JPS6024712B2 - Cellulase production method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing cellulase using Acremonium bacteria.

Cellulase is a general term for a group of enzymes that catalyze the enzymatic reaction system that breaks down cellulose into glucose, cellobiose, and cellooligosaccharides.
It is composed of enzymes called Cx enzyme, B-glucosidase, exo-8-glucanase, endo-B-glucanase, and cellobiase, but its actual nature is still unclear.

It is derived from microbial origin that produces cellulases, producing crystalline cellulose, carboxymethylcellulose (CMC)
This is due to the presence of a wide variety of enzymes with different modes of action on cellodextrins, cellooligosaccharides, cellobiose, etc., and the structural complexity of natural cellulose.
However, cellulase is a complex enzyme that decomposes cellulose into its constituent sugars through the harmonious interaction of these multiple enzymes. In recent years, cellulases have attracted attention from the perspective of effectively utilizing biomass resources, and have been actively researched. However, cellulases produced by well-known microorganisms such as Trichoderma and Asbergillus do not have sufficient decomposition power for natural cellulose, and cannot completely decompose cellulose into glucose, producing cellobiose and even more cellulose oligomers. There were problems such as producing a large amount of sugar. Furthermore, conventionally known cellulases usually have poor thermostability and can only react at about 45 to 50q0 in long-term saccharification reactions, so they are susceptible to contamination with bacteria during saccharification. There was a danger that. The present inventors have searched extensively for microorganisms in the natural world in search of cellulase-producing bacteria that have excellent decomposition power for crystalline cellulose and saccharification power to glucose. A filamentous fungus identified as belonging to the genus Acremonium was isolated.

In order to use the cellulase produced by this bacterium industrially, we have continued to conduct intensive research into improving the production titer. As a result, we have found that when cellulase-producing bacteria of the genus Acremonium are cultured in the presence of polyethylene glycol, the cellulase is reduced. It was confirmed that the production volume will increase significantly. The present invention has been made based on this knowledge. That is, the present invention relates to a method for producing cellulase, which comprises culturing cellulase-producing bacteria of the genus Acremonium to produce cellulase in the presence of polyethylene glycol.

The details of the present invention will be explained below.

Examples of the polyethylene glycol used in the present invention include polyethylene glycol 1000, polyethylene glycol 200u, polyethylene glycol 40,
Various types with different degrees of polymerization are known, such as 00,
All of these can be effectively applied to the present invention, but especially polyethylene glycol 100
Those with a low degree of polymerization were more effective.

Polyethylene glycol is usually added at 0.0% to the medium.
It is added in an amount of about 0.01 to 5%, preferably about 0.05 to 0.5%.

When Acremonium bacteria are cultured in a medium to which polyethylene glycol has been added, the production amount of cellulase is significantly increased compared to the case without the addition of polyethylene glycol. For example, among cellulases, avicelase activity is 1.1 to 1.
5 times, carboxymethyl cellulase increases 1.5 to 2.9 times, and 3-glucosidase increases 1.3 to 2 times. The mycological properties of the cellulase-producing bacteria used in the present invention are as follows.

Growth: Growth is fast on malt extract agar, reaching a diameter of 7 cm in 30,007 days.

The colony is initially white and later becomes slightly yellowish. The aerial hyphae are loosely swollen, wool-like, and sometimes form rope-like hyphae. In the later stages of cultivation, the underside of the colony becomes pinkish-brown to reddish-brown. Almost the same growth was observed on Abec agar, but the aerial mycelia were slightly more bulging. Growth pH range is 3
．． 5 to 6.0, the optimum pH is around 4, and the growth temperature range is 15
qo to 43 qo, and the optimum growth temperature is around 30 qo. Morphology: The diameter of the hyphae is 0.5 to 2.5 mm, colorless, and septa are observed in the hyphae. In addition, the hyphal surface is smooth.
Conidia: The ability to form conidia is extremely unstable and is abolished by subculture on Abek's agar and malt extract agar media.

When observed during isolation, the conidiophores protrude from the sides of the aerial hyphae and are colorless. Conidia are subglobose (2.5-5 x 2
~4.5 m), grain-faced, colorless, and the chains are very loose and easily dispersed. Regarding the above mycological properties, W. GamS's "Cephalosporlumareige Schi"
mmelpilge” P84, G. Fisher (19
1971) and C. Day. Dickinson. Mycol
．． As a result of referring to 115PI0 (196 Mother King), Pape thought that it was appropriate to consider this fungus to be a filamentous fungus closely related to the genus Acremonium. Note that Acremonium bacteria have not been known to have strong cellulase productivity, and the strain of the present invention produces a strong and characteristic cellulase. Celluloliticus (Acremo)
mmmcell mountain oyticus). In addition,
This bacterium has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as FERMP-6867. The genus Acremonium is a genus name recently adopted after a detailed study by Cams and a reexamination of the genus previously described as the genus Cephalosporium. Bacteria of the genus Acremonium that produce strong true cellulases, ie, avicelase and FPase, capable of acting on cellulose are completely unknown prior to the present invention.

