JP3508937B2 - Novel endo-type neutral cellulase and its production method - Google Patents

Description

Detailed Description of the Invention

[Background of the Invention]

TECHNICAL FIELD The present invention relates to a novel cellulase and a method for producing the same. More specifically, it relates to a novel endo-type neutral cellulase obtained from a microorganism belonging to the genus Scopraliopsis and a method for producing the same.

[0002]

2. Description of the Related Art In recent years, remarkable progress has been made in clothing detergents and dish detergents, and in particular, a method of decomposing dirt with various hydrolases has been introduced to remarkably increase the detergency. ing. Here, it is considered that these enzymes play a role of directly decomposing the dirt and assisting a cleaning component such as a surfactant by lowering the molecular weight of the dirt.

Protease, lipase, amylase, cellulase and the like are used as hydrolytic enzymes to be added to the detergent composition. Among them, cellulase is an enzyme that has recently been put to practical use, and it acts to cut off the non-crystalline portion of the cellulose fiber such as cotton at the end to elute the dirt inside the fiber and enhance the cleaning effect. Therefore, as the cellulase used for such a purpose, one having a high end property is preferable in order not to damage the fiber body as much as possible. Furthermore, since detergents generally exhibit neutrality to alkalinity, neutral-alkaline cellulases that exhibit the above-mentioned high endo-activity in neutrality to alkalinity are desired.

Cellulase satisfying such conditions includes alkaline cellulases produced by alkalophilic Bacillus (Japanese Patent Publication No. 50-28515, Japanese Patent Application No. 61-257776).
Alternatively, a cell alkaline cellulase obtained from alkaline actinomycetes (Japanese Patent Publication No. 3-65150) is also known.

However, all of these are alkaline enzymes which show the optimum pH for alkaline, and there has been a strong demand for the development of neutral cellulases which can be used in neutral detergents. On the other hand, generally, as a microorganism expressing a strong cellulase, filamentous fungi such as mold occupy the majority, and since filamentous fungi are easy to culture, development of useful cellulase as described above produced by filamentous fungi is desired. Was there.

[Summary of the Invention] [0006] The inventors of the present invention, as a result of searching mainly for filamentous fungi such as fungi, search for a microorganism that produces a cellulase with maximum neutrality and high endogeneity, and belongs to the genus Scopraliopsis. The present inventors have found that a filamentous fungus efficiently produces a desired novel cellulase by aerobic culture, and completed the present invention.

[0007]

SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide a novel cellulase which exhibits maximum activity in the vicinity of neutrality and has high endogeneity, and a method for producing the same.

Another object of the present invention is to provide a novel strain having the above-mentioned novel cellulase-producing ability.

[0009]

The novel cellulase according to the present invention has the following physicochemical properties. (a) Action and substrate specificity: It acts specifically on cellulose such as carboxymethylcellulose (CMC), and its β-1,4-glycosidic bond is cleaved by an endo-type mechanism to cause a significant decrease in viscosity, Almost no production of reducing sugar is observed. (b) Optimum pH: The optimum pH is 6.0 to 7.0. (c) Stable pH: 30 ° C for 30 minutes,
It is stable at pH 7.0 to 12.0. (d) Optimum temperature: The optimum working temperature is about 50-60 ° C. (e) Stable temperature: It is stable up to 55 ° C. under the treatment conditions of pH 9.0 and 10 minutes. (f) Molecular weight: about 22,000 (SDS electrophoresis) (g) Isoelectric point: about 3.7 to 3.8.

The method for producing the novel cellulase according to the present invention comprises a step of culturing a microorganism belonging to the genus Scopraliopsis and having the cellulase-producing ability, and separating the novel cellulase from the culture obtained in the step. It is one that encloses the process.

Furthermore, a novel strain having the above-mentioned novel cellulase-producing ability according to the present invention is Scopulariopsis.
Brevi Kauris TOTO-9221 strain (FERM-P-
13285).

The cellulase according to the present invention has an optimum pH in the vicinity of neutrality and acts with high endogeneity. Therefore, it can be added to laundry detergents and softeners for the purpose of enhancing the cleaning effect, or can be used in a wide range of fields as research reagents and the like. Further, the strain according to the present invention efficiently secretes the above cellulase to the outside of the bacterial cell, and is therefore advantageous in that it can be efficiently produced by a simple process.

