JPS61162178A - Improved process for producing cellulase - Google Patents

Abstract

PURPOSE:To increase the productivity of cellulase in the culture of a cellulase- producing mold, by carrying out the culture in the presence of lecithin. CONSTITUTION:A cellulase-producing strain belonging to Acremonium genus, Trichoderma genus, Aspergillus genus, Penicillium genus, etc. such as Acremonium cellolyticus (FERM BP-685) is cultured in a medium containing a cellulose-containing component such as cellulose, avicel, etc. as a carbon source and added with a nitrogen source, metallic salt, etc. In the above process, the medium is added with 0.1-1% lecithin. The lecithin may be the one separated from animals or vegetables or produced by synthetic means. After the cultivation by conventional method, the objective cellulase is separated from the culture liquid.

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical Field] The present invention relates to a method for producing cellulase using filamentous fungi.

[Prior art]

Cellulase is a general term for a group of enzymes that catalyze an enzymatic reaction system that breaks down cellulose into cellooligosaccharides such as glucose or cellobiose. Depending on its mode of action, cellulase can be classified into C1-enzyme, Cx-enzyme, β-glucanase, Efso β-glucanase, and Endo-glucanase. It is composed of a group of enzymes called by various names such as β-glucanase and cellobiose.

In recent years, cellulases have attracted attention from the perspective of effective utilization of biomass resources, and research on the saccharification of cellulose has been actively conducted. Since microorganisms do not have sufficient ability to produce cellulase, the production cost of the enzyme is high, and it has not been possible to use it as an enzyme source for obtaining resources such as glucose from cellulose.

The present inventors previously discovered a cellulase-producing bacterium (Acremonium) that has excellent decomposition power for crystalline cellulose and ability to convert it into glucose, as well as excellent thermostability.
A bacterium of the genus nium) was newly isolated. In order to use the cellulase produced by this bacterium industrially, we have continued to conduct intensive research into culturing methods for this bacterium, and have found that when cultured in the presence of lecithin, the production of cellulase, especially endo-
β-glucanase ((carboxymethyl cellulase (C
It was found that the production of MCase) and β-glycosidase could be significantly increased. The present invention has been made based on this knowledge.

〔the purpose〕

Therefore, an object of the present invention is to provide an improved method for producing cellulase.

〔composition〕

The present invention involves culturing filamentous fungi that have the ability to produce cellulase,
The present invention provides a method for producing cellulase, which comprises culturing in the presence of lecithin.

The present invention will be explained in detail below.

Lecithin is the common name for phosphatidylcholine (phospholipid), and is a substance that is widely present in the animal and plant kingdoms, such as egg yolks, blood cells, and plant seeds such as soybeans.

However, since lecithin has a simple structure, not only natural products but also synthetic products have been obtained.

As lecithin used in the present invention, in addition to those collected from animals and plants, synthetic products can also be used, but lecithin made from soybeans is used in large quantities.

Moreover, it can be obtained at low cost.

Lecithin is sufficiently effective when added in small amounts, but usually 0.1
When ~1% is added, compared to when no additive is added.

Among cellulases, carboxymethyl cellulase (
The production amount of CMCase) and β-glucosidase is 20~
It will increase by about 30%.

The filamentous fungi used in the present invention include Trichoderma,
In addition to cellulase-producing bacteria such as the genus Aspergillus, Penicillium, and Sborotrichum, cellulase-producing bacteria of the genus Acremonium newly isolated by the present inventors can be mentioned.・Celloriteika Yoi cremonium ce
lluloliticus). This bacterium is nR
55p-I, has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology.

A summary of the mycological properties of this bacterium is as follows.

Growth: Growth is fast on malt extract agar, reaching a diameter of 70 μm in 7 days at 30°C. The colony is initially white and later becomes slightly yellowish. The aerial hyphae are loosely swollen, wool-like, and sometimes form cotton-like hyphal bundles.

In the later stages of cultivation, the underside of the colony becomes pinkish-brown to reddish-brown.

Almost the same growth was observed on Zapek agar, but the growth of aerial mycelia was smaller. Growth pH range is 3.5-6.
Optimum PI at 0 (is around 4, growth temperature range is 15°C to 4
At 3°C, the optimum growth temperature is around 30°C.

Morphology: The diameter of the hyphae is 0.5 to 2.5 μm, colorless, and septa are observed in the hyphae. In addition, the hyphal surface is smooth.

Conidia: The ability to form conidia was very unstable and was easily lost by subculture with Czapek agar and malt extract agar. Il at the time of separation! On examination, the conidiophores protrude from the sides of the aerial hyphae and are colorless.

Conidia are subglobular (2.5-5 x 2-4.5 μm), smooth, colorless, and have very loose chains and are easily dispersed.

