JPH04104788A - Cellulase and production thereof - Google Patents

Abstract

NEW MATERIAL:A cellulase having the following properties. Optimum pH; 6.0-8.0 when CMC is used as a substrate. Optimum temperature; 50 deg.C. Stable pH; stable to pH6-11 when treated at 30 deg.C for 30min. Molecular weight; 55000+ or -2000 (electrophoresis). Isoelectric point; 4.6+ or -0.2 (isoelectric focusing). Effects of surfactant; having >=90% residual activity even when treated at 30 deg.C for 2hr in the presence of a sodium straight-chain alkylbenzenesulfonate at 3000ppm concentration (pH9.0). USE:An enzyme for detergents. PREPARATION:A microorganism such as Bacillus SD403 strain (FERM P-11647), belonging to the genus Bacillus and having the ability to produce the cellulase is cultured at 25-40 deg.C for 12hr to 3 days, preferably by a liquid culture method.

Description

[Detailed description of the invention] [Industrial application fields] The present invention relates to a novel cellulase and a method for producing the same.

[Prior Art] The development of cellulases has heretofore been advanced with the aim of effectively utilizing biomass resources, particularly cellulose resources, but cellulases for biomass have not necessarily been utilized on an industrial scale.

On the other hand, one of the new industrial uses of cellulase is
Cellulase is attracting attention as an enzyme for detergents because it is said to be effective in improving detergency. Cellulases for detergents are alkaline cellulases that function under highly alkaline conditions, since the pH of cleaning solutions is highly alkaline under normal washing conditions.
Alternatively, the cellulase must have alkaline resistance, and furthermore, it must function stably against anionic surfactants, which are one of the detergent ingredients.

Alkaline cellulase produced by microorganisms includes a method of collecting cellulase A by culturing an alkalophilic bacterium belonging to the genus Bacillus (Japanese Patent Publication No. 5O-28515), and a method of culturing an alkaliphilic bacterium belonging to the genus Cellulomonas to produce alkaline cellulase 301-A. Method (Japanese Unexamined Patent Publication No. 58-22468
6), a method for producing alkaline cellulase K by culturing alkalophilic Bacillus bacteria KSM-635 (Japanese Unexamined Patent Publication No. 6
3-109776) etc., but as an alkali-resistant cellulase, -58
8 (Japanese Unexamined Patent Publication No. 64-37286),
A method for producing -597 in cellulase by culturing Bacillus bacteria (Japanese Unexamined Patent Publication No. 1-112982) has been reported. However, these conventionally known cellulases do not necessarily have sufficient stability against anionic surfactants, which are detergent ingredients, so there is a desire to develop enzymes that are more stable against anionic surfactants. There is.

[Problem to be solved by the invention g] The present invention has an optimal pH in a neutral range, but has sufficient activity even in an alkaline region compared to the activity at the optimal pH, and The present invention aims to provide a cellulase that is stable even against anionic surfactants and is suitable as a detergent enzyme.

[Means for Solving the Problems] The present inventors conducted a search by isolating and culturing a large number of microorganisms in order to obtain cellulases having the above-mentioned properties.
We have discovered that Bacillus 5D403 strain, a bacterium belonging to the genus Bacillus isolated from the soil in the lower part of Tokyo, produces a novel cellulase that has excellent properties as a detergent enzyme, leading to the completion of the present invention.

That is, 1) the present invention is based on 1) carboxymethyl cellulose (CM
The optimum pH when C) is used as a substrate is 6, ○ ~ 8.0,
The optimum temperature is approximately 50°C, and 2) the stable pH range is 30°C.
, when treated for 30 minutes, the pH is 6 to 11, 3) the molecular weight is 55, OOO+2000 when measured by SDS-polyacrylamide electrophoresis, 4) the isoelectric point is measured by polyacrylamide electrophoresis,
4.6±0. 2, and 5) in the presence of 3000 ppm sodium linear alkylbenzenesulfonate (pH
A novel cellulase that is extremely stable against surfactants, with a residual activity of more than 90% even after treatment at 30°C for 2 hours at 9,0), and a microorganism of the genus Bacillus that has the above-mentioned cellulase-producing ability. It is an object of the present invention to provide a method for producing the above-mentioned cellulase, which is characterized by culturing the cellulase in a medium and collecting the desired cellulase from the culture.