[Detailed Description of the Invention] Novel Cellulase-Producing Strain The novel cellulase according to the present invention can be produced by using a microorganism. Particularly preferably, the cellulase according to the invention is of the genus Scopulariopsis, in particular Scopulariopsis brevicaulis T.
Produced by OTO-9211 strain. This strain was isolated from the air of a general house in Hiroshima City by the inventors. This strain is a filamentous incomplete bacterium and has mycological properties as shown below. I. Morphological properties 1 Large conidia: None 2 Conidia: Surface rough, spherical, 8 μm, concatenated 3 Conidia Form: ring-shaped II. Growth state in various media 1 Potato dextrose agar medium (29 ° C, 11 days culture) Colony diameter: 60-82mm Velvet, light brown, flat, surface colorless 2 Tuapec agar medium (29 ° C, 11 days culture) Colony diameter: 45mm Velvety, light yellowish brown, colorless surface III. Physiological properties 1 Growth pH range: 3.0 to 11.0 Optimal growth pH: 4.0 to 10.0, alkali tolerance 2 Growth temperature range: 10 to 50 ° C Optimal Growth temperature: 25 to 35 ° C. 3 Neutral cellulase-producing ability.

The present strain having the above-mentioned mycological properties is a genera of Hyphomycetes (1980) by Carmichael et al.
) And the General of GLBarron
Hyphomy Ceteth From Soil (The Genera of H
As a result, it was found that the strain was a morphologically belonging to the genus Scopraliopsis. In addition, since the conidia have a rough spherical shape, this strain can be used for Scopulariopsis brevicauris (Scopular
iopsis brevicaulis) TOTO-9221.
In addition, this strain has been deposited under the deposit number 13285 (FERM-P-13285) at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology.

Culture Conditions The medium for culturing the above-mentioned strain does not have to be special, and an ordinary medium can be used. Carbon sources such as various types of cellulose (paper, pulp, cellulose powder, cotton, etc.), cellulose derivatives (carboxymethyl cellulose, cellulose acetate, etc.), processed cellulose products (phosphoric acid-swelling cellulose, alkali-swelling cellulose, etc.), or wheat, wood flour, etc. A natural substrate, glucose or the like can be used. As the nitrogen source, inorganic substances such as nitrates and ammonium salts, urea, peptone, dry yeast, yeast extract, soybean powder, corn steep liquor, casein, meat extract, amino acid and the like can be used. In addition to these carbon and nitrogen sources, various salts such as magnesium salts,
You may add potassium salt, sodium salt, phosphate etc. as needed. Furthermore, the pH of the medium may be made alkaline by adding separately sterilized sodium carbonate or sodium hydrogen carbonate.

Culture is carried out in such a medium at a medium temperature of 20.
It is carried out at -50 ° C, preferably 25-35 ° C for 3 to 7 days under aerobic stirring or shaking.

The novel cellulase according to the present invention is mainly secreted and accumulated in the culture medium by the culture under the above conditions.

Collection of Enzyme In order to collect and purify the enzyme of the present invention from the above culture solution, known purification methods can be used alone or in combination. Since the present enzyme is mainly secreted outside the cells (in the culture solution), a crude enzyme solution can be easily obtained by removing the cells by filtration or centrifugation.
This crude enzyme is further purified by known methods such as salting out with ammonium sulfate; precipitation with organic solvents such as methanol, ethanol and acetone; adsorption with Avicel: ultrafiltration; gel filtration chromatography; ion exchange chromatography, Other various chromatographies can be used alone or in combination for purification.

The preferred purification method is as follows.
First, 70% saturated ammonium sulfate is added to the culture filtrate for salting out, and the obtained precipitate is dissolved in a buffer solution. Then Sephadex
Gel filtration that also serves as desalting is performed using a G-75 (manufactured by Pharmacia) column. This active fraction is subjected to ion exchange chromatography with DEAE-Toyopearl 650M (manufactured by Tosoh Corporation) (elution with a gradient of 0 to 0.5 M NaCl). After concentration of this active fraction, chromatofocusing with a PBE94 (Pharmacia) column (elution with a pH gradient) is performed to obtain a purified enzyme that is uniform by SDS electrophoresis.

