JPH0655138B2 - CMCase ▲I▼ - Google Patents

Description

Detailed Description of the Invention [Industrial Application Field] The present invention relates to a novel CMCase, and more specifically to a novel enzyme, CMCase I, which has an optimum pH on the alkaline side.

PRIOR ART

Cellulase converts cellulose and related polysaccharides into glucose,
It is understood as a general term for enzymes called _C1 enzymes, C _X enzymes and β-glucosidases, or exo-β-glucanases, endo-β-glucanases, cellobiases, etc., depending on their mechanism of action. For many years, cellulase research has been conducted solely for the purpose of making effective use of biomass resources, and sources of cellulase have been sought in molds such as Trichoderma, Aspergillus, Acremonium, and Humicola. However, cellulases of microbial origin, including mold, have diversity in the action specificity and physicochemical properties of their constituent enzymes, and the actual situation has not yet been clearly defined.

Among the above cellulases, those with particularly high activity against carboxymethylcellulose (CMC), i.e., _CX enzyme activity, are called CMCases. Recently, new industrial uses of cellulases including CMCases have been reported, for example:
Its use as an additive component in laundry detergent compositions is being developed. However, in the case of cellulases produced by microorganisms in nature, most of them are unstable and inactivated at alkaline pH levels, i.e., so-called acid cellulases (optimal pH for activity is 4 to 6).
However, the reality is that there are very few so-called alkaline cellulases that have maximum activity on the alkaline side and also have resistance, which are the requirements for a laundry detergent composition.

Specifically, with regard to alkaline cellulases that can be used in laundry detergent compositions, methods for producing alkaline cellulases derived from alkalophilic microorganisms include a method in which bacteria belonging to the genus Bacillus are cultured and cellulase A is extracted from the medium (JP Patent Publication No. 50-28515), a method in which alkalophilic bacteria belonging to the genus Cellulomonas are cultured to produce alkaline cellulase 301-A (JP Patent Publication No. 58-2246), and a method in which alkaline cellulases derived from alkalophilic microorganisms are cultured and cellulase A is extracted from the medium (JP Patent Publication No. 58-2246).
86), and a method for producing carboxymethylcellulase by culturing alkaliphilic Bacillus No. 1139 (Ho
Rikoshi et al., J. Gen. Microbiol., vol. 131, p. 3339 (1985)), and a method for producing alkaline cellulase using Streptomyces species (JP-A-61-1986).
Only one such technique is known (Publication No. 483).

[Problem that the invention aims to solve]

Therefore, there has been a demand for a new alkaline cellulase which has an optimum pH on the alkaline side and functions as an enzyme for washing clothes.

[Means for solving the problem]

The present inventors have been searching for alkaline cellulase-producing microorganisms in nature and have now discovered that a microorganism belonging to the genus Bacillus collected from soil in Haga County, Tochigi Prefecture,
The present invention has been completed by discovering that a novel alkaline cellulase K, which is effective as an additive component in laundry detergent compositions, can be produced and that further purification of said alkaline cellulase gives novel CMCases I and II as its main components.

That is, the present invention provides a novel enzyme, CMCase I.

The microorganisms used in the production of CMCase I of the present invention exhibit the following bacteriological properties. The media used for classifying the strains below are the following media 1 to 24, which contain an appropriate amount of 1.0% by weight _of separately sterilized sodium carbonate ( _Na2CO3 ) unless otherwise specified.

