JPS59166082A - Preparation of glucocellulase - Google Patents

Abstract

PURPOSE:To prepare novel glucocellulase, by cultivating a specific microorganism belonging to the genus Acremonium to give beta-glucocellulase separating and purifying it. CONSTITUTION:Novelly separated Acremonium cellulolyticus (FERM P6867) is cultivated in a medium containing cellulase as a carbon source, to give a large amount of cellulase consisting of three kinds of enzyme groups of C1 enzyme, Cx enzyme, and beta-glucosidase. The culture filtrate is desalted and concentrated by hollow fiber (amiron H1-P5) subjected to DEAE-Sepharose (CL-6B), column chromatography (NaClO 1M gradient), and gel filtration of chromatofocusing (pH 6 4) and Bio-gel, to give novel glucocellulase. This enzyme acts on not only cellooligosaccharide and cellodextrin, but also on avicel and hydrolyzes it into glucose.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing glucocellulose, which is characterized by culturing Acremonium bacteria.

Cellulose is made by replacing cellulose with glucose or Hirobi A-
It is a general term for enzymes 11)' that catalyze the enzymatic reaction system that decomposes S-tV) into lo-Δoligosaccharides. -glucanase, endo-β-glucano-
Although there are enzymes called by various names such as cellulase and cellulase U, their true nature is still not clear.
Dex 1-3 for phosphorus, oligosaccharide, cellobiose, etc.
The fact that natural cellulose is a water-insoluble substance with a tough 4-structure, and that hirulose has physical interactions with hemicellulose, lignin, etc. Cellulase has chemically complex bonds, and its depiction itself is not clear. However, cellulase converts cellulose into its constituent sugars by the harmonious interaction of these multiple enzymes. It is a complex enzyme that breaks down into

In recent years, cellulases have attracted attention from the perspective of effectively utilizing biomass resources, and have been actively researched. Until now, it has been known that various #I bacteria and filamentous fungi produce cellulases. However, due to the microbial origin of cellulases, their action characteristics are significantly reduced, and no two cellulases can be said to be the same. For example, the well-known cellulose of the genus Trichoderma, such as Lycoderma reesei, acts on highly crystalline cellulose such as Avihil, but it produces a large amount of cellobiose as a decomposition product. β broken down into glucose
- It is well known that glucosidase activity is extremely low. It is also known that Aspergillus R bacteria often produce cellulose, but the cellulases produced by Aspergillus bacteria are usually made from water-soluble cellulose derivatives such as carboxymethylcellulose, cellobiose and cellooligosaccharides. Although it works well on substrates for low molecules, it is said to have a weak effect on highly crystalline cellulose.

The present inventors have discovered that the decomposition power for natural cellulose is excellent;
As a result of extensive searches for microorganisms in the natural world in search of cellulase-producing bacteria with excellent saccharification ability to glucose, Acremonium was isolated from soil.
Hirulase, which is produced by filamentous fungi thought to belong to the genus Ium), is highly crystalline and acts well against lulase.
It was confirmed that this was almost completely broken down into glucose. As a result of C1 separation and purification of the β-gulunisidase enzyme, which is involved in the production of glucose, we found that it acts not only on cellooligosaccharides and lerodex l-phosphorus, but also on avicel. A novel Lerula L that decomposes and produces only glucose
admitted that it was.

The present inventors named this novel cellulase glucocellulase. The present invention has been made based on this knowledge.

That is, the present invention relates to a novel method for producing glucocellulase, which is characterized by culturing Acremonium bacteria, which are responsible for the ability to produce cellulase.

The present invention will be explained in more detail below.

Conventionally, β-glucosidase produced by cellulase-producing bacteria such as Trichoderma and Aspergillus has been known as an enzyme that decomposes cellobiose and cellooligosaccharides. These conventionally known β-glucosidases are enzymes with fairly broad substrate specificity that can decompose cello-oligosaccharides larger than cellobiose and setriose, or low-molecular-weight insoluble lerodextrins with an average polymerization rate of about 20. However, it has been said that it has no or little effect on Avicel, which has a high molecular weight and a very high degree of crystallinity (AgriO).

[3iol, CI+em, , 43 p 1
791 1979 and A, rcl+.

Biocl) em, 5iopHys, , 1 '1
1 p445 1965). However, the glucolerage produced by the present invention hydrolyzes not only cellobiose but also cellooligosaccharides and cellodex 1-phosphorus, which have a higher degree of polymerization than cellobiose, to phosphorus. do. The most distinctive feature of this enzyme is that it can act on high molecular weight, insoluble, highly crystalline cellulose such as Avicel, and the only decomposition product is glucose. Table 1 shows the Km value and maximum rate for various cellooligosaccharides of glucose cellulase produced by bacteria of the genus Acremonium, indicating that this enzyme acts well on substrates with a higher degree of polymerization. I get 4.