Properties of the enzyme The properties of the cellulase according to the present invention are as shown below. In addition, in the following, the activity measuring method means the following method.

(Activity measuring method) 50 mM phosphate buffer (pH
0.5% CMC dissolved in 7.0) is used as a substrate, and its viscosity reduction is measured by Ostwald viscometer. Add an appropriate amount of enzyme solution to 5 ml of substrate solution and
React at. Then, the flow rate of the solution is measured at regular intervals to determine the specific viscosity. Under these conditions, 1
The amount of enzyme required to cleave 1 μM of substrate once per minute is defined as 1 unit of cellulase activity. The formula for calculating the activity unit is shown below. Cellulase activity (unit / ml) = a / η sp a: substrate, solvent, constant determined by the temperature and the like (0.735 in this condition) eta _sp: reduction ratio of specific viscosity per minute enzyme reaction time (1) Action And substrate specificity Cellulose derivatives such as CMC were used as a substrate to carry out an enzymatic reaction according to the activity measurement method to examine the decrease in viscosity and the amount of reducing sugar produced. As a result, this enzyme acted on various celluloses and endoly cleaved the β-1,4-glycoside bond to cause a rapid decrease in viscosity, but almost no production of reducing sugars was observed.

(2) Optimum pH and stable pH Using this enzyme, an enzymatic reaction was carried out in the range of pH 4.0 to 9.0 according to the activity measuring method. As the buffer, Britton-Robinson broad range buffer was used. Its relative activity is as shown in Figure 1A. From this, it can be seen that the optimum pH of the present enzyme is about 6.0 to 7.0 at 30 ° C.

Further, the present enzyme is added to a predetermined pH buffer solution,
The residual activity was measured after holding at 30 ° C. for 30 minutes. Britton-Robinson buffer (pH 3.0-
12.0) and KCl-NaOH buffer (pH 13.0). The result is as shown in B of FIG. From this, it can be seen that the stable pH range of the present enzyme is pH 7.0 to 12.0 under the treatment condition of 30 ° C. for 30 minutes.

(3) Optimum temperature and stable temperature Using this enzyme, an enzymatic reaction was carried out according to the activity measuring method under the temperature conditions of pH 7.0 and 30 to 70 ° C. Its relative activity is as shown in Figure 2A. From this, it can be seen that the optimum working temperature of the present enzyme is about 50 to 60 ° C.

Further, the present enzyme was added to a predetermined buffer solution (pH 9.0), and the remaining activity was measured after holding for 10 minutes under the temperature condition of 30 to 70 ° C., and the residual activity was measured. The result is shown in FIG. 2B. This shows that this enzyme is stable up to 55 ° C.

(4) Molecular weight The molecular weight of this enzyme was measured by SDS electrophoresis, and the molecular weight was about 20,000 to 22,000.

(5) Isoelectric point The isoelectric point of the present enzyme was measured by the isoelectric focusing method, and the isoelectric point was about 3.5 to 3.8.

[0028]

EXAMPLES The present invention will be described in more detail with reference to the following examples, which should not be construed as limiting the invention thereto.

Example 1 Preparation of crude enzyme powder Scopulariopsis brevicauris TOTO-921
400 ml of preculture liquid for 1 strain (28 ° C, 3 days shaking culture)
CMC (Na salt) 1%, glucose 3%, yeast extract 0.5%, NaNO ₃ 0.3%, K ₂ HPO ₄ 0.1
_{%, MgSO 4 · 7H 2 O} 0.05%, KCl 0.
_{05%, FeSO 4 · 7H 2} O 0.001%, Tri
TonX-100 medium containing 0.1% (pH 7.0)
The jar fermenter containing 20 L was inoculated and cultured at 28 ° C. for 3 days at a stirring rate of 300 rpm and an aeration rate of 20 L / min. After completion of the culture, the culture solution was adjusted to 8,000 rpm, 10
The cells were removed by centrifugation for a minute. The obtained supernatant is freeze-dried to obtain 0.0025 units / mg of crude enzyme powder 40.
0 g was obtained.