Regarding the above mycological properties, 11. Gam5's rcep
halosporium art, ige Sch
immelpilgeJ p84, G, Fishe
r (1971) and C.H. Dickinson,
My col.

was identified.

This bacterium has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology under the name FERM Bl)-1,8.

Cellulase produced by this bacterium has the following enzymatic properties.

(A) Enzymatic properties of Avicelase (1) Action It acts on highly crystalline insoluble cellulose such as cellulose powder, Avicel, and absorbent cotton to produce reducing sugars such as glucose and cellobiose.

(2) Action pH and optimum action pH The action pH range of this enzyme is 2-8. The optimum working pH is about 4.
5 was recognized.

(3) Stable pH The stable pH range was about 3.5 to about 6 when left at 45° C. for 20 hours under citric acid-phosphate buffer.

(4) Action temperature range and optimum action temperature This enzyme acts at high temperatures up to approximately 90°C, but the optimum action is when reacted for 10 minutes under 1% Avicel and 0.05M acetate buffer (pH 4, 5). The working temperature was found to be about 65°C.

(5) Thermostable enzyme under 0.05M acetate buffer (pH 4, 5)
As a result of heat treatment at each temperature for 10 minutes, the enzyme was approximately 60%
It was hardly inactivated at temperatures up to 65°C, about 50% when heated at 65°C for 10 minutes, and about 80% when heated at 70°C for 10 minutes.

(6) Inhibitor Among various heavy metal ions, it is strongly inhibited by 1+mM or more of mercury ion and copper ion. Furthermore, it is inhibited by about 80% by the SH inhibitor parachlormercury-benzoate at 1N.

(7) Purification method This enzyme is extracted from the culture filtrate using Holofiber (Amicon HI).
-P5), followed by column chromatography (NaCQ O → IM gradient) using DEAE-Sepharose (CL-6B) and re-chromatography using the same column (Na (10 → 0.6M)). It can be refined.

(8) Molecular Weight The molecular weight measured by gel filtration using a Bio-gel (A 0.5m) column was approximately 140,000.

(9) Activity measurement method Avicel suspension at 0.5% concentration in 0.1M acetate buffer (
An appropriate amount of enzyme solution was added to pH 4, 5) 0.5 mΩ, the total volume was made up to 1.0 mQ with distilled water, and the reaction was carried out at 50°C. The reducing sugar produced was measured by the Somogyi-Nelson method.

Under these conditions, the amount of enzyme that generates a reducing power corresponding to 1 μmol of glucose per minute is defined as 1 unit (6-IIB)
Enzymatic Properties of CMCase (1) Multicomponent CMCase CMCase is separated by disk electrophoresis into at least four components, each of which is distinguished by its molecular weight and isoelectric point. CMCase I has a molecular weight of approximately 160,000 and an isoelectric point of 5.
．． 08, similarly below ■ is approximately 160,000.4.95.
m is about 120, 000.4.60, ■ is about 120
, 000.4.48, and CMCase consists of a complex of these isozymes.

(2) Components (CMCase ■ and ■
) and a component (CMC) that produces very little glucose and decomposes it into cellooligosaccharides with more than zero bioses.
Aze ■, ■) exist.

(3) Action pH and Optimal Action pH The action pH range of the CMCase complex was approximately 2 to 8, and the optimum action pH was found to be approximately 4.5.

(4) Stable pH The stable pH range of the CMCase complex when left at 45°C for 20 hours under citrate-phosphate buffer is 13.
It was 5 to about 6.

(5) Action temperature range and optimal action temperature This CMCase complex can be grown at high temperatures up to about 90°C.
) for 10 minutes, the optimum working temperature is approximately 6
It was observed at 5°C.

(6) Thermostable enzyme under 0.05M acetate buffer (pH 4, 5),
As a result of heat treatment at each temperature for 10 minutes, this enzyme was approximately 60℃
There is almost no deactivation at temperatures up to
0% inactivation.

(7) Inhibitor Among various metal ions, it is strongly inhibited by 1mM or more of mercury ion and copper ion.

(8) Purification method This enzyme is extracted from the culture filtrate using holofiber (Amico JHI-
After desalting and concentrating using ps), DEAR-Sepha 1
It can be purified into each component by column chromatography (1aCQ O→IM gradient) using @-S (CL-6B), re-chromatography using the same column, and chromatofocusing.

(9) Activity measurement method 1% CMC solution dissolved in 0.1M acetate buffer (pH
4,5) An appropriate amount of enzyme solution was added to 0.5+sQ, the total volume was adjusted to 1.0+*Ω with distilled water, and the reaction was carried out at 50°C. The reducing sugar produced was measured by the Somogyi-Nelson method.

