JPH0322148B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing filamentous protoplasts and aims to significantly increase the formation rate of filamentous fungal protoplasts. In recent years, with the development of tissue culture science, two types of cultured cells are treated with cell wall lytic enzymes so that they can come into contact with each other to obtain cells (protoplasts) with their cell walls removed, and these are fused under appropriate conditions. ,
Subsequently, a technology was developed to obtain new cells by regenerating the cell wall around the fused cells, and in the brewed food industry, several methods of breeding soy sauce koji mold by protoplast fusion and breeding methods of brewing koji mold were known. There is. However, when forming protoplasts of Aspergillus oryzae, the rate of protoplast formation is extremely low when conventional enzymes derived from the genus Trichoderma or cell wall lytic enzymes such as commercially available cellulases are used. This has been a major obstacle to the development of protoplast fusion technology. Therefore, the present inventors conducted various studies on methods for obtaining protoplasts with a high formation rate, and found that
Conidia of fungi belonging to filamentous fungi are cultured in a liquid nutrient medium to obtain germinated spores whose length is 5 to 15 times the outer diameter of the original spore (at the start of culture), which is then transformed into β
It was found that enzymatic treatment with cell wall lytic enzymes including -1,3-glucanase and chitinase significantly increases the formation rate of filamentous fungal protoplasts, and based on this knowledge, the present invention was completed. That is, the present invention cultivates filamentous fungal spores in a liquid nutrient medium to obtain germinated spores with a length of 5 to 15 times the original spore outer diameter, which is then treated with β-1,3-glucanase. This is a method for producing filamentous fungal protoplasts, which is characterized by enzymatic treatment with a cell wall lytic enzyme including chitinase. The present invention will be explained in detail below. First, the filamentous fungal spores used in the present invention include, for example, aspergillus for brewing used in the production of soy sauce, miso, sake, etc., as well as filamentous fungi used in the production of enzyme preparations for general reagents and pharmaceuticals. Examples include conidia of bacterial strains. Specific examples thereof include Aspergillus, Rhizopus, Mucor, and Penicillium. Next, the nutrient medium for liquid culture of filamentous fungi may be any medium as long as it contains the nutrient source required by the strain belonging to the filamentous fungi, and a synthetic medium, a semi-synthetic medium, or a natural medium may be used. As a synthetic medium, crucose, satucarose, etc. are used as a carbon source, and ammonium sulfate, ammonium nitrate, etc. are used as a nitrogen source. As a specific example, 0.5% yeast extract and 0.2% casamino acids are added to Czapek medium, and the PH
6.0. In addition, the composition of the natural medium includes cracked wheat as a carbon source, etc.
Examples of nitrogen sources include skim milk and defatted soybean flour. 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. Wear. As for the culture method, pure culture was carried out in the above-mentioned appropriate medium, such as Suapetsuk medium to which 0.5% yeast extract and 0.2% casamino acid were added, the pH was adjusted to around 6.0, and the medium was heat sterilized by a conventional method. Conidia of filamentous fungi are inoculated, and most of the germinated spores (combined length of spores and hyphae) are 5 to 15 times longer than the outer diameter of the original spores (at the start of culture) at 15 to 35℃. Cultivation is carried out aerobically by standing, shaking, aerobic cultivation, etc. for a sufficient period of time to double the size, for example, 5 to 15 hours in the case of Aspergillus belonging to the genus Aspergillus. In the present invention, it is extremely important to use germinated spores that have grown to a length 5 to 15 times the original spore outer diameter. That is, when it is less than 5 times, it is hardly possible to form protoplasts of filamentous fungi, and on the other hand, when it exceeds 15 times, the formation rate of protoplasts of filamentous fungi becomes extremely low, which is not preferable. Next, in order to obtain protoplasts from the germinated spores of filamentous fungi thus obtained, the germinated spores are
When reacting cell wall lytic enzymes containing 1,3-glucanase and chitinase, it is extremely important to use β-1,3-glucanase and chitinase together.
When each is used alone, it is not possible to increase the protoplast formation rate of filamentous fungi. As the chitinase, an enzyme agent having chitinase activity is used, such as chitinase No. 100466 manufactured by ICN, USA, chitinase manufactured by Sigma Corporation, USA,
No. C-6137, Chitinase Fungal No. 100290 sold by Seikagaku Corporation, 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 Bacillus circulans IAM1165 and Sputomyces 0143 (FERM P-7276). preferable. These enzymes may be used alone or in combination. The crude enzyme prepared from Bacillus circulans IAM1165 and the crude enzyme prepared from Streptomyces strain 0143 were obtained as follows. [] Method for preparing crude enzyme from Bacillus circulans IAM1165 A medium (PH7.2) consisting of 1% meat extract, 1% polypeptone, 2% wet bacterial cells of Aspergillus oryzae and water (PH7.2) was placed in a 5-volume Erlenmeyer flask and sterilized under high pressure. was inoculated with 10 ml of the inoculum of Bacillus circulans IAM1165, which had been precultured (48 hours at 30°C) in the same medium as above, except that no wet bacterial cells were added, and cultured with shaking (170r) at 30°C for 48 hours. .pm)
did. To obtain the crude enzyme from the culture solution obtained in this way, centrifuge it and separate and obtain the supernatant.
Ammonium sulfate was added to this and salting out was carried out by a conventional method, and the resulting precipitate fraction was collected. This was dissolved in 25 ml of water, placed in a cellophane tube, and dialyzed against distilled water at 5° C. overnight. The resulting dialyzed solution was freeze-dried to obtain a crude enzyme. [] Crude enzyme preparation method from Streptomyces 0143 Medium composition 1 KH ₂ PO ₄ 0.2% Yeast extract 0.05% KCl 0.05% Glucose 0.25% MgSO ₄・7H ₂ O 0.1% Neopeptone 0.1% NaCl 0.05% NaNO ₃ 0.1% Aspergillus oryzae Wet bacterial cells 2.1% Distilled water 1.0 PH 5.0 Streptomyces 0143 precultured in medium 1 (PH5.0) with the above composition and the same composition as this.
(FERM P-7276) inoculated with 10 ml of inoculum solution, 30
The culture was carried out with shaking (170 rpm) at ℃ until the pH of the culture solution rose to 7.0 (7 days). Thereafter, crude enzyme was obtained from the culture solution in exactly the same manner as the method for obtaining crude enzyme from the culture solution of Bacillus circulans described above. 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, the two crude enzymes act on the agar plate medium containing the washed and homogenized koji mold cells,
It was also confirmed that all of the crude enzymes lysed the cell walls of filamentous fungi, as they formed transparent halos. Next, we will explain the conditions for treating germinated spores of filamentous fungi with a cell wall lytic enzyme. In other words, the concentration of cell wall lytic enzyme used is a concentration sufficient to lyse the cell wall of germinated spores of filamentous fungi and obtain complete protoplasts. For example, in the case of a crude enzyme derived from Bacillus circulans, it is preferably 10 to 100 mg/ml, preferably 25 to 50 mg/ml.
mg/ml. The concentration of chitinase used in combination is 1 to 10 mg/ml, preferably 3 to 5 mg/ml. The enzyme treatment time dissolves the cell wall of filamentous fungi,
It is necessary to allow sufficient time to obtain protoplasts, and usually 30 minutes to 6 hours is preferable. Also PH
is preferably 6.0 to 7.0, particularly preferably 6.5. As is clear from the above description, according to the present invention, the formation rate of fungal protoplasts can be significantly increased. Therefore, the present invention has the effect of greatly contributing to the development and advancement of protoplast fusion technology for filamentous fungi. Hereinafter, the present invention will be explained in detail by showing examples. Example 1 The filamentous fungi shown in Table 1 below were each inoculated into a slant medium (rice malt juice agar medium) in a test tube, and cultured at room temperature until sufficient conidial adhesion occurred. I got it. 1 x 10 ⁷ conidia/ml of each of the following conidia.
It was dispersed and suspended in a 0.01% sorbitan fatty acid ester solution "Sorgen TW-60 (manufactured by Dai-ichi Kogyo Seiyaku Co., Ltd.)" so that Next, 1 ml of this dispersed suspension of conidia was added to a liquid nutrient medium [Zapek medium with 0.5% yeast extract and 0.2% casamino acids to adjust the pH to 6.0, and this was diluted 10 times with water. ] Inoculate 30ml,
In a 150 ml Erlenmeyer flask, culture with shaking at 30°C for a sufficient period of time until the length of the germinated spores becomes about 10 times the original spore outer diameter to obtain each germinated spore suspension. The suspension was further centrifuged (4500 g, 20
minutes) and germinated spores were isolated from there. Next, the germinated spores thus obtained were thoroughly washed with water, and 1 ml of cell wall lysing enzyme solution was added thereto.
Enzyme treatment was performed by gently shaking (50 to 60 cycles per minute) at 27°C for 2 hours to obtain protoplasts of filamentous fungi. The cell wall lytic enzymes used here were a crude enzyme derived from Bacillus circulans IAM1165 and a commercially available chitinase (manufactured by ICN, USA), each at 30% volume per 1 ml.
It was obtained by dissolving in the ratio of mg and 5 mg and aseptically treating it. When the protoplast formation rate of the filamentous fungi obtained was investigated, the results shown in Table 1 were obtained.

