JPS62208279A - Production of cellulase - Google Patents

Abstract

PURPOSE:To produce highly active cellulase, by cultivating a microorganism, belonging to the genus Trichoderma and having enhanced induction ability of cellulase and beta-galactosidase activity. CONSTITUTION:A microorganism, e.g. Trichoderma reesei LW376 (FERM BP-980), belonging to the genus Trichoderma and having enhanced induction ability of cellulase and beta-galactosidase activity is cultivated in a culture medium containing lactose or a substance containing the lactose, e.g. whey, in an amount of 3-8% expressed in terms of the lactose as a carbon source at 20-33 deg.C, preferably 28-30 deg.C and 4-6pH under aerobic condition for 4-10 days to collect the aimed cellulase from the resultant cultivation filtrate.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a method for producing cellulases. In this specification, cellulase refers to exo-β-glucanase (BO2,2
, 1, 19), endo-β-glucanase (IC3,
2,1,4) and β-glufusidase (BO3,2,1
, 21), which is an enzyme that decomposes cellulose into glucose.

Conventional technology Trichoderma reesei (Trichoderma reesei), which has an enhanced ability to induce cellulase by lactose,
rees*i) MCG-77 (NRTLL 112
36) (hereinafter referred to as MCG-77) to lactose 1
A method for obtaining cellulase by culturing in a medium of ~2.5 mm is known [Japanese Patent Publication No. 500890/1989, Biotechnology Ann R.
eehnology and Blo@ngin@er
ing) 26°3887 (1979)).

Problems to be Solved by the Invention An object of the present invention is to provide an improved method for producing highly active cellulases.

Means for Solving the Problems According to the present invention, a microorganism belonging to the genus Trichoderma and having an enhanced ability to induce cellulase by lactose and β-galactosidase activity is cultured in a medium, and cellulase is produced and accumulated in the culture, A method for producing cellulase is provided, which comprises a step of collecting cellulase.

Any microorganism belonging to the genus Trichoderma that has an enhanced ability to induce cellulase by lactose and β-galactosidase activity can be used for the purpose of the present invention.

The bacterial strain described in the Examples is Trichoderma reesei (T
richormar @5sei) LW376
(PE MBP-980) (hereinafter referred to as LW376) was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology on February 1, 1986, pursuant to the provisions of the Strike Treaty.

The mycological properties of Trichoderma reesei are published by the Second International Mycological Humbres (2n
d International Myaology
alCongress), U.S. 7o Lida, Tough/Q,
August 1977, page 618.

The bacterial strain used in the method of the present invention can be obtained, for example, by the following method. A microorganism belonging to the genus Trichoderma and capable of producing cellulase is subjected to a known mutation induction treatment such as ultraviolet irradiation or the use of a mutagenic agent such as nitrosoguanidine, and a strain with a high ability to induce cellulase by lactose is selected from the treated strains. For example, practical methods for this purpose include:
Trichoderma reesei 13G-3-7 (hereinafter referred to as PC-3-7) was used as a parent strain and cultured on a Strauss agar slant medium in Botetoide at 25° C. for 7 days to allow sufficient spore formation. The generated spores were suspended in physiological saline (approximately 1 to 3 x 107 spores/IM) and reacted with N-methyl-No-nitro-N-ditho△guanidine (300 μl).
l/R1, pH 7, 0, 30°C, 30-120 minutes). Spores were collected from this spore suspension by centrifugation, washed thoroughly, diluted to 100-300 spores/dK per plate, and applied to an agar plate containing lactose as a carbon source as shown in Table 1. After culturing for 2 days at
．． Add 5% phosphoric acid swollen cellulose agar and incubate at 45℃ for 1 hour.
Bring to bed temperature for 0-24 hours. A zona pellucida is formed around colonies that produce cellulase. Then, a desired strain with increased cellulase activity was selected based on the 1 bright zone. As shown in Table 2 below, the ability of the parent strain 1 to form a zona pellucida is weak.

Table 2 shows a comparison of the properties on the agar medium between the T and W376 strains and the parent strain.

