JPH02215381A - Novel cellulase and production thereof - Google Patents

Abstract

PURPOSE:To obtain a cellulase, having the optimum pH on a high alkali side and suitable as an enzyme for detergents stable even to nonionic surfactants by respectively specifying the optimum pH, optimum temperature, stable pH, influence of a surfactant, molecular weight and isoelectric point. CONSTITUTION:A microorganism, consisting of a bacterial strain of Bacillus SD401, separated from soil in TOKYO Metropolis, belonging to the genus Bacillus and having the ability to produce the above-mentioned cellulase is cultured in a culture medium. The objective cellulase is then collected from the afore- mentioned culture. The above-mentioned cellulase has about 9.5-10.0 optimum pH and about 55 deg.C optimum temperature if CMC is used as a substrate and a stable pH region of pH6-11 in treatment at 30 deg.C for 30min. The afore- mentioned cellulase has characteristics, such as extremely stable even to surfactants, such as >=90% residual activity in treatment at 30 deg.C for 2hr in the presence of straight-chain alkylbenzenesulfonates (pH9.0) in 3000ppm concentration.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel cellulase, a method for producing the same, and a cellulase-producing microorganism.

[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 must be alkaline cellulases that function under highly alkaline conditions, since the pH of washing solutions is highly alkaline under normal washing conditions. It must also function stably against anionic surfactants, which are one of the detergent ingredients.

Alkaline cellulase produced by microorganisms can be obtained by culturing alkalophilic Bacillus bacteria to collect cellulase A (Japanese Patent Publication No. 5O-28515), or by culturing alkaliphilic bacteria belonging to the Cellulomonas genus to obtain alkaline cellulase 30.
1-A method for producing A (Japanese Unexamined Patent Publication No. 58-224686),
Method for producing alkaline cellulase K by culturing 5M-635 in alkaliphilic Bacillus bacteria (JP-A-63-1
09776) etc. have been reported.

However, these conventionally known cellulases do not necessarily have sufficient stability against anionic surfactants, which are one of the detergent ingredients, so there is a desire to develop enzymes that are more stable against anionic surfactants. There is.

[Problems to be Solved by the Invention] The present invention aims to provide a cellulase suitable as a detergent enzyme that has an optimum pH on the highly alkaline side and is stable against anionic surfactants. be.

[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 discovered that Bacillus strain SD401, a bacterium belonging to the genus Bacillus isolated from the soil in the lower Tokyo area, produces a new cellulase that has excellent properties as a detergent enzyme.
The present invention has now been completed.

That is, the present invention provides 1) carboxymethylcellulose (CM
The optimum pH when C) is used as a substrate is 9.5 to 10.0.
The optimum temperature is approximately 55°C, and 2) the stable pH range is 30°C.
, pH 6-11 when treated for 30 minutes, 3) 30
It is extremely stable against surfactants, with a residual activity of more than 90% even after treatment at 30°C for 2 hours in the presence of 00 pp■ sodium linear alkylbenzene sulfonate (pH 9,0). ) The molecular weight is 52.000 when measured by SDS-polyacrylamide electrophoresis.
±2000, and 5) a novel cellulase whose isoelectric point is 4.0 or less when measured by polyacrylamide electrophoresis; a microorganism of the genus Bacillus having the above-mentioned cellulase-producing ability is cultured in a medium, and the culture is The present invention aims to provide a method for producing the cellulase described above, which is characterized by collecting the desired cellulase from a cellulase; and a novel strain of the genus Bacillus having the ability to produce the cellulase described above.

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

Production 1 The microorganism used for the production of the novel cellulase of the present invention is a bacterium belonging to the genus Bacillus that can produce cellulase having the above-mentioned properties, and has the following properties.

(a) Form ■Cell shape and size ■Cellular polyplasia ■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 0.3~0.5X 2.8~ It is 5.2 μm.

Does not show polyplasia.

Has periflagella.

Forms spores.

Its shape is oval; The size is 1.0~1.5x It is 1.8 to 2.2 μm.

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

Also, the color tone of the village is yellow-white. ■Meat juice agar slant medium ■ Broth liquid medium ■Meat juice gelatin puncture culture ■Lidomas milk (c) Physiological properties reduction of nitrate ■Denitrification reaction ■MR test ■vP test ■Generation of indole Production of hydrogen sulfide ■Hydrolysis of starch ■ Benefits of citric acid ■Use of inorganic nitrogen sources [Phase] Formation of pigment ■Urease ■Oxidase The color is translucent.

