JPH03130072A - Production of microbial cellulase - Google Patents

Abstract

PURPOSE:To obtain the title cullulase higher in crystalline cellulose decomposing activity and filter paper decomposing activity in comparison with CMC decomposing activity by culturing a specific Bacillus circulans in a culture medium and producing and accumulating cellulase in the culture medium. CONSTITUTION:Bacillus circulans LP-547 strain (FERM P-10970) is cultured in a culture medium and a cellulase having higher crystalline cellulose composing activity and filter paper decomposing activity in comparison with CMC decomposing activity is produced and accumulated and then collected. Furthermore, the culture is preferably e.g. carried out at about pH7.0-9.0 and 30-37 deg.C for 24-90hr.

Description

DETAILED DESCRIPTION OF THE INVENTION 1) Industrial Field of Application The present invention provides a new bacterial strain that has a cellulase-producing ability with higher crystalline cellulose-degrading activity and filter paper-degrading activity than CMC-degrading activity, and The present invention relates to a method for producing the cellulase used.

(2) Conventional technology As part of the comprehensive development of biomass energy in recent years,
Many attempts have been made to utilize enzymatic saccharification of cellulose resources, and pulp beating using cellulase agents (Japanese Unexamined Patent Application Publication No. 1989-1999)
-126395> and modification of wheat flour (Unexamined Japanese Patent Publication No. 1-171
Development of new uses for cellulases such as 647) is also progressing.

A cellulase that can be applied to such a wide range of purposes requires a cellulase that exhibits high crystalline cellulose decomposition activity or filter paper decomposition ability. This method requires a long cultivation time, and also has the drawbacks of complicating the purification process because many kinds of contaminant enzymes are produced in the culture solution.

Regarding cellulases produced by actinomycetes, there have been reports of the genera Thermomonospora, Microbispora, Streptomyces, etc., but none of them have reached the stage of practical use.

(3) Problems to be solved by the invention Certain types of bacteria secrete enzyme proteins into the culture solution, but the types of enzymes produced outside the bacterial body are usually only 2 or 3 types, and the purpose of This is relatively advantageous when trying to specifically obtain a desired enzyme.

There are many things.

As aerobic bacteria that produce cellulases, Cellulomonas, Cellovibrio, Pseudomonas, and Bacillus strains are known, but the cellulases produced by these bacteria are generally thought to have low effects on crystalline cellulose. ing.

Exceptionally, Cellulomonas rada strain C84 (JP-A-58-1
01691) has been reported to have relatively strong Avicel-degrading activity. However, this CB4 strain also has low P-degrading activity ((J,
Ferment, Technol, vol. 61. p,
379-382 (1983); Fermentation and Industry vol.
44. p., 753-764 (1986)), and this point is difficult in practical application.

Regarding cellulases produced by bacteria of the genus Bacillus; alkaline-resistant cellulase, alkaline cellulase
-28515. Japanese Patent Publication No. 58-224686. JP-A-63-146786. J, Gen, Micr
obiol, vol, 131. Most of the studies focus on p,3339 degrading activity and β-glucosidase activity, and few focus on effects on crystalline cellulose or filter paper.

M, A, O, Trevino et al. reported that Bacillus circulans isolated from contaminated bacteria at a CMC production factory has a weak filter paper degrading activity (3,93 Filter paper u) together with endoglucanase (CLIC degrading enzyme).
However, it has been reported that nits/+g protein)
(Appl, Microbiol.

Biotechnol, vol, 31. p, 1
46 (1989)), and when converted to the enzyme unit system of the present invention to be described later, the Q, 056 FPU/ml is extremely low, and it is difficult to say that it has a practical low P decomposition activity.

Others, JP-A-61-35784. JP-A-63-1
Regarding cellulases such as 46786, there are literatures that refer to their effects on filter paper and Avicel, but the degree of their activity is weak or unclear, and it is not practically useful because both the effective pH and the stable pH are (A filter paper-degrading activity-producing bacterium that has an optimum range around 4 to 5, exhibits almost no activity at pH 7 or higher, and can act in neutral or slightly alkaline conditions has not been obtained.

(4) Means for solving the problem The present inventors newly isolated a cellulase producing strain that is an aerobic bacterium and exhibits high p-low decomposition activity or crystalline cellulose decomposition activity in the neutral range. As a result of our intensive search, we found an excellent strain LP-547 from a field in Shimizu City, Shizuoka Prefecture.
I got the stock.

Furthermore, by examining the culture conditions and medium conditions for this strain, we discovered the possibility of industrially producing an enzyme equivalent to cellulase from filamentous fungi within 2 to 4 days of culture. It was recognized that it could also be used in the neutral range, where it did not work, and the present invention was completely defeated.

