JPH09163980A - Production of cellulase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently obtain the subject enzyme useful for saccharifying a cellulose, by culturing a mutant of Trichoderma reesei capable of producing a cellulase in a medium containing a crystalline cellulose, etc., culturing a fungus of a specific enzyme activity in a supernatant liquid. SOLUTION: A mutant belonging to Trichoderma reesei [e.g. Trichoderma reesei PCD-19 (FERM P-8172)], capable of producing a cellulase, is cultured in a 2 liter flask containing 500ml of a medium comprising 10.0g of a crystalline cellulose, 1.0g/l of a polypeptone and 0.5gll of a yeast essence as main components at 28 deg.C for 4 days and cultured in a 5 liter fermenter containing 60g/l the crystal line cellulose, 1.0g/l of the polypeptone and 0.5g/l of the yeast essence as main components at 28 deg.C at 1VVM at 450rpm for 7 days while adjusting pH. The culture solution is centrifuged, a fungus having >=13 units/ml of β-glucosidase activity in a supernatant liquid is cultured in a medium and the prepared product is collected to give the objective cellulase.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing cellulase. In the present specification, cellulases include exocellobiohydrolase (hereinafter referred to as C ₁ enzyme), endo-β-glucanase (hereinafter referred to as C _x enzyme) and β.
-A general term for an enzyme system composed of glucosidase, which is an enzyme that decomposes cellulose into glucose.

[0002]

2. Description of the Related Art In recent years, the establishment of an efficient saccharification method of cellulose has been demanded for the effective use of cellulose resources. The enzyme source for cellulase necessary for saccharification, Trichoderma reesei (Trichoderma reesei) QM6 _{a (A}
TCC 13631) and a series of mutant strains, such as Trichoderma reesei QM9414 (ATCC2
6921) [Biotechnology & Bioen
gineering Symposium), 6 , 9-20 (1976)] is the most promising. C _{1 of} cellulase produced by these strains
And _Cx enzyme activity is extremely potent compared to those from other types of microorganisms. However, β-glucosidase has only a weak activity [enzyme and
Microvial Technology (Enzyme and Microbi
al Technology), 2 , 91-102 (1980)]. When cellulose is saccharified using cellulases produced by these strains, cellobiose accumulates. In order to decompose this accumulated cellobiose into glucose, addition of β-glucosidase of Aspergillus niger or Aspergillus forensis has been studied [Canadian Journal of Microbiology (Can
adian Journal of Microbiology), 23 , 139-147 (1977)]
.

Therefore, in order to enhance the β-glucosidase activity of Trichoderma reesei, a culture method has been investigated [Applied and Environmental Microbiolo (Applied and Environmental Microbiolo
gy), 31 , 648-654 (1976), and biotechnology
And bioengineering (Biotechnology & Bio
engineering, 23 , 1837-1849 (1981)], and various mutant strains have been tried [FEMS Symposium No. 13 , 405-416 (1982),
JP-A-59-17984]. However, the β-glucosidase activity of the cellulase obtained in these studies is still insufficient for efficiently saccharifying a high concentration of cellulose. Further, in order to inexpensively perform saccharification of cellulose, it is necessary that not only β-glucosidase activity but also C ₁ enzyme activity and C _x enzyme activity are high.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide an improved method for producing cellulase, particularly a method for producing high β-glucosidase activity.

[0005]

The present invention provides: (a) a mutant strain belonging to Trichoderma reesei, (b) capable of producing cellulase, (c) crystalline cellulose 10.0
After culturing at 28 ° C. for 4 days in a 2 liter flask containing 500 ml of a medium containing g / l, polypeptone 1.0 g / l and yeast extract 0.5 g / l as main components, the culture solution is crystalline cellulose 60 g / l polypeptone , 1.0 g / l
And while adjusting the pH in a 5 liter fermenter containing 3 liter of a medium containing 0.5 g / l of yeast extract as a main component, 2
After centrifuging the culture broth cultured at 8 ° C., 1 VVM, 450 rpm for 7 days, a microorganism having a β-glucosidase activity of 13 units / ml or more in the culture supernatant is cultivated in a medium, and the obtained cellulase is collected. The present invention relates to a method for producing cellulase, which comprises steps.