Grows in a spreading pattern.

turbidity liquefy Coagulation and peptonization do not have negative negative negative negative negative negative positive negative Nitrate and ammonia Use mucilage salt.

negative negative unclear 0 catalase [Strain] Growth range (pH) (temperature) ■Attitude towards oxygen @O-F test positive Grows at 8.0 but 7.1 It does not grow in

Although it grows in 10.8.

12.3 will not grow.

Grows at 15℃ and 40℃ However, it does not grow at 50°C.

Aerobic fermentation (Fer+mentation) Oxidation of 0w and production of gas Oxidation Gas L-arabinose D-xylose D-glucose D-mannose D-fructose D-galactose Maltose Sucrose Lactose (lO) Trehalose 10-(11
) D-Sorvit (12) Mannitol - (13) Inosit - (14) Glycerin - (15) Starch
Bacteriology (Bergey's Manual
of Systematic Bacteriolo (
y), and The Genus Bacillus (The Gen
As a result of referring to Bacillus (US Bacillus), it was found that the heavy two were Bacillus alcalophilus (Bacillus alkalophyllus), which is a spore-bearing bacterium.
It was recognized that it is a bacterium related to S. lus alcalophilus). However, the Bersey's Manual
NCIB 104, a standard strain of Bacillus arcalophilus described in the Department of Systematic Bacteriology
38 and Juryo have different bacteriological properties, such as growth conditions on broth agar and lidmus milk, and production of acid from galactose, and are clearly new strains. It was named Bacillus SD401.
It was deposited as Microtech Research Institute No. 10527.

Note that the microorganism used in the present invention is not limited to the Bacillus SD401 strain mentioned above, but may be any strain as long as it has the productivity of cellulase having the characteristics described later in the present invention. It also includes mutant strains and strains improved by genetic engineering techniques.

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; for example, glucose, maltose, starch, CMC, etc. are used. As a nitrogen source, any assimilable nitrogen compound or one containing it may be used, such as ammonium salts, peptone, soybean flour, defatted soybean flour, and the like. Also,
Salts such as phosphates and magnesium salts are used as the inorganic salts. 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 vary somewhat depending on the medium composition, but conditions are selected that are most advantageous for producing cellulase, which is the target product. Culture temperature is 25-3
The temperature range is 5°C, the culture time is about 12 hours to 3 days, and the culture may be terminated when cellulase production reaches its maximum.

The pH of the medium is preferably 8 or higher, and 9 to 10 is suitable for cellulase production. Through such culturing, the desired cellulase can be obtained in the culture solution.

Kametoyo 1 Urasha Cellulase was collected from the culture solution obtained in this way according to the conventional method for collecting enzymes.

Can be separated and purified. i.e. filtration method.

A supernatant or filtrate can be obtained by separating the bacterial cells and medium solids by a known appropriate method such as centrifugation. 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, cellulase can be purified using purification methods such as adsorption/desorption using an ion exchange resin, gel filtration, or the like alone or in combination.

The detailed properties of the cellulase of the present invention will now be described.

-゛■ Carboxymethyl cellulase (CMCase) activity Carboxymethyl cellulose (CMC) was dissolved in M/20 sodium carbonate-M/20 boric acid-potassium chloride buffer (p
Measurement is performed using a solution dissolved in H9,0) as a substrate.

Specifically, 1% CMC (pH 9,0) 1.0 d1M
0.1 ml of enzyme solution was added to a substrate solution consisting of 0.9 ml of /20 sodium carbonate-M/20 boric acid-potassium chloride buffer (pH 9,0) and reacted at 30°C for 15 minutes. Hydroxybenzoic hydrazide (4
-Hydroxybenzoic hydrazide
) method (pHBAH method) to quantify reducing sugars. That is, 1.0 ml of pHBAH reagent was added to 2.0 ml of reaction solution.
1 was added thereto, and the mixture was heated at 100° C. for 10 minutes to develop color. After cooling, colorimetric determination was performed at a wavelength of 410 nm. For the enzyme titer, one unit was defined as the amount of enzyme that produced reducing sugar equivalent to 1 μmol of glucose per minute under the above conditions.

■ Avicelase activity Avicel (Merck & Co.) l'l/20 sodium carbonate -
A suspension in M/20 boric acid-potassium chloride buffer (pH 9,0) was used as a substrate for measurement. Specifically, 10
After 0 reaction, 0.1 ml of enzyme solution was added to 9 ml of Avicel (pH 9,0) and reacted at 30°C for 300 minutes.

Add 1.0 ml of pHBAH reagent to 2.0 ml of the reaction solution.

