JPH0787971A - Calboxymethyl cellulase and microorganism producing the enzyme - Google Patents

Abstract

PURPOSE:To obtain the subject enzyme having a wide active pH region, stable in a surfactant, excellent in alkali resistance and useful e.g. as washing agent additive by culturing a bacterial strain belonging to the genus Bacillus and producing a calboxymethyl cellulase 5812. CONSTITUTION:The objective carboxymethyl cellulase 5812 is obtained by culturing a bacterial strain belonging to the genus Bacillus and producing carboxymethyl cellulase 5812 [e.g. Bacillus sp. PKM-5812 (FER P-12573)] separated from the soil collected in Philippine. This cellulase has the following features: strongly active on carboxymethyl cellulose and lichenan and also active on cellulose powder, phosphoric acid dilated cellulose, cellobiose, p-nitrophenyl-beta-D- glucopyranoside, etc. ; active at pH 4. 2 to 12.5 and optimal pH at 7.7 and 9.5 ; stable at pH 4.8 to 12; free from activity loss by heating at 60 deg.C for 10 min; free from activity inhibition by a surfactant; molecular weight (gel filtration method) of 26000+ or -1000.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel carboxymethylcellulase, more specifically a novel enzyme carboxymethylcellulase 5812 which has a wide working pH range and a wide working temperature range, and is extremely excellent in pH stability. Regarding microorganisms.

[0002]

Cellulase consists of a complex enzyme system that catalyzes an enzymatic reaction that decomposes cellulose and its similar polysaccharides into glucose, cellobiose, or cellooligosaccharides. Depending on its mechanism of action, C ₁ enzyme, C _x enzyme, and β enzyme. -Glucosidase, or exo-β-glucanase, endo-β-glucanase, is understood as a general term for enzymes called by the names such as cellobiase. For many years, the history of cellulase research has been carried out exclusively for the purpose of effectively utilizing biomass resources, and for example, molds such as Trichoderma, Aspergillus, Acremonium, and Humicola are sought for their sources. However, cellulases of microbial origin, including molds, have a variety of action specificities and physicochemical properties of their constituent enzyme groups, and it is hard to say that the actual situation has been clarified.

Of the above cellulases, one having a particularly high action on carboxymethylcellulose, that is, an endo-type hydrolyzing action is called carboxymethylcellulase. Recently, as a novel industrial use of cellulase containing carboxymethylcellulase, for example, washing for clothes. The use as an additive component of a drug composition has been developed. However, the cellulase produced by microorganisms in nature is
Most of the above-mentioned microbial sources have an acidic pH.
Has optimum activity and deactivates at alkaline pH,
So-called alkaline cellulase and alkaline-tolerant cellulase, which are so-called acid cellulases (optimal pH of 4 to 6) and which have the maximum activity on the alkaline side and are resistant, which are the requirements for the detergent composition for clothing. The reality is that there are very few.

That is, as a conventional method for producing an alkali cellulase and an alkali resistant cellulase which can be used as a detergent composition for clothes, a method of collecting cellulase A by culturing an alkalophilic Bacillus bacterium (Japanese Patent Laid-Open No.
No. 28515), a method of culturing an alkalophilic bacterium belonging to the genus Cellulomonas to produce alkaline cellulase 301-A (JP-A-58-224686), culturing alkalophilic Bacillus No. 1139 to produce carboxymethylcellulase. Method of production (F. Fukumor
i, T. Kudo and K.K. Horikoshi,
J. Gen. Microbiol . , 131, 3339
(1985)) and a method of producing an alkaline cellulase using a species of Streptomyces (JP-A-61-1).
No. 9483) was reported, and none of them was suitable for industrial fermentation production.

On the other hand, in recent years, Bacillus sp. (Bacillus sp.K which is a kind of alkalophilic microorganisms
SM-635) (Microtechnology Research Institute No. 8872) efficiently produces carboxymethylcellulase suitable as a detergent composition for clothes, and further selects culture conditions to give a higher yield. It was reported that carboxymethylcellulase can be obtained and industrial fermentative production becomes possible.