【table】

[Table] Example 2 Aspergis sojae as a test bacterial strain
Protoplasts of Aspergillus oryzae were obtained by enzymatic treatment in exactly the same manner as in Example 1, except that ATCC42251 was used and the enzyme listed in Table 2 below was used as the cell wall lytic enzyme. The results are shown in Table 2.

【table】

[Table] From the results in Table 2, it can be seen that when forming koji mold protoplasts, the cellulase agents that were conventionally used,
The formation rate of the crude enzyme originating from Trichoderma harzianum is extremely low, and the crude enzyme originating from Pacillus circulans and the crude enzyme originating from Streptomyces, both of which have high β-1,3-glucanase activity, have a high formation rate when used alone. Although the formation rate is low at less than 50%, it can be seen that when chitinase is used in combination with these crude enzymes, the formation rate increases significantly to over 70%. Example 3 In the above Example 1, Aspergillus sojae 2165 (FERM P-7280) was used instead of Aspergillus sojae ATCC42251, and the lengths of the germinated spores to be subjected to enzyme treatment are listed in Table 3 below. Protoplasts of Aspergillus oryzae were obtained in exactly the same manner as in Example 1, except that the same procedure was used as in Example 1. The results are shown in Table 3.

[Table] From this result, the protoplast formation rate is extremely low when the length of the germinated spore is less than 5 times the original spore outer diameter, and becomes lower again when the length exceeds 15 times the original spore outer diameter.
On the other hand, it can be seen that the protoplast formation rate of Aspergillus aspergillus becomes significantly higher at 5 to 15 times the magnification.

Claims (1)

[Claims] 1. Cultivate filamentous fungal spores in a liquid nutrient medium to obtain germinated spores whose length is 5 to 15 times the original spore outer diameter, which are then subjected to cell wall lysis containing β-1,3-glucanase and chitinase. A method for producing filamentous fungal protoplasts, which is characterized by enzymatic treatment with an enzyme.