GB2232983A

GB2232983A - Alkaline cellulase extracted Bacillus

Info

Publication number: GB2232983A
Application number: GB9012000A
Authority: GB
Inventors: Shitsuw Shikata; Katsuhisa Saeki; Tadashi Yoshimatsu; Hiromi Kubota; Hiroko Jhokura; Shuji Kawai; Susumu Ito; Youko Nomura; Showya Ohkubo
Original assignee: Kao Corp
Current assignee: Kao Corp
Priority date: 1989-06-02
Filing date: 1990-05-30
Publication date: 1991-01-02
Anticipated expiration: 2010-05-30
Also published as: GB2232983B; HK105494A; GB9012000D0; MY105832A

Abstract

Alkaline cellulases named K-64, K-19, and K-520; microorganisms of the genus Bacillus producing these alkaline cellulases; and each process for producing them are disclosed. The alkaline cellulases have an optimum pH range at an alkaline region and are stable at a wide pH range. The enzymes of the present invention are not adversely affected by the addition of detergent components such as surfactants, proteases, chelating agents, and the like, and, accordingly, can effectively be used as a detergent component.

Description

TITLE OF THE INVENTION ALKALINE CELLULASE, MICROORGANISM PRODUCING THE SAME, AND PROCESS FOR PRODUCING THE SAME BACKGROUND OF THE INVENTION Field of the Invention: This invention relates to novel alkaline cellulases, the microorganisms producing the same, and processes for producing the alkaline cellulases.

Descrintion of the Backaround Art: Conventionally, studies on cellulase which is a cellulose-decomposable enzyme have been directed, for the most part, to the effective utilization of biomass resources, particularly of cellulose resources. The major supply sources of cellulase have been placed on fungi and wide varieties of strains isolated as the cellulaseproducing microorganisms have been reported. Typical examples of these strains are the fungi belonging to the genera such as Aspergillus, Penicillium, Trichoderma, Fusarium, Humicola, Acremonium, and the like.Examples of these strains other than fungi are bacteria belonging to the genera such as Pseudomonas, Cellulomonas, Ruminococcus, Bacillus, and the like, the actinomycetes such as Streptmyces, Thermoactinomyces, and the like.

Studies on a new application of cellulase as an additive to detergents for washing clothes have lately attracted considerable attention (Japanese Patent Laid-open Nos. 49279/1984, 23158/1985, and 36240/1985). The cellulase produced by natural microorganisms is almost classified as, namely, the neutral or acidic cellulase exhibiting a foremost and stable enzymatic activity in neutral or acidic conditions. There is few alkaline or alkali-resistant cellulase having a good stability and exhibiting maximum activity at a pH of alkaline region at which cloth washing is performed effectively when the cellulase is incorporated into a detergent composition for clothing. The alkaline cellulases herein mean those having an optimum pH in an alkaline region.

Several processes are known for producing an alkaline cellulase. Until recently, such processes have been typified by a process for collecting the cellulase A through culturing an alkalophilic microorganism belonging to the genus Bacillus (Japanese Patent Laid-open No. 28515/1975), a process for producing the alkaline cellulase 301-A through culturing of a microorganism belonging to the genus of Cellulomonas (Japanese Patent Laid-open No. 224686/1983), a process for obtaining a carboxymethyl cellulase through culturing of an alkalophilic strain, Bacillus sp. No. 1139 [Fukumori F., Kudo T., and Horikoshi K.; J. Gen. Microbiol., 131, 3339 (1985)1, and a process for producing an alkaline cellulase with the use of a microorganism belonging to the Streptomyces (Japanese Patent Laid-open No. 19483/1986).

These processes for producing an alkaline or alkaliresistant cellulase are nevertheless unsuitable for industrial fermentative production.

Recently, it has been found that a novel microorganism Bacillus sp. KSM-635 (FERM BP-1485, Japanese Patent Application No. 109771/1988), a sort of an alkalophilic microorganism, sufficiently yields the alkaline cellulase K (Japanese Patent Application No. 109776/1988) which can be adapted to a detergent component for clothing, so that the fermentative production of alkaline cellulase has been realized in an industrial scale. It has also been found that an alkaline cellulase could be produced by neutrophilic microorganisms which could be easily cultured (Japanese Patent Application Nos. 137677/1988, 240785/1988, 240777/1988, 240786/1988, and 37285/1989, and S. Kawai et al., Agric. Biol.Chem., 52, 1425(1988)); and an alkaliresistant cellulase capable of keeping high activities in an alkaline condition could be produced also by the abovementioned neutrophilic microorganisms (Japanese Patent Application Nos. 141586/1988, 146786/1988, 273474/1988, 273475/1988, 279790/1988, and 37286/1989).

