JPS59166081A - Preparation of cellulase - Google Patents

Abstract

PURPOSE:To obtain cellulase in high yield, by cultivating a specific microorganism belonging to the genus Acremonium. CONSTITUTION:A microorganism belonging to the genus Acremonium was not known to have strong cellulase productivity conventionally, Acremonium cellulyticus (FERM P6867) having strong cellulose productivity is separated. This microorganism is cultivated in a medium containing cellulose as a carbon source aerobically under aerobic conditions, to give a large amount of cellulase. Cellulase is a combined enzyme consisting of three kinds of enzyme groups of C1 enzyme, Cx enzyme, and beta-glucosidase. By harmonized interaction of the enzyme groups, natural cellulose is completely hydrolyzed into glucose.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing lulase from the genus Acre.

Cellulase converts cellulose into glucose or L! ]] It is a general term for a group of enzymes that catalyze the enzymatic reaction system that decomposes biose and L7[1 oligosaccharides, and due to their mode of action, CI
There are enzymes called by various names such as BW14, Cx enzyme, β-glucosider 1, exo-β-glucanase, endo-β-glucanase, and kerobiase, but their actual nature is not yet clear. Due to the origin of microorganisms that produce cellulase, it is possible to produce crystalline cellulose, carboxymedylalyl [coase, lerodex-1-phosphorus,
This is due to the existence of a wide variety of enzymes with different modes of action on cellulose, cellulose, etc., and the complexity of the 4hi structure of cellulose. However, cellulase is a complex enzyme that decomposes C-lulose into its constituent sugars, such as glucose, through coordinated phase L1 action of these multiple enzymes.

In recent years, cellulase has attracted attention from the perspective of effective utilization of biomass resources, and has been actively studied.
However, the cellulases of the genus Trichoderma and Aspergillus, which are conventionally known for garlic, either do not have sufficient decomposition power for natural cellulose, or cannot completely decompose cellulose into glucose, resulting in a large amount of rerobiose-11) relo-oligosaccharides. There were problems such as generating. Furthermore, most of the conventionally known cellulases have poor thermostability, so they can only be reacted at a temperature of about 45 to 50°C during long-term saccharification reactions, and as a result, contaminants may be present during saccharification. There was a risk of contamination.

The present inventors have searched extensively for microorganisms in the natural world in search of cellulase-producing bacteria that have excellent decomposition horns for crystalline cellulose and have a strong ability to saccharify old glucose. Separated from Acremonium (Acrc
Cellular U, which is produced by a filamentous fungus identified as belonging to the genus Moniun+, has a strong decomposition power against crystalline cellulose.
Shika:b-glucosidase activity of b.
It was found that this enzyme is extremely strong in saccharification, being significantly stronger than known cellulases such as those produced by Trichoderma reegei, and capable of almost completely decomposing cellulose into glucose. Furthermore, even though the cellulase produced by Canteen has a thermostability of J3 (9), it can be saccharified at a temperature 5 to 10°C higher than the saccharification temperature when using conventional cellulase.

For this reason, bacterial contamination during saccharification almost never occurs.
Once available, it has been recognized that this is a novel enzyme with significant technological and economical benefits. The present invention was made based on this knowledge.

In other words, the present invention relates to a method for producing cellulase, which comprises culturing Acremonium bacteria having a high ability to produce hillase.

The present invention will be explained in more detail below.

The cellulase produced by the present invention exhibits excellent saccharification ability even for highly crystalline cellulose such as Avicel, and the sugar composition of the product obtained by the saccharification reaction is
Most of it is occupied by glucose. This means that n)! This can be cited as a major feature of the accumulation. Table 1 shows the FPase filter decomposition activity, CMC, which forms 4M of the cellulase produced in the present invention.
Fig. 3 compares typical contents of Aase and β-curcosidase with those of Trichoderma reesei.

As is clear from the table, the cellulase (β-glucosidase activity) produced by the present invention is significantly higher than that of Lycoderma leii. In addition to this, the β-glucosidase produced in the present invention has broader substrate specificity than conventionally known β-glucosidases, and has a wide range of substrate specificities, including cellobiose, hylotriose, rerote 1- This enzyme is a novel β-curcosidase that acts on cellooligosaccharides such as laose and relopentase, cellodextrin, and even high-molecular cellulose such as avihil, decomposing them into glucose. It is thought that this is largely due to the fact that the inhibitory effect of glucose, which is a product of , is small.

