JPS6024710B2 - Method for producing cellulase - Google Patents

Description

[Detailed description of the invention] The present invention relates to a method for producing cellulase using basidiomycetes.

Cellulase has been widely used as an enzyme that decomposes fibers in fields such as the food industry, feed, sewage treatment, and medicine.

Therefore, the present inventors conducted a wide range of searches for cellulase-producing bacteria from the natural world in order to discover and extract cellulase-producing microorganisms that exhibit strong enzymatic activity against plant fibers. Copri
The present invention was completed based on the knowledge that basidiomycetes belonging to the genus N.

That is, the present invention is a method for producing cellulase, which comprises inoculating a cellulose-containing medium with a strain belonging to the genus Coprinus and having cellulase-producing ability, culturing the cellulase, and collecting cellulase from the culture.

The present invention will be explained in detail below. First, the bacteria used in the present invention may be any bacteria as long as they belong to the genus Coprinus and have cellulase production facilities, and may also be variants or mutant strains of these bacteria.

Specific examples of bacteria belonging to the genus Coprinus and having cellulase-producing ability include Coprinus cinereus (C.
oprin female cmereus) KK-37M, etc.

The mycological properties of Cobrinus cinereus 37M are as shown below.

In addition, the mycological properties are generally ``The F plate g N B (Cha
pter23)” Ale ShigeruunderH. Smith (A
academicpress. 1973) and "Standard Primary Color Encyclopedia Complete Works 14 Fungi" co-authored by Mutsuya Imazeki, Tsuguo Hongo, and Keisuke Tsubaki (Korisha Publishing, 1977). Mycological properties of Coprinus cinereus KK-37M Growth status in each tower site (all cultured at 25°C) ■ Active growth on malt extract agar tower site on the 7th day, the diameter of the colony was 65 x 65 It is white, soft, and fluffy.

On the 14th day, the seeds grew all over the Petri dish (85 x 85 side).
The center is circular with a diameter of about 5 snouts and has fluff-like hyphae, and the surrounding area is covered with extremely short hyphae.

■ Potato/glucose agar After 7 days of growth, the colonies are about 83 x 83 candles (diameter), and are white, soft, and have an extremely dense fluff-like appearance.

On the 14th day, it was 85 x 85 legs in diameter, 2 to 3 sides thick, white hair-like mycelium was growing densely, and brown 0.1 cm was growing around the periphery of the colony.
Forms lateral sclerotia.

■ Growth on the Abec agar medium on day 7 was poor, with thin mycelia extending to about 83 x 83 sides, but no colony formation was observed.

On the 14th day, there is almost no change in the growth of mycelia, but brown sclerotia of about 0.1 rib size are sparsely formed on the entire surface of the chalet.

■ On the 7th day of Sabouraud's agar tower, a small amount of growth was observed, and the colonies were about 45 x 45 sides, white and soft, and long-haired hyphae formed in slightly coarse clusters.

On the 14th day, the flower showed medium growth (75 x 75 ribs), the mycelium was white and slightly long, the center was swollen, the petals were stacked as a whole, and the underside was yellow.

■ On the 7th day of YpSs agar plating, extremely vigorous growth was observed, and the colonies were about 83 x 83 sides, white, fluffy, and fluffy, with brown 0.05 mm in size in the medium.
Forms many sclerotia about the size of one rib.

On the 14th day, it grew on the entire surface of the Petri dish (85 x 85 skin), the mycelia were grayish and fluffy, sclerotia were formed on the entire surface, the underside was brown, and the surface of the colony was white for the formation of fruiting bodies. The primordium of 1 is formed to a solid degree.