Also, shown in FIG. 1 is the time course of the degradation of 1% Avicel by purified glucohirulase, which shows that Avihil is clearly degraded. In addition, as a result of identifying the decomposition products at that time using Paperc [1 MAdography], it became clear that the reaction products consisted only of glucose from the early stage of the reaction.

The glucocellulase of the present invention is Acremonium (Acr
It is produced extracellularly by culturing a microorganism belonging to the genus Acremonium (Acremonium cellulolyticus), for example, which the inventors isolated from soil and identified as Acremonium cellulolyticus.

The mycological properties of Acremonium cellulolyticus are as shown below.

Growth: Growth is fast on malt extract agar, reaching a diameter of 70+i1+1 in 7 days at 30°C. I'll reach you. The colony is initially white and later becomes slightly yellowish. 3. Aerial mycelium, yoyu. It has a raised wool-like appearance and sometimes forms cotton-like mycelial bundles.

In the later stages of cultivation, the back side of the colony becomes pinkish brown or reddish brown.

Almost the same growth was observed on Czapek agar, but the growth of aerial mycelium was less. Growth p1-1 range is 3.
5 to 6.0, the optimal pl-1 is around 4, and the growth temperature range is
The optimum growth temperature is around 30°C, between 15°C and 43°C.

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

Conidia and the ability to form conidia were extremely unstable and disappeared easily after subculture on Czapek agar and malt extract agar media. When observed during isolation, the conidiophores protrude from the sides of the aerial hyphae and are colorless.

The conidia are spherical (2.5-5 x 2-4.5 μm), smooth, colorless, and the chains are very loose and easily dispersed.

Regarding the above mycological properties, W. Qams' “Ccpl
+alosporiumartige Scbimm
elpilc+eJ P84, G, Fisber
(1971) and C, I7 Dickinson, M.
ycol, Papers 115 PIO (19
As a result of referring to 1968), the water bottle is Acre[nium (Ac
We believe that it is appropriate to consider it to be a filamentous fungus closely related to the genus rcmonium. It should be noted that the genus Acremonium was not previously known to have strong cellular productivity.
In addition, since the strain of the present invention produces a cellulase with strong horns and characteristics, the present strain is classified as Acremonium hirlolyticus (A Crelli (ill!ulllce)).
1.61 olyticus). The water head has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as FERM P=6867.

Acremonium flexi is a local name that was adopted in recent years after a detailed study by Ga+ns and a reduction in the genus previously described as Cephalosporium. Therefore, there have been no descriptions of chelulase-producing bacteria using the local name of the genus Acremonium. Furthermore, Leph7.L1 Suborium bacteria that produce strong true cellulose agents that can act on crystalline cellulose, such as noavirerase and Page, as shown in the present invention, had not been known before the present invention. Not known at all.

In addition, the purification method and enzymatic quality of glucocellulase produced by Mokukan are as shown below.

(1) Action: Acts on lelo-oligosaccharides such as salysin, cellobiose, lerotriose, cellotetraose, cellopentaose, and cellohexaose, completely decomposing them into glucose1. It also acts on high molecular weight cellulose, but has almost no effect on CMC (carboxymethylhirulose) or 1-IEC (hydroxyedyllerulose). Salicin, cellobiose, cello1-liose, cellotetraose, cellopentaose, and lelohekilyose were 0.51111M.

(2) Effect p)-1 and optimal effect D t-1 Effect of this enzyme pf- (range 2 to 8, optimum action pH was observed at about 4.5 (Figure 2 (a)) 3) Stable pH Stable 1) H range was about 3.5 to about 5 when left at 45° C. for 20 hours under citric acid-phosphate buffer. (Figure 2 (C)) (4) Action temperature range and optimal action temperature This enzyme acts at high temperatures up to approximately 90°C, but 1%
Salicin, 0.05 M acetate buffer (DI-14,5)
The optimum temperature of action when reacted for 10 minutes under
It was observed at 5°C. (*N2 diagram (b)) (5) Under thermal stability 0.05 M acetate buffer (1) l-14,5),
As a result of heat treatment at each temperature for 10 minutes, this enzyme was approximately 65℃
There was almost no inactivation at temperatures up to 70° C. for 10 minutes, about 40% inactivation occurred at 70° C. for 10 minutes, and more than 90% inactivation occurred at 80° C. for 10 minutes. (Figure 2(d)) (6) Inhibitor Among various heavy metal ions, mercury and copper ions above 1mMg are strongly inhibited. Also, glucose
Lactones act as competitive inhibitors on substrates.