That is, the sugar composition of the cellulose decomposed product by the enzyme of the present invention is that glucose in the resulting reducing sugars is 98 to 100%.
On the other hand, it has been reported that the ratio of cellobiose:glucose is 1:1 to 1:3 in saccharified products by Trichoderma reesei cellulase.

(J, Fcrment, 1-Echnol, vol. 54, p. 267 (1976), etc.) In order to decompose this hirobiose into glutinose, Aspergillus sp. Nigger's β
-Use of curcosidase (this is a common practice).

(For example, Applied MiCrObiOlooy
, Vol. 16, p. 419 (1968), etc. However, since the cellulase of the present invention contains a sufficient amount of β-glucosidase, there is no need to add β-glucosidase from other sources, and it can completely (decompose hillulose into glucose). be.

Notes to Table 1; (1) Cellulase data of Trichoderma reesei QM 941/l is based on M9 Mandel et al. [3iot
ech, [3ioenz, , XX I I 12
009 <1981> was cited.

(2) FPase decomposes filter paper pieces (IX6cm) to produce reducing sugars.The measurement method is J. Ferm, Tech., Vol. 54, 267.
On page 197B, the activity is shown in international units (1
The amount of enzyme that produces a reducing power equivalent to 1 μn+ol of glucose per minute). It is thought that Avicelase and FPase exhibit the same enzymatic action.

Cellulani 1 produced by Trichoderma reesei, illustrated in m1'8, has a ratio of FP7-se:CMCase:β~glucosidase of 1:17, s: 0.06, whereas the production of the present invention The equivalent ratio of 1:1 cellulase is 1:12, 1:5.4. As described above, the cellulase of the present invention has a composition ratio of FPase (or) bicerase): CMCase: β-glucosidase [usually 1:10 to 20: 3 to 10], and β-
It is an enzyme with a significantly high composition ratio of glucosidase.

The mycological properties of the cellulase-producing bacteria used in the present invention are as follows.

Growth: Growth on malt extract agar is rapid, reaching a diameter of 7 Qmm in 7 days at 30°C. The village is initially white and later becomes slightly yellowish. The aerial hyphae grow loosely and become woolly, sometimes forming thin hyphal bundles.

In the later stages of cultivation, the village demon mask takes on a pinkish-brown to reddish-brown color.

C on Zapek agar showed similar growth, but the growth of aerial mycelium was less. Growth pH range is 3.5-,
At 6.0, the optimum pH is around 4, and the growth temperature range is 15℃~
At 43°C, the optimum growth temperature is close to 30°C.

Morphology: The diameter of the hyphae is 0.5 to 2.5 μm1, colorless and septa are observed on the hyphae1, and the surface of the hyphae is clear.

Conidia and the ability to form conidia were extremely unstable and were easily lost by subculturing on Czapek agar and malt extract agar media. Observation on 6th day of isolation revealed that the conidiophores protruded from the sides of the vaporized hyphae and were colorless and transparent.

Conidia are slender spherical (2.5 to 5 x 2, 4.5 μm)
It has 87 faces, is colorless, and the chain is very loose and easily dispersed.

Regarding the above mycological properties, W, Gam5 [Cepb
alospori LImartige Scl+im
melpi1geJ Pa4, G, F 1sher
(1971) and Hi C, I-1, Dicl (inso
n, Mycol, Paners 115 Pl
0 (7968), we concluded that it is appropriate to consider woody fungi to be filamentous fungi closely related to the genus Acremonium. It should be noted that the Acremonium genus has not been known to have strong hillase productivity, and the strain of the present invention produces a strong and characteristic cellulase. Cellulolyticus (ACremOniUmcel I
ulolyticus). J5, water bottle,
"r:RM p-6867-(, deposited with the National Institute of Microchemical Technology, Agency of Industrial Science and Technology).

The genus Acremonium is a name recently adopted by GamS after a detailed investigation and re-examination of the genus previously listed as the genus Lephalosborium. Therefore, using the station name of Acremonium
There is no description of this cellulase-producing bacterium yet. Moreover, prior to the present invention, no Cephalosporium bacterium that produces strong true cellulases, ie, avirerase and FPase, capable of acting on crystalline cellulose as in the present invention was known.

Cellulase produced by wood fungi is an enzyme that acts on Avicel, which is a highly crystalline cellulase due to its action characteristics, that is, the C1 enzyme represented by avirerase or FPase, and acts on carboxymethyl cellulose (CMC).
It is a complex enzyme system mainly consisting of three enzyme groups: cx enzyme represented by so-called C, MCase, and β-glucosidase that acts on cellooligosaccharides such as cellobiose. The harmonious interaction of these enzymes makes it possible to completely decompose natural cellulose into glucose. The properties of these enzymes are described below.

