JPS6043954B2 - Method for producing cellulase - 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 enzyme reaction system that decomposes cellulose into glucose, cellobiose, and zero oligosaccharides.
There are enzymes called by various names such as x-enzyme, β-glucosidase, exo-β-glucanase, endo-β-glucanase and cellobiase, but their true nature is still unclear.

This is due to the existence of a wide variety of enzymes with different modes of action on crystalline cellulose, carboxymethyl cellulose, zero dextrin, zero oligosaccharides, cellobiose, etc., depending on the origin of the microorganism that produces cellulase, and the structural complexity of cellulose. ing. However, cellulase is a complex enzyme that decomposes cellulose into its constituent sugars, such as glucose, through the harmonious interaction of these multiple enzymes. In recent years, cellulases have attracted attention from the perspective of effective utilization of biomass resources, and have been actively researched.
There have been problems such as insufficient decomposition power for natural cellulose or inability to completely decompose cellulose into glucose, producing large amounts of cellobiose and zero-oligosaccharides.

Furthermore, most of the conventionally known cellulases have poor thermostability, so in long-term saccharification reactions, 4
The reaction could only be carried out at temperatures of about 5 to 50'C, and therefore there was a risk of contamination with various bacteria during saccharification. The inventors
As a result of extensive searches for microorganisms in the natural world in search of cellulase-producing bacteria with excellent decomposition power for crystalline cellulose and strong ability to saccharify glucose into glucose, we isolated them from soil and identified them as belonging to the genus Acremonium. Cellulases produced by filamentous fungi have strong decomposition power for crystalline cellulose, and this enzyme has significantly stronger β-glucosidase activity than conventionally well-known cellulases such as Trichoderma reesei. It was recognized that this enzyme is extremely capable of saccharification, being able to almost completely decompose cellulose into glucose.

Furthermore, since the cellulase produced by this bacterium is excellent in thermostability, it can be saccharified at a temperature 5 to 10° C. higher than the saccharification temperature when using conventional cellulases. As a result, bacterial contamination during saccharification hardly occurs, and it has been recognized that this enzyme is a novel enzyme that brings significant technical and economical benefits. 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 Acremonium bacteria capable of producing cellulase.

The present invention will be explained in more detail below. The cellulase produced by the present invention shows excellent saccharification ability even for highly crystalline cellulose such as Avicel, and the sugar composition of the product obtained by the saccharification reaction is mostly composed of glucose. occupied.

This can be cited as a major feature of the enzyme according to the present invention. Table 1 shows FPase (filter paper degrading activity), CM, which constitutes the cellulase produced according to the present invention.
The typical contents of Case and β-glucosidase are compared with those of Trichoderma reesei. As is clear from the table, the cellulase produced according to the present invention has significantly higher β-glucosidase activity than that of Trichoderma ♦ reesei.

This is considered to be the reason why most of the products produced when cellulose is saccharified by the enzyme of the present invention are glucose. In addition to this,
The β-glucosidase produced in the present invention has broader substrate specificity than conventional β-glucosidases, and includes cellooligosaccharides such as cellotriose, cellotetraose, cellopentaose, etc. in addition to cellobiose, and zero. It is a novel β-glucosidase that acts on dextrin and even high-molecular cellulose such as Avicel, decomposing it into glucose, and this enzyme is also less inhibited by the product glucose. It is thought that they are largely involved. In other words, the sugar composition of the cellulose decomposed product produced by the enzyme of the present invention is 98 to 100% glucose among the reducing sugars produced, whereas the saccharified product produced by Trichoderma reesei cellulase has a ratio of cellobiose niglucose of 1. :1 to 1:3
It is reported that.

(J. Ferment. TechrlOI., Volume M,
26th cape (Cape 197, etc.), and it is common practice to use β-glucosidase from Aspergillus niger, which has an excellent decomposition ability for cellobiose, in combination to decompose this cellobiose into glucose ( For example, AppliedMicrOblOlOgy, Volume 1, Page 419 (1968), etc.). However, since the cellulase of the present invention contains sufficient β-glucosidase, there is no need to add β-glucosidase from other sources, and cellulose can be completely decomposed into glucose. Remarks: (1) The cellulase data of Trichoderma reesei QM94l4 is from M. Mandels et al. BiOtech.
BiOenz. , XX■, P2OO9 (1981).