'2) Physiological properties ■ Optimum growth conditions PH; 6.0-9.0 Temperature: 25-40q0 ■ Growth range PH; 4.5-10.5 Temperature; 20-45°C Next, the mycological conditions described above Regarding the taxonomic position of Coprinus cinereus KK-37M, which has
ngi WB (Chapter 23)” Ale Shigeru and
erH. Smith (Academicpress.1
973) and "Standard Primary Color Illustrated Works 14 Fungi" co-authored by Rokuya Imazeki, Tsuguo Hongo, and Keisuke Tsubaki (Korisha Publishing, 1977), this strain was compared with the classification, and it was found that this strain has a scallop-like protrusion on the septum of the hyphae. However, the fruiting bodies formed on the YpSe medium are long in shape when young and covered with white hairs that fall off easily, the folds are acidic on the stem and have a blade-like appearance, and the spores have a cap when mature. The strain becomes lobed along the liquefied streaks overnight, and the spore print is blackish-purple-brown, with large lateral cystidia on the cap, indicating that the strain belongs to the genus Coprinus.

Furthermore, the stem of the fruiting body of this strain is covered with silky scales,
The base of the stem is swollen and slightly curled upwards, and each basidia contains 4 spores, and the spores germinate in an oval shape (6 to 7 x 9 to 11 buds). It is determined to be Coprinus cinereus because it has holes. The above-mentioned umbrella gradually changes from cream color to dark purple when young, and turns blackish-purple when mature, and the optimum temperature for growth is 3.
From the fact that fruiting bodies were actively formed between 7 and 40 oo, especially around 370, this strain was identified as a new strain of Coprinus cinereus, and this strain was named Coprinus cinereus KK-37M.

The above-mentioned Coprinus cinereus KK-37M was submitted to the National Institute of Microbiology, Agency of Industrial Science and Technology, with the National Institute of Industrial Science and Technology Research Institute No. 6072 (
FERMP-6072).

Next, the soil used for cellulase production in the present invention includes, for example, oak paper, Avicel, absorbent cotton, sawdust,
It is essential to contain cellulose such as okara, rice straw, soy sauce oak, coffee grounds, fallen raw husks, etc., and if necessary, polysaccharides such as starch, monosaccharides and oligosaccharides, beptone as a nitrogen source, meat A medium containing extracts, cornstarch liquor, yeast, ammonium salts, organic or inorganic nitrogen compounds such as urea, other inorganic salts such as phosphates, magnesium salts, iron salts, vitamins, and growth-promoting substances is used. It will be done.

The culture medium may be either a liquid medium or a solid medium, and the culture pH' is usually 5.5 or higher, preferably in the range of 6 to 9. The culture temperature was 25 to 40 qo, preferably around 30 to 30 qo, and the culture period was usually about 2 to 10 days. After completion of the culture, in the case of a solid culture, the culture is extracted with water or a buffer solution by a conventional method, and in the case of a liquid culture, the culture is filtered through a sieve or centrifuged in a conventional manner to obtain a crude enzyme solution.

The physicochemical properties of the present enzyme (crude enzyme solution) obtained by the above-mentioned operation are as follows.

'11 Action and substrate specificity Avicel CMC-
It acts on Na, absorbent cotton, sawdust, okara, rice straw, soy sauce lees, rice husks, peanut shells, coffee grounds, etc., and produces reducing sugars.

'21 Optimum pH and Stable pH The optimum pH of this enzyme is PH6.0 (5
000), and the stable pH range is pH 4 as shown in Figure 2.
~9 (30oo, 2 unique inter-processing).

In addition, in the pH range of 5 to 8, 50% or more of the activity remained even after heat treatment at 50 qo for 4 hours. Leg titer measurement method Cellulase activity was measured using a method that measures the increase in reducing sugar produced using washi paper as a substrate (M. Man.
dele & D. S erg, J, Ferm
ent. Technol・, 54, 267.1976)
.

That is, 50 female monkey papers (Whatman M.1) were used as a substrate, and to this was added 0.5 ml of enzyme solution and citrate buffer (6.0 ml of bait, 0.5 ml of enzyme solution).
After adding 1 M) 1' and carrying out an enzyme reaction at 5000 for 60 minutes, dinitrosalcylic acid reagent 3' was added and heated in a boiling room for 5 minutes to develop color. To this, 16 parts of water was added, and the absorbance at 55 m was measured and expressed as the amount of glucose. The enzyme unit has 1 enzyme activity that produces a reducing power equivalent to 1 mountain of glucose in a reaction of 50 o 1 hour.
It was taken as a unit.