(△) Enzymatic properties of abisylase (1) Action: Reruulose powder, Avicel, j] (2) Acts on highly crystalline insoluble hiruulose to produce reducing sugars such as glucose and cellulose. .

(2) Effect p1-1 and optimal effect p1-1 Effect of this enzyme 1) The H range was 2 to 8, and the optimum effect p+-+ was found to be about 4.5. (Figure 1(a)) (3) Stable pH The stable p1-1 range was about 3.5 to about 6 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 1 This enzyme acts at high temperatures up to approximately 90°C, but 1%
Avirel, when reacted for 10 minutes under 0.05 M acetate buffer (pH 4, 5), the optimum working temperature is approximately 65°C.
was recognized. (Figure 1(b)) (5) Thermostability The enzyme was heat-treated for 10 minutes at each temperature in a 0.05M acetate buffer (pl-14,5).
There was almost no inactivation at temperatures up to 0°C, about 50% when heated at 65°C for 10 minutes, and about 80% when heated at 70°C for 10 minutes. (Figure 1(d)) (6) li,
l] 1m1v among various harmful agents ions! It is strongly inhibited by the above mercury ions and copper ions. In addition, the SH inhibitor parachlormercury benzoei 1 ~ caused about 80% IVI damage at 1111M.

(7) Purification method: This enzyme is extracted from the culture filtrate using Borofiber (Amicont II).
-P5), followed by column chromatography (DEAE-Sepharose (CL-6B)).
NaCI O → 1M Re-chromatography using the same column as Grady Gene (NaCI O → 0.6M)
This allows for further purification.

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

(9) Activity measurement method 0.5% in 0.1M Hei-1 acid buffer (pt-1/1.5)
Avicel suspension (pI-14,5) at a concentration of 0.5 m
Add an appropriate amount of enzyme solution to 1 ml, and dilute the total volume with distilled water. The reaction was carried out at 50°C. The reducing sugar produced was measured by the Somogyi-Nerlan method.

In this strip A'1, 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 method of CMCase CMCase is separated into at least four components by disk electrophoresis, and each component is distinguished by its molecular weight and isoelectric point. CMCase I has a molecule ω of approximately ieo, ooot"-isoelectric point of 5.'08, and similarly, ■ of approximately 160,000°4
．． 95, mha approximately 120,000. 4.60,
There are about 120,000.4.48r of IVt, i, and CMCase consists of a complex of these isoIJ-im.

(2) Components (CMCase I and ■) that act on soluble cellulose derivatives such as carboxymethyl cellulose (CMC) and decompose it into glucose and cellobiose, etc. and cellooligosaccharides that produce very little glucose and are more than cellobiose. A component that has the effect of decomposing into CM
Case ■, IV) exists.

(3) Effect p1-1 and optimum effect pI-1 The action pH range of the CMOase complex was approximately 2 to 8□, and the optimum effect DI-1 was found to be approximately 4.5. (Figure 1 (a.
)) 4) Stability L) Stability of the CMCase complex when exposed to l-1 at 45°C for 20 hours under l-1 citric acid phosphate buffer 1) H range from about 3.5 to about 6 there were.

(Figure 1 (C)) (5) Action temperature range and optimal action temperature This CMCase complex is active at high temperatures up to about 90°C, but at 1% C
MC, 0.05M acetate buffer (pl-14,5> (7
)The optimum temperature for reaction after 10 minutes of heating is approximately 65.
℃ was observed. (Figure 1 (b)) (6) Add the thermostable enzyme to 0.05 M acetate buffer (1) H/1.!i
) 17) Below, each fiAI'J-Q was heat-treated for 10 minutes and the results showed that this enzyme was hardly inactivated at temperatures up to about 60'C, and about 40% was inactivated by heating at 65'C for 10 minutes.
(TL/70% deactivated by heating for 10 minutes at 770°C. (Figure 1 (d)) be done.

(8) tFfi production method This enzyme is extracted from the culture filtrate by holofiber (+-11-P5
) After desalting and concentrating, re-chromatography using a column of EA E-Sepharose (CL-68) and the same column with 1 M gradientene (NaCI O->1 M gradientene 1~) and chromatography using the same column. It can be purified into each component by casing.

