JPS59166083A - Preparation of cellulase - Google Patents

Abstract

PURPOSE:To prepare cellulase in high yield, by cultivating a specific microorganism belonging to the genus Acremonium in a medium containing polyethylene glycol. CONSTITUTION:Novelly separated Acremonium cellulolyticus (FERM P6867) is cultivated in a medium containing cellulose as a carbon source and 0.01-5wt% (especially 0.05-0.5wt%) polyethylene glycol, and production of cellulose is extremely increased by comparison with the case where no polyethylene glycol is added to the medium. Polyethylene glycol 1000, polyethylene glycol 2000, polyethylene glycol 4000, etc. having different polymerization degree may be cited as the polyethylene glycol, any of them can be used, and especially polyethylene 1000 having low polymerization degree is effective.

Description

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

For cellulose, cellulose is converted into glucose or RerobiΔ-
Su or L? ['' is a general term for a group of enzymes that catalyze the enzymatic reaction system that decomposes them into oligosaccharides, and depending on their mode of action, C1 enzymes, Cx enzymes, β-glucosider U, or exo-β
It is made up of enzymes called by various names such as -glucanase, endo-β-glucanase, and hyrobiase, but their true nature is still unclear. Due to the microbial origin that produces cellulases, it can be used to produce crystalline cellulose, carboxymethylcellulose (CMC), cellodex i~
This is due to the presence of a wide variety of enzymes with different modes of action on phosphorus, cellooligosaccharides, cellobiose, etc., and the structural complexity of natural cellulose. However, cellulases require harmonious interactions between these multiple enzymes.
It is a complex enzyme that decomposes cellulose into its constituent sugars by leaching.

In recent years, cellulases have attracted attention and have been actively researched from the perspective of making effective use of biomass sources. However, cellulases produced by well-known microorganisms such as Trichoderma and Aspergillus do not have sufficient decomposition power for natural cellulose, and cannot completely decompose cellulose into glucose, producing cellobiose and more. There are problems with 1ζ, such as the production of large amounts of cellooligosaccharides. Furthermore, conventionally known cellulases usually have poor thermostability and can only be used for long-term saccharification reactions at temperatures of about 45 to 50°C, so they are often contaminated 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. Monium (Acremo)
A filamentous fungus identified as belonging to the genus Nium was isolated. In order to use the cellulase produced by this bacterium industrially, we have continued our 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, cellulase is produced. However, it was observed that the production of The present invention has been made based on this knowledge.

Specifically, 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 present invention will be described in detail below.

As the polyethylene glycol used in the present invention, there are various known polyethylene glycols (for example, polyethylene glycol 1000, polyethylene glycol 2000°, polyethylene glycol 4000, etc.) with different degrees of purity, and all of these can be used in the present invention. Although it can be applied effectively, polyethylene glycol of about 1000 was particularly effective.

Polyethylene glycol is usually added at 0.0% to the medium.
It is added in an amount of about 0.01 to 5% culm, preferably about 0.05 to 0.5%. When Cremonium bacteria are cultured in such a medium to which polyethylene glycol is added, the production of cellulase significantly increases compared to the case without the addition of polyethylene glycol. For example, the talkability of cellulase is 1.1 to 1.5 times higher, and that of carboxymethyl cellulase is 1.
．． 5-2.9 times, β-glucosidase increases 1.3-2 times.

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

Growth: Growth on malt extract agar is fast, reaching a diameter of 701101 in 7 days at 30°C. The village was initially white and later became slightly yellowish. The aerial hyphae are loosely raised and wool-like, sometimes forming thin hyphal 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 Zapek agar, but the growth of aerial mycelium was less. Growth 1) H range to 3.5-
At 6.0, the optimum pH is close to 4', and the growth temperature range is 15℃.
~43°C, and the optimum working temperature is close to 30°C (q).

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.

Conidia and the ability to form conidia were extremely unstable and were easily lost by subculturing on Czapek agar and malt extract agar media. When observed during separation, the conidiophores are colorless and protrude from the side of the aerial kunihito.

The conidia are subglobular (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' r Cep
halosporiumartige 3chimm
elpi1gcj P84,G. Fisl+er
(1971) and C, l-1, DiCkillS
On, Mycol, Papers '115 P
10 (1968), we concluded that it is appropriate to consider the canteen to be a filamentous fungus closely related to the genus Acremonium. In addition, since Acremonium genus Acremonium has not been known to have a strong cellulase productivity, and the strain of the present invention produces a strong cellulase characteristic of potash, the water bottle should be made of Acremonium hirloliteino. Jsu(ACrenlOniUmc
el 1ulolyticus). The water bottle has been deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology as Fl=RIVI P-6867.