In the method of the present invention, the mutant strain Trichoderma reesei PCD-10 (FERM P-8172) is used.
Or Trichoderma Reisei CDU-11 (FERM
P-8173) is preferred.

The mycological properties of Trichoderma reesei are:
EG Simmons, Abstract Second International Mycolonic Congress (EG
Simmons, Abst. 2nd International Mycological Cong
ress) Tampa, Florida, USA, August 1977, 618
Page].

The mutant strain used for the purpose of the present invention can be obtained, for example, by the following method. A microorganism that has cellulase-producing ability and belongs to Trichoderma reesei is subjected to a known mutagenesis treatment such as ultraviolet irradiation or the use of a mutagenizing agent such as nitrosoguanidine, and a strain having a high cellulase-inducing ability by cellobiose from the treated strain. Choose. As a practical method for this, for example, QM9414 as a parent strain
(ATCC 26921) is used and cultured on a potato dextrose agar slant medium at 25 ° C. for 7 days to sufficiently form spores. The spores formed are suspended in physiological saline (about 1 to 3 × 10 7 cells / ml) and reacted with N-methyl-N′-nitro-N-nitrosoguanidine (300 μg / ml).
ml, pH 7.0, 30 ° C., 30 to 120 minutes). Spores were collected from this spore suspension by centrifugation, washed well,
Dilute to 100-300 spores / ml per plate, apply to the agar plate containing cellobiose shown in Table 1 as a carbon source, incubate at 30 ° C. for 2 days, and add 50 mM acetate buffer on the grown colonies. 0.5% including pH 5.0
Add phosphoric acid swollen cellulose agar to 10-2 at 45 ° C
Incubate for 4 hours. Since a zona pellucida is formed around the cellulase-producing colonies, a mutant strain having enhanced C ₁ enzyme activity and C _x enzyme activity can be selected. In addition, 50 mM acetate buffer pH 5.0, and 30%
Add 0.05% 4-methylumbelliferyl-β-D-glucoside agar containing glucose, and add 2-16 at 45 ° C.
Insulate for hours. When irradiated with ultraviolet rays, fluorescence is generated around colonies with high β-glucosidase activity, so
This makes it possible to select a mutant strain having an increased β-glucosidase activity. Table 2 shows a comparison of the properties of the mutant strain thus obtained and QM9414.

[0009]

[Table 1] Composition of agar medium Cellobiose 5 g Yeast extract 1 g (NH ₄ ) ₂ SO ₄ 2 g KH ₂ PO ₄ 4 g Na ₂ HPO ₄ 6 g MgSO ₄ / 7H ₂ O 200 mg F ₂ SO ₄ / 7H ₂ O 1 mg CaCl ₂ .2H ₂ O 1 mg Triton X-100 [manufactured by Hanai Chemical Co., Ltd.] 1 g agar 20 g H ₃ BO ₃ 10 μg MnSO ₄ · 4H ₂ 0 10 μg ZnSO ₄ · 7H ₂ O _{70 μg CuSO 4 · 5H 2 O} 50 μg (NH 4) 6Mo 7 0 24 · 4H 2 O 10 μg water 1l (pH 5.5)

[0010]

[Table 2] Comparison of properties between mutant strain and QM9414 (comparison of properties on agar medium) (Note) Strain 1: QM9414. Strain 2: PCD-10. Strain 3: CDU-
11 A: potato dextrose agar medium. B: Cellobiose-phosphate swollen cellulose agar medium. C: Cellobiose-4-methylumbelliferyl-β-
D-Glucoside agar medium.