Composition of the classification test medium (in % by weight): Medium 1: Bacto peptone, 0.5; meat extract, 0.3; Bacto agar, 1.5 Medium 2: Bacto peptone, 0.5; meat extract, 0.3 Medium 3: Bacto peptone, 0.5; meat extract, 0.3; salt, 7.0 Medium 4: Bacto peptone, 0.5; meat extract, 0.3; Bacto gelatin, 20.0 Medium 5: Bacto toritoma milk, 10.5 Medium 6: Bacto peptone, 0.5; meat extract, 0.3; _KNO3 , 0.1 Medium 7: Bacto peptone, 0.7; glucose, 0.5; salt, 0.5 Medium 8: Bacto peptone, 3.0; meat extract, 0.3; sodium thiosulfate, 0.005; cysteine hydrochloride, 0.02; ferric ammonium citrate. , 0.05; bacto agar, 0.5 Medium 9: bacto peptone, 1.5; meat extract, 0.4; lactose, 1.0; sucrose, 1.0; glucose, 1.0; salt, 0.5; sodium thiosulfate, 0.008; sodium sulfite, 0.04; ferrous sulfate, 0.02; phenol red, 0.002; bacto agar, 1.5 Medium 10: bacto peptone, 1.5; yeast extract, 0.5; soluble starch, 2.0; K ₂ HPO ₄ , 0.1; bacto agar, 1.5; MgSO ₄ · 7H ₂ O, 0.02 Medium 11: ammonium phosphate, 0.1; potassium chloride, 0.02; yeast extract, 0.05; MgSO ₄ · 7H ₂ O, 0.05 Medium 12: Potassium monohydrogen phosphate, 0.1; Ammonium dihydrogen phosphate, 0.1; Sodium citrate, 0.2; _MgSO4 · _7H2O , 0.03; Salt, 0.5; Bromthymol blue, 0.0024; Bacto agar, 1.5 Medium 13: Yeast extract, 0.05; Cysteine hydrochloride, 0.01; Sodium citrate, 0.3; Salt, 0.5; Sodium thiosulfate, 0.008; Ammonium ferric citrate, 0.04; Glucose, 0.02; Potassium dihydrogen phosphate, 0.15; Phenol red, 0.0012; Bacto agar, 1.5 Medium 14: Ammonium phosphate, 0.1; Potassium dihydrogen phosphate, 0.05; Sodium citrate Medium 15 _: Yeast extract, 0.05; Na2SO4, 0.1; _KH2PO4 , _0.1 ; glucose, 1.0; inorganic nitrogen source, appropriate amount ^* *Sodium nitrate was added to 0.25%, sodium nitrite _to 0.2025%, ammonium _chloride to 0.158%, and ammonium phosphate to 0.195% ( _each equivalent to 0.0412 N%).

Medium 16: Yeast extract, 0.05; _Na2SO4 , _0.1 ; _KH2PO4 , _0.1 ; glucose, 1.0; inorganic nitrogen _source , appropriate amount ^** ; _CaCl2.2H2O , _0.05 ; _MnSO4.4-6H2O , 0.001; _FeSO4.7H2O , _0.001 (filter sterilized); _MgSO4.7H2O , _0.02 (filter sterilized) **Sodium nitrate was added to 0.25%, sodium nitrite to 0.2025%, ammonium chloride to 0.158%, and ammonium phosphate to 0.195% (each equivalent to 0.0412 N%).

Medium 17: King A medium "Eiken" (Eiken Chemical Co., Ltd.), indicated amount Medium 18: King B medium "Eiken" (Eiken Chemical Co., Ltd.), indicated amount Medium 19: Potato dextrose agar medium "Eiken" (Eiken Chemical Co., Ltd.), indicated amount Medium 20: Bactopeptone, 0.25; salt, 0.25; yeast extract, 0.25; mannitol, 0.5; bacto agar, 2.0 Medium 21: Urea medium "Eiken" (Eiken Chemical Co., Ltd.), indicated amount Medium 22: Bactopeptone, 0.1; salt, _0.5 ; _KH2PO4 , 0.2; yeast extract, 0.05; glucose, 0.1; urea, 2.0; phenol red, 0.001 Medium 23: Bactopeptone, 0.5; yeast extract, 0.5; _K2HPO Culture medium 24: Yeast extract, _0.5 ; glucose, 1.0; casein (Harmerstein, Merck), 0.5; _K2HPO4 , _0.1 ; _MgSO4.7H2O , 0.02; _Bacto agar, 1.5 (Mycological properties) _1. Microscopic observation results: The size of the fungus is a rod-shaped bacterium with a size of 0.5-1.2 μm x _1.5-4.0 μm, and an endospore (0.7-1.2 μm x 1.0-2.0 μm) is attached to one end of the fungus.
It produces a periflagellate. Motility: positive. Gram staining: positive.