However, the above-mentioned examples are enumerated as those of alkaline or alkali-resistant cellulases suitable for industrial fermentative production, further development of alkaline cellulases having various features and the microorganisms producing the alkaline cellulases have been desired.

In view of this situation, the present inventors have carried out extensive studies in order to obtain natural microorganisms capable of producing alkaline cellulases, and, as a result, found that a group of microorganisms belonging to the genus Bacillus which was discovered by the inventors in the soils in Nasu-gun, Tochigi-ken, Japan, was capable of producing a novel alkaline cellulase which was effective as a detergent component for clothing. This finding has led to the completion of the present invention.

SUMMARY OF THE INVENTION Accordingly, an object of this invention is to provide the novel alkaline cellulases K-64, K-19, and K-520, and microorganisms belonging to the genera Bacillus sp. KSM-64, KSM-l9, and KSM-520 which are all capable of producing the above alkaline cellulases respectively.

A specific object of the present invention is to provide an alkaline cellulase K-64 possesses the following physicochemical characteristics: (A) Activity Acts well on cellulosic materials such as carboxymethyl cellulose (CMC), cellulose, filter paper, avicel, and the like, dissolves them and produces reducing sugars.

(B) Substrate specificity Besides CMC, Acts on cellulose powder, phosphoric acid swollen cellulose, avicel, filter paper, p-nitrophenyl-glucoside, and p-nitrophenylcellobioside.

(C) Active pH range and optimum pH Has an active pH range on CMC of 3 - 13.0 with an optimum pH range being 8.5 - 10.0 and even in pHs ranging 7.0 - 12.0 has a relative activity of more than 50% of the activity of the optimum pH range.

(D) pH stability Is quite stable at pHs ranging 5 - 10.5 and even in pHs ranging 3 - 11 has an activity of more than 50% of the activity of the optimum pH, when CMC is a substrate.

(E) Optimum temperature Is capable of acting at wide temperatures ranging 10 - 700C with an optimum temperature being 500C and even in temperatures ranging 35 - 600C has more than 50% of the activity of the optimum temperature.

(F) Molecular weight Has peaks of activities at approximately 180,000 + 10,000 and 80,000 + 2,000 (measured by means of the gel filtration method using Bio-Gel A 0.5m), when CMC is a substrate.

(G) Effect of metal ions Is adversely affected by the addition of Hg2+ ion and activated by the addition of Co2+ and Mn2+ ions, when CMC is a substrate.

(H) Effect of surfactants Exhibits almost no inhibitive effects on enzymatic activities by linear alkylbenzene sulfonate, alkyl sulfate, polyoxyethylene alkyl sulfonate, a-olefin sulfonate, acarboxymethyl alkylsulfonate, secondary alkyl sulfonate, dodecyl sulfonate, soap, and/or polyoxyethylene secondary alkylether, when CMC is a substrate.

(I) Effect of proteases Possesses resistance to proteases, when CMC is a substrate.

(J) Effect of chelating agents Exhibits no inhibitive effects on enzymatic activities by EDTA, EGTA, citric acid, sodium tripolyphosphate, zeolite, when CMC is a substrate.

Another object of the present invention is to provide an alkaline cellulase K-19 possesses the following physicochemical characteristics: (A) Activity Acts well on cellulosic materials such as carboxymethyl cellulose (CMC), cellulose, filter paper, avicel, and the like, dissolves them and produces reducing sugars.

(B) Substrate specificity Besides CMC, acts on cellulose powder, phosphoric acid swollen cellulose, alkali swollen cellulose, avicel, filter paper, p-nitrophenylglucoside, and p-nitrophenylcellobioside.

(C) Active pH range and optimum pH Has an active pH range on CMC of 4.5 - 12.5 with an optimum pH range being 9 and even in pHs ranging 7.0 - 11.0 has a relative activity of more than 50% of the activity of the optimum pH range.

(D) pH stability Is quite stable in pHs ranging 4.5 - 11.0 and even in pHs ranging 4.0 - 12.0 has an activity of more than 50% of the activity of the optimum pH, when CMC is a substrate.

(E) Active temperature and optimum temperature Is capable of acting at wide temperatures ranging 10 - 700C with an optimum temperature being 500C and even in temperatures ranging 35 - 600C has more than 50% of the activity of the optimum temperature.