[0006]

However, the above-mentioned Bacillus sp. KSM-635-derived carboxymethylcellulase has an optimum action temperature of about 40 ° C,
Although suitable for low-temperature washing, there is a demand for an enzyme that can be used as a detergent at higher temperatures. Also,
Generally, the higher the pH, the better the detergency of a detergent. Therefore, carboxymethylcellulase having higher stability at higher pH is required.

The present invention therefore operates at high temperatures,
The purpose of the present invention is to obtain carboxymethylcellulase having a wide working pH range and excellent stability especially at high pH.

[0008]

[Means for Solving the Problems] The present inventor has eagerly searched for a carboxymethylcellulase-producing microorganism in the natural world, and has now been searching for a bacterium belonging to the genus Bacillus collected from soil in Los Banios, Laguna Province, Philippines. A microorganism belonging thereto produces a novel carboxymethylcellulase 5812 effective as an additive component of a detergent composition for clothes, and the carboxymethylcellulase 5812 has a wider action pH range than conventional carboxymethylcellulase, and is thermostable. Was found to be excellent, and particularly stability at high alkaline pH was found, and the present invention was completed.

Carboxymethylcellulase 58 of the present invention
The microorganism that produces 12 has the following mycological properties.
The medium used for separating and culturing the enzyme-producing bacterium is as follows.
It is C. Also, Na ₂ CO ₃ was added after being sterilized separately from other components. Medium A (PCMC medium): Polypeptone (manufactured by Nippon Pharmaceutical Co., Ltd.), 20 g; carboxymethyl cellulose (C
MC, Sanyo Kokusaku Pulp Co., Ltd., 10 g; Yeast extract (manufactured by Difco Laboratories), 1 g; KH ₂ PO ₄
(Wako Pure Chemical Industries, Ltd.), 1 g; NaCl (Merck), 5 g; Na ₂ CO ₃ (anhydrous, Iwai Chemical Co., Ltd.)
5g; MgSO ₄ .7H ₂ O (Wako Pure Chemical Industries, Ltd.)
0.2 g; Trypan blue dye (Merck),
0.075 g; agar (manufactured by Difco Laboratories), 15 g (1.0 L with deionized water) Medium B: Medium A without trypan blue dye. Medium C (CMC-peptone medium): CMC, 10 g; polypeptone, 20 g; yeast extract, 0.5 g; KH ₂ P
O ₄ , 1 g; NaCl, 5 g; Na ₂ CO ₃ , 5 g (1.0 l with deionized water)

(Mycological properties) 1. Microscopic observation results: The size of the bacterial cells was 0.6 to 0.
8 μm × 1.5-3.3 μm rod-shaped spores, and an elliptical spore (0.7-1.0 μm ×) near the center or end of the cells.
1.0-1.6 μm). 2. Motility; positive, with flagella. 3. Gram stain; positive.

4. Growth state in various media: (1) Nutrient agar medium (nutrient agar plate medium) The shape of the colony is circular, its surface is rough, and its periphery is wavy. The color tone of the settlement is white to extremely pale yellow. (2) Nutrient liquid medium (nutrient broth medium) Grow. (3) NaCl tolerance in nutrient broth Grow with 5% NaCl and grow with 7% NaCl and 10% NaCl
Does not grow in 1 (4) Gelatin medium Liquefy gelatin. (5) Litmus milk medium The upper layer of the culture solution is weakly decolorized.

5. Physiological properties: (1) Reduction of nitric acid to nitrite Negative. (2) Denitrification action Negative. (3) MR test Negative. (4) VP test positive. (5) Formation of indole Negative. (6) Generation of hydrogen sulfide Positive. (7) Hydrolysis of starch Negative. (8) Casein hydrolysis positive. (9) Utilization of citrate Simons Citrate agar plate medium; positive. Christensen citrate agar plate; positive. (10) Use of inorganic nitrogen source Negative. (11) Dye formation King A medium; negative. King B medium; negative. Potato dextrose Agar Eiken; Negative. Mannitol and salt agar; negative. (12) Urease negative. (13) Oxidase positive. (14) Catalase positive. (15) Range of growth Growth temperature; 15 to 45 ° C. Optimum growth temperature; 25 to <45 ° C. Growth pH; 4.7 to 10 Growth optimum pH; 6.0 to 9.5 (16) Attitude toward oxygen It grows up anaerobically and anaerobically. (17) OF test Fermentation (F). (18) Use of propionic acid Positive. (19) Tyrosine degradability is negative. (20) Deamination of phenylalanine Negative. (21) Acid production from sugar: positive; D-ribose, L-arabinose, D-xylose, D-glucose, mannitol negative; D-mannose, D-fructose, maltose, sucrose, trehalose, inositol, glycerol, D-
Galactose, lactose, sorbitol, dextrin, raffinose, starch (22) G + C content of DNA 42.6 mol%.