(7-')) Ft'i production method This enzyme is extracted from the culture filtrate with small fibers (amine l-1).
After desalting and concentrating using DEAE-Sepharose (CL-6B) and column Chroma 1 to Graphi (
Na C1O → 1M gradientene 1~), chromatofocusing (pi-16 → 4) and Bio-(tel (
It can be electrophoretically purified to homogeneity by gel filtration using A 0.5 m ).

(8) Molecular Weight The molecular weight measured by gel filtration using Bio-get (A 0.5m ) was about 240,000.

(9) Activity measurement method 1%'liricin solution dissolved in 0.1M acetate buffer (
1) H4,! i) Add appropriate enzyme solution to o, sm+ and suspend with distilled water.1. Om+ and the reaction was carried out at 50°C. Then, the produced glucose was measured by Sochiky-Nerlan 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.

In order to produce glucocellulase using Acremonium bacteria of the present invention, cellulose, Avicel, cotton,
Using cellulose or relulose-containing substances such as barbu, barbeque, and wheat gluten as a carbon source, and using this as a nitrogen source, pair 1 to
Using a liquid or solid medium containing an organic or inorganic nitrogen source such as soybean flour, soybean meal, meat extract, yeast extract, nitrate, ammonium salt, urea and a small amount of gold n salt, at 20-40°C.
The cells are then cultured aerobically for about 2 to 15 days. Glucocellulase is an enzyme produced outside the bacterial body, so in the case of liquid culture, it is extracted with the filtered supernatant after cultivation, and in the case of solid culture, it is extracted with water or an appropriate inorganic salt solution after cultivation. By doing so, it can be collected as an 8M solution.

Then, if necessary, it is salted out or powdered with an organic solvent.

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

Example 1 Cell[]-244% K1-12PO41.2%, Baku1~Pep]~n 1%, Zn SO4・7H201X
10=%, K N 03 0.6%, MlISO4
・7H201X 10-3%, 1 hydrogen 0.2%, C
uSO4・5 days 201×10-3%, K(40,16
%, Li->40 lx 10-2%, M(] SO
4.71120 0.12%, p1~14.0, After sterilizing the medium by a conventional method, 30% of Acremonium cellulolyticus (FERMP-6867) was used.
The cells were cultured aerobically for 8 days at ℃. The glucocellulase produced in the supernatant obtained after culturing and before centrifugation was measured and found to be 19.1 u/ml.

iJ>, avirerase and carbo produced simultaneously,
Coconut Methyllerur U was 4.6 u/ml and 35.7 LI/nll, respectively.

[Brief explanation of the drawing]

Figure 1 shows crystalline cellulose produced by glucocellulase produced by Acremonium bacteria. The graph shows the change in the decomposition of the molecule over time. Figure 2 shows (a) optimal action p1-1, (l]) optimal action temperature, <C,) l) H stability, and <d) thermostability of glucocellulase produced by Acremonium bacteria. τ
There is. J'-'y・() 1・・ ] Patent applicant Director of the Agency of Industrial Science and Technology tj Makoto Saka, 1i
J-i,,□・゛. Kanhara tribe 1. Chang 11・,,,,,−,−,,＞,i
S l +”3 Reaction time PjI (H strange) Office application procedure manual (spontaneous) February 9, 1980 Mr. Commissioner of the Japan Patent Office 1, Indication of the case Patent application No. 384 of 1982
32 No. 2, Title of invention Method for producing glucocellulase Name (114) Director of I Industry Technology Institute
1) Hirobe 5, Date of amendment order f' Voluntary action 6, Number of inventions increased by amendment (N/A)
Paper 1, special gtf claims are amended as follows. "A method for producing glucocellulase, which is characterized by culturing a bacterium of the genus Acremonium that produces glucocellulase, and collecting glucocellulase from the culture." 2. 1 conidium, 1 minute, on page 6, line 9 of the specification. "The ability to form conidia is corrected to the ability to form conidia and biconidia." 3. “Easily disappears and IC0” on page 6, line 10 of the specification
1 disappears. ” is corrected. 4. On page 7 of the specification, lines 6-7, “Recently adopted...
...No description yet. Furthermore, the term ``the present invention'' has been amended to ``the present invention, which has been adopted in recent years and has a name of the genus Iζ.'' 5. On page 7, lines 9-10 of the specification, 1. "Cephalosporium" is corrected to "Acremonium." 6. In the 7th line of page 10 of the specification, "to extract" is corrected to "to extract." 411

Claims (1)

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