(F) Molecular weight Has peaks of activities at about 120,000 + 10,000 and 34,000 + 2,000 (small peak), (measured by means of the gel filtration method using Bio-Gel A 0.5m), when CMC is a substrate.

(G) Effect of metal ions Is adversely affected by the addition of Hg2+, and Pb2+ ions and activated by the addition of Cho2+ and Mn2+ ions, when CMC is a substrate.

(H) Effect of surfactants Exhibits almost no inhibitive effects on enzymatic activities by linear alkylbenzene sulfonate, alkyl sulfate, polyoxyethylene alkyl sulfonate, a-olefin sulfonate, acarboxymethyl alkylsufonate, secondary alkane sulfonate, dodecyl sulfonate, soap, and/or polyoxyethylene secondary alkylether, when CMC is a substrate.

Still another object of the present invention is to provide an alkaline cellulase K-520 possesses the following physicochemical characteristics: (A) Activity Acts well on cellulosic materials such as carboxymethyl cellulose (CMC), cellulose, filter paper, avicel, and the like, dissolves them and produces reducing sugars.

(C) Active pH range and optimum pH Has an active pH range on CMC of 3 - 13.0 with an optimum pH range being 8.5 - 9.5 and even in pHs ranging 7.0 - 11.0 has a relative activity of more than 50% of the activity of the optimum pH range.

(D) pH stability Is quite stable in pHs ranging 6.0 - 11.0 and even in pHs ranging 5 - 11.5 has an activity of more than 50% of the optimum pH, when CMC is a substrate.

(F) Molecular weight About 170,000 + 10,000 and 80,000 + 2,000 (measured by means of the gel filtration method using Bio-Gel A 0.5m), when CMC is a substrate.

(G) Effect of metal ions Is adversely affected by the addition of Hg2+ Cd2+, and Pb2+ ions and activated by the addition of Co2+ and Mn2+ ions, when CMC is a substrate.

(H) Effect of surfactants Exhibits almost no inhibitive effects on enzymatic activities by linear alkylbenzene sulfonate, alkyl sulfate, polyoxyethylene alkyl sulfonate, a-olefin sulfonate, secondary alkane sulfonate, soap, and/or polyoxyethylene secondary alkylether, when CMC is a substrate.

Other objects, features and advantages of the invention will hereinafter become more readily apparent from the following description.

BRIEF DESCRIPTION. OF THE DRAWINGS Fig. 1 is a drawing showing the relation of relative activity vs. reaction pH of the alkaline cellulase K-64 of this invention against CMC.

Fig. 2 is a drawing showing the relation of treating pH vs. residual activity of the alkaline cellulase K-64 of this invention against CMC.

Fig. 3 is a drawing showing the relation of reaction temperature vs. relative activity of the alkaline cellulase K-64 of the present invention.

Fig. 4 is a drawing showing the relation of treating temperature vs. residual activity of the alkaline cellulase K-64 of the present invention.

Fig. 5 is a drawing showing the relation of relative activity vs. reaction pH of the alkaline cellulase K-19 of this invention against CMC.

Fig. 6 is a drawing showing the relation of treating pH vs. residual activity of the alkaline cellulase K-19 of this invention against CMC.

Fig. 7 is a drawing showing the relation of reaction temperature vs. relative activity of the alkaline cellulase K-19 of the present invention.

Fig. 8 is a drawing showing the relation of treating temperature vs. residual activity of the alkaline cellulase K-19 of the present invention.

Fig. 9 is a drawing showing the relation of relative activity vs. reaction pH of the alkaline cellulase K-520 of this invention against CMC.

Fig. 10 is a drawing showing the relation of treating pH vs. residual activity of the alkaline cellulase K-520 of this invention against CMC.

Fig. 11 is a drawing showing the relation of treating temperature vs. relative activity of the alkaline cellulase K-520 of the present invention.

Fig. 12 is a drawing showing the relation of treating temperature vs. residual activity of the alkaline cellulase K-520 of the present invention.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS Mycological characteristics of these microorganisms are now discussed. The following 21 culture media (Media 1 to 21) are used for the classification of strains. They all contain 1.0% by weight of sterilized sodium carbonate (Na2C03).

Compositions of the culture media used (% by weight) Medium 1: nutrient broth, 0.8; Bacto agar, 1.5 Medium 2: nutrient broth, 0.8 Medium 3: nutrient broth, 0.8; Bacto gelatin, 20.0; Bacto agar, 1.5 Medium 4: Bacto litmus milk, 10.5 Medium 5: nutrient broth, 0.8; KNO3, 0.1 Medium 6: Bacto peptone, 0.7; NaCl, 0.5; glucose, 0.5 Medium 7: SIM agar medium, an amount prescribed.