The genus Acremonium is a name adopted in recent years after a detailed examination by Qams and a reexamination of genera previously described as the genus Cephalosporium. Therefore, there have been no descriptions of cellulase-producing bacteria using the local name of the genus Acremonium. Furthermore, Cephalosporium bacteria that produce strong true cellulases that can act on crystalline cellulose, such as Avicelage and l-Pase, as shown in the present invention, were completely unknown prior to the present invention. C isn't there.

The cellulase produced by the water bottle is a dehydrator that acts on Avihil, a highly crystalline cellulose, due to its action characteristics. J or Avicella L'u: or C represented by FPase.
It is a complex enzyme system consisting of 13 types of enzymes, including CxtW, represented by the so-called CMCase, which acts on one enzyme, carboxymethyl cellulase (CMC), and β-glucosider L, which acts on cellooligosaccharides such as cellobiose. The harmonious interaction of these enzymes makes it possible to completely break down natural cellulose into glucose. The properties of these enzymes are described below.

(A) Enzymatic properties of Avicelase (1) Action Avicelase acts on highly crystalline insoluble cellulose such as cellulose powder, Avicil, and absorbent cotton to produce reducing sugars such as glucose and cellobiose.

(2) Action pl-1 and optimal action pl-1 The action pH range of this enzyme is 2 to 8, and the optimum action pl-1 is approximately 4.5. (Figure 1 (a)) (3)
Stable pH Stable pHl values ranged from about 3.5 to about 6 when exposed to a citric acid-phosphate solution for 20 hours at 45°C.

(Fig. 1 (C)) (4) Action temperature range and optimal action temperature This enzyme works at high temperatures up to about 90°C, but it is effective at high temperatures up to about 90°C. The optimum working temperature when reacting for 10 minutes under 5) is approximately 65℃.
was recognized. (Figure 1 (b)) (5) Add the thermostable enzyme to 0.05 M acid-resistant buffer (pl-1 4.5)
As a result of heat treatment at each temperature for 10 minutes under
About 50% and 70'C after 10 minutes of heating.

Approximately 80% of the activity was inactivated by heating for 10 minutes. (Figure 1(d))
(6) Mercury ion J5 of 1mM or more behind various small metal ions of 1iIII harmful agent
Strongly inhibited by copper ions. Also, 5HII
However, some parachlormercury benzoei 1 also caused about 1% of the 1tlJ damage at 1mM.

(7) Purification method The enzyme was desalted and concentrated from medium M filtration II1 through a small fiber (Amicon 1st edition-P5), and then subjected to column chromatography (NaCl O → 1M gradientene 1~) and rechromatography using the same column (NaCl O → 0.6
~1) allows for further purification.

(8) Molecule M Bio-gel (A 0.5m) Molecular weight measured by overspraying Gel'aG with J Lamb is approximately 140,00.
It was 0.

(9) Activity measurement method 0.1Ml! Add an appropriate amount of enzyme solution to 0.5 ml of 0.5% Avicel compound (i)l-14,5) in IT acid buffer, make the total volume 1.0-ml with distilled water, and add 50" The reaction was carried out at C. The amount of reducing sugar produced was measured by the Somogyi-Nelson method.

Under these conditions, the enzyme Minami that produced reduced horns corresponding to 1 μmol of glucose per minute was defined as one unit.

(B) Enzymatic properties of CMCase (1) Multi-component method of CIVICase CMCase is separated into at least four components by disk electrophoresis, each of which is distinguished by its molecular weight and isoelectric point. CMCase I has a molecular weight of about 160,000 and an isoelectric point of 5.08, and similarly, ■ is about 160,000.4.95.111 tcl.
: approx. 120,000.4,60, IV Ha approx. 120,0
00. There are 4.48 isozymes, and CMCase consists of a composite of these isozymes.

(2) A component (CMCase) that acts on soluble cellulose derivatives such as CMC and decomposes them into glucose, cellobiose, etc.
and (2), and a component (CtvlCase (2), IV) which only generates glucose very little and has the action of decomposing it into cellooligosaccharides greater than cellobiose.