Regarding the above-mentioned mycological properties,
Manual of Systematic Bacteriology (Bergey's Manual of Sys)
As a result of reference to Volume 2 of the Bacterial Bacteriology, this strain was identified as Bacillus ( B.
It was determined to be a member of the genus acillus ).

Since the mycological properties of this strain do not match any of the known Bacillus, this strain was judged as a new strain and named Bacillus spp PKM-5812. Deposited to Institute of Microbial Technology, Institute of Technology.

Carboxymethylcellulase 58 of the present invention
To obtain 12, the above Bacillus SPKM-
5812 or its mutant strain may be cultured in a medium, and the obtained culture may be separated and purified by a conventional enzyme purification method.

In the fermentative production of carboxymethylcellulase 5812, a suitable medium is sterilized by heating or the like, and then Bacillus spp PKM-5812 (Microtechnical Laboratory No. 12573) is inoculated, preferably at 15 ° C to 45 ° C. Culture may be carried out at 25 ° C to 43 ° C for 1 to 4 days with shaking or aeration and stirring. Good results are obtained when the pH is adjusted to neutral to pH 10. Foaming sometimes occurs, but it can be eliminated by adding an appropriate defoaming agent.

For production of carboxymethylcellulase 5812, a nitrogen source and a carbon source which can be assimilated may be appropriately combined and contained in the culture medium, and both nutrient sources are not particularly limited. For example, as a nitrogen source, corn gluten meal, soybean powder, corn steep liquor, casamino acid,
Organic nitrogen sources such as yeast extract, pharma media, sardine meal, meat extract, peptone, hypro, ajpower, coffee meal, cottonseed meal meal, cultivator, amiflex and adipron, zest, azix can be mentioned. Further, as a carbon source, rice husks, rice flour, filter paper, general papers,
In addition to plant fiber such as sawdust, waste sugar concentrate, invert sugar, CMC, Avicel, cellulose cotton, xylan, pectin, assimilable carbon source, for example, ribose, arabinose, xylose, glucose, mannose, fructose, maltose, sucrose , Mannitol, inositol, glycerol, and assimilable organic acids such as acetic acid and citric acid. That is, any medium in which these nitrogen source and carbon source are appropriately combined may be used, and the above nutrient sources are not particularly limited. Others, phosphoric acid, M
A medium containing inorganic salts such as g ²⁺ , Ca ²⁺ , Mn ²⁺ , Zn ²⁺ , Zn ²⁺ , Co ²⁺ , Na ⁺ , K ⁺ and, if necessary, inorganic and organic micronutrient sources is appropriately selected. Then used.

From the thus obtained culture, carboxymethylcellulase 5812 can be obtained according to a general means of collection and purification. That is, cells are separated from the culture by centrifugation, filtration, or the like, and the cells and the culture supernatant are separated by a conventional means such as salting out method, isoelectric focusing method, solvent precipitation method (methanol, ethanol). , Isopropanol, etc.) to precipitate the protein, or it is concentrated by ultrafiltration to obtain a crude enzyme solution of carboxymethylcellulase 5812. For example, ammonium sulfate (30 to 100% saturated fraction) is used in the salting-out method, and solvent precipitation is performed using, for example,
After precipitating the enzyme in 75% ethanol, it is also possible to obtain a freeze-dried powder by filtering, centrifuging, or desalting. As a desalting method, a general method such as dialysis or gel filtration using Sephadex or biogel is used.

This crude enzyme solution can be used as it is, but if necessary, known methods such as hydroxyapatite chromatography, ion exchange chromatography such as DEAE-Sephadex or DEAE-Biogel, and Sephadex or Biogel can be used. It is also possible to purify by combining appropriate molecular sieve gel chromatography, and use as a purified enzyme.

The carboxymethylcellulase 5812 (crude enzyme) thus obtained has the following properties. The enzyme activity was measured according to the following method.