As the carbon source of the medium in the present invention, cellulose powder, cellobiose, filter paper, general paper, sawdust, bran, chaff, bagasse, soybean meal, coffee meal, starch, lactose and the like are used. As the nitrogen source, inorganic ammonium salts such as ammonium sulfate and ammonium nitrate, organic nitrogen-containing substances such as urea, amino acids, meat extract, yeast extract, polypeptone and proteolytic products are used. As inorganic salts,
_{KH 2 PO 4, MgSO 4 ·} 7H 2 O, CaCl 2 · 2H 2 O, Fe 2 Cl 3 · 6H 2 O, MnCl
_{2 · 4H 2 O, ZnSO 4} · 7H 2 O and the like are used. If necessary, a medium containing organic micronutrients is used. The culture of the strain can be solid culture as well as liquid culture. A conventional aeration and agitation culture apparatus is used for liquid culture, and the culture temperature is 20 to 33 ° C., preferably 28 to 3 using the above medium.
Culturing at 0 ° C. and a culture pH of 4 to 6 gives the highest cellulase activity in 4 to 10 days. Then, the 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 it is as a crude enzyme solution. The enzyme activity is measured by a colorimetric method after the enzyme reaction as shown below.

(1) C ₁ enzyme activity Using 150 mg of Avicel SF as a substrate, 1.0 ml of enzyme solution, 0.2 M, pH 5.0 acetate buffer 4.0 was added thereto.
Add each ml and react at 45 ° C. for 1 hour. 10
The reaction is stopped by heating at 0 ° C. for 10 minutes. The reducing sugar is colorimetrically determined by the 3,5-dinitrosalicylic acid method. The amount of enzyme that releases 1 μmol glucose per minute is defined as one enzyme unit.

[0013] (2) C _x enzyme activity 0.2 M, prepared with 1% carboxymethylcellulose sodium salt solution in acetate buffer pH 5.0.
To this, an equal amount of an appropriately diluted enzyme solution is added and reacted at 45 ° C for 30 minutes. After heating at 100 ° C. for 10 minutes to stop the reaction, the reducing sugar is colorimetrically determined by the 3,5-dinitrosalicylic acid method. The amount of enzyme that releases 1 μmol glucose per minute is defined as one enzyme unit.

(3) β-Glucosidase activity As a substrate solution, 2 mM of p-nitrophenyl-β-D-glucopyranoside dissolved in an acetate buffer of 0.05 M and pH 5.0 is prepared. 20 ml of enzyme solution to 1.0 ml of substrate solution
μl is added, and the mixture is reacted at 45 ° C. for 10 minutes.

The reaction is stopped by adding 2.0 ml of 1M sodium carbonate solution. The amount of enzyme that liberates 1 μmol of p-nitrophenol in 1 minute is defined as 1 enzyme unit. Further, a crude enzyme preparation can be obtained from the obtained supernatant by using a known method such as freeze-drying, salting out with ammonium sulfate and precipitation with an organic solvent.

[0016]

Example 1 Trichoderma reesei QM9414 (A
TCC26921), PCD-10 (FERM P-8)
172) and CDU-11 (FERM P-8173) strains on a potato dextrose agar slant medium at 25
Cultivate at ℃ for 7 days to fully form spores. Part 1 Medium 50 having the composition shown in Table 3 using platinum loops as cellobiose as a carbon source
Inoculate a 300 ml Erlenmeyer flask containing 30 ml,
The cells were cultivated with shaking at 7 ° C for 7 days. The culture solution was filtered on the 7th day, and the cellulase activity of the supernatant was determined. Table 4 shows the results.

[0017]

TABLE 3 cellobiose composition flask medium 10.0 g of polypeptone 1.0 g yeast extract _{0.5 g KH 2 PO 4 2.0 g} (NH 4) 2 SO 4 1.5 g MgSO 4 · 7H 2 O 0 .3 g CaCl ₂ .2H ₂ O 0.3 g Tween 80 [manufactured by Hanai Chemical Co., Ltd.] 1.0 ml Trace element liquid * 1.0 ml Tartrate buffer 50 mM water 1 l (pH 4.0) * Trace Elemental solution H ₃ BO ₄ 6 mg (NH ₄ ) ₆ Mo ₇ 0 ₂₄ .4H ₂ O 26 mg FeCl ₃ .6H ₂ O 100 mg CuSO ₄ .5H ₂ O 40 mg MnCl ₂ .4H ₂ 0 8 mg ZnSO _4. 7H ₂ O 200 mg Water 100 ml