The cellulase of the present invention, its production method, and the novel strain used therein will be explained in more detail below.

The microorganism Bacillus 5D403 strain used for the production of the novel cellulase of the present invention has the following properties.

(a) Form ■Cell shape and size ■Cell pleomorphism ■Motility ■Spores ■Durham stainability ■Anti-acidity (b) In each of the following media growth condition ■Meat juice agar plate medium It is a bacillus and its size The size is 0.5~0.9X 1.4~ It is 3.2 μm.

Does not exhibit polymorphism.

Has periflagella.

Forms spores.

Its shape is oval; The size is 0.8~1.0× It is 1.0-1.5 μm.

positive negative The village is circular and the front The surface is raised and the center is It is slightly raised.

Also, the color tone of the village is yellow-white. The color is translucent.

■Meat juice agar slant medium ■ Broth liquid medium ■Meat juice gelatin puncture culture ■Lidomas Milk (c) Physiological properties ■Reduction of nitrates ■Denitrification reaction ■MR test ■vP test ■Generation of indole ■Generation of hydrogen sulfide ■Hydrolysis of starch ■Use of citric acid ■Use of inorganic nitrogen sources [Phase] Formation of pigment ■Urease 0 oxidase @catalase [Strain] Growth range (pH) Grows in a spreading pattern.

turbidity liquefaction coagulation negative negative negative negative negative negative positive negative Nitrates and ammonia Use mucilage salt.

negative negative positive positive Grows at 6.8, but 5.7 (temperature) [Phase] Attitude towards oxygen [Phase]〇-F test Oxidation of O-saccharides and production of gas L-arabinose D-xylose D-glucose D-mannose D-fructose D-galactose maltose sucrose lactose trehalose D-Tsurupinto It does not grow in

Grows at 12.1.

Grows at 20℃ and 45℃ grows at 15℃ and 50℃ do not.

Aerobic Fermentation (12) D-manninto + (13) Inodite + (14) Glycerin + (15) Starch Bergey's Ma
nual of SystematicBact, er
iology), and The Genus Bacillus (The
As a result of referring to Genus Bacillus, this bacterium is a spore-forming bacillus Bacillus firmus.
It was recognized that it is a bacterium related to S. firIIlus). However, the Bersey's Manual of
The standard strain of Bacillus firmus described in Systematic Bacteriology ^TCC14575 and this bacterium have different mycological properties, such as swelling of sporangia and production of acid from mannitol. It was recognized that it was a new strain, and it was therefore named Bacillus 5D403 and deposited as Fiber Science and Technology Research Institute No. 11647.

The microorganism used in the present invention is not limited to the above-mentioned Bacillus 5D403 strain, but may be any strain as long as it has the productivity of cellulase having the characteristics described later in the present invention. Bacillus 5D403 also includes natural or artificial mutant strains and strains improved by genetic engineering techniques.

11. In producing the cellulase of the present invention, there are no special requirements for the method of culturing the microorganisms as described above, and conventional methods can be used as appropriate.

As a nutrient source for the culture medium, a wide variety of nutrients commonly used for culture can be used. The carbon source may be any assimilable carbon compound or one containing it, such as glucose, maltose, starch, CMC, etc. Any nitrogen compound that can be assimilated as a nitrogen source or one containing it may be used, such as ammonium salts and peptone.

Soybean flour, defatted soybean flour, etc. are used. Further, as the inorganic salts, salts such as phosphates and magnesium salts are used. In addition, various organic and inorganic substances necessary for bacterial growth and enzyme production, or substances containing them, such as vitamins and yeast extract, can be added to the medium.