That is, the present invention provides the obtained cellulase-producing strain LP-547 that exhibits high filter paper decomposition activity or crystalline cellulose decomposition activity, and a method for producing cellulase using this strain.

Cellulase producing bacteria used in the present invention LP-54
The 7 strains have the following mycological properties.

Motion by biperiflagella.

Durham staining: positive Culture form: Developed on broth agar plate culture.

The surface is smooth and the six colors are translucent and white.

Spores: Mainly formed at one end, oval in shape.

State of sporangium: Swelling.

■Physiological properties Growth temperature: Growth pH: Oxygen requirement: Starch decomposition = P Paper decomposition: Tyrosine decomposition: Deamination of 7-enylalanine: 20-40℃ ( Optimal 35-40"C) 6.0-9.0 (maximum*7.0-8.0) Absolute aerobic positive Positive Negative Negative Indole production: Negative Hydrogen sulfide production: Positive v- p reaction: Negative VP culture pH: 6 or less (pF[5,28> Catalase reaction: Positive Cytochrome oxita 9-70 reaction: Negative Acid production from xylose, mannifol) : Positive Assimilation of citric acid : Negative Due to the above mycological properties, this strain was included in "Bergy's Manual of Systematic Bacteriology" Volume 2 (Bergy's Manual of Syst
ema-tic Bacteriology vol.
2 (1986)), it was identified as Bacillus circulans, and Bacillus circulans was identified as Bacillus circulans.
The strain designated as P. culans LP-547 has been deposited with the National Institute of Microbial Technology, Agency of Industrial Science and Technology, and its deposit number is Microbiological Research Institute No. 10970.

In addition, in the section on Bacillus circulans in the above manual, there is a statement that some strains weakly degrade cellulose, but this is based on the results on CMC-containing plates and does not relate to the novelty of the present invention. To obtain, Bacill
This is carried out by inoculating the S. us circulans strain LP-547 into a nutrient-containing medium, culturing it according to a conventional method, and collecting the enzyme accumulated in the medium.

For the production of this enzyme, Bacillus circula
In addition to the ns LP-547 strain, natural or artificial mutant strains thereof can also be used as long as they have the ability to produce the enzyme. LP-5
General methods of artificial mutation are used to obtain the 47 artificial mutant strains, but for example, ultraviolet irradiation, gamma ray irradiation such as cobalt-60, chemical mutagenic agents, etc. are possible, as well as genetic engineering methods. The gene for this enzyme was cloned by
It is also possible by introducing the ability to produce this enzyme into other microorganisms.

Although normal bacterial culture methods can be used to culture these strains, liquid culture is generally preferred.

As the carbon source for the medium, cellulose-based substances such as natural cellulodimethylcellulose such as cotton, cotton thread, sawdust, bran, soybean meal, rice straw, and rice bran, cellulose derivatives such as methylcellulose and ethylcellulose, and cellooligosaccharides are preferable, but starch, Polysaccharides such as dextrin, white rice bran and corn meal, trisaccharides such as lactose and sucrose, and monosaccharides such as glucose, fructose and mannose can be used, and combinations thereof can also be used.

As a nitrogen source, Cornstave force (C3L),
Malt extract, casein, meat extract, peptone, inorganic ammonium salts, etc. can be used.

Also, KHzPOq, MgSO4, Fe5Oa,
MnSO4, CaCl2゜COCl2. KCI,
Inorganic salts such as NaC1, organic trace elements such as vitamins, and Tween 40. A surfactant such as Tween 80° Span 80 can be added if necessary. In addition, the initial pH of the culture can be varied over a wide range from 7.0 to 9.0.

The production of this enzyme is carried out using a culture solution containing the above nutrients, etc.
Alternatively, the extract can be used directly as an enzyme solution, but culture solution, bacterial cells, culture solution concentrate, or salting out using ammonium sulfate, common salt, etc., ion exchange chromatography, isoelectric precipitation, Partially purified products or purified specimens obtained by purification methods such as solvent fractionation and adsorption chromatography alone or in combination can also be used for the purpose of utilizing the present enzyme.

Cellulase activity was measured using the method of Mandels et al. (B: otechnol, Bioeng, 5yap
,, vol, 6. p, 2l-33 (1976>). However, the buffer solution contains acetic acid f! Using street liquid,
The pH was set to 6.0. A specific method for analyzing filter paper decomposition activity is shown below.

0.511+7) diluted enzyme solution was added to 1i1 0.05M acetic acid) 1fij solution (pH 6,0), and a piece of filter paper adjusted to 50 mg (about I x 6 cm of Whatman Island filter paper) was added and mixed. After reacting at 50"C for 1 hour, the amount of reducing sugar produced in the solution was quantified as glucose by the dinitrosalicylic acid method. 1 enzyme unit (FPU/
al) was carried out according to the fermentation method that produced reducing sugars equivalent to 1 μll 1 ol of glucose per minute.