Under these conditions, one unit is the amount of enzyme that generates a reducing power equivalent to 1 μmol of glucose per minute.
- Enzymatic properties of glucosidase (1) Action: Acts on cellooligosaccharides such as salicin, cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose, decomposing them into glucose. In addition, this enzyme acts on polymeric cellulose such as Avicel, but hardly acts on CMC or HEC (hydroxyethyl cellulose). The Km values for salicin, cellobiose, serotri/fi4-s, cellotetraose, cellopentaose, and cellohexaose are 3JO12, 26, 1, ■9.0.82, 0.
52 SO L TE 0.51mM't''.

(2) Action pH and optimal action pH The action pH range of this enzyme is 2-8. The optimum working pH is about 4.
5 was recognized.

(3) Stable pH The stable pH range was about 3.5 to about 5 when left at 45° C. for 20 hours under a citric acid-phosphate buffer.

(4) Action temperature range and optimum action temperature This enzyme acts at high temperatures up to approximately 90°C, but the optimum action is when reacted for 10 minutes under 1% salicin and 0.05M acetate buffer (PH4, 5). The working temperature was found to be about 65°C.

(5) Thermostability Under 0.05M acetate buffer (pH 4, 5) at each temperature for 10 minutes (as a result of heat treatment, one enzyme was hardly inactivated at high temperatures up to about 65℃, and 70℃ , deactivated by heating at 80°C for 10 minutes at approximately 41)°C, and 9°C by heating at 80°C for 10 minutes.
More than 0% deactivation occurred.

(6) Inhibitor course - δ-lactone acts as a competitive inhibitor against the substrate.

(7) Purification method This enzyme is extracted from the culture filtrate using holofiber (formerly Amicon-P).
After desalting and concentration using 5), column chromatography (N
It can be electrophoretically purified to homogeneity by aCΩ0→IM gradient) chromatofocusing (pH 6→4) and gel filtration using Bio-gel (A 0.5m).

(8) Molecular weight The molecular weight measured by gel filtration using Bio-gel (AO, 5+s) was about 240,000.

(9) Activity measurement method 2% salicin solution dissolved in 0.1M acetate buffer 'p
An appropriate amount of the enzyme solution was added to 0.5 m (1) of H4,5), the remaining volume was made up to 1.0 mQ with distilled water, and the reaction was carried out at 50°C. The produced glucose was then determined by the Somogyi-Nerlan method.

Under these conditions, the amount of enzyme that produced a reducing power equivalent to lμ1 dol of glucose per minute was defined as one unit.

For the cultivation of cellulase-producing bacteria, pure cellulose such as cellulose, Avicel, cotton, or cellulose-containing materials such as bagasse and wheat gourd are used as carbon sources, and nitrates, ammonium salts, urea, etc. are used as nitrogen sources. This is a medium in which inorganic nitrogen or organic nitrogen sources such as peptone, yeast extract, meat extract, and soybean meal are used alone or in combination, and metal salts such as manganese and zinc are added as supplementary medium raw materials. However, approximately 0.1 to 1% lecithin is added to this medium. Culture may be either solid culture or liquid culture, but usually π) 40°C, 2
Cultured aerobically for ~15 days.

Diurulase is an enzyme produced outside the bacterial body; in the case of liquid culture, it is added to the filtered supernatant after culture, and in the case of solid culture, it is added to water or an appropriate salt solution after culture. The enzyme solution extracted can be precipitated with ammonium sulfate or sodium sulfate, or an organic solvent such as acetone or alcohol may be added to precipitate the cellulase, followed by separation and drying to obtain an enzyme powder.

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

Example 1 Cellulose 4%, K2HPO41.2%, Bactopeptone 1%, ZnS044H201X 10-3%, KN
O30.6%, MnSO44H201XIO-”%,
Urea 0.2%, CuSO4・5H201XIO-3%,
KCQ O, 16%, MgSO4・7H200, 12
% (pH 4.0) and a medium to which 0.5% soybean lecithin was added, 20 m each of Q 200+
a0 seeds and cultured aerobically at 30°C for 12 days. Avicelase and carboxymethyl cellulase (cellulase) produced from the supernatant obtained by centrifugation after culturing.
CMCase) and β-glucosidase activities were measured. The results were as shown in Table 1.

Table 1 [Effects] As is clear from the table, when cultured with the addition of lecithin, the production amounts of avicelase, CMCase, and β-glucosidase all increased compared to the case without the addition of lecithin. for the production of Aase and β-glucosidase.
It showed a remarkable effect.

Claims (1)

[Claims] (1) A method for improving the production of cellulase, which comprises culturing a filamentous fungus capable of producing cellulase to produce cellulase in the presence of lecithin.