JPS61162179A - Improved method 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 betaine. 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 about 0.1-1% betaine. The betaine may be the one separated from animals or vegetables or produced from glycine 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 their mode of action, cellulase can be classified into C1-enzyme, Cx-enzyme, β-glycosidase, Efso β-glucanase, endo- It is called by various names such as β-glucanase and cellobiose. It is composed of at least three enzyme groups.

In recent years, cellulases have attracted attention from the perspective of effective utilization of biomass resources, and saccharification of cellulose has been actively researched. Since the production capacity of cellulase is not sufficient, the production cost of the enzyme is high, and it has not been used for saccharification of biomass resources.

The present inventors have previously developed a cellulase-producing bacterium (Acremonylem), which has excellent decomposition power for crystalline cellulose and conversion ability to glucose, and also has excellent thermostatic properties.
A bacterium of the genus Niu■) was isolated. In order to industrially utilize the cellulases produced by this fungus, we have continued to conduct intensive research into methods for culturing microorganisms, and we have found that when cellulase-producing filamentous fungi are cultured in the presence of betaine, cellulase production increases. In particular, it has been recognized that the production of endo-β-glucanase (carboxymethylcellulase (CMCase)) and β-glycosidase can be significantly increased.The present invention was made based on this knowledge. be.

〔the purpose〕

Accordingly, one object of the present invention is to provide an improved method for producing cellulases.

〔composition〕

The present invention is directed to the production of cellulase by culturing filamentous fungi capable of producing cellulase.

The present invention relates to a method for producing serrase, which is characterized by culturing in the presence of betaine.

The present invention will be explained in detail below.

Betaine is a general term for trialkyl amino acids and is an amphoteric electrolyte containing quaternary ammonium. Betaine is an animal,
It exists widely in plants, and is particularly abundant in sugar beet molasses, but it can also be easily obtained by chemical synthesis from glycine.

Betaine is sufficiently effective when added at 0.01% or less to the culture medium, but normally when added at around 0.01 to 1%,
When no additives are used, the production of becellulase, especially carboxymethyl cellulase and β-glucosidase, is 10-50%.
%To increase.

The filamentous fungi used in the present invention include Trichoderma,
In addition to cellulase-producing bacteria such as those of the genus Aspergillus, Penicillium, and Sporotrichum, cellulase-producing bacteria of the genus Acremonium newly isolated by the present inventors can be used. This bacterium is FERM8PJ
15' and 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 m in one day at 30°C. The colony is initially white and later becomes slightly yellowish. The aerial hyphae are loosely swollen, wool-like, and form 1 o'clock knitted 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.
At 0, the optimum pH is around 4, and the growth temperature range is 15℃ to 43℃.
The optimum growth temperature is around 30'C.

Morphology: The diameter of the hyphae is 0.5 to 2.5μ layered, 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 on Czapek agar and malt extract agar. When observed at the time of isolation, the conidiophores protrude from the sides of the vaporized hyphae and are colorless.

The conidia are subglobose (2.5-5 x 2-4.5 μm), smooth, colorless, and are very loosely chained and easily dispersed.

Regarding the above mycological properties, V, Gam5 rcep
halosporium artige Schimm
elpilgeJ p84, G. Fisher (19
71) and C. H. Dickinson, Myco.
l.

Papers 115 plO (1968), this bacterium was treated with Acremonium cellulolitica re+wo.
nium cellulolyticus).

The enzymatic properties of cellulase produced by this bacterium are as shown below.

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

Produces reducing sugars such as cellobiose.

(2) Action p)l and optimal action p.

The action pH range of this enzyme is 2 to 8, and the optimum action 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 about 90°C, but when reacted for 10 minutes in 1% Navicel and 0.05M acetate buffer (pH 4, 5), The optimum working temperature was found to be around 65°C.

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

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

(7) Purification method This enzyme is extracted from the culture filtrate using holofiber (amicone) I.
After desalting and concentration using DEAE-Sepharose (CL-6B) (ΔacQo → IM gradient) and re-chromatography using the same column (NaCΩ0 → 0.6M), it was further purified. can do.

(8) Molecular Weight The molecular weight measured by gel filtration using a Bio-gel (A 0.5m) column was about 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) O15+mA, the total volume was adjusted to 1.0+afi 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 produced a reducing power equivalent to 1 μmol of glucose per minute was defined as 1 unit.

(B) 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, ■ is approximately 160,000.4.95,
■ is approximately 120,000.4.60, ■ is approximately 120,0
00.4.48, and CMCase is composed of a complex of i isozymes and 1! do.

! 2) Components (CMCase I and
) and a component (CMC) that produces very little glucose and decomposes it into cellooligosaccharides greater than cellobiose.
Aze ■, ■) exist.

(3) Action pH and optimum 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 approximately 3.
It was 5 to about 6.