The culture may be either liquid or solid culture, but liquid culture is preferred. Culture conditions for liquid culture differ somewhat depending on the medium composition, but the most advantageous conditions for producing cellulase, which is the target product, are selected. Culture temperature is 25-4
The temperature range is 0°C, the culture time is about 12 hours to 3 days, and the culture may be terminated when cellulase production reaches the maximum.

The pH of the medium is preferably 8 or higher, and 8 to 9 is suitable for cellulase production. Through such culturing, the desired cellulase can be obtained in the culture solution.

Cellulase can be collected from the culture solution thus obtained and purified according to a conventional method for collecting enzymes. That is, bacterial cells and medium homomorphs are separated by a known appropriate method such as a filtration method or a centrifugation method,
A supernatant or filtrate can be obtained. Cellulase is obtained by concentrating these separated liquids and spray-drying or freeze-drying, or by a salting-out method in which soluble salts are added and precipitated without concentration, or by a precipitation method in which a hydrophilic organic solvent is added and precipitated. be able to. To further purify the enzyme, purification methods such as adsorption/desorption using an ion exchange resin, gel filtration, etc. may be used alone or in combination.
Cellulase can be purified.

1Rin J11 The detailed properties of the cellulase of the present invention will be described.

' ■ Carboxymethyl cellulase (CMCase) activity Carboxymethyl cellulose (CMC) was dissolved in M/20 sodium carbonate-M/20 boric acid-potassium chloride buffer (p
H9.0) is used as a substrate for measurement.

Specifically, 0.5% CM C (pH 9,0) 0.9
Add 0.1 ml of enzyme solution to 1 ml of substrate solution.

The reaction was carried out at 30°C for 15 minutes. After the reaction, 4-hydroxybenzoink hydrazide (4-1ydrOxybenzo
Reducing sugars were quantified by the ichhydrazide method (PHBAH method). That is, P was added to 1.0 ml of the reaction solution.
Add 3.0 ml of HBAH reagent and incubate at 100°C for 10 minutes.
The color was developed by heating, and after cooling, colorimetric determination was performed at a wavelength of 4] Onm.

For the enzyme titer, one unit was the amount of enzyme that produced reducing sugar equivalent to 1 μmol of glucose per minute under the conditions listed in h.

■ Phosphoric acid swelling cellulose decomposition activity method of Tomita et al. (Tomita, Y. et al.: J
, Ferment.

Avicel (Merck & Co., Ltd.) treated with Avicel (Merck & Co., Ltd.) was treated with A/20 boric acid-potassium chloride buffer (PH9) as phosphoric acid swollen cellulose.
, 0) was used as a substrate for measurement. in particular.

0.25% phosphoric acid swollen cellulose (pH 9,0) 0.
0.1 ml of enzyme solution was added to 9 ml and reacted at 30° C. for 120 minutes. After the reaction, add PHBAH to 1.0 ml of the reaction solution.
3.0 ml of reagent was added, heated at 100°C for 10 minutes to develop color, and after cooling, colorimetric determination was performed at a wavelength of 410 nm.

What is the enzyme titer?

The amount of enzyme that produced reducing sugar equivalent to 1 μmol of glucose per minute under the above conditions was defined as 1 unit.

■ P-nitrophenyl β-D-glucopyranoside (pNPG), which is a synthetic substrate for β-glucosidase and p-nitrophenyl cellobioside decomposition activity, and P-nitrophenyl β-D-
Measurement was performed using cellobioside (pNPC) as a substrate. Specifically, 1.5 ml of M/20 sodium carbonate-M/20 boric acid-potassium chloride buffer (PH9,0), 50 m
Add 0.1 ml of enzyme solution to 1 ml of substrate solution of MpNPG or 5mM pNPCO, and add 0.1 ml of enzyme solution to 30'C1
The reaction was allowed to proceed for 60 minutes, and the liberated P-nitrophenol was determined colorimetrically at a wavelength of 400 nm. Under these conditions, the amount of enzyme that caused 1 μmol of P-nitrophenol to Mli in 1 minute was defined as 1 unit.