Table 1 Composition of agar medium Lactose 5II yeast extract
1y (NH4), 5o42 F KH, PO44# No・ToIPO・
6 pMgSO4・7H2020
0Yu2 FeS04・7H, Ol 1'9 CsiCA! 2-2H, 01jip Miltriton 100 (Gyudon Chemical Co., Ltd. #) ]
,! Agar 2
0 F+1. Bo,
10μgMnSO,'4H,O''
10μyZnSO4”7
11,0 70μyCuSO
4' 5H,050pl/ (N)14), MO,O,,・4)1,0
1011g water l l (P) (5,5) Table 2 (Note) 1)+: Forms a village.

2) +: Good conidia settlement.

3)-2 Not allowed.

4) ++-: Clearly recognized with the naked eye.

+: Slightly visible to the naked eye.

As the carbon source of the culture medium in the present invention, lactose and substances containing it are used, and these materials include, for example, cellulose powder, glucose, cellobiose, paper, paper, sawdust, wheat bran, and rice hulls. , bagasse, soybean meal,
Coffee grounds, starch, etc. may be used in combination. Examples of lactose-containing substances include whey.

The concentration of lactose or lactose-containing substances in the medium is in the range of 3 to 8% in terms of lactose.

Nitrogen sources include inorganic ammonium salts such as ammonium sulfate and ammonium nitrate, urea, amino acids, meat extract, and yeast extract. T? I
Organic nitrogen-containing substances such as J-peptone and protein decomposition products are used. Inorganic salts include KH, PO2, Mg804-
7H,01Ca(J,"2H,0,F'e(J,-6H
,01MnC11・4 H! O1Z! l5044H,O
etc. are used. If necessary, a medium containing organic micronutrients is used. The above-mentioned bacterial strains can be cultured not only in liquid culture but also in solid culture. A normal aerated stirring culture device is used for liquid culture, and the culture temperature is 20 to 33°C, preferably 28 to 30°C, using the above medium (C1 culture p).
(If cultured for 4 to 6 days, the cellulase activity will be i&high after 4 to 10 days. Then, the bacterial cells are removed from the culture solution by a known method such as centrifugation or filtration to obtain a supernatant. This supernatant can be used as this it crude enzyme solution.

Further, a crude enzyme preparation can be obtained from the obtained supernatant liquid by using known methods such as freeze drying, ammonium sulfate salting out, and precipitation using an organic solvent.

Next, the method for measuring #unactivated will be explained.

(1) Exo-β-glucanase activity Avicel 8F
150 M9 as a substrate, and enzyme solution 1.01.0
．． A 2 M, pH 5.0 acetate buffer 4.0- was added to each, and the mixture was reacted at 45°C for 1 hour.

The reaction is stopped by heating at 100° C. for 10 minutes. 3゜5
- Colorimetric determination of reducing sugars by the dinitrosalicylic acid method.

1 minute K] The amount of enzyme that releases μmol of glucose is 1
Defined as enzyme quantity level (Unit).

(2) Endo-β-glucanase activity 0.2 M
Prepare a 1-tyl carboxymethyl cellulose sodium salt solution dissolved in vinegar @ mild 4E solution with SpH 5.0. Add 21+ volumes of the appropriately diluted fermentation solution to this and react at 45°C for 30 minutes. After stopping the reaction by heating at 100°C for 0 minutes, reducing sugars are determined colorimetrically by the 3°5-dinitrosalicylic acid method. The amount of n volume that releases 1 μmol of glucose per minute is defined as one enzyme amount (Unit).

(3) Prepare 2mM p-nitrophenyl-β-D-glucopyranosyl dissolved in 0.05M, pH 5.0 acetate buffer as a β-glucosidase activity substrate solution. Substrate solution 1.0ulIc#*solution 2
Add 0μ and react at 45°C for 10 minutes.

Stop the reaction by adding 2.0 ml of I Fl sodium carbonate solution.
).

(4) Prepare β-galactosidase lactopyranosi f. Substrate solution 1.0ml 1C enzyme solution 20μ! Add and react at 45°C for 10 minutes.

The reaction is stopped by adding 2.01 Kt IMR sodium acid solution. The amount of enzyme that releases 1 fi mat of 0-nitrophenol per minute is defined as 1 enzyme amount ([Jnit).