(5) Action temperature range and optimum action temperature The CMCase complex with the optimum action temperature 5 is susceptible to high temperatures up to about 90°C, but it is not necessary to use 1% CMC, 0.05M acetate buffer (pH 4,
5) The optimum operating temperature when reacting for a lower Ld minute was approximately 65°C.

'(6) Thermostable This enzyme was prepared 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.

After desalting and concentrating with DEAE-P5)
It can be purified into each component by column chromatography (NaCQ O→IM gradient) using Sepharose (CL-6B), rechromatography using the same column, and chromatofocusing.

(]9) Activity measurement method 1% CNC solution dissolved in 0.1M acetate buffer (pi
(4,5) An appropriate amount of enzyme solution was added to 0.5 mΩ, the total amount was made up to 1.0 mg 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 produced a reducing power equivalent to 1 μlll01 glucose per minute was defined as 1 unit.

(C) Enzymatic properties of β-glucosidase (1) Action Acts on cellooligosaccharides such as salicin, cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose, decomposing them into glucose. Furthermore, this enzyme acts on polymeric cellulose such as Avicel, but hardly acts on CMC or HEC (hydroxyethyl cellulose). The +w values for salicin, cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose are 3.40.2.26.1.19.0.82.0.52, respectively.
And it was 0.51mM. 2) Action pH and optimum action pH The action pH range of this enzyme is 2 to 8, and the optimum action pH is about j5.
was recognized.

(3) Stable pH Stable pi (range was about 3.5 to about 5) 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% salicin and 0.05M acetate buffer (pH 4, 5). The working temperature was found to be about 65°C.

(5) Thermostability As a result of heat treatment for 710 minutes at each temperature under 0.05M acetate buffer C pH 4,5) (7), one enzyme was hardly inactivated at high temperatures up to about 65°C, and at 70°C, Heating at 80°C for 10 minutes deactivated it by about 40°C, and heating it at 80°C for 10 minutes deactivated it by about 40°C.
More than 0% deactivation occurred.

, C6) Inhibitor Among various heavy metal ions, 1+*M or more of mercury ion and copper ion are more strongly inhibited. Glue-delta-lactone also acts as a competitive inhibitor against the substrate.

(7) Purification method This enzyme is extracted from the culture filtrate using Holofiber (Amicon HI).
-P5), followed by column chromatography (Nall O→IM gradient) using DEAε-Sepharose (CL-6B), chromatofocusing (p)+6→4), and Bio-gel (A 0
．． 5m) can be electrophoretically purified to homogeneity.

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

(9) Activity measurement method 2% salicin solution (p
H4,5) Add an appropriate amount of enzyme solution to 0.5 mm, and make the total volume 1.5 mm with distilled water. OmQ was used, and the reaction was carried out at 50°C. The produced glucose was then measured by the Somogyi-Nerlan method.

Under these conditions, the amount of enzyme that generated a reducing power that phased 1 μmol of glucose per minute was defined as 1 unit.

For culturing cellulase-producing bacteria, pure cellulose or cellulose-containing substances such as cellulose, avicel, cotton, bagasse, and wheat cranes are used as carbon sources, and nitrogen sources such as nitrates, ammonium salts, urea, etc. Use either or a combination of inorganic nitrogen or organic nitrogen sources such as peptone, yeast extract, meat extract, soybean meal. Furthermore, as a supplementary medium raw material, a medium to which metal salts such as manganese and zinc are added is used, and approximately 0.01 to 1% betaine is added to this medium. Culture may be either solid culture or liquid culture, but usually
Cultured aerobically at 20-40°C for 2-15 days.

Cellulase is an enzyme produced outside the bacterial body, so in the case of liquid culture, use the sterilizing solution obtained by filtration or centrifugation after culturing, and in the case of solid culture, use water or an appropriate solution after culturing. The enzyme solution extracted with a salt solution is precipitated with ammonium sulfate or sodium sulfate, or an organic solvent such as acetone or alcohol is 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%, 2HPO41.2%, Bactopeptone 1%, ZnSO4-7H201XIO""3%, KN
O30.6%, Mn5O4・7H201X 10-'
%, urea 0.2%, CuS04 ・5H201X10
″″3%, KCQ 0.16%, MgSO4・7H20
A medium containing 0.12% (pH 4) and a medium to which 0.03% betaine was added were sterilized by a conventional method, and then inoculated and cultured aerobically at 30°C for 12 days. After culturing, the cellulase avicelase, CMCase, and β-glucosidase activities produced in the supernatant obtained by centrifugation were measured. The results were as shown in Table 1.

Table 1 Effects] As is clear from the table, when betaine was added, the production amounts of avicelase, CMCase, and β-glucosidase all increased significantly compared to the case without addition.

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 betaine.