Example 2 Preparation of Purified Enzyme A jar fementor containing 22 L of the same medium as in Example 1 was added to Scopraliopsis brevicauris TOTO-.
400 ml of the pre-medium solution of strain 9211 was inoculated. After culturing this in the same manner as in Example 1, the supernatant liquid 20 was centrifuged.
L was obtained. The activity of this supernatant solution at pH 7.0 was 0.
It was 048 units / ml. Then, ammonium sulfate powder was added to this supernatant until it became 70% saturated, and the mixture was allowed to stand overnight for 8.0
The precipitate was recovered by centrifugation at 00 rpm for 10 minutes. This precipitate is added to 50 mM Tris-HCl buffer (pH
It was dissolved in 7.0) and applied to a Sephadex G-75 (Pharmacia) column which had been equilibrated with the same buffer solution in advance for gel filtration. Active fractions are concentrated and desalted before DEAE
-Ion exchange chromatography was performed with Toyopearl 650M (Tosoh). Elution of enzyme is 0-0.5
It was performed by the gradient method of M NaCl. Then, the active fraction was concentrated and desalted, and then subjected to PBE94 column for chromatofocusing, and the enzyme was eluted by the pH gradient method. 10 units / m by collecting the active fraction
40 ml of the purified enzyme solution (1) was obtained. This showed a single band on SDS electrophoresis and was confirmed to be homogeneous as an enzyme protein.

[0031]

EFFECTS OF THE INVENTION Since the cellulase exhibits maximum activity in the vicinity of neutrality and has a high end property, it can be added to a detergent for clothes or the like for the purpose of enhancing the cleaning effect.

[Brief description of drawings]

1 is a graph showing (A) optimum pH and (B) stable pH range of cellulase according to the present invention.

FIG. 2 is a graph showing (A) optimum pH action temperature and (B) stable temperature range of the cellulase according to the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Hiroshi Sato 2-1-1 Nakajima, Kokurakita-ku, Kitakyushu City, Fukuoka Prefecture Totoki Equipment Co., Ltd. (72) Inventor Motoo Arai Kamodaidai, Sakai City, Osaka Prefecture 3-2-20-204 (72) Inventor Kenji Minami 82-82 Nakano, 21 Nakano, Higashiosaka-shi, Osaka (56) References World J. Microbio l. Biotechnol. , 1990, Vol. 6, No. 1, pages 64-6 (58) Fields investigated (Int.Cl. ⁷ , DB name) C12N 9/14-9/46 JSTPlus (JOIS) BIOSIS / WPI (DIALOG)

Claims (5)

(57) [Claims] 1. A Scopulariopsis brevicauris (Sc
opulariopsis brevicaulis) TOTO-9221 cellulase having the following physicochemical properties. (a) Action and substrate specificity: It acts specifically on cellulose such as carboxymethylcellulose (CMC), and its β-1,4-glycoside bond is cleaved by an endo-type mechanism to cause a significant decrease in viscosity, Almost no production of reducing sugar is observed. (b) Optimum pH: The optimum pH is 6.0 to 7.0. (c) Stable pH: Stable at pH 7.0 to 12.0 under the treatment condition of 30 ° C. for 30 minutes. (d) Optimum temperature: The optimum working temperature is about 50-60 ° C. (e) Stable temperature: It is stable up to 55 ° C. under the treatment conditions of pH 9.0 and 10 minutes. (f) Molecular weight: about 20,000 to 22,000 (SDS
Electrophoresis method) (g) Isoelectric point: about 3.5 to 3.8. 2. A method comprising culturing a microorganism belonging to the genus Scopraliopsis and having the cellulase-producing ability according to claim 1, and separating the cellulase from the culture obtained in the step. Method for producing cellulase. 3. The culture of the microorganism has a pH of 7.0 to 11.0.
3. The method according to claim 2, which is carried out neutral to alkaline. 4. The method for producing cellulase according to claim 2, wherein the microorganism is Scopulariopsis brevicauris strain TOTO-9221. 5. Scopraliopsis brevicauris TOTO-92 having cellulase-producing ability according to claim 1.
11 strains (FERM P-13285).