Medium 8: TSI agar medium (manufactured by Eiken Chem.

Co.,Ltd., Japan), an amount prescribed.

Medium 9: Bacto peptone, 1.5; yeast extract, 0.5; soluble starch, 2.0; K2HPO4, 0.1; MgSO4o7H2O, 0.02; Bacto agar, 1.5 Medium 10: Koser's medium, an amount prescribed.

Medium 11: Christensen's medium (manufactured by Eiken Chem. Co., Ltd., Japan), an amount prescribed.

Medium 12: the medium including the following compositions (1) and (2) to which added are nitrogen sources consisting of sodium nitrate, sodium nitrite, ammonium chloride, and ammonium phosphate in such an amount as that the total amount of nitrogen is 0.0412N% by weight in the medium.

(1) yeast extract, 0.05; Na2SO4, 0.1; KH2PO4, 0.1; glucose, 1.0 (2) yeast extract, 0.05; Na2SO4, 0.1; KH2PO4, 0.1; glucose, 1.0; CaCl2g2H2O, 0.05; MziSO4c4-6H2O, 0.01; FeSO4.7H2O, 0.001; MgSO4e7H2O, 0.02 Medium 13: King A medium "Eiken" (manufactured by Eiken Chem. Co., Ltd., Japan), an amount prescribed.

Medium 14: King B medium "Eiken" (manufactured by Eiken Chem. Co., Ltd., Japan), an amount prescribed.

Medium 15: urea medium "Eiken" (manufactured by Eiken Chem. Co., Ltd., Japan), an amount prescribed.

Medium 16: cytochrome-oxidase test filter paper (manufactured by Nissui Pharmaceutical Co., Ltd., Japan) Medium 17: 3% aqueous hydrogen peroxide Medium 18: Bacto peptone, 0.5; yeast extract, 0.5; glucose, 1.0; K2HPO4, 0.1; MgSO4c7H2O, 0.02 Medium 19: Bacto peptone, 2.7; NaCl, 5.5; glucose, 0.5; K2HPO4, 0.3; bromthymol blue, 0.06; Bacto agar, 1.5 Medium 20: (NH4)2HPO4, 0.1; KCl, 0.02; MgSO4*7H2O, 0.02; yeast extract, 0.05; sugar, 1.0 Medium 21: casein, 0.5; Bacto agar, 1.5; yeast extract, 0.5; glucose, 1.0; K2HPO4, 0.1; MgSO4.7H2O, 0.02 Mycoloqical characteristics of Bacillus sp.KSM-64 (1) Observation under microscope The cells are gram-positive rods of a size of 0.5 - 1.2 Wm x 1.2 - 6.6 Wm, with a cylindrical endospore (0.7 - 1.0 gm x 1.5 - 2.5 Clam) forming at their subterminals. They have flagella and are motile. Gram's staining is positive.

(2) Growth in various culture media (a) Nutrient broth-agar plate (Medium 1) Growth of cells is good. The colony has a circular shape, with its surface being smooth and its peripheral end being smooth. The color of the colony is milky and glossy.

(b) Liquid nutrient broth (Medium 2) Growth of cells is good.

(c) Stab culturing in nutrient broth-gelatin (Medium 3) Growth of cells is good. Liquefaction of gelatin is observed.

(d) Litmus milk medium (Medium 4) Milk coagulation and peptonization are not observed. Litmus discoloration is undeterminable because the medium is an alkaline medium.

(3) Physiological characteristics (a) Nitrate reduction and denitrification (Medium 5) Nitrate reduction: positive Denitrification: negative (b) MR test (Medium 6) Undeterminable (c) VP test (Medium 6) Positive (d) Production of indole (Medium 7) Negative (e) Production of hydrogen sulfide (Medium 8) Negative (f) Hydrolysis of starch (Medium 9) Positive (g) Utilization of citric acid (Medium 10, Medium 11) Positive in Koser medium and undeterminable in Christensen's medium (h) Utilization of inorganic nitrogen sources (Medium 12) Nitrate and ammonium salts are both utilized.

(i) Discoloration (Medium 13, Medium 14) Yellow color is observed in King B medium.