(2) FPase is an enzyme that decomposes a piece of filter paper (1 x 6 cm) to produce reducing sugars, and the measurement method is as described in J. Ferm
．． Tech. The activity is expressed in international units (the amount of enzyme that produces a reducing power equivalent to 1 μMOI of glucose per minute). Note that avilase and FPase are thought to exhibit the same enzymatic action. The cellulase produced by Trichoderma reesei illustrated in Table 1 is FPaseniCMCaseniβ-
While the ratio of glucosidase is 1:17.5:0.06, the same ratio of cellulase produced by the present invention is 1:17.5:0.06.
:12.1:5.4.

As described above, in the cellulase of the present invention, the composition ratio of FPase (or Avicelase': CMCaseni β-glucosidase) is usually 1:10 to 20:3 to 10, and the composition ratio of β-glucosidase is significantly high. The mycological properties of the cellulase-producing bacteria used in the present invention are as follows.

Growth: Growth is fast on malt scratched agar, reaching a diameter of 70T$t in 7 days at 30'C.

The colony is initially white and later becomes slightly yellowish. The aerial hyphae are loosely raised and wool-like, sometimes forming rope-like hyphal bundles. In the later stages of cultivation, the underside of the colony becomes pinkish-brown to reddish-brown. Growth on Tuapetsk agar is almost the same, but the growth of aerial mycelia is smaller. Growth pH range is 3
．． 5 to 6.0, the optimal pH is around 4, and the growth temperature range is 1.
The optimum growth temperature is around 3 generations, ranging from 5°C to 43°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.
Monoconidia: Conidia formation ability is very unstable and disappears by subculture on Czapetsk agar and Malt's scratch agar medium. When observed during isolation, 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 very loosely chained and easily dispersed. Regarding the above mycological properties, W. Gams 1Cepha10s
p0riumartigeSchimme1pi1? J
P84,G. Fishe,. r (1971) and C.R.
H. Dickirls On, MycOI. Papers
As a result of referring to ll5PlO (19B), it was considered appropriate to consider these two bacteria to be filamentous fungi closely related to the genus Acremonium.

It should be noted that the Acremonium genus has not been known to have a strong cellulase productivity, and the strain of the present invention produces a strong and characteristic cellulase. Therefore, this bacterium is known as Acremonium celluloliticus (Acre
It was named mOniunlcellOlyticljs). In addition, this bacterium is known as FERMP-6867.
It has been deposited at the Institute of Biological Technology, Agency of Industrial Science and Technology. The genus Acremonium is a genus name that was recently adopted after a detailed study by Gams and a reexamination of the genus previously described as the genus Cephalosporium. Powerful true cellulases that can act on cellulose, i.e. avirase and FP
Bacteria of the genus Acremonium that produce Aase were completely unknown prior to the present invention. The cellulase produced by this bacterium is
Due to its action properties, it acts on the highly crystalline cellulase Avicel, i.e., the C1 enzyme represented by Z in Avicelase or FPase, and carboxymethyl cellulose (C
MC), expressed by the so-called CMCase, and β, which acts on cellooligosaccharides such as cellobiose.
- Glucosidase is a complex enzyme system consisting mainly of Z types of enzyme groups.

Through the harmonious interaction of these enzymes, it is possible to completely break down natural cellulose into glucose. The properties of these enzymes are described below. Enzymatic properties of J 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 optimal action PH The action PH range of this enzyme is 2 to 8, and the optimum action PH is about 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 a citric acid-phosphate buffer.

(Fig. 1c) (4) Action temperature range and optimum action temperature This enzyme acts at high temperatures up to about 90°C, but it can be used in one powder under 1% Avicel and 0.05M acetate buffer (PH4.5). The optimum working temperature was found to be about 65°C when the reaction was carried out for a while.

