JPS63287480A - Production of mold protoplast - Google Patents

Abstract

PURPOSE:To efficiently form a mold having chitosan in a cell wall into a protoplast, by using a cell wall lytic enzyme chitosanase. CONSTITUTION:A mold belonging to the genus Rhizopus, Mucor, Mortierella, etc., or the genus Trichoderma, etc., of the order Moriliales is cultivated in a culture medium to form spores, which are then transplanted into a liquid culture medium to collect just germinated young spores. Mycelia are washed and subsequently suspended in a buffer solution and previously prepared cell wall lytic enzyme containing chitosanase produced by Bacillus pumilus (FERM P-8814) is added in an optional order and gently shaken at 15-30 deg.C, preferably 20-25 deg.C for 1-8hr, preferably 3-7hr to form a protoplast. beta-1,3-Glucanase, cellulase, chitinase, etc., are used alone or in combination of two or more thereof as the cell wall lytic enzyme other than the chitosanase.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing filamentous fungal protoplasts, and more particularly to a method for converting filamentous fungi into protoplasts using chitosanase.

Filamentous fungi, along with yeast, have been used for a long time in the production of foods, medicines, etc. by fermentation methods, but there is an increasing demand for breeding them into industrially useful strains.

[Conventional technology]

To this end, it is possible to use genetic recombination techniques and cell fusion techniques, and converting filamentous fungi into protoplasts is an important issue.

Conventionally, methods for producing filamentous fungal protoplasts include β-1,
3-Method using glucanase, chitinase and cellulase (JP-A No. 60-75281, No. 60-1644)
77, No. 61-7318) is known.

[Problem that the invention seeks to solve]

However, unlike bacteria such as Escherichia coli, filamentous fungi have tough cell walls, so it is not easy to lyse them. Many reports have been made so far regarding microorganisms that produce enzymes involved in the cell walls of filamentous fungi, but none have yet been fully satisfactory. In particular, when forming protoplasts of filamentous fungi having chitosan in their cell walls, enzymes originating from the genus Trichoderma or Streptomyces or commercially available cellulases are commonly used.

When cell wall lytic enzymes such as chitinase and β-1,3-glucanase are used, the rate of protoplast formation is extremely low, and this is a major obstacle to the development of protoplast fusion technology and genetic recombination technology for filamentous fungi. Ta.

The present invention relates to a method for producing filamentous fungal protoplasts, which aims to efficiently increase protoplast formation of filamentous fungi using chitosanase.

[Means for solving problems]

The present inventors investigated the protoplast formation of filamentous fungi that have chitosan in their cell walls, and found that by using the cell wall lytic enzyme chitosanase, the cell wall can be efficiently removed under mild conditions to achieve the desired protoplast formation. They discovered this and completed the present invention.

That is, the present invention relates to a method for producing filamentous fungal protoplasts, which is characterized by using chitosanase in converting filamentous fungi having chitosan in their cell walls into protoplasts.

As the chitosanase used in the present invention, any chitosanase derived from a microorganism that produces chitosanase can be used regardless of its origin. Furthermore, the microorganisms may originate from variants or mutant strains of these microorganisms. For example,
There is a chitosanase described in Japanese Patent No. 1-207780. As chitosanase, the culture solution of the enzyme-producing bacteria or its culture filtrate may be used, and furthermore, the filtrate may be dialyzed after salting out ammonium sulfate, or the crude enzyme obtained by adding a solvent such as ethanol or acetone. Various forms can be used, such as a powder, a solution of the powder in water or a buffer solution, and an active fraction fractionated by gel filtration, ion exchange, or the like.

Next, the filamentous fungi may be those that have chitosan in their cell walls, such as Mucorales (Mucorales).
) are filamentous fungi that belong to the order, specifically Rhizopus spp.
Examples include filamentous fungi belonging to the genus Mucor, genus Mortierella, etc., and filamentous fungi belonging to the order Moriliales, such as filamentous fungi belonging to the genus Trichoderma.

Protoplastization of filamentous fungi according to the present invention involves culturing filamentous fungi on a suitable medium, such as a solid medium or an agar medium, to form spores, transplanting the spores into a liquid medium, and collecting young mycelia that have just germinated. .

After washing the hyphae, they are suspended in a buffer solution, a cell wall lytic enzyme containing chitosanase prepared in advance is added thereto in any order, and the mixture is incubated at 15-30°C, preferably 20-25°C for 1-8 hours, preferably 3 This is done by gentle shaking for ~7 hours. Here, any known cell wall lytic enzymes other than chitosanase can be used, such as β
Enzymes such as -1,3-glucanase, cellulase, and chitinase can be used alone or in combination of two or more together with chitosanase. Note that the origin of these enzymes does not matter.

〔Example〕

Next, the present invention will be explained in detail with reference to examples.

Example 1 Mortierella Isabelina
isaberina) I F O8187 was cultured at 25°C for one week on MY agar medium (consisting of 3 g of yeast extract, 5 g of polypeptone, 3 g of malt extract, 10 g of glucose, 11 g of water and 20 g of agar) to form spores. Physiological saline was added to this to prepare a spore suspension, and this suspension was mixed into CBS-M u medium (consisting of 20 g of glucose, 3 g of meat extract, 10 g of polypeptone, 0.5 g of potassium phosphate, and 11 g of water). Inoculate the
Young hyphae immediately after germination were obtained by culturing with shaking at 30°C for 16 hours.