2. Growth state in various media: Nutrition agar medium (medium 1) The colony shape is circular and the surface of the colony is flat.
The colonies are white to yellow in color and semi-transparent, and glossy.

Broth liquid medium (medium 2) Grows and becomes turbid.

7% salt broth liquid medium (medium 3) Grows and becomes turbid.

Meat juice gelatin stab culture (medium 4) No growth.

Triton milk medium (medium 5) Milk coagulation and peptonization: negative. Also, because medium 5 is alkaline, no discoloration of litmus was observed.

3. Physiological properties: Nitrate reduction and denitrification reaction Nitrate reduction: positive, denitrification reaction: negative (medium 6).

MR test (medium 7) Because the medium is alkaline, no change in methyl red was observed and a judgment was not possible.

VP test (medium 7) positive.

Indole production (medium 8) Negative for both reaction with indole production test filter paper ("Nitsan", manufactured by Nissui Pharmaceutical Co., Ltd.) and color reaction with Kovac's reagent.

Hydrogen sulfide production (Medium 9) Negative.

Hydrolysis of starch Even when the plate agar medium, medium 10, was acidified with 4N hydrochloric acid, the usual detection method using iodine reaction gave a negative result. In the liquid medium 11, the assimilation of soluble starch was negative.

Citric acid utilization Medium 11: positive.

Medium 12 (Cosa (Simmons) citric acid agar plate medium): negative. A liquid medium prepared by removing the agar from Medium 12 and adding 0.05% yeast extract was positive.

Medium 13 (Christensen's agar plate medium): Positive. However, due to its alkalinity, there was no change in the phenol red.

Medium 14: Negative (no growth). Remove the agar from medium 14.
Positive in liquid medium containing 0.05% yeast extract.

Utilization of inorganic nitrogen sources Medium 15: Nitrate, nitrite, and ammonia all ranged from negative to positive.

Medium 16: This medium contains trace metal salts and is positive for nitrate and nitrite. Ammonium chloride: positive. Ammonium phosphate: positive.

Pigment production: No growth on King A medium (medium 17), so determination was impossible.

Light yellow (no fluorescence) on King B medium (medium 18).
The strain grew on potato dextrose agar medium (medium 19) and mannitol yeast extract agar medium (medium 20), but was negative for pigment production.

Urease Medium 21: No growth. Phenol red was removed from Medium 21, and the bacteria was cultivated. Ammonia production was confirmed using Nessler's reagent, but the result was negative.

Medium 22 (Christensen's urea medium with yeast extract added): After removing the phenol red from the medium and culturing the bacteria, the production of ammonia was confirmed using Nessler's reagent, but the result was negative. Also, in anticipation of cytotoxicity, the urea concentration of Medium 22 was reduced to 0.
Tests were performed at 1, 0.2, 0.5, 1.0, and 2.0%, but the results were negative.

Oxidase: Positive or negative, but not clear.

Catalase positive.

Growth range (medium 23) The growth temperature range is 20 to 45°C, the optimum growth temperature range is 29
To investigate the pH range for growth, we changed the initial pH _{with Na2CO3} and found that the pH range for growth was 8 to 1.
1. _The optimum pH range for growth was _9.5-10.2 .
When the pH was adjusted to the optimum pH, the growth rate was significantly reduced.
was about 9.

Attitude towards oxygen: Aerobic.

O-F test: No discoloration due to alkaline nature. Grows only in aerobic conditions.

Sugar utilization (medium 11) Available carbon sources: D-ribose, L-arabinose,
D-xylose, D-glucose, D-mannose, D
-Fructose, maltose, sucrose, trehalose, D-
Mannite, inositol, glycerin Unavailable carbon sources: D-galactose, lactose, D-sorbitol, starch, dextrin, raffinose Hydrolysis of casein (medium 24) The bacteria was grown on agar plate medium 24 and 30% trichloroacetic acid was poured in to judge the results, but no clear zone was formed around the bacteria, so the result was negative.