11 Side 1I (1) Action This enzyme acts well on cellulose such as carboxymethyl cellulose (CMC) and phosphoric acid-swollen cellulose, hydrolyzing and dissolving β-glucoside bonds.

(2) Substrate specificity In addition to its main activity on CMC, the enzyme also has activity on phosphoric acid-swollen cellulose, which accounts for about 0.48% of the activity. p-nitrophenyl-β-D-glucopyranoside (pNPG), p-nitrophenyl-β-
It has almost no activity against D-cellobioside (pNPC).

(3) Optimal pH Br1tton-Robins
CMC using a broad range buffer (pH 5-11)
When measured using P as a substrate, the 1 action p and H ranges over a very wide range from 5 to 11, and the optimum pH is 6. It is 0 to 8.0. The relationship between reaction pH and relative activity is shown in Figure 1.6 (4) Stable pH range Under different pH buffers, the reaction mixture was left at 30'C for 30 minutes and measured using CMC (pH 9,0) as a substrate. In this case, the stable pH is 6-11. FIG. 2 shows the treatment pH and residual activity.

(5) Optimal temperature When measuring CMC as a substrate, the optimal temperature is approximately 50°C
Figure 3 shows the relationship between reaction temperature and relative activity.

(6) Thermal stability M/20 sodium carbonate-M/20 boric acid-potassium chloride buffer (pH 9,0) at various temperatures (5-65°C)
When measured using CMC as a substrate after heat treatment for 30 minutes, the activity does not deactivate at all until around 40°C, and it has about 60% residual activity at 55°C. FIG. 4 shows the relationship between heat treatment temperature and residual activity.

(7) Influence of metal ions and chelating agents Cu” (5
In addition to being inhibited by P-chloroflucribenzoic acid (5mM), it is also slightly inhibited by EDTA (5mM), but is not affected by EDTA (5mM).

(8) Molecular weight The molecular weight obtained by SDS-polyacrylamide gel electrophoresis is 55,000±2,000.

(9) As a result of measurement by isoelectric focusing polyacrylamide gel electrophoresis, the result was 4.6±0. It is 2.

(10) Effect of surfactant Sodium linear alkylbenzene sulfonate 3000
ppm (pH 9,0) at 30°C for 2 hours, there is almost no inactivation. FIG. 5 shows the relationship between treatment time and residual activity.

This enzyme is a strain of Bacillus 5D403 belonging to the genus Bacillus.
It is produced by Japanese Patent Publication No. 50-28515, Japanese Patent Application Publication No. 63-109776, Japanese Patent Application Publication No. 64-37288,
When compared with the alkaline cellulase and alkaline-resistant cellulase described in JP-A-1-112982.

The molecular weight of this enzyme is 55,000±2,000, while the molecular weight of the alkaline cellulase and alkali-resistant cellulase is 15,000. 30000.180000.
27000, 30000. They are clearly different enzymes in that they have a molecular weight of 40,000 and other physicochemical properties.

[Effects of the Invention] The cellulase of the present invention has an optimum pH of 6°O to 8.0, but even at a highly alkaline pH of 9, the optimum pH is about 80°.
% relative activity, and maintains a relative activity of about 50% of the optimum pH in a wide pH range from pH 5.5 to 9.5. It also has sufficient activity even at low temperatures. Furthermore, it has high stability against anionic surfactants, which are detergent ingredients.
It can be used as an enzyme for detergents to enhance detergent power.

Next, the present invention will be explained in more detail by giving representative examples.'' Peptone 1%, sodium chloride 0.5%, dipotassium phosphate 0.5%, magnesium sulfate 0.05% A liquid medium consisting of 0.5% yeast, 0.5% yeast, 15% CMC, and 1.0% sodium carbonate was dispensed into test tubes and sterilized by a conventional method. This was inoculated with the 5D403 strain, and cultured with shaking at 35°C for 40 hours. When the culture solution was centrifuged and the cellulase activity of the supernatant was measured, it was 0.1.
It was 1 J/ml.