Heat to develop color at 100℃ for 10 minutes, and after cooling, wavelength 41
Colorimetric determination was performed at 0 nm. For the enzyme titer, one unit was defined as the amount of enzyme that produced reducing sugar equivalent to 1 μmol of glucose per minute under the above conditions.

■ 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 carried out using cellobioside (pNPC) as a substrate. Specifically, M/20 sodium carbonate-M/20 boric acid-
Potassium chloride buffer (pH 9,0) 1.5ml, 50+
Add enzyme solution 0.1 to a substrate solution consisting of 1 ml of mMPNPO or 5mM pNPCO, and incubate at 30°C for 300 min.
After reacting for minutes, the liberated P-nitrophenol was
Colorimetric determination was carried out using a 0n phoenix. 1μmo per minute under these conditions
The amount of enzyme that liberated 1 of p-nitrophenol was defined as 1 unit.

(1) Action This enzyme acts on carboxymethyl cellulose (CMC) and highly crystalline Avicel.

Hydrolyzes β-glucoside bonds. It also acts on the synthetic substrate p-nitrophenyl cellobioside to liberate p-nitrophenol.

(2) Substrate specificity This enzyme has CMC, Avicel, p-nitrophenyl β-
If CMCase activity, which acts on D-cellobioside (pNPC) and does not act on p-nitrophenyl β-D-glucopyranoside (pNPG), is 100, avicelase activity is 3, and pNPC degrading activity is 0. It shows the relative activity of 3.

Substrate Relative activity CMC100 Avicel 3 pNPG 0 pNPC0,3 (3) Optimal pH Britton-Robins
CM on) using a wide range & IWI solution (pH 6-11)
When measured using C as a substrate, it is 9.5 to 10.5.

The relationship between reaction pH and relative activity is shown in FIG.

(4) Stable pH range: Leave at 30°C for 30 minutes under buffers with different pH values,
When measured using CMC (pH 9,0) as a substrate, the stable pH is 6 to 11. Figure 2 shows the heat treatment pH and residual activity.

(5) Optimum temperature When measuring CMC as a substrate, the optimal temperature is approximately 55°C.
It is. The relationship between temperature and relative activity of Ji Oyu is shown in Figure 3.

(6) Thermal stability A/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 at
It has approximately 30% residual activity at 5°C.

FIG. 4 shows the relationship between heat treatment temperature and residual activity.

(7) Effects of enzyme inhibitors, metal ions and chelating agents
It is inhibited by M) and activated by Ca'' (5mM).EDTA (5mM)
It is not affected by

(8) Effect of surfactant Sodium linear alkylbenzene sulfonate 3000
FIG. 5 shows the relationship between the treatment time and the residual activity, which shows that there is almost no inactivation even when treated in the presence of (J)H9,O) for 2 hours at 30°C in the presence of ppm.

(9) Molecular weight The molecular weight obtained by SDS-polyacrylamide gel electrophoresis is 52,000±2,000.

(10) Isoelectric point The isoelectric point is 4.0 or less as measured by polyacrylamide gel electrophoresis.

This enzyme/element is Bacillus SD40, a strain belonging to the genus Bacillus.
Produced by one strain, optimal pH for high alkaline pH range
It has the following: Special Publication No. 50-28515 and
When compared with the alkaline cellulase described in JP-A-83-109778, the molecular weight of this enzyme is 520.
00±2000, whereas the molecular weight of the alkaline cellulase is 15000.30000 and 1soooo
They are clearly different enzymes in that they differ in their chemical properties and other physicochemical properties.

[Effects of the Invention] The cellulase of the present invention has a high optimum pH of 9.5 to 10.5, and has a relative activity of about 70% of the optimum pH even at pH 11, and about 60% of the relative activity at pH 6. It has relative activity and maintains its activity over a wide pH range.

It also has sufficient activity even at low temperatures. Furthermore, since it has high stability against anionic surfactants, which are detergent components, it can be used as an enzyme for detergents, and its detergency can be enhanced.

Next, the present invention will be described in more detail with reference to typical examples.

Real (medicine) - 1% peptone, 0.5% sodium chloride, 0.1% dipotassium phosphate, 0.05% magnesium sulfate.

Yeast extract 0.5%, cellobiose 0.5%, CMC0
, 5% and sodium carbonate 0.5% was dispensed into test tubes and sterilized by a conventional method. SD-40 for this
One strain was inoculated and cultured with shaking at 35°C for 25 hours. The culture solution was centrifuged and the cellulase activity of the supernatant was measured and found to be 0.20/ml.