The cellulase produced by this bacterium is an enzyme that acts on Avicel, which is a highly crystalline cellulose, due to its action characteristics, such as C, an enzyme represented by Avicelase or FPase,
It is a complex enzyme system consisting of three main enzyme groups: a Cx enzyme represented by so-called CMCase, which acts on carboxymethylcellulose (CMC), and 8-glucosidase, which acts on cellooligosaccharides such as cellobiose.

Through the harmonious interaction of these enzyme groups,
Natural cellulose can be completely broken down into glucose. The properties of these enzymes are described below. W 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.'21 Action pH and optimum action p
H The action pH range of this enzyme is 2 to 8, and the optimum action is approximately 4.5.
was recognized.

(Figure 1a) [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.

(Fig. 1c) '4} Action temperature range and optimal action temperature This enzyme acts at high temperatures up to about 90°C, but it can be reacted for 10 minutes under 1% Avicel 0.08M acetate buffer (pH 4.5). The optimum working temperature was found to be around 65°C.

(Figure 1b) [5} The thermostable enzyme was prepared under 0.08M acetate buffer (pH 4.5).
As a result of heat treatment at each temperature for 10 minutes, this enzyme was approximately 60q
There was almost no inactivation at temperatures up to 100° C., about 50% at 65° C. for 1 minute, and about 80% at 70° C. for 10 minutes.

(Figure 1d) [6] Inhibitor Among various heavy metal ions, it is strongly inhibited by mercury ion and copper ion at 1mM or more.

It is also inhibited by approximately 80% at lmM by the SH inhibitor parachlormercury benzoate. {
7} Purification method This enzyme is purified from cultured monkey fluid using holofiber (Amicon HI-
After desalting and concentration using DEAE-sepharose (CL-string), it was subjected to force-lamb chromatography (Na
Further purification can be achieved by rechromatography using the same column (CI○→IM gradient) (NaCI O→0.8M).

'8- Molecular Weight The molecular weight measured by the gel filtration method using a Bio-gel (AO.Same) power ram was about 140,000.

{91 Activity assay method Apicel suspension at 0.5% concentration in 0.1M acetate buffer (
An appropriate amount of enzyme solution was added to a solution with a pH of 4.5) 0.5, the total volume was adjusted to 1.0 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 mol of glucose per minute was defined as 1 unit. '
B} Enzymatic properties of CMCase '11 Multicomponent CMCase CMCase is separated into at least four components by disk electrophoresis, and each component is distinguished by its molecular weight and isodew point.

CMCase 1 has a molecular weight of approximately 160,000 and an isodew point of 5.
08 and below, 0 is approximately 160,000, 4.95, m is approximately 120,000, 4.60, W is approximately 120,000,
4.48, and from the composite of these isozymes, CM
Case is made up of. Acts on soluble cellulose derivatives such as '2' carboxymethyl cellulose (CMC),
A component that decomposes this into glucose, cellobiose, etc. (
CMCase 1 and 0) and components (CMCase m, W) that produce very little glucose and have the effect of decomposing it into cellooligosaccharides of cellobiose or higher.

[3} Working pH and Optimal Working pH The working pH range of the CMCase complex was approximately 2 to 8, and the optimum working pH was found to be about 4.5.

(Figure 1a) '4} Stable pH The stability range of the CMCase complex when left for 20 hours at 4500 in a citric acid-phosphate buffer is approximately 3.
It was 5 to about 6.

(Figure 1C) {5} Working temperature range and optimum working temperature This CMCase complex works at high temperatures up to about 90 qo, but it is not recommended to use 1% CMC, 0.09 M acetate buffer (pH 4.
The optimum working temperature was found to be about 65 oo when reacted for 10 minutes under 5).

(Fig. 1b) The '6' thermostable enzyme was dissolved in 0.08 M acetate buffer (pH 4.5).
As a result of heat treatment at each temperature for 10 minutes, this enzyme was approximately 6
There is almost no deactivation at temperatures up to 0qo, and at 65qC, 1
About 40% was lost by heating at 70° C. for 10 minutes, and about 70% by heating at 70° C. for 10 minutes.

(Fig. 1d)'7) Inhibitor Among various heavy metal ions, it is strongly inhibited by mercury ion and copper ion at 1mM or more.

[8' Purification method This enzyme was extracted from cultured monkey fluid by holofiber (Amicon HI-
After desalting and concentration using DEAE-sepharose (CL-set), it was subjected to column chromatography (Na
It can be purified into each component by rechromatography using the same column (CIO → IM gradient) 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 skin, the total volume was made up to 1.0' with distilled water, and the reaction was carried out at 50:00.

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 French mol of glucose per minute was defined as 1 unit.

Enzymatic properties of C1 8-glucosidase [1] Action: Acts on cellooligosaccharides such as salicin, cellobiose, cellotriose, cellotetraose, cellobentaose, 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 Km values for salicin, cellobiose, celltriose, cellotetraose, cellobentaose, and cellohexaose are 3.40, 2.26, and 1.16, respectively.
They were 0.82, 0.52 and 0.51 mK. ■
Action range and optimum action pH The action range of this enzyme was 2 to 8, and the optimum action pH was found at approximately 4.5.