[4) Range of suitable temperature for action The range of suitable temperature for action of this enzyme is 45 to 55 °C as shown in Figure 3.
0 (pH 6.0).

'5} Conditions for inactivation due to pH, temperature, etc. As shown in Figure 4, it is stable up to approximately 50 qC under the condition of pH 6.0.

[6} Inhibition and activation This enzyme is inhibited by heavy metals such as mercury and silver, and activated by manganese, cobalt, barium, etc.

'7} Purification method The method for purifying this enzyme is to add 0.09M Na2HP04 (crude enzyme solution) obtained by centrifuging the culture solution of this bacterium.
The mixture was adsorbed onto DEAE-Sephadex (manufactured by Pharmacia, Sweden) which had been sufficiently equilibrated with a solution (adjusted to pH 8.0 with citric acid).

Then, after washing thoroughly with the buffer, elution is performed with the buffer containing 0.9M sodium chloride. The eluate was filtered through an ultrafilter membrane UM-1.
0 (manufactured by Amicon, USA) and concentrated, and the concentrated solution was equilibrated with the above buffer solution.
50 (Pharmacia, Sweden) for purification. [81 Molecular weight This enzyme is C, monoenzyme, Cx-
It consists of enzymes such as enzymes and 8-glucosidase, and has an average molecular weight of 10,000 to 100,000.

The cellulase obtained by the present invention has an extremely wide stable pH range of 4 to 9, and has particularly strong enzymatic activity even at around pH 8.
It is also widely used in the food industry, etc., and the present invention is extremely significant industrially.

The present invention will be specifically illustrated by examples below. Example Avicene; 1% (W/V), (NH4)2S04; 0.
14% (W/V), K ratio P04; 0.20% (W/V)
, Urea; 0.03% (W/V), CaC12; 0.03
% (W/V), MgS04 7 days 20; 0.03% (W
/V), Beptone: 0.10% (W/V), Yeast extract; 010% (W/V), Tween 80; Z of 0.1, F
eS04・7th 280; 0.00005% (W/V),
MnS04・Day 20; 0.00016% (W/V), Z
nS04/7 days 20: The top of the liquid medium 50 containing 0.000014% (W/V) and CoC12: 0.00002% (W/V) was placed in a 500M folded flask and heat sterilized by a conventional method. After that, 20 minutes of 5% Na2C03 solution was added to this to adjust the pH of the medium to 8.5.
Coprinus cinereus KK-37M
No. 072, FERMP-6072) was inoculated, and 3
The total amount (40') of the culture solution obtained by culturing seeds in a rotary shaker for 3 days at 0qo was added to liquid medium 1 with the above composition.
The culture medium was then placed in a 5-container Erlenmeyer flask with a sterilizer, and the pH of the medium was adjusted to 8.5 with a 5% Na2C03 solution, followed by culturing in a rotary shaker at 30:00 for 7 days.

Hashi liquid 80 obtained by centrifuging the cultured solution by a conventional method
After adding 2,400 g of cold alcohol to 0' and leaving it for 1 day and night, the precipitated fraction was collected and then dried under reduced pressure by a conventional method to obtain the present crude enzyme powder 59.

The enzymatic activity of the crude enzyme was 1.4×1 pih. /It was evening.

[Brief explanation of drawings]

Figure 1 shows the normal pH of this enzyme, Figure 2 shows the stable range, and Figure 3 shows the normal pH of the enzyme.
The figure shows the range of suitable temperature for action, and Figure 4 shows the conditions for inactivation due to temperature (
pH 6.0). O diagram O 2 diagram Z 5 diagram Power 4 diagram

Claims (1)

[Claims] 1. A method for producing cellulase, which comprises inoculating a cellulose-containing medium with a strain belonging to the genus Coprinus and having cellulase-producing ability, culturing the cellulase, and collecting cellulase from the culture.