(A) Carboxymethylcellulase activity:
The activity was measured by the DNS method (J. R. Summer and
G. F. Somers, Laboratory Ex
periments in Biological C
chemistry, Academic Press, N
ew York, U.S.A. S. A. , 1944). That is, glycine-NaOH buffer (pH 9.
0) 1.1% (w / v) CMC (A0 in 0.1 M)
1MC) substrate solution (0.9 ml) and appropriately diluted enzyme solution (0.1 ml) were added and reacted at 40 ° C. for 20 minutes, and then 0.05% (w / v) 3,5-dinitrosalicylic acid (Wako Pure Chemical Industries, Ltd.) 1 ml (manufactured by K.K.) was added, and immediately placed in a boiling water bath for 5 minutes and then cooled in an ice water bath. 4 ml of deionized water was further added to this, and then colorimetric determination was carried out by the absorbance at 535 nm. The enzyme titer was defined as 1 unit of the amount of enzyme that produces a reducing sugar corresponding to 1 μmole of glucose per minute under the above conditions.

(B) Hydrolytic activity on cellulose substrate: Sodium hydroxide swollen cellulose and phosphoric acid swollen cellulose can be prepared by the method of Tomita et al. (Y. Tomit).
a, H.A. Suzuki and K.M. Nisizaw
a, J. Biochem . , 78, 499 (197
5)) was used. The produced reducing sugar was measured by the same operation as in (a) except that a substrate solution containing a cellulose substrate was used and the reaction temperature was 30 ° C.

(C) Hydrolytic activity on polysaccharide: The reducing sugar produced was measured by the same procedure as in (a) except that a substrate solution containing a polysaccharide was used and the reaction temperature was 30 ° C.

(D) Hydrolytic activity for p -nitrophenyl-β-D-glucopyranoside (PNPG: Sigma) and p -nitrophenyl-β-D-cellobioside (PNPC: Sigma): pH 7.0. In 0.1 M phosphate buffer solution (1), an appropriately diluted enzyme solution was added to a substrate solution containing PNPG or PNPC, 8 mM, and reacted at 30 ° C for 20 minutes. The produced p -nitrophenol was measured at 400 nm and the amount produced was determined.

(Enzymatic Properties) (1) Action The present enzyme has an enzymatic activity on CMC.

(2) Substrate specificity As shown in Table 1 below, this enzyme has a strong activity against CMC and lichenan. Also, cellobiose, PNP
It is also active against G, PNPC, cellulose powder and phosphate swollen cellulose.

[0027]

[Table 1]

(3) Working pH and optimum pH The working pH range of this enzyme is 4.2 to 12.5.
There are two at about 7.7 and about 9.5 (Fig. 1). PH 1
It has an activity of about 90% of the maximum activity at 0.0, and the activity exceeds about 30% even at pH 11.5.
The activity is maintained at about 10% even at 2.5.

(4) pH stability This enzyme was added to McClubain buffer (pH 3 to 7), 0.1 M.
Tris-HCl buffer (pH 7-9), 0.1 M glycine-sodium hydroxide buffer (pH 8-11), 0.1.
1 M potassium chloride-sodium hydroxide buffer (pH 9
~ 14) overnight at 5 ° C and then at pH 6.0.
1 M sodium phosphate buffer and pH 9.0 0.1
The residual activity was measured in M glycine-sodium hydroxide buffer and the pH stability was investigated. As a result, this enzyme is extremely stable in both pH ranges from pH 4.8 to 12 and hardly inactivated (Fig. 2).

(5) Action temperature and optimum temperature The carboxymethylcellulase activity of this enzyme is adjusted to pH 9.0.
Of glycine-sodium hydroxide buffer. Its working temperature is in a wide range of 10 to 70 ° C, and the optimum temperature is around 55 ° C (Fig. 3).

(6) Thermostability After the enzyme was pretreated at each temperature for 10 minutes, the residual activity was measured in 0.1 M glycine-sodium hydroxide buffer solution at pH 9.0 to determine the thermostability. Examined. As a result, 60 ℃
Almost no deactivation until heating, and some activity remained after heating at 70 ° C (Fig. 4). Under these conditions, Ca ²⁺
No effect was observed.