(Figure 1b) (5) Thermostable This enzyme was prepared under 0.05M acetate buffer (PH4.5).
As a result of heat treatment for one powder at each temperature, this enzyme was hardly inactivated at temperatures up to about 60°C, 65%, about 50% after heating for 1 gin, and 70°C for 1 gin. Approximately 8% of the activity was lost.

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

It is also inhibited by approximately 80% at 1 mM by the SH inhibitor parachlormercury benzoate. (
7) Purification method This enzyme is purified from the culture filtrate by holofiber (Amicon HI-
After desalting and concentration using DEAE-Sepharose (CL-σL), column chromatography (Na
Further purification can be achieved by rechromatography using the same column (NaClO-+0.6M gradient) and ClO→1M gradient).

(8) Molecular weight The molecular weight measured by gel filtration using a BiO-Gel (AO.5rrl) column was about 140,000.

(9) Activity measurement method 0.5% concentration of Avicel suspension (PH4.5) in 0.1M acetate buffer (PH4.5)
Add an appropriate amount of enzyme solution to 111 and adjust the total volume to 1.0 rT with distilled water.
1I, 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 generated a reducing power equivalent to 1 μM 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 5.6.
8. Similarly, ■ becomes about 16,000. 4.9\■ becomes about 12.
000 to 4.6 to ■2 is about 12,000 to 4.48, and CMCase is composed of a composite of these isozymes. (2) Carboxymethyl cellulose (CMC)
The third component (CMCase I and ■) acts on soluble cellulose derivatives such as and decomposes it into glucose and cellobiose, etc., and the component (CMCase I and CMCase (■, ■) exists.

(3) Action PH and optimal action PH3. The effective pH range of the CMCase complex was approximately 2 to 8, with the optimum effective pH being found to be approximately 4.5.

(Figure 1a) (4) Stable PH The stable PH range of the CMCase complex when left for 204 hours at 45°C under citrate-phosphate buffer is about 3.5 to about 6. Ta.

(Figure 1c) (5) Operating temperature range and optimum operating temperature This CMCase complex acts at high temperatures up to about 90°C, but at 1% CMClO. O5M acetate buffer (PH4.5
) The optimum temperature for reaction is approximately 65
℃ was observed.

(Figure 1b) (6) Thermostable This enzyme was prepared under 0.05M acetate buffer (PH4.5).
As a result of heating one powder at each temperature, this enzyme was hardly inactivated at temperatures up to about 60°C, about 40% when heated at 65°C with one powder, and about 40% when heated with one powder at 70°C. 70%
Deactivated.

(Figure 1 d) (7) Inhibitor Strongly inhibited by 1mM or more of mercury ion and copper ion among various heavy metal ions (8) Purification method This enzyme is removed from the culture filtrate using a holofiber (HI-P5). After concentrating the salt, DEAE. It can be purified into each component by column chromatography (NaClO→1M gradient) using Sepharose (CL-6B), rechromatography using the same column, and chromatofocusing.

(9) Activity measurement method 1% CMC solution (PH
4.5) An appropriate amount of enzyme solution was added to 0.5r]11, the total volume was adjusted to 1.0rT11 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 generated a reducing power equivalent to 1 μM of glucose per minute was defined as 1 unit.

Enzymatic properties of β-glucosidase (2) 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 Km values for salicin, cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose are 3.40, 2.26, and 1.19, respectively.
They were 0.82, 0.52 and 0.15rr1M.
(2) Action PH and optimal action PH The action PH range of this enzyme is 2 to 8, and the optimum action PH is about 4.
5 was recognized.

(Figure 1a) (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.

(Figure 1 c (4) Action temperature range and optimum action temperature This enzyme acts at high temperatures up to about 90'C, but under 1% salicin and 0.05M acetate buffer (PH 4.5) The optimum operating temperature for one-powder reaction was found to be about 65°C.

(Fig. 1b) (5) Thermostability As a result of heat treatment of one powder at each temperature in 0.05M acetate buffer (PH4.5), this enzyme showed almost no effect at high temperatures up to about 65°C. Approximately 4 hours after heating 70'Cll powder without deactivation
0% inactivation was achieved, and more than 90% inactivation was achieved by heating one powder at 80°C.