To see ■l Soy flour 4%, sodium chloride 0.5%, dipotassium phosphate 0.1%, magnesium sulfate 0.02%, yeast extract 0.5%, amylose 0.5%, CMCO, 1%, A liquid medium containing 0.3% sodium carbonate was placed in a 5 L culture tank and sterilized by steam. This was inoculated with the 5D403 strain that had been cultured in advance, and cultured with aeration at 35° C. for 48 hours. This culture solution was centrifuged to obtain a supernatant. The cellulase activity of this supernatant was 0.14 [1/ml].

Firstly, the cellulase of the present invention was purified from the culture supernatant obtained in Example 2.

4 by cold ethanol precipitation method from 500 ml of culture supernatant.
A 0-65% fraction of precipitate was obtained. This precipitate was dissolved in 10mM Bis-Tris buffer (pH 7.0), and DEAE equilibrated with 10mM Bis-Tris buffer (pH 7.0).
(diethylaminoethyl) anion exchange resin column (
52 mmφ, 20 cm) and eluted with a sodium chloride concentration gradient (0 to 1 M). The active fraction eluted in the same procedure was concentrated and desalted using an ultrafiltration membrane, and then transferred to a phenyl Sepharose CL4B column (20 mmφ, 20 cm
) and the column was soaked in 10mM Bis-Tris buffer (
After washing with pH 7.0), it was eluted with a concentration gradient of isopropanol (0-60%). At least one or more active fractions were obtained in each of the washed fraction with 10 mM Bis-Tris buffer (pH 7,0) and the fraction eluted with the concentration gradient of Impropatool (0 to 60%). Ta. Fractionation, activity, and absorbance (A
280) is shown in FIG. After concentrating and desalting the main component, CMCase (fraction numbers 26 to 48), using an ultrafiltration membrane, 10 mM Bis-Tris buffer (pH 7,
Sephadenx G-75 column (25
mmφ, 90 cm) and eluted with the same buffer to obtain an active fraction, which was used as a purified enzyme preparation.

This purified enzyme preparation was colorless, and when tested by polyacrylamide gel electrodynamic method, it was confirmed to be single.

Using this purified enzyme preparation, the optimum pH, pH stability, optimum temperature, thermal stability, and stability against surfactants were investigated, and the results are shown in FIGS. 1 to 5.

[Brief explanation of drawings]

FIG. 1 is a graph showing the relationship between reaction pH and relative activity. FIG. 2 is a graph showing treatment pH and residual activity. FIG. 3 is a graph showing the relationship between reaction temperature and relative activity. FIG. 4 is a graph showing the relationship between heat treatment temperature and residual activity. Figure 5 shows linear alkali sodium benzene sulfonate 3
1 is a graph showing stability in the presence of 000ppI11. FIG. 6 is a graph showing fractionation and activity on phenyl sepharose CL4B. 9 animals 1 Figure 2 Figure 2 Encounter pH Patent applicant Showa Denko Co., Ltd. yPS3 diagram 54 Figure: I & Logic amount degree ('C) Kogin 5 diagram angle qe;

Claims (1)

[Claims] 1. Cellulase having the following properties: (1) Optimal pH, optimal temperature When carboxymethyl cellulose (CMC) is used as a substrate, the optimal pH is 6.0 to 8.0, optimal The temperature is approximately 50°C. (2) Stable pH Stable at pH 6 to 11 when treated at 30°C for 30 minutes. (3) Molecular weight The molecular weight measured by SDS-polyacrylamide electrophoresis is 55,000±2,000. (4) Isoelectric point The isoelectric point measured by isoelectric focusing is 4.6±0.2. (5) Effect of surfactant Even after treatment in the presence of 3000 ppm of sodium linear alkylbenzene sulfonate (pH 9.0) at 30°C for 2 hours, it has a residual activity of 90% or more. 2. The method for producing cellulase according to claim 1, which comprises culturing a microorganism belonging to the genus Bacillus and having the ability to produce cellulase according to claim 1 in a medium, and collecting the desired cellulase from the culture.