Add a certain amount of enzyme solution to 1ml of 0.1M vinegar'#1mi solution (pH 6.0), heat at 50℃ for 2 to 3 minutes, and add 5m
Varanitrophenyl-β-D-cellobioside (111) was added (1,5a+1) and reacted for 20 minutes. After 9 reactions, 1M sodium carbonate was added to 1111 to stop the reaction, and the absorbance at 410 nm was determined to be free. Quantify the amount of varanitrophenol.

One enzyme amount (U/ml) was defined as the amount of enzyme that releases 1 μmol of varanitrophenol per minute.

The enzyme solution obtained by the above method had the following properties.

(2) Action pH and Stability As shown in Figure 1, the optimum relationship between activity and pH at 50°C was pF[5.5 to 7.0. Also,
pl when held at 50° C. for 1 hour (the stable range was <<, pH 5.5 to 9.0 as shown in FIG. 2).

(2) Temperature of action and stability Regarding the relationship between activity and temperature in the case of reaction at pH 6.0 and 1 hour, as shown in FIG. 3, 50°C was optimal. In addition, when maintained at pi (6,0) for 30 minutes, the activator and inhibitor of this enzyme were 1 aM of 5nC15L, Zn5O,...
and 10mM NiCl, approximately 60-701.
Inhibition close to 1001 is caused by 1mM CuCIz, LOtxM ZnSO4.

On the other hand, the cellulase agent originating from the genus Trichoderma is Cu″!
”, since it is not inhibited by Zn 1°, this enzyme is considered to be clearly different from that of the genus Trichoderma (Table 1).

Table 1 <Example-1> One platinum loop of Bacillus circulans LP-547 was inoculated into 20+I glucose-containing meat extract medium.

WJ shaking culture was performed at 28° C. for 2 days to prepare a preculture solution.

This preculture solution was mixed with 100+ al of production medium (KC71
1! Fuku w-1002/, cornstarch liquor 0.03, urea 0.15G
, monopotassium phosphate 0.1%.

Twin-800, 1X, pF47.0> was inoculated at a concentration of 2, and cultured at 30°C for 4 days.

Bacterial cells and residue were removed by centrifugation, and the filter paper fraction activity in the obtained P solution was measured, and it was found to be 0.80 F.
It was PU/ml.

<Example-2> The culture P solution obtained in Example 1 was collected, and the molecular weight cutoff was 1 (1°0
0
5 FPU/ml, CMC decomposition activity 30.4 U/m
It was l.

Note that β-glucosidase activity and caseinolytic activity were not detected.

2.5L production medium (KC Flock!-100251
;, Corn staple liquor IJ Ammonium nitrate 0.5J Magnesium nitrate O,O3
;=, Urea 0.1";, Monopotassium phosphate 0
．． 1. Inoculated a 5L mini-jar fermenter containing 1% Twin-800, pH 9.0 to a concentration of 4%, and incubated at 37°C, 250 rpm, 1
The cells were cultured in vva+ for 4 days.

Bacterial cells and residues were removed by centrifugation, and the filter paper decomposition activity in the obtained R solution was 0.88 FPU/ml, cellobiohydrase activity was Ojl U/ml, and CMC.
The degrading activity was 8.910/ml.

<Example-4> 1 g of door paper was added to 30 ml of 0.1 M phosphate buffer (
In addition to the culture solution obtained in Example 3 and the desalted concentrate of the culture solution of Trichoderma reesei QM-9414, the enzyme of the genus Luma was prepared according to Mandels et al. According to the original note, 50mM
(measured using a citric acid buffer pif of 4.8) and allowed to react at 50° C. with gentle stirring. The results are shown in Figure 5. The ability of the enzyme of the present invention to decompose filter paper was determined by Trichoderma riichii. A new bacterial strain has been provided, and a new method for producing cellulase using this strain has been established.

[Brief explanation of the drawing]

FIG. 1 shows the relationship between the activity of this enzyme and pF. Figure 2 shows the PH stability of this enzyme. FIG. 3 shows the relationship between the activity of this enzyme and temperature. Figure 4 shows the temperature stability of this enzyme. FIG. 5 shows the time course of low P decomposition at p) 17.0 of the present enzyme (song!! 1) and Trichoderma cellulase (curve 2).

Claims (2)

[Claims] (1) A bacterial strain Bacillus circulans LP-547 (Ba
cillus circulans LP-547) Microtechnical Research Institute No. 10970. (2) Bacillus circulans LP-547 strain (Ba
cil-lus circulans LP-547)
10970 was cultured in a medium, and C was added to the medium.
1. A method for producing cellulase, which comprises producing and accumulating cellulase having higher crystalline cellulose degrading activity and filter paper decomposing activity than MC decomposing activity, and collecting the cellulase.