The mycelium was collected by centrifugation (3000 rpm, 15 minutes), washed twice with 0.01M sodium phosphate buffer (pH 7.0) containing 0.4M sorbitol, and then added to the same buffer with a wet bacterial weight of 50 μm. The suspension was suspended so that the concentration was 11/11. On this mycelial suspension, Bacillus pamilus
us) BN-262 (FERM P-
Chitosanase crude enzyme powder (Patent Application No. 61-207780) produced by Chitosanase 8814) 2, OU/ml. Chitinase (manufactured by Sigma) 21U/ll11, β-glucuronidase (manufactured by Sigma) 2,0000/mj! , Finecellzyme (manufactured by Yakult) was added at a concentration of 6 U/mβ, and while shaking gently in a 23°C water bath,
Samples were taken over time and observed under a microscope. Protoplasts began to appear 1 hour after the start of the reaction and reached the maximum level after 3 hours. The number of protoplasts at that time is I
It was 10' pieces/ml. Also, chitinase.

When β-glucuronidase and chitosanase were used in combination, the number was 4×10′/lal. On the other hand, when only chitinase including chitosanase, β-glucuronidase, and fine cellzyme were used, the number of protoplasts was 4 x 10'/lal;
For β-glucuronidase alone, the concentration was 2×10′ cells/lsl (see Table 1).

The method for preparing the chitosanase crude enzyme powder used in this example is as follows:
It was carried out as follows. Bacillus pamilus BN-2
62 (FERM P-8814) was centrifuged (6000 rpm) to remove bacterial cells and obtain a culture supernatant. Ammonium sulfate was added to this to make it 80% saturated, and the resulting salting-out precipitate was thoroughly dialyzed with a cellophane tube and freeze-dried to obtain a crude enzyme powder.

Example 2 Mucor Musedo (Mucor 肚蒀ωATCC9836
was cultured on MY agar medium (same as in Example 1) at 25°C for one week to form spores.

Next, physiological saline was added to this to prepare a spore suspension, this spore suspension was inoculated into CBS-Mu medium (same as in Example 1), and cultured with shaking at 28°C for 18 hours. Young mycelia were obtained immediately after germination. Centrifugation (3000 rpm, 1
After washing twice with 0.1 M acetate buffer (pH 5, 6) containing 0.5 M sorbitol (for 5 minutes), add the mycelium to the same buffer at a wet bacterial weight of 100 μ/all. Suspended.

Bacillus pamilus BN-262 (
FERM P-8814) chitosanase crude enzyme solution 2
．． OU/+al, chitinase (manufactured by Sigma) 21U/m
1. β-glucuronidase (manufactured by Sigma) 2.00
0U/+11. Fine Cellzyme (manufactured by Yakult) 6
The sample was taken out over time while gently shaking in a water bath at 23° C., and observed under a microscope.

Protoplasts begin to appear 1 hour after the start of the reaction, and 3
．． The maximum was reached after 5 hours. The number of protoplasts at that time was 6×10′/vsl. Also, chitinase,
When β-glucuronidase and chitosanase were used in combination, the concentration was 2×10 h/ml. On the other hand, chitinases include chitosanase.

When using only β-glucuronidase and fine cellzyme, the number of protoplasts is 8 x 10'/ll11
In the case of only chitinase and β-glucuronidase, it was 2×10' pieces/l111 (see Table 1).

The chitosanase crude enzyme solution used in the Zero Example was prepared as follows. Bacillus pamilus BN-262
(FEPN P-8814) was centrifuged to remove bacterial cells and obtain a culture supernatant. This was fractionated using SP-)Yopearl 650M and Toyopearl HW-55 to obtain an active fraction (crude enzyme solution).

Example 3 Trichoderma sp, K
A spore suspension was prepared by the method of Example 2 using I-29 (ATCC 32086). This spore suspension was converted into CBS
-Mu medium (same as Example 1) was inoculated and cultured with shaking at 28°C for 40 hours to obtain young mycelium immediately after germination.

Centrifugation 11iii (300, Orpm, 15 minutes)
Collect the mycelia and add 0.1
After washing twice with M acetate buffer (pH 5, 6), the cells were suspended in the same buffer at a wet weight of 100 μ/lan.

This mycelial suspension contains chitosanase and chitinase.

Example 1 of β-glucuronidase and finecellzyme
Protoplast formation was carried out at the same concentration of 1 reaction site. The number of protoplasts obtained reached a maximum of 7 after 4 hours.
It became X10' pieces/ll11.

On the other hand, if chitosanase is not included, lXl0' pieces/l
11 (see Table 1).

Example 4 Rhizopus japonicus stool is l
Protoplast formation was performed using the method of Example 3 using 35 (ATCC 24863). The number of protoplasts obtained reached a maximum of 1×10S/rsll after 4 hours.

On the other hand, if chitosanase is not included, 2XIO' pieces/m
1 (see Table 1).

〔Effect of the invention〕

According to the present invention, filamentous fungi having chitosan in their cell walls can be efficiently converted into protoplasts. Therefore, the method of the present invention is useful for breeding filamentous fungi that are used industrially in the fields of food, medicine, etc.

Claims (3)

[Claims] (1) A method for producing filamentous fungal protoplasts, which comprises using chitosanase in converting filamentous fungi having chitosan in the cell wall into protoplasts. (2) Filamentous fungi that have chitosan in their cell walls are Mucorales (
Order Mucorales or Mori
2. The method according to claim 1, wherein the method belongs to the order A. liales). (3) Chitosanase is produced by Bacillus pamylus (￥Baci)
llus pumilus￥)BN-262(FERM
8814).