(3) Effect 1) H and Optimal Effect 1) Effect of HCMCase Complex 1) H ranged from 2 to 8, and optimum effect 1) H was found to be approximately 4.5. (Figure 1 (a)> (4
) Stability 1) l-1 The stable p1-1 range of the CMCase complex when left at 45° C. for 20 hours under citrate-phosphorus M salt buffer was about 3.5 to about 6.

(5) Working temperature range and optimum working temperature This CM CL
The i'-f complex acts at high temperatures up to about 90°C, but
1% CMC, 0.05M 1 acid buffer (optimum effect when reaction is stopped for 10 minutes under pH 4, 5>) crystallization temperature is approximately 65°C
He was defeated in t2. (Figure 1(b))
As a result of heat treatment at each temperature for 10 minutes under
Heating for 10 minutes deactivated about 10%, and heating at 70° C. for 10 minutes deactivated about 70%. (FIG. 1(d)) (7) Inhibitor Among various heavy metal ions, it is strongly inhibited by mercury ion J3 and copper ion of ln+M and lower.

(8) Purification method This enzyme is purified from the culture filtrate by borofiber (Amicon I)1.
After desalting and concentration using DEAE-Sepharose (CL-6B), rechromatography using the same column (NaCI O → 1M gradient 1) and chromatography using the same column and chromatography A-cassing. It can be purified into each component.

(9) Activity measurement method 1% CMc solution dissolved in 0.1M acetate buffer (
Add an appropriate amount of enzyme solution to pt-14,5>0.5ml,
The total volume was made up to 1.0 ml 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, one unit of enzyme was used to generate a reducing power equivalent to 1 μmol' of glucose per minute.

<C) Enzymatic properties of β-glucosidase 71 (1) Action 1 Acts on cellooligosaccharides such as naricin, xerobiose, L'DI-lyase, lerotetraose, lerobentaose, cellotetraose, and breaks down into glucose. In addition, this enzyme acts on Avicel's J:Una high molecular cellulose, but hardly acts on CMC or 1-IEC (hydrogythyl cellulose). Zarysin, L-RobiA-se, cellotriose, cellotetraose,
The Km values for L-ropentase and lerohexaose are 3.40, 2.2G, and 1.19.0, respectively.
82.0.52 Soshi T: 0,5111M tear.

(2) Effect 1) l-(and optimum effect 1))-1 The action pH range of this enzyme was 2 to 8, and the optimum action pH was found to be about 4.5. (Figure 1(a)) (3> Stability 1) H The stable ejection range was about 3.5 to about 5 when left at 45° C. for 20 hours under a citric acid-phosphate buffer. (Fig. 1 (C)) 4) The action temperature of 1%1 and 114% of the enzyme acts at high temperatures up to about 90°C.
Nor'lysine, 0.05 M acetate buffer (pH 4,!i)
The optimum working temperature is approximately 65
℃ was observed. (Figure 1(b)) (5> Thermostability As a result of heat treatment at each temperature for 10 minutes under 0.05 M acetate buffer (pl-14,5), the enzyme was heated at approximately 65°C.
There was almost no deactivation at high temperatures up to 70° C., approximately 40% deactivation occurred when heated at 70° C. for 10 minutes, and more than 90% deactivated when heated at 80° C. for 10 minutes. (Figure 1 (d)) (6) Inhibitor: Among various heavy metal ions, mercury ion i of 11IIM or more
It is strongly inhibited by l'i, I and 14-ion. In addition, it acts as a competitive inhibitor against glucose-δ-lactone (1) and reverses the action of -C.

(7) Purification method This enzyme is purified from culture solution d using poro fiber (Amicon l-1
After desalting and concentration using 1-P5), column chromatography (NaCI O → 1M gradientene 1 to 1) using DEAE-Sepharose (CL-6B) and chroma l-
Focusing (pH 6→4) and Bio-gel (
It can be electrophoretically purified to homogeneity by gel filtration using A 0.5 m).The molecular weight measured by the gel filtration method using Bio-get (A 0,5 m) is approximately 240. ,000.

(9) Activity measurement method 2% salicin solution (1%) dissolved in 0.1M acetate buffer
) H4,5) An appropriate amount of enzyme solution was added to 0.5 ml, the total fi was adjusted to 1.0 tnl with distilled water, and the reaction was carried out at 50°C.