Auxotrophy: biotin (or desthiobiotin).

The above mycological properties are described in the Burgess Manual.
of Determinative Bacteriology (Berge
According to the results of the 8th edition of "Y's Manual of Determinant Bacteriology", this strain is recognized as a species of the genus Bacillus, which is a spore-forming rod-shaped bacillus. However, since this strain cannot grow at neutral pH and grows well only at high alkaline pH, it is considered to be a type of bacteria, Horikoshi and Akiba ("Alkaloph
ilic Microorganism”, Japan Scientific Society Pre
As a so-called alkaliphilic microorganism, as claimed by the Japanese Society for Bacillus Subtilis, SS (Tokyo, 1982), it is provisionally differentiated from conventional bacilli that grow in neutral conditions.

Since the bacteriological properties of this strain do not coincide with those of any of the known alkaliphilic bacilli, it was determined to be a novel strain, named Bacillus sp. KSM-635, and deposited at the Fermentation Research Institute, Agency of Industrial Science and Technology under Microbial Research Institute Deposit No. 8872.

To obtain the CMCase I of the present invention, the above-mentioned Bacillus sp. KSM-635 or a mutant thereof is cultured in a medium to obtain alkaline cellulase K, which is then separated and purified by a conventional enzyme purification method.

In the fermentation production of alkaline cellulase K, a suitable medium is sterilized by heating or the like, and then Bacillus sp. KSM is added.
-635 (FERM P-8872) was inoculated and incubated at 22°C to 40°C.
C., preferably at 26.degree. C. to 37.degree. C., for 1 to 4 days with shaking or aeration and agitation.Good results can be obtained by adjusting the pH to 8 to 11.Since the fermentation production medium is alkaline, foaming may occur in some cases, but this can be eliminated by adding an appropriate antifoaming agent.

For the production of alkaline cellulase K, a suitable combination of assimilable nitrogen and carbon sources may be added to the culture medium.
There is no particular limitation on the sources of both nutrients. For example, the nitrogen source may be inorganic nitrate, ammonium sulfate, ammonium chloride, ammonium phosphate, sodium nitrate, corn gluten meal, soybean flour,
Corn steep liquor, casamino acids, yeast extract, pharmamedia, sardine meal, meat extract, peptone, hypro, ajipower, corn soybean meal, coffee grounds, cottonseed meal, cultivator, amiflex, ajipron, zest, ajitux, etc.
Carbon sources include rice husk, wheat bran, filter paper, general paper, sawdust,
In addition to vegetable fibers such as molasses, invert sugar, carboxymethylcellulose (CMC), avicel, cellulose cotton, xylan, and pectin, assimilable carbon sources such as ribose, arabinose, xylose, glucose, mannose, fructose, maltose, sucrose, trehalose,
Examples of nutrient sources include mannite, inositol, glycerin, and assimilable organic acids, such as acetic acid and citric acid. In other words, any medium that appropriately combines these nitrogen sources and carbon sources may be used, and the above-mentioned nutrient sources are not particularly limited. Other examples include phosphoric acid, Mg ²⁺ , Ca ²⁺ , Mn ²⁺ , Zn ²⁺ ,
A suitable medium is selected and used, which contains inorganic salts such as Co ²⁺ , Na ⁺ , and K ⁺ , and, if necessary, inorganic and organic trace nutrient sources.

Alkaline cellulase K can be obtained from the thus obtained culture by, for example, following the general procedures for obtaining and purifying enzymes, as shown in the Examples below.

That is, the culture is centrifuged, filtered, or the like to separate the bacterial cells, and the bacterial cells and the culture filtrate are separated by a conventional separation means.
For example, alkaline cellulase K can be obtained by precipitating proteins by salting out, isoelectric precipitation, or solvent precipitation (methanol, ethanol, isopropanol, etc.), or by concentrating the protein by ultrafiltration (e.g., Diaflow Membrane YC, manufactured by Amicon Co., Ltd.).
~70% saturation fraction), solvent precipitation, for example, in 75% ethanol, followed by filtration or centrifugation,
It is also possible to prepare a lyophilized powder by desalting the product, which can be carried out by a general method such as dialysis or gel filtration using Sephadex G-25 or the like.