(7) Effect of metal ion Each metal ion preparation shown in Table 2 and CMC were mixed with an enzyme solution, and carboxymethylcellulase activity was adjusted to pH 9.0.
In 0.1 M glycine-sodium hydroxide buffer solution at 40 ° C. for 20 minutes. Among the various metal ions tested, 1 mM Hg ²⁺ inhibited the enzyme activity by about 75%. 1 mM Co ²⁺ promotes enzyme activity by about 50%,
Mn ²⁺ promoted the enzyme activity by about 50%. Incidentally, the other metal ions shown in the same table showed some inhibition or promotion, and no influence by the other metal ions was observed.

[0033]

[Table 2]

(8) Effect of Surfactant The surfactants shown in Table 3 were mixed with the enzyme solution, and the carboxymethylcellulase activity was adjusted to 40% in 0.1 M glycine-sodium hydroxide buffer solution of pH 9.0. It measured at 20 degreeC for 20 minutes. From the results shown in Table 3, the present enzyme is sodium dodecyl sulfate (SDS), linear alkylbenzenesulfonate sodium (LAS), alkyl sulfate sodium salt (AS), polyoxyethylene alkyl sulfate sodium salt (ES), α-olefin. Sodium sulfonate (AOS), α-sulfonated fatty acid ester sodium salt (α-SFE), fatty acid salt (soap), polyoxyethylene alkyl ether hardly inhibited the activity, or accelerated the activity. Above all, SD
21% of S and 23% of fatty acid salt promoted enzyme activity.

[0035]

[Table 3]

(9) Effect of Inhibitors The inhibitors shown in Table 4 were mixed with an enzyme solution and the carboxymethylcellulase activity was adjusted to 0.1 M glycine-pH 9.0.
The measurement was carried out at 40 ° C. for 20 minutes in a sodium hydroxide buffer. The activity of this enzyme was 9 by N- bromosuccinimide.
It is significantly inhibited by 7%, but the activity is not inhibited by other inhibitors.

[0037]

[Table 4]

(10) Effects of chelating agent, thiol reagent, additives and proteinases Each reagent shown in Table 5 was mixed with an enzyme solution to give a carboxymethylcellulase activity of 0.1 M glycine-pH 9.0.
The measurement was carried out at 40 ° C. for 20 minutes in a sodium hydroxide buffer. The activity of this enzyme was somewhat inhibited by EDTA or EGTA. On the other hand, 1 mM 2-mercaptoethanol promoted the enzyme activity by 52% and dithiothreitol promoted by 84%. In addition, alcalase, maxatase, savinase,
With proteinases such as the protease API-21, inactivation of the enzyme was not observed under the reaction conditions.

[0039]

[Table 5]

(11) Molecular Weight The method of Shikata and Nizawa (S. Shi
kata and K.K. Nizawa, J .; Bio
chem . , 78, 499 (1975)), a gel filtration method using Biogel A-0.5m was performed, and the molecular weight of this enzyme was 26,000 ± 1,000.
Met.

[0041]

EFFECT OF THE INVENTION Carboxymethylcellulase 5 of the present invention
812 has a feature that the working pH range is extremely wide as 4.2 to 12.5 and that it is extremely stable up to pH 12. Further, it has a wide working temperature of 10 to 70 ° C., good thermal stability, and high temperature and high alkali cleaning. Furthermore, it is scarcely inhibited even by detergent-containing components such as surfactants, metal ions and proteinases. Therefore, the enzyme of the present invention can be advantageously used as a blending component of a detergent composition.

[0042]

EXAMPLES The present invention will be described in more detail below with reference to examples.

Example 1 About 0.5 g of a soil sample collected in Los Banios, Laguna Province, Philippines was suspended in 9 ml of sterile physiological saline,
It heat-processed at 10 degreeC for 10 minutes. This heat-treated sample was appropriately diluted, smeared on an agar plate [PCMC medium (medium A)], and cultured at 30 ° C. for 3 days. Then, bacteria having transparent spots around the colony were smeared on the same plate medium and cultured at 30 ° C. for 3 days to form colonies again.

According to the above-mentioned method, Bacillus sp.
No.).