(Figure 1a) [3] Stability - Stable pH range was about 3.5 to about 5 when left at 490 for 20 hours under citric acid-phosphate buffer.

(Figure 1c) {4} Action temperature range and optimal action temperature This enzyme acts at high temperatures up to about 90%, but under 1% salicin and 0.09M acetate buffer (pH 4.5)
The optimum working temperature was found to be about 6yo when reacted for minutes.

(Figure 1b) ■ Thermostability As a result of heat treatment at each temperature for 10 minutes under 0.09M acetate buffer (pH 4.5), this enzyme was hardly inactivated at high temperatures up to about 65°C. , about 40% deactivated by heating at 70°C for 10 minutes, and 90% by heating at 80qo for 10 minutes.
More than % was inactivated.

(Fig. 1d) '6} Inhibitor Among various heavy metal ions, it is strongly inhibited by mercury ion and copper ion at 1mM or more.

Furthermore, glucose-6-lactone acts as an anti-inhibitor against the substrate. {7) Purification method This enzyme is purified from the culture solution using holofiber (Amicon HI-
After desalting and concentration using DEAE-Sepharose (CL-Han), it was subjected to lamb chromatography (Na
By using CIO → IM gradient), chromatography (pH 6-4), and gel filtration with Bio-gel (AO. The molecular weight was approximately 240,000 as determined by gel filtration using a .

'9' Activity assay method 1% dissolved in 0.1M acetate buffer
An appropriate amount of the enzyme solution was added to a salicin solution (pH 4.5) at a concentration of 0.5, the total volume was adjusted to 1.0 with distilled water, and the reaction was carried out at 50°C.

The produced glucose was then measured by the Somogyi-Nelson method. Under these conditions, the amount of enzyme that produced a reducing power equivalent to 1 rmol of glucose per minute was defined as 1 unit.

The B-glucosidase of the present invention includes salicin and cellobio-glucosidase.
The enzyme has a greater affinity for oligosaccharides such as cellohexaose and cellobentaose than for small molecular substrates such as cellophentaose, and this enzyme also acts on high molecular weight substrates such as Avicel.

In particular, the existence of an enzyme with substrate specificity that can degrade cellobiose and degrade Avicel to a considerable extent was revealed for the first time by the present invention, and furthermore, the fact that all the products are glucose indicates that the enzyme produced by this bacterium is 8-glucosidase is a novel 8-glucosidase that has not been previously known, and the present inventors named this enzyme glucocellulase because it has action characteristics very similar to glucoamylase on starch. Thus, the cellulase produced by the Acremonium bacterium of the present invention is a novel 8-cellulase.
-A novel cellulase complex enzyme agent containing glucosidase.

Next, the details of the present invention will be explained with reference to Examples.

Example 1 Cellulose 4%, KH2P04 1.2%, Bactobeptone%, ZnS〇4.7 days 201×10-3
%, KN〇30.6%, MnS04・7 days 201×10
-3%, urea 0.2%, CuS04・8LOI×10-
3%, KCIO. 16%, pH 4.0, M ratio 04/7 ratio 00.12%, and a medium to which polyethylene glycol 1000 was added in an amount of 0.05 to 0.4%, 200M of each 20% Place in a Erlenmeyer flask,
After sterilization by a conventional method, Acremonium cellulolyticus (FERM P-6867) was inoculated and cultured aerobically for 8 days at 30 pm.

After culturing, the supernatant obtained by centrifugation was assayed for the produced cellulase activities of avicelase, carboxymethyl cellulase (CMCase), and 8-glucosidase. The results were as shown in Table 1. As is clear from Table 1, the addition of 0.05% polyethylene glycol 1000 increased apicelase by 1.2 times compared to the case without addition.

CMCase was increased 2.1 times by addition of 0.4% amount of polyethylene glycol 1000. And 8
- Glucosidase increased 1.7 times by addition of 0.4% amount of polyethylene glycol 1000. Example
2 Acremonium celluloticus (FER
M P-6867) was inoculated and cultured aerobically at 3000 °C for 6 days.

After culturing, the supernatant obtained by centrifugation was assayed for the avicelase, CMCase, and 8-glucosidase activities of the produced cellulases. The results obtained are shown in Table 2. As is clear from the table, all of the tested polyethylene glycols 1000, 2000, and 4000 were effective for the production of avicelase, CMCase, and 8-glucosidase, but polyethylene glycol 1000 was more effective among them.

Table 2

[Brief explanation of drawings]

Figure 1 shows the cellulases Avicelase, CMCase, and 8-glucosidase produced by Acremonium (FERMP General 67), a...optimum action pH, b.
. . . Optimum operating temperature, c . . . thermal stability, and d . . . thermal stability. Next'Kaede

Claims (1)

[Claims] 1. A method for producing cellulase, which comprises culturing cellulase-producing bacteria of the genus Acremonium to produce cellulase in the presence of polyethylene glycol.