Furthermore, this alkaline cellulase K is converted to CMCase I.
To obtain the above compound, for example, separation and purification may be carried out by appropriately combining hydroxyapatite chromatography, ion exchange chromatography using, for example, DEAE-Sephadex or DEAE-cellulose, and molecular sieve gel chromatography using, for example, Sephadex or Biogel.

The CMCase I thus obtained has the following properties:

(1) Action This enzyme has _CX enzyme activity acting on carboxymethylcellulose (CMC). However, it also acts on phosphoric acid-swollen cellulose, and has specific action on crystalline cellulose (cellulose cotton) and Avicel, a highly crystalline cellulose (i.e., Avicelase), and C enzyme activity such as filter paper disintegration activity (FPase).
It also has _β -glucosidase activity that acts on cellobiose and cellooligosaccharides.
It also acts slightly on nitrophenyl cellobioside to liberate p-nitrophenol.

(2) Substrate specificity The main activity of this enzyme is CMCase activity, of which approximately 0.
The enzyme had avicelase and FPase activities ( _C1 activity) of 3%, and the activity for decomposing the artificial substrate p-nitrophenyl cellobioside was about 1.5-1.8% of that of the enzyme (Table 1).
On the other hand, it had no decomposition ability for xylan, amylose, dextrin, pectin, inulin and curdlan.

(3) Active pH and Optimal pH The active pH range of this enzyme is 3 to 12.5.
The range is 6 to 11.5 (Figure 1). The optimum pH is about 9.5.

In addition, the pH of action against p-nitrophenyl cerbioside
The range was 4-11 with an optimum pH of about 7.

(4) pH stability The enzyme was incubated at 30°C for 1 hour at each pH level and the residual activity was measured to examine the pH stability.
2, it was extremely stable and did not become inactivated (Figure 2).

(5) Titer measurement method CMCase activity 0.2 ml of CMC (2.5%), 0.5 M glycine buffer (pH 9.0) 0.
Add 0.1 ml of enzyme solution to a substrate solution consisting of 0.1 ml of PBS and 0.1 ml of deionized water.
1 ml was added and reacted at 40°C for 20 minutes.
5-dinitro-salicylic acid
The amount of reducing sugar was determined by the DNS method. That is, 1 ml of DNS reagent was added to 0.5 ml of the reaction solution, and the solution was incubated for 5 minutes.
The color was developed by heating at 00°C, and after cooling, 4.5 ml of deionized water was added for dilution. This was then colorimetrically quantified at a wavelength of 532 nm.
The enzyme activity was defined as 1 unit, which was the amount of enzyme that produces reducing sugars equivalent to 1 μmol of glucose per minute under the above conditions.

p-Nitrophenyl cellobioside decomposition activity 100 μmol
A suitable amount of CMCase was reacted at 30°C in 1.0 ml of a reaction mixture containing phosphate buffer (pH 7.0) and 0.1 μmol p-nitrophenyl cellobioside (Sigma), and then 1 M Na ₂ CO ₃ was added.
0.3 ml of the solution and 1.7 ml of deionized water were added in sequence, and the liberated p-nitrophenol was colorimetrically quantified at 400 nm.
The amount of enzyme that liberates 1 μmol of p-nitrophenol per minute was defined as one unit.

Avicelase and FPase activity In 2 ml of reaction solution, 20
mg of Avicel (Merck) or in chunks, width 0.5 cm
m, 5 cm long filter paper piece (cellulase activity test filter paper,
Toyo No. 51-special) was added and the avicelase and FPase activities were measured. The amount of enzyme that liberates 1 μmol of reducing sugar (calculated as glucose) per minute under these conditions was defined as 1 unit.

Protein quantification was performed using a Bio-Rad Protein Assay Kit (Bio-Rad) with bovine serum albumin as the standard protein.