Sougantou 2 Soy flour 2%, Sodium chloride 0.5%, Dipotassium phosphate 0.1%, Magnesium sulfate 0.02%, Yeast extract 0.5%, Maltose 0.5%, CMG O, 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 SD-401 strain that had been cultured in advance, and cultured with aeration and stirring at 35°C for 35 hours. This culture solution was centrifuged to obtain a supernatant. The cellulase activity of this supernatant was 1. It was IU/ml.

1.3 L of this supernatant was concentrated using an ultrafiltration membrane and freeze-dried to obtain a crude enzyme with a cellulase specific activity of 100 ulz.

The cellulase of the present invention was purified from the crude enzyme obtained in Example 2.

4 g of crude enzyme was added to 200 ml of 10mM sodium carbonate-10mM boric acid-potassium chloride buffer (pH 9).
A 20-70% fraction of precipitate was obtained by the ammonium sulfate precipitation method. This precipitate was dissolved in the above buffer solution, desalted using an ultrafiltration membrane, and then 10 mM Bis-Tris buffer (p
The active fraction was purified using a CM (carboxymethyl) cation exchange resin column (25 mmφ, 30 cm) equilibrated with H7,0), and the obtained active fraction was further purified using the same CM cation exchange resin column. After concentrating and desalting the active components eluted in the same procedure using an ultrafiltration membrane, 10 mM Bis-Tris buffer (p
DEAE (diethylaminoethyl) anion exchange resin column (25 mmφ, 36 cm) equilibrated with
) and the concentration gradient of sodium chloride (0 to LM)
It was eluted with There are at least 3 CMCases in the crude enzyme.
Ingredients were present. The relationship between fractionation and activity using DEAE anion exchange resin is shown in FIG. The main component, CMCase (fraction numbers 122 to 141), was concentrated using an ultrafiltration membrane and further purified using the same DEAE anion exchange resin. Both active components eluted in the same procedure were desalted using an ultrafiltration membrane, and after lyophilization, 10mM Bis-Tris buffer (pH 7) was added.
, 0) and diluted with 10mM Bis-Tris buffer (pH 7).
It was adsorbed onto a phenyl sepharose CL4B column equilibrated with
M) was eluted. The active fraction eluted in the same procedure was desalted using an ultrafiltration membrane and freeze-dried. In this process 2.2
A lyophilized sample of 1.0 mg was obtained.

This freeze-dried specimen was white in color, and when tested by polyacrylamide gel electrophoresis, it was confirmed to be single.

Using this freeze-dried sample, 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.

Back mom A A washing test was conducted on the cellulase of the present invention.

(a) Preparation of contaminated cloth Place about 200 ml of activated carbon in a milk drum and mix with pure water to make an aqueous solution. It was applied uniformly to a cotton cloth in a pallet, air-dried, rubbed with a sponge 20 times to remove excess activated carbon, cut into 5 cm x 5 cm pieces, and used for a cleaning test.

(b) Cleaning test Cleaning was carried out using a turbotometer (Terg-0-To+aet
I went to er). The cleaning conditions were 12 rpm and 30'C.

For 10 minutes, detergent (JIS phosphorus-free) concentration 1330p
It was set as pm. When the crude enzyme obtained in Example 2 was added at a concentration of LoIIg, an improvement of 5% in white charcoal and 8% in washing efficiency was observed compared to the case without addition.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the relationship between reaction pH and relative activity. FIG. 2 is a graph showing heat treatment pH and residual activity. Figure 313 is a graph showing the relationship between reaction temperature and relative activity. FIG. 3 is a graph showing the relationship between heat treatment temperature and residual activity. Figure 5 shows linear alkali sodium benzene sulfonate 3
2 is a graph showing stability in the presence of 000 pp+a. FIG. 6 is a graph showing the fractionation and activity in the first DEAE anion exchange resin. Figure 1 Figure 2

Claims (1)

[Claims] 1. A novel cellulase having the following properties (1) Optimal pH, optimal temperature When carboxymethyl cellulose (CMC) is used as a substrate, the optimal pH is 9.5 to 10.5, optimal The temperature is about 55℃
It is. (2) Stable pH Stable at pH 6 to 11 when treated at 30°C for 30 minutes. (3) 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. (4) Molecular weight The molecular weight measured by SDS-polyacrylamide electrophoresis is 52,000±2,000. (5) Isoelectric point The isoelectric point measured by isoelectric focusing is 4.0 or less. 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 target cellulase from the culture. 3. A microorganism capable of producing cellulase according to claim 1. 4. The bacterial strain is Bacillus SD401 strain (Feikoken Bacteria Collection No. 105)
The microorganism according to claim 3, which is No. 27).