The degree of cellulase induction from lactose of a Trichoderma reesei mutant strain is evaluated by performing liquid culture using 1% lactose as a carbon source and measuring the enr-β-glucanase activity in the supernatant. That is, the mutant strain is cultured on a Strawberry agar medium at 25° C. for 7 days. The live spores were inoculated into a 30,011 Lt capacity Erlenmeyer plastic tube containing 50 tu of the medium shown in Table 3, and cultured with shaking at 28° C. for 7 days. The culture solution is centrifuged and the endo-β-glucanase activity in the supernatant is determined. The results are shown in Table 4.

Table 3 Lactose 10. Q p polybutone 1.0 p yeast extract
0.51KH2PO42,0# (NH4)2So41.5, litMtrSO4-
771200,31 C&C12-2 Summer'zo
O, 31/Tween 80 [manufactured by Gyudon Kagakuyaku Co., Ltd.] 1. o Ni trace element solution *1.0 Benitartrate buffer 50mM water IA' (PH4,0) *Trace element solution H3BO361n9 (NH4)6Mo, 024'4H202619FeC1
3-6H20100ll9 CuSO4-5H2040Q MnGl, -4H□0
8! R9ZoSO4・7Hz0
200 R9 water
100m 4th
Table LW376 25PC, -3-7
15 MCG-7711 Next, examples will be shown.

Example I LW376 was mixed with potato dextrose agar medium.
The cells were cultured at 5°C for 7 days. The generated spores were transferred to medium 5 in Table 5.
A 300rnl Erlenmeyer flask containing 01nl was inoculated and cultured with shaking at 28°C for 4 days.

This culture solution was then applied to a 21 Erlenmeyer flask containing 500 ml of a medium of the same composition and cultured with shaking at zs°C for 4 days. Furthermore, add this culture solution to the medium 2.51 in Table 6.
The seeds were inoculated into a 51 jar fermentor containing
VVM). The pH was adjusted by adding 3N NH40H.
i was adjusted to a range of 4.0 to 4.5.

Table 5 Avicel 10. OF Polypeptone 1.01 Yeast engineered beef o,s gKH2PO
42, OF (NH4)2So, 1.5 N
MgSO4・71120 0.3
．． 9C&IC12・2[f20
o-31 Tween 80 (manufactured by Gyudon Chemical Co., Ltd.) 1
．． 0 Ni trace element liquid (same as Table 3) 1.0 m
l Water IJ (pHs, s) Table 6 Lactose 40. OIi polybutone 1. Oi yeast engineering school
o, syKH2PO,□,. 1 (N) (,)28041.5 N Mg804-7H200,3,9' CaCJ z ・2) rzo O
-31 Adeca, Nord LG-109 [manufactured by Asahi Denka Co., Ltd.] 6. Om trace element liquid (same as Table 3) 1. Qll to water l i<pH
s, s) On the fourth day, the culture solution was centrifuged to obtain a supernatant. The endo-β-glucanase activity in the supernatant was 110 U7'lj, the endo-β-glucanase activity was 15 U/iu, the β-glufusidase activity was 2.70 L/d, and the β-galactosidase activity was 3.107 m. .

The supernatant 1 was then salted out with 70% ammonium sulfate, desalted using Sephadex G-25 (manufactured by Pharmacia Fine Chemicals), and lyophilized to obtain a crude enzyme preparation of OI. This standard en? -β-glucanase activity is 8.
IU/1169, exo-β-glucanase activity is 0,
86 U/19, β-glucosidase activity is 0.14 U
/1119 and β-galactosidase activity is 0.170
/ It was da.

MCG-77 and PC-3-7 were used as controls and cultured in the same manner as above. Table 7 shows the enzyme activity in the culture solution.

Effects of the Invention Highly active cellulases can be obtained by the method of the present invention.

Claims (2)

[Claims] (1) From the step of culturing a microorganism belonging to the genus Trichoderma that has enhanced cellulase inducibility and β-galactosidase activity with lactose in a medium, producing and accumulating cellulase in the culture, and collecting cellulase from the culture. A method for producing cellulase. (2) The amount of lactose or its content in the medium is 3 to 8
% (in terms of lactose).