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

Furthermore, glucose-delta-lactone acts as a competitive inhibitor against the substrate. (7) Purification method This enzyme is extracted from the culture filtrate using holofiber (Amicon HI-
After desalting and concentration using DEAE-Sepharose (CL-6B), column chromatography (N
aClO→1M gradient) and chromatofocusing (PH6-4) and BiO-Gel (AO.5rrl)
can be electrophoretically purified to homogeneity by gel filtration.

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

(9) Activity measurement method 1% dissolved in 0.1M acetate buffer
An appropriate amount of the enzyme solution was added to 0.5n11 of salicin solution (PH4.5), the total volume was made up to 1.0n11 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 generated a reducing power equivalent to 1 μM of glucose per minute was defined as 1 unit.

The β-glucosidase of the present invention has a greater affinity for oligosaccharides such as cellohexaose and cellopentaose than for small molecular substrates such as salicin and cellobiose, and the enzyme has a higher affinity for oligosaccharides such as cellohexaose and cellopentaose than for small molecule substrates such as salicin and cellobiose. It also acts on molecular weight substrates.

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

In order to produce the cellulase by Acremonium bacteria of the present invention, cellulose, Abysse. The carbon source is cellulose or cellulose-containing materials, such as cotton, bagasse, and wheat gluten, and a small amount of organic or inorganic nitrogen sources such as nitrates, ammonium salts, urea or peptone, and yeast-induced nitrogen sources are used as the nitrogen source. The cells are cultured aerobically at 20 to 40° C. for about 2 to 15 days using a liquid or solid medium containing a metal salt.

Cellulase is an enzyme produced outside the bacterial body, so in the case of liquid culture, it can be extracted with the supernatant liquid filtered after culturing, and in the case of solid culture, it can be extracted with water or an appropriate inorganic salt solution after culturing. It can be collected as enzyme solution 7. Next, the present invention will be explained in detail with reference to Examples.

Example 1 Cellulose 4%, bactopeptone 1%, potassium nitrate 0.6%, urea 0.2%, potassium chloride 0.16%, magfunesium sulfate 0.12%, monopotassium phosphate 1.2%
, zinc sulfate 1 x 10-3%, manganese sulfate 1 x 10-3
% and copper sulfate 0-3% (PH4.O) 20r
Pour T11 into a 200rT11 Erlenmeyer flask, sterilize it using a conventional method, and then remove Acremonium celluloliticus (FER).
MP-6867) was inoculated and cultured with aeration at 30°C for 6 days.

After culturing, bacteria were removed using a centrifuge, and the resulting supernatant was measured for avicelase activity, CMCase activity, and β-glucosidase activity, each of which was 3.1 u/M.
ll was 26.3u/ml and 13.8u/ml. Example 2 In Example 1, Acremonium celluloliticus (FERMP-6867) was inoculated into a medium to which 2% Bonito Sorbul was added instead of Bactopeptone.
Aerated culture was carried out at 0°C for 8 days.

After culturing, the centrifuged supernatant was treated with Avicelase,
As a result of measuring CMCase and β-glucosidase activities, they were 3.9L1/Mll45.5u/ml and 2
It was 1.1 u/ml. Add twice the volume of cold acetone to the culture supernatant, and centrifuge the resulting precipitate (9000
rpm 11 powder), dissolved in a small amount of water, and insoluble materials were removed to obtain a clear concentrated enzyme solution.

The recovery rates of Avicelase, CMCase and β-glucosidase were 91.9%, 71.7% and 96%, respectively.
．． It was 8%.

[Brief explanation of drawings]

Figure 1 shows the cellulases Avicelase, CMCase and β produced by Acremonium (FERMP-6867).
- Regarding glucosidase, a...optimal action P
H. ．． b...Optimum working temperature, c...P
H stability and d...indicates thermal stability.

Claims (1)

[Claims] 1. A method for producing cellulase, which comprises culturing cellulase-producing bacteria of the genus Acremonium and collecting cellulase from the culture.