[0018]

[Table 4] Cellulase production in flask culture by various strains

[0019]

[Example 2] Trichoderma reesei QM9414, P
Each strain of CD-10 and CDU-11 is cultured on a potato dextrose agar slant medium at 25 ° C. for 7 days to sufficiently form spores. The 1 platinum loop was prepared by using Avicel PH301 (manufactured by Asahi Kasei Co., Ltd.) instead of cellobiose in the medium having the composition shown in Table 3 and further removing the tartrate buffer solution.
Inoculate a 300 ml Erlenmeyer flask containing 30 ml,
The cells were cultured at 4 ° C with shaking for 4 days. This is a medium of the same composition 500m
A 2 liter Erlenmeyer flask containing 1 liter was inoculated and shake-cultured at 28 ° C for 4 days. Main culture solution is Avicel PH301 60g
/ L, Adecanol LG-10 instead of Tween 80
9 (manufactured by Asahi Denka Co., Ltd.) was added to a 5 l fermenter containing 6 g / l of a medium of the same composition and cultured at 28 ° C. Ventilation is 1
VVM, stirring is 450 rpm, and the lower limit of pH is 2N
The pH was adjusted to 4.0 by NH4OH for 3 days after the start of the culture, and then the pH was raised to 5.5.
Adjusted with H. The culture broth was centrifuged on the 7th day, and the cellulase activity in the supernatant was determined. Table 5 shows the results.

[0020]

[Table 5] Cellulase production by various strains in a 5 l fermenter

[0021]

Example 3 Trichoderma reesei C obtained in Example 2
Of the supernatant of DU-11, 1 l was salted out with 70% saturated ammonium sulfate, desalted using Sephadex G-25 (Pharmacia Fine Chemicals), and further freeze-dried to obtain 30 g of a crude enzyme preparation. Got The C ₁ enzyme activity was 0.72 unit / mg, the C _x enzyme activity was 6.5 unit / mg, and the β-glucosidase activity was 0.50 unit / mg. Finely pulverize cane bagasse and suspend it in 0.3N NaOH, 120 ° C, 1
It was treated for 5 minutes. After washing and neutralization, the cells were suspended in 0.1 M acetate buffer (pH 5.0) to a concentration of 150 g / l. Further, the above crude enzyme preparation was added so as to have a concentration of 10 g / l and allowed to act at 45 ° C. for 24 hours. The reaction solution was centrifuged, and the supernatant was added to Shodex Ionpack C-81.
1 (manufactured by Showa Denko) and the like using a method using high performance liquid chromatography (elution with 0.1% H ₃ PO ₄ at 60 ° C., detection by refractometer). 69g glucose
/ L and xylose were produced at 31 g / l, but the presence of cellobiose or oligosaccharide was not recognized at all.

[0022]

The cellulase can be obtained in high yield.

Claims (3)

[Claims] 1. A mutant strain belonging to (a) Trichoderma reesei, which has the ability to produce (b) cellulase,
(c) 28 g in a 2-liter flask containing 500 ml of a medium containing crystalline cellulose 10.0 g / l, polypeptone 1.0 g / l and yeast extract 0.5 g / l as main components.
After culturing at 4 ° C. for 4 days, while adjusting the pH in a 5 liter fermenter containing 60 liters of crystalline cellulose, 1.0 g / l of polypeptone, and 3 liters of a medium containing 0.5 g / l of yeast extract as main components. After centrifugation of the culture broth at 28 ° C., 1 VVM, 450 rpm for 7 days, β in the supernatant was removed.
-A method for producing cellulase, which comprises a step of culturing a microorganism having a glucosidase activity of 13 units / ml or more in a medium and collecting the obtained cellulase. 2. The microorganism is Trichoderma reesei PCD.
The method according to claim 1, which is -10 (FERMP-8172). 3. The microorganism is Trichoderma reesei CDU.
-11 (FERMP-8173).