(6) Active temperature and optimum temperature The active temperature range of CMCase I is 10 to 60°C. The optimum temperature range is 22 to 53°C. The optimal active temperature of this enzyme is 40°C. This enzyme is also sufficiently resistant to low temperatures, and retains about 50% of its activity even at a temperature as low as 20°C (Figure 3).

(7) Temperature stability: Approximately 50% of the activity remains even after heating at 50°C for 30 minutes (in glycine buffer, pH 9.0).

(8) Influence of metals Metals and ions change the physicochemical properties of CMC, especially its viscosity, so it is self-evident that when CMC is used as a substrate, the activity of the enzyme component of the present invention will not accurately reflect the reaction kinetic factors of alkaline cellulase K. Therefore, the influence of metals on the enzyme activity was investigated using the p-nitrophenyl cellobioside decomposition activity of CMCase I as an indicator. As a result, Zn2 ⁺ , Co2 ⁺ , Ni
²⁺ , Cu ²⁺ and Hg ²⁺ inhibited the activity of CMCase I. On the other hand, Mn ²⁺ and Ba ²⁺ slightly activated the activity of CMCase I.

(9) Effect of chelators CMCase I reacts with chelators such as EDTA and EGT.
No inhibition was observed by A, NTA, STPP or zeolite.

(10) Effect of sugars As in (8), p-nitrophenyl cellobioside was added to CMC.
The effects of various sugars as substrates for α-aspartase I were examined. Cellobiose inhibited both enzyme components and caused product inhibition.
The β-glucosamine, N-acetylglucosamine, ribose, arabinose, sorbose, xylose, fructose, galactose, and glycerol showed a type of inhibition (t inhibition) in the β-glucosamine-induced β-glucosamine complex. However, other disaccharides, such as lactose and maltose, had no effect. The monosaccharides glucosamine, N-acetylglucosamine, ribose, arabinose, sorbose, xylose, fructose, galactose, and glycerol showed no effect.
Glucose, its derivatives 3-O-methyl-D-glucose, α-methyl-β-D-glucose, α-methyl-D-glucoside, α-methyl-β-mannoside, 2
-Deoxyglucose or other sugars such as rhamnose did not inhibit the activity.

(11) Effect of salt concentration Phosphate buffer, bicine (BICINE-N)
a) Using a Tris-HCl buffer as a buffer and sodium chloride (0-250 mM) as an ionic strength regulator, the effect of ionic strength on CMCase I was investigated using p-nitrophenyl cellobioside decomposition activity as an index.
No significant relationship was observed between ionic strength and enzyme activity, such as promotion or inhibition.

(12) Effect of surfactants: sodium linear alkyl benzene sulfonate (LAS),
Sodium alkyl sulfate (ES), sodium polyoxyethylene alkyl sulfate (E
S), sodium α-olefin sulfonate (AO
S), α-sulfonated fatty acid ester sodium salt (α-SFE), sodium alkylsulfonate (SA
The activity was hardly inhibited by polyoxyethylene secondary alkyl ether, fatty acid salts (sodium salts), dimethyldialkylammonium chloride and taurocholic acid.

(13) Molecular weight: The molecular weight measured by gel chromatography (Toyopearl 55S; Toyo Soda) was 145,000±10,000.

(14) UV absorption spectrum: The enzyme was dissolved in bicine-sodium buffer and the UV absorption spectrum was measured. The maximum absorption was found at about 280 nm, and the differential absorption spectrum showed the presence of a shoulder absorption at 290 nm (5th
(Figure). (15) Detection of sugars When the purified enzyme protein was subjected to a color development test using the phenol-sulfuric acid method, it had a maximum absorption at 480 nm. This result indicates that the enzyme contains sugars. Sugars were detected by gas chromatography using the alditol-acetic acid method, and it was found to contain N-acetylglucosamine as a constituent sugar. The enzyme contained 1.3-4.0% by weight of the relevant sugar component.