(9) Activity measurement method 1% CMC'a solution dissolved in 0.1M acetate buffer (
To 0.5 ml of pf-14,5 was added an enzyme solution of a caterpillar, the volume was made up to 1.0 ml with distilled water, and the reaction was carried out at 50°C. The reducing sugar produced by C1 was measured by the Somoky-Nelson method.

In this section f'F, the enzyme unit that generates a reducing power equivalent to 1 μmol of glucose per minute was defined as one unit.

(C) Enzymatic properties of β-glucosidase (1) Action: Acts on cellooligosaccharides such as lyricin, cellobiose, seol-lyase, lysine-laose, zeropentaose, and cellohexaose. It decomposes into glucose.Although this enzyme also acts on various polymeric relases of Avisil, it has very little effect on CMC and l-1#c (hydroxyedyl cell [course)].
The K11l values for lyricin, e-robiose, tro-1-liose, lerotetra-A-ose, leropentaose, and serohegi-1)-ose were 3.40 and 2.2, respectively.
6.1.19.0.82.0.52 and 0.51mf
I was drowsy in vl.

(2) Action pH and optimal action 1) 8 Production of this enzyme January] 1-1 range is 2 to 8, optimal action p+
-+ was observed at about 4.5. (Figure 1(a)) <3) Stable p1-1 The stable pH range was about 3.5 to about 5 when left at 45°C for 20 hours under citric acid-phosphate buffer. (
Figure 1 (C) The optimum working temperature was found to be about 65°C when the reaction was carried out for 10 minutes at 1°C. (Figure 1 (b)) (5) Thermostability As a result of heat treatment at each temperature for 10 minutes under 0.05M acetate buffer (1) l-14,5), this enzyme was stable at temperatures up to approximately 65°C. At a high temperature of , almost no deactivation occurred, when heated at 70°C for 10 minutes, about 40% of the activity was lost, and when heated at 80°C for 10 minutes, more than 90% of the activity was lost. (Figure 1 (d)) (6) Inhibitor: 1mM or more of mercury ion J3 among each sand mold metal ion
J: Strongly inhibited by copper ions. Glucose-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 II-
P'5), 1 3io-gel (
It can be purified to electrophoretic homogeneity by gel filtration at AO, 5m).

(8) The molecular fit measured by the glue 6a filtration method using the molecule ω 3io-get (A 0.5111 > is about 240.00
It was 0.

(9)) Activity measurement method 1% lyricin solution (p
t14.5) Add the appropriate amount of enzyme solution to 0.5 ml, and add distilled water to complete the solution.1. The reaction was carried out at 50°C.

The produced glucose was then measured by the Somogyi-Nelson method.

In this strip A'1, one unit of alcoholic acid that can generate a reducing power equivalent to 1 μIII (+1) glucose per minute was defined as one unit.

The β-glucosidase of the present invention has a greater affinity for oligosaccharides such as celloheklyose and hyropentaose than for small molecule substrates such as lyricin and rerobiose, and the enzyme has a greater affinity for oligosaccharides such as celloheklyose and hyropentaose. It also acts on high molecular weight substrates. In particular, the existence of an enzyme with substrate specificity capable of degrading bios and a considerable amount of cellulose was revealed for the first time by the present invention, and all of the products are glucose. The present inventor discovered that the β-glucosidase produced by Honkan is a novel β-glucosidase C' that has not been previously known, and has action characteristics very similar to the glucoamylase found in starch. named this enzyme glucohirurage.

Thus, Lerula L produced by the Acremonium bacterium of the present invention is a novel cellulose complex enzyme agent containing a novel β-glucosidase.

In order to produce Lerula 1 using bacteria belonging to the genus Acre[nium] of the present invention, a lelurose-containing material such as lelurus, avicel, cotton, bagasse, wheat gluten, etc. Using a liquid or solid medium containing carbon as a carbon source, an organic or inorganic nitrogen source such as nitrate, ammonium salt, urea or peptone, yeast extract, and a small amount of metal jAa as a nitrogen source, C, 2-150
Feed aerobically for about 18 hours. Cellulase is an enzyme produced outside the bacterial body, so in the case of liquid culture, the supernatant liquid filtered after culturing, and in the case of solid culture, after culturing,
It can be collected as an enzyme solution by extracting and covering with water or an appropriate saline solution.