Example 2 Bacillus sp. PKM-obtained in Example 1
5812 was subjected to slant culture using medium B (medium A from which trypan blue dye was removed). Then, the obtained colonies were inoculated into 5 ml of CMC-peptone medium (medium C) placed in a 50 ml test tube, and cultured at 30 ° C. for 2 days while shaking at 300 rpm. Furthermore, 500 ml
50 ml of the same medium in a Sakaguchi flask was inoculated with 1 ml of the above culture and shaken at 120 rpm for 3
The cells were cultured at 0 ° C for 3 days.

Cells were removed from the thus obtained culture broth by centrifugation, and carboxymethylcellulase 58 was added.
12-containing culture supernatant was obtained.

Example 3 The culture supernatant obtained in Example 2 was treated with ammonium sulfate (pH was kept neutral with dilute aqueous ammonia), and the protein precipitated at 90% saturation was centrifuged (11,000 ×). g ). This precipitate was dissolved in 20 mM phosphate buffer of pH 7.0 and dialyzed against the same buffer at 5 ° C. overnight.

Bradford's method (M. Bradf
ord, Anal . Biochem . , 72, 248
(1976)), the protein concentration was measured by a Bio-Rad protein assay kit (manufactured by Bio-Rad Laboratories) using bovine plasma gamma / globulin as a standard, and the specific activity was 7.73 units (U) / mg protein. To obtain a crude enzyme solution.

Example 4 The crude enzyme solution obtained in Example 3 was subjected to the Davis method (BJ Davis, Ann . N. Y. Acad .
Sci . , 121, 404 (1964)) and polyacrylamide gel electrophoresis (PAGE) at 9% (w /
v) Polyacrylamide slab gel (70mm × 80m)
m, 1.0 mm thickness) using 150 mM Tris-glycine buffer (pH 8.8) as a running buffer. Electrophoresis was performed at room temperature for about 2 hours at 10 mA per gel. The method of Oakley et al. (BR Oakley,
D. R. Kirsch and N.M. R. Morri
s, Anal . Biochem . , 105, 361 (1
980)), the protein was detected by silver staining (silver staining kit, Kanto Kagaku).

Then, the protein band having carboxymethylcellulase activity was activity-stained with Congo red. That is, the method of Beguin (P. Begui
n, Anal . Biochem . , 131, 333 (1
983), the electrophoresed slab gel was placed on the surface of a CMC-agar plate and left at 37 ° C. for 30 minutes. CMC
-The composition of the agar plate is 2% (w / v) CMC (Su
nrose A10MC), 3% (w / v) NaCl,
10% (v / v) 0.5 M glycine-sodium hydroxide buffer (pH 9.0) and 0.8% (w / v) agar (manufactured by Difco) were used. This plate is 0.1% (w
/ V) 15 with Congo Red (Wako Pure Chemical Industries, Ltd.) solution
Staining was carried out for one minute at room temperature, followed by destaining with 0.1 M NaCl solution for 15 minutes. As a result, the relative mobility (Rm) of 0.
A protein band having carboxymethylcellulase activity was detected at position 43.

Example 5 With respect to the crude enzyme solution obtained in Example 3, Wrigley
Method (CW Wrigley, Methods E
nzymol . , 22, 559 (1971)), and the isoelectric point (pI) was determined by PAGE. 100 μl of each enzyme solution (1 mg / ml) was added to 5% (w) of acrylamide gel containing 13.3% (w / v) glycerol and 6.3% (v / v) Pharmalite (Pharmacia: pH range 3-10). / V) about 15 at a constant voltage of 200 V in the column
Electrophoresed for hours. The anode is the upper chamber (20 mM
The H ₃ PO _4), the cathode was connected to the lower chamber (0.1 M sodium hydroxide). 0.2% (w /
v) Protein stained with Comassie Brilliand Blue R-250 (Merck). The carboxymethylcellulase activity was stained with the activity according to Example 4, the relative mobility of the active band was measured, and the pI value was determined from a standard curve of known proteins to be pH 4.5.

[0052]

[Brief description of drawings]

FIG. 1 Carboxymethyl cellulase 5812 of the present invention
2 is a view showing a pH-activity curve of the.

FIG. 2 Carboxymethyl cellulase 5812 of the present invention
It is a figure which shows the pH stability of.

FIG. 3 Carboxymethyl cellulase 5812 of the present invention
It is drawing which shows the temperature-activity curve of.