(16) Protease resistance Detergent proteases, such as API (Showa Denko), Maxatase (Gist), and Alcalase (Novo) were coexisted with the present enzyme (0.0002 to 0.1% by weight).
The residual activity of the sample preincubated for 2 hours was measured, and no inactivation was observed, indicating that the enzyme has strong resistance to proteases (Secondary
table).

The above-mentioned properties of the CMCase I of the present invention can be compared with those of known cellulases as follows.

This enzyme has an optimum pH in the high pH range, and is effective in treating Trichoderma, Penicillium, Aspergillus (Kazutoshi Nishizawa, "Cellulase" (Tokyo Nankodo, published in 1974)), Acremonium (JP Patent Publication 59-16608) and other genus.
1) It is clear that it is different from cellulases having an optimum pH on the neutral to acidic side, such as those of molds represented by the genus Humicola (JP Patent Publication 61-16316).

Also, the cellulase disclosed in Japanese Patent Publication No. 50-28515 and the cellulase disclosed in J. Gen. Microbiol, Vol. 131, p. 3339 (198
In comparison with the cellulase reported in 5), the molecular weight of this alkaline cellulase is 145,000±10,000, whereas the molecular weight of the alkaline cellulase in JP-B-50-28515 is 15,000 to 30,000, and the molecular weight of Bacillus No.
It is clearly distinguished from the present CMCase I in that it has a molecular weight of 92,000 and in that it contains sugar as a constituent component.

[Action and Effect of the Invention]

The CMCase I of the present invention has a relative activity of about 75 to 80% of the optimum pH even at pH 11, and is therefore an enzyme group that exerts sufficient activity at the most alkaline side among the alkaline cellulases previously studied.
Although the pH is strongly on the alkaline side, pH 3.5
It is also unique in that it has activity in the strong acidic range both before and after. There has never been a cellulase that is stable and active in such a wide pH range.
A cellulase which has sufficient activity even at relatively low temperatures (around 15°C) and which also has strong resistance to surfactants, chelating agents and various proteases has not been known up to now, and has been discovered for the first time by the present invention.

Therefore, the CMCase I of the present invention exhibits a strong cleaning effect even at low temperatures and can be effectively used as an enzyme added to laundry detergents, as well as for biomass and other applications.

[Example]

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

Example 1. Soil from Ichikai-machi, Haga-gun, Tochigi Prefecture was suspended in sterilized physiological saline.
The heat treatment was performed at 80°C for 30 minutes. The heat treatment solution was appropriately diluted and applied to a master plate (1% meat extract (Oxoid), 1% bactopeptone (Difco), 1% NaCl)
The _bacteria were smeared on _{agar containing} 0.1% _KH2PO4 , 0.5% _Na2CO3 (sterilized separately), and 1.5% Bacto agar, and cultured at 30°C for 3 days to form colonies. The bacteria were transferred to a master plate containing 2% CMC by replica method, and colonies were allowed to form again. After that, a Congolais dye solution was poured on the plate, and colonies with transparent periphery were detected. The corresponding colonies were selected from the master plate, and high-titer CMCase producing bacteria were screened.

By the above-mentioned method, the Bacillus sp. KS of the present invention can be obtained.
M-635 (FERM P-8872) was obtained.

Example 2. Bacillus sp. KSM-635 (FERM P-8872)
1.5% meat extract, 0.5% yeast extract, 1% CMC, 0.1
The bacteria were cultured in a liquid medium _{containing 0.75} % _KH2PO4 and 0.75% _Na2CO3 at 34°C for 2 days.
Cold ethanol (-10°C) was slowly added to precipitate the protein, and the precipitate was dissolved in a minimum amount of sterile deionized water, neutralized with dilute acetic acid, dialyzed against running water for 15 hours, and freeze-dried to obtain 8.6 g of enzyme powder. The enzyme activities in the resulting dry powder are shown in Table 3.

Example 3. In Example 2, fish extract was used instead of bactopeptone.
Bacillus sp. KSM-6 was added to the medium at 1.5%
35 (FERM P-8872) was inoculated and cultured at 30°C for 3 days with shaking. After the culture, the supernatant was centrifuged and
The MCase activity was measured and found to be 3,000 units/g.

