 ^-2 . The diploid obtained here is further subjected to mutation treatments such as ultraviolet irradiation, and a strain with a high target enzyme titer is selectively isolated, resulting in an excellent koji mold with both a high enzyme titer and a high growth rate. Obtainable. One strain thus obtained, which is excellent in protease production and has a high growth rate, has been deposited with the Microtech Institute as Aspergillus sawya SD, FERM P No. 5956. Next, an experiment was conducted using the method described above.

[Table] Growth rate: Based on the giant colony diameter (mm) when cultured at 30℃ for 100 hours on an agar plate with rice malt juice (sugar content 10) prepared by a conventional method. Alkaline protease activity u/g〓Koji: 150ml
Take 5g of wheat in an Erlenmeyer flask and add 4ml of tap water.
was added, kneaded well, and sterilized by steaming in an autoclave for 30 minutes. One platinum loop of spores was inoculated into this medium and cultured at 30°C for 3 days to produce koji. A 10-fold amount of water was added and the enzyme was extracted at 5°C overnight. Protease activity was measured according to the Anson-Hagiwara method. u/g = Expressed in koji. Creation of diploids from H and M strains: By the method described above, H strains are separated from each other, and H strains and M
Diploids were constructed using three combinations of M strains and M strains. We will call them the HH strain, HM strain, and MM strain. The growth rate of these bacteria and the alkaline protease activity u/g of the koji are shown in Figure 1. With HH strain
MM strains have properties that are not much different from their parent strains,
The HM strain showed intermediate between the parents. Furthermore, we selected several strains from HH strain, HM strain, and MM strain and subjected them to UV irradiation in an attempt to isolate bacteria that produced a significant amount of alkaline protease. As a primary screening, the Helo ratio [casein lysis circle/colony diameter,
H.SEKINE, S.NASUNO &N.IGUCHI;
Agr. Biol. Chem., 33, 1477 (1969)] selected bacteria that were superior to the parent strain. As a result, we were able to isolate a significant amount of alkaline protease producing bacteria from the HM and MM strains.
It could not be isolated from the HH strain. (Figure 2) In particular, the growth of the bacteria that produce a large amount of alkaline protease derived from the HM strain is almost the same as that of the M strain.
Even when scaled up, it was able to fully demonstrate its capabilities. The survival rate of the above mutation is desirably 10 ^-3 or less, and with a survival rate of about 10 ^-2 (or less) in the case of a normal mutation, it was almost impossible to obtain a significant amount of alkaline protease-producing bacteria. Also, chemical mutating agents such as nitrous acid could be used in the same way as UV. When the M strain (haploid) was used as the parent strain and mutation and screening were carried out in the same manner, it was possible to obtain bacteria that produced a significant amount of alkaline protease. However, the efficiency was extremely poor compared to diploid strains (HM strain, MM strain (Figure 2)). (Figure 3) Furthermore, all bacteria with sufficiently high alkaline protease production Growth was considerably inferior, and when scaled up, it was not much different from the M strain, or in fact could only show lower values (see Figure 1).

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Claims (1)

[Scope of Claims] 1. A diploid strain is created by using at least one of the parent strains from a highly enzyme-producing Aspergillus strain obtained by mutation, and the resulting diploid strain is further mutated to create a more advanced strain. A method for breeding Aspergillus oryzae, characterized by obtaining a Aspergillus oryzae strain with significant enzyme production. 2 In Aspergillus oryzae, the enzymes that increase enzyme productivity are alkaline protease, neutral protease,
The method according to claim 1, wherein the enzyme is one or more selected from acid protease, leucine aminopeptidase, acid carboxypeptidase, glutaminase, amylase, cellulase, and pectin transeliminase.