Then, the produced glucose was measured by Somogyi-Nelson measurement.

Under these conditions, the amount of enzyme that produced a reducing power equivalent to 1 μl Ilol of glucose per minute was defined as 1 unit.

The β-glucosidases of the present invention are more suitable for cellohexade than small molecular groups such as xyricin and elopiose.
This enzyme has a high affinity for Δribosaccharides such as burnopentaose, and also acts on polymeric substrates such as alleles. In particular, the present invention revealed for the first time the existence of an enzyme with substrate specificity that can decompose semibiose and to a considerable extent Achrel, and furthermore, the product is glucose, which is a major improvement in production. This β-glucosidase is a novel β-glucosidase that has not been previously known, and the present inventors have named this enzyme glucocellulase because it has similar action characteristics to glucoamylase on raw flour.

2, the cellulase produced by the Acremonium bacterium of the present invention is a novel cellulase complex enzyme agent containing a novel β-glucosidase.

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

Example 1 Cellulose 4%, KII2 PO4 1.2%, Baku1~Peptone 1%, Zn'SO4・7H20ix i
Q-:]%, KNO30,6%, to411804
・71-120 1x 10-co%, urea
0.2%, Cu 804 ・5H20ix 10.
-3%, KCQ O, 16%, 1) l-14,0, M
! J804・7H200, 12%, and a medium containing 0.0% polyethylene glycol 1000.
05-0.4% suspended medium, 20ml each
Pour into a 0m1 Erlenmeyer flask 1, sterilize it using a conventional method, and add Acremonium cellulolyticus (FERM P-6).
867) and cultured aerobically at 30°C for 80 hours.

After culturing, the supernatant obtained by centrifugation was measured for abilerase, carboxymethyllerulase (CMCase), and β-glucosidase activities of the produced xerulase. The results are shown in Table 1. IC0 As is clear from Table 1, the addition of 0.05% polyethylene glycol 1000 resulted in a 1.2-year increase in J. The number increased to 8. CMCase increased 2.1 times upon addition of 0.4% amount of polyethylene glycol 1000. And β-glucosidase is 0.4% polyethylene glycol 1000
It increased 1.7 times by adding .

Example 2 Acremonium cellulolyticus (FER
MP-6867) was inoculated and cultured aerobically at 30°C for 6 days. After culturing, the supernatant obtained by centrifugation was measured for the avicelase, CMCase, and β-glucosidase activities of the produced cellulases. (1) The results were 8J- as shown in Table 2.

As is clear from the table, the polyethylene glycol tested was 1ooo.

Both 2000 and 4000 are Avicelase, CMC
of which -b polyethylene glycol 1000 was more effective.

Table 2 Figure 1 shows Acremonium (FFRM, P-6867>
(a) Optimal action pH for Avirella-1, CMCase and β-glucosidase of L-lulase produced by
1 (b) optimal working temperature, (c) pl-1 stability and (d) thermal stability.

(Spontaneous) February 9, 1980, Commissioner of the Patent Office 1, Indication of the case Patent Application No. 384 of 1982
34 No. 2, Title of the invention Cellulase production method 5, Amendment order Q-day] Self-discrimination Sheet 1, page 2 of the specification, line 4, ``Nataga. But'' was changed to ``
became. However, it is corrected to "However."

2. Change “because it cannot be carried out” on page 2, line 11 of the specification to “
This is corrected to "because it cannot be done."

3. In the third row of page 3 of the interlayer 1 pile, correct J to "produce cellulase" to "produce cellulase".

4. On page 4, line 11 of the specification, "conidia, conidium-forming ability" is corrected to "conidia: conidium-forming ability."

5. 1 on page 4, line 12 of the specification easily disappeared. ” is corrected to “disappear.”

6. “Recently adopted
...No description yet. Furthermore, "the present invention" is corrected to "the present invention, which is a recently adopted station name."

7. In the specification, page 5, line 12 to page 13, line 1, "1 Cephalosporium" is corrected to "1 Acremonium."

8. "Multi-component method" on page 8, line 4 of the specification is corrected to "multi-component property."

9. Ming! 1111, page 9, line 18, "chromatofocusing" is corrected to "chromatofocusing".

10. Correct "Refining to homogeneity" in the first line of page 12 of Akira f@F5 to "Refining to homogeneity."

11. Correct "12% salicin solution" on page 2, line 4 of the specification to "1% salicin solution."

Claims (1)

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