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

Example 1 Cellulose 4%, Bactopeb 1-1%, potassium nitrate 0.
6%, urea 0.2%, potassium chloride 0.16%, W11 acid magnezi 1 cum 0.12%, phosphate 1 potassium 1.2%,
Zinc sulfate 1×10-3%, manganese sulfate 1×10-”%
and a medium containing 1x10-3% copper sulfate (pl-14.0)
Pour 20ml into a 200ml Erlenmeyer flask and sterilize Acremonium cellulolyticus (FERM) using a conventional method.
P-6867> and aerated at 30°C for 6 days.
I made a profit of 8. After culturing, bacteria were removed using a centrifuge, and the resulting supernatant was measured for avicelase activity, CMCase activity, and β-glucosidase activity.
3. Iu/ml, 2G, 3 u/ml and 13.8
u/ml.

Example 2 Acremonium cellulolyticus (FERMP-68, 67) was inoculated into a medium to which 2% Bonito Sorbul was added in place of Bac1 to peptone in J3 of Example 1.
The cells were incubated at 0° C. for 8 days with aeration. After culturing, avicelase, CMCno7-ase and β were extracted from the centrifuged supernatant.
- As a result of measuring glucosidase activity, 3.
9 Ll/ni1.45.5 U/ml and 2
1. I U /Inl.

The culture supernatant was incubated with 2 volumes of cold aletone, and the resulting precipitate was centrifuged at 11111 m (9000 rpm, 1
The enzyme solution was collected by 0 minutes), dissolved slightly in water, and insoluble materials were removed to obtain a clear layered enzyme solution. Avicelase, CM
The respective recoveries of Case and β-glucosidase were 91.9%, 71.7%, and 96.8%.

[Brief explanation of the drawing]

Figure 1 shows Aku-1-no-7nium (FFRM P-6867).
Regarding cellulases such as avicelase, CMC age, and aβ-glucosidase produced by ), (optimal action 1)
Hl (b) optimal working temperature, (C) pl-1 stability and (d) thermal stability are shown. 4 (a)' Procedural Notes (Spontaneous) February 9, 1980 Commissioner of the Patent Office 1, Indication of the Case Patent Application No. 384 of 1982
31 No. 2, Title of invention Method for producing cellulase Name (114) Director of the Agency of Industrial Science and Technology Kawa 1
)Yuube i'', ;. , :'l; , France shi□2; 5. Date of amendment order Voluntary 6. Number of inventions increased by amendment None.
Paper 1, the scope of claims is amended as follows. "Cultivating Acremonium bacteria that produce cellulase,
2. "Only one reaction can be carried out on page 2, line 9 of the specification" in "A method for producing cellulase characterized by collecting cellulase from cultured bamboo shoots" has been corrected to "Only one reaction can be carried out." 3. "In terms of usage" on page 3 of the specification, lines 2-3
amended to ``1 also in terms of usage''. 4. On page 3, line 15 of the specification, amend rFPase (paper-powered paper-degrading activity) to ``I'FPase (filter paper-degrading activity)''. 5. Change "It is something that is being done. (For example)" to "It is something that is being done (
For example, correct it to ``. 6. Line 7 from the bottom of No. 5 of the specification (7) rM, Man
del et al. [3iotech, Bioenz. , XX [I2009 (1981) J rM, 1vl
andels et al. Biojech. Bioenz,, XXm, D 2009 (1981
) Correct to J. 7. Change "purify reducing sugar" in the fifth line from the bottom of page 5 of the specification to "
Corrected to "generates reducing sugars." 8. On page 6, line 10 of the specification, 1 "Growth, malt extract" is corrected to "Growth: malt extract." 9. In the fourth line from the bottom of page 6 of the specification, [Morphology, diameter of hyphae J] is corrected to "morphology: diameter of hyphae." 10. In the second line from the bottom of page 6 of the specification, ``The ability to form conidia and conidia'' is corrected to ``The ability to form conidia and biconidia.'' 11. In the first line from the bottom of page 6 of the specification, “Easily disappears,
Ta. ” disappears by 1. ” is corrected. 12. 4th to 3rd lines from the bottom of page 7 of the specification [Recently adopted...no description yet. Furthermore, the present invention] is [
This is a station name adopted in recent years, and has been corrected to ``The present invention''. 13. "Cephalosporium" on page 7, line M1 from the bottom of the specification to page 8, line 1 is corrected to "acremonium." 14, “Multi-component method” in Book 1, page 10, line 12 of Mei ■
Corrected to ``multi-component nature''.

Claims (1)

[Claims] Acremonium bacteria that produce cellulase were cultured, and J
8 A method for producing cellulase characterized by collecting cellulase from a nutrient solution