FIG. 4 Carboxymethyl cellulase 5812 of the present invention
It is a figure which shows the temperature stability of.

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] January 9, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] 0032

[Correction method] Change

[Correction content]

(7) Effect of metal ion Each metal ion preparation shown in Table 2 and CMC were mixed with an enzyme solution, and carboxymethylcellulase activity was adjusted to pH 9.0.
In 0.1 M glycine-sodium hydroxide buffer solution at 40 ° C. for 20 minutes. Among the various metal ions tested, 1 mM Hg ²⁺ inhibited the enzyme activity by about 73% . 1 mM Co ²⁺ promotes enzyme activity by about 50%,
Mn ²⁺ promoted the enzyme activity by about 50%. Incidentally, the other metal ions shown in the same table showed some inhibition or promotion, and no influence by the other metal ions was observed.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0036

[Correction method] Change

[Correction content]

(9) Effect of Inhibitors The inhibitors shown in Table 4 were mixed with an enzyme solution and the carboxymethylcellulase activity was adjusted to 0.1 M glycine-pH 9.0.
The measurement was carried out at 40 ° C. for 20 minutes in a sodium hydroxide buffer. The activity of this enzyme is significantly inhibited by N - bromosuccinimide at 97%, but not by other inhibitors.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0050

[Correction method] Change

[Correction content]

Then, the protein band having carboxymethylcellulase activity was activity-stained with Congo red. That is, the method of B'eguin (P. B'egui
n , Anal . Biochem . , 131, 333 (1
983), the electrophoresed slab gel was placed on the surface of a CMC-agar plate and left at 37 ° C. for 30 minutes. CMC
-The composition of the agar plate is 2% (w / v) CMC (Su
nrose A10MC), 3% (w / v) NaCl,
10% (v / v) 0.5 M glycine-sodium hydroxide buffer (pH 9.0) and 0.8% (w / v) agar (manufactured by Difco) were used. This plate is 0.1% (w
/ V) 15 with Congo Red (Wako Pure Chemical Industries, Ltd.) solution
Staining was carried out for one minute at room temperature, followed by destaining with a 1.0 M NaCl solution for 15 minutes. As a result, the relative mobility (Rm) of 0.
A protein band having carboxymethylcellulase activity was detected at position 43.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location C12R 1:07) (72) Inventor Marietta N. Ballendor, Lower Miser, Misamis Oriental, Philippines (No address) (72) Inventor Nelly Em. Rabnos Philippines, Misamis Oriental, Gracia (No address) (72) Inventor Shinta Ueda Cagayan de Orocit, Gusa, Pkeiai (No address) Philippines

Claims (2)

[Claims] 1. A carboxymethylcellulase 5812 having the following physicochemical properties. (1) Action Hydrolyzes carboxymethyl cellulose. (2) Substrate specificity It strongly acts on carboxymethylcellulose and lichenan, and is against cellulose powder, phosphate swollen cellulose, cellobiose, p -nitrophenyl-β-D-glucopyranoside and p -nitrophenyl-β-D-cellobioside. Works. (3) Working pH and optimum pH The working pH is 4.2 to 12.5, and there are two optimum pHs at about 7.7 and about 9.5. (4) pH stability When kept at 5 ° C overnight, it hardly deactivates at pH 4.8-12. (5) Working temperature and optimum temperature The working temperature is 10 to 70 ° C, and the optimum temperature is around 55 ° C. (6) Thermal stability Even when heated at 60 ° C for 10 minutes, it hardly deactivates, and at 70 ° C
Some activity remains after heating. (7) Effect of surfactant Sodium dodecyl sulfate (SDS), linear alkylbenzenesulfonate sodium (LAS), alkyl sulfate sodium salt (AS), polyoxyethylene alkyl sulfate sodium salt (ES), α-olefin sulfone Sodium acid salt (AOS), α-sulfonated fatty acid ester sodium salt (α-SFE), fatty acid salt (soap), and polyoxyethylene alkyl ether do not inhibit the activity. (8) Molecular weight (gel filtration method with Biogel A-0.5 m) 26,000 ± 1,000 2. A microorganism which belongs to the genus Bacillus and produces the carboxymethylcellulase 5812 according to claim 1.