Example 4 The culture supernatant 1 obtained in Example 3 was purified according to the following procedure to obtain CMCase I. The purification was carried out by streptomycin treatment, ammonium sulfate fractionation (30-75% saturated precipitate fraction), preparative high performance liquid chromatography (e.g., SW 3000 G column (Toyo Soda)), DE
AE - Toyopearl (Toyo Soda) Chromatography,
The product was purified by chromatography on hydroxyapatite (Seikagaku Kogyo Co., Ltd.) and then by DEAE-Toyopearl chromatography again. In the sixth step of purification, the product was eluted with a linear gradient of NaCl (0.25M NaCl to 0.35
When elution was performed at 100 M NaCl, the order of elution rate was
and CMCase II were eluted, yielding 24 mg of CMCase I.
The CMCase I thus obtained was isolated and obtained.
After electrophoresis according to the method described in the paper on page 404 (1964), it was confirmed that a single band was given by staining with Coomassie Prilliant Blue.

Example 5. The CMCase I and II obtained in Example 4 were subjected to sodium dodecyl sulfate electrophoresis in the usual manner. The results are shown in Figure 6.
It has been recognized that CMCase I and II are aggregates of molecular species with molecular weights ranging from a minimum of 30,000±2,000 to a maximum of 120,000±15,000, and it is believed that the CMCase I and CMCase II of the present invention are enzyme proteins with molecular weights of at least 30,000±2,000 tightly associated by some kind of physicochemical interaction.
55,000 to 65,000.

[Brief description of the drawings]

Fig. 1 is a diagram showing the relationship between reaction pH and relative activity of the CMCase I of the present invention. Fig. 2 is a diagram showing the relationship between treatment pH and residual activity of the CMCase I of the present invention. Fig. 3 is a diagram showing the relationship between reaction temperature (pH 9.
4 is a graph showing the relationship between the treatment temperature (pH 9.0) and the relative activity of CMCase I of the present invention.
Fig. 5 is a graph showing the UV absorption of CMCase I of the present invention. Fig. 6 is a graph showing the results of sodium dodecyl sulfate electrophoresis of CMCase I and II of the present invention.

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Claims (2)

[Claims] [Claim 1] CMCase I having the following physicochemical properties. (1) Action: In addition to _Cx enzyme activity acting on carboxymethylcellulose, it has weak _C1 enzyme activity and β-glucosidase activity. (2) Substrate specificity: Acts on carboxymethylcellulose, crystalline cellulose, Avicel, cellobiose and p-nitrophenyl cellobioside. (3) Working pH and optimum pH: Working pH is 3 to 12.5, with the optimum pH being approximately 9.5. (4) pH stability: When kept at 30°C for 1 hour, it is not inactivated at pH 5 to 12. (5) Working temperature and optimum temperature: Working temperature is 10 to 60°C, with the optimum temperature being 22 to 53°C. (6) Effect of chelating agents: EDTA, EGTA, NTA, STPP and zeolite do not inhibit the activity. (7) Effect of surfactants: Linear alkylbenzenesulfonic acid sodium salt (LAS) is not inactivated.
S), alkyl sulfate sodium salt (AS), polyoxyethylene alkyl sulfate sodium salt (ES), sodium α-olefin sulfonate (AO
S), α-sulfonated fatty acid ester sodium salt (α-SFE), sodium alkylsulfonate (SA
S), polyoxyethylene secondary alkyl ether, fatty acid salts (sodium salts), dimethyldialkylammonium chloride and taurocholic acid do not inhibit the activity. (8) Protease resistance It has strong resistance to proteases. (9) Molecular weight (gel chromatography method) The molecular weight of this enzyme is 145,000 ± 10,000. (10) UV absorption spectrum This enzyme has a maximum absorption at 280 nm and a shoulder absorption at 290 nm, and contains sugar components. Claim 2: It is obtained by isolating from the culture of Bacillus sp. KSM-635.
CMCase I as described in paragraph .