(j) Urease (Medium 15) Negative (k) Oxidase (Medium 16) Positive (1) Catalase (Medium 17) Positive (m) Growth range (Medium 18) Growth temperature: 20 - 400C, Optimum growth temperature: 30 - 370C Growth pH range: 7 - 11 Optimum pH range: 10.5 (n) Behavior on oxygen Aerobic (o) O-F test (Medium 19) Undeterminable because the medium is an alkaline medium. Cells can grow only in an aerobic condition.

(p) Sugar utilization (Medium 20, +: cells can grow, -: cells do not grow) (i) L-arabinose + (ii) D-xylose + (iii) D-glucose + (iv) D-mannose + (v) D-fructose + (vi) D-galactose + (vii) Maltose + (viii) Sucrose + (ix) Lactose + (x) Trehalose + (xi) D-sorbitol (xii) D-mannitol + (xiii) Inositol + (xiv) Glycerol + (xv) Starch + (q) Growth in a medium containing sodium salt (a modification of Medium 1) Cells can grow in the presence of a 7% of sodium chloride, but can not grow in the presence of a 10% sodium chloride.

(r) Hydrolysis of casein ( Medium 21) Positive (s) G + C content (Tm method) About 37.7 mol% for the DNA of the microorganism of the present invention.

Mycolocrical characteristics of Bacillus sP. KSM-19 (1) Observation under microscope The cells are gram-positive rods of a size of 0.5 - 1.2 Rm x 1.2 - 5.8 Clam, with an oval endospore (0.7 - 1.2 zm x 1.2 - 1.7 Wm) forming at their subterminals. They have flagella and are motile. Gram's staining is positive.

(23 Growth in various culture media (a) Nutrient broth-agar plate (Medium 1) Growth of cells is good. The colony has an irregular shape, with its surface being smooth and its peripheral end being wavy. The color of the colony is milky and glossy.

(b) Liquid nutrient broth (Medium 2) Growth of cells is good.

(3) Physiological characteristics (a) Nitrate reduction and denitrification (Medium 5) Nitrate reduction: positive Denitrification: negative (b) MR test (Medium 6) Undeterminable (c) VP test (Medium 6) Positive (d) Production of indole (Medium 7) Negative (e) Production of hydrogen sulfide (Medium 8) Negative (f) Hydrolysis of starch (Medium 9) Positive (g) Utilization of citric acid (Medium 10, Medium 11) Positive in Koser's medium and undeterminable in Christensen's medium (h) Utilization of inorganic nitrogen sources (Medium 12) Nitrate and ammonium salts are both utilized.

(j) Urease (Medium 15) Negative (k) Oxidase (Medium 16) Positive (1) Catalase (Medium 17) Positive (m) Growth range (Medium 18) Growth temperature: 20 - 370C, Optimum growth temperature: 30 - 370C Growth pH range: 7 - 11 Optimum pH range: 10.5 (n) Behavior on oxygen Aerobic (o) O-F test (Medium 19) Undeterminable because the medium is an alkaline medium. Cells can grow only in an aerobic condition.

(p) Sugar utilization (Medium 20, +: cells can grow, -: cells do not grow) (i) L-arabinose + (ii) D-xylose + (iii) D-glucose + (iv) D-mannose + (v) D-fructose + (vi) D-galactose + (vii) Maltose + (viii) Sucrose + (ix) Lactose + (x) Trehalose + (xi) D-sorbitol (xii) D-mannitol + (xiii) Inositol (xiv) Glycerol + (xv) Starch + (q) Growth in a medium containing sodium salt (a modification of Medium 1) Cells can grow in the presence of a 7% of sodium chloride, but can not grow in the presence of a 10% sodium chloride.

(r) Hydrolysis of casein ( Medium 21) Positive (s) G + C content (Tm method) About 38.0 mol% for the DNA of the microorganism of the present invention.

Mycolopical characteristics of Bacillus sP. KSM-520 (1) Observation under microscope The cells are gram-positive rods of a size of 0.5 - 1.2 Wm x 1.2 - 5.0 pin, with an oval endospore (0.7 - 1.2 Rm x 1.2 - 1.6 Wm) forming at their subterminals. They have flagella and are motile. Gram's staining is positive.

(2) Growth in various culture media (a) Nutrient broth-agar plate (Medium 1) Growth of cells is good. The colony has an irregular shape, with its surface being smooth and its peripheral end being wavy. The color of the colony is milky and glossy.

(b) Liquid nutrient broth (Medium 2) Growth of cells is good.

(d) Litmus milk medium (Medium 4) Milk coagulation and peptonization are observed.

Litmus discoloration is undeterminable because the medium is an alkaline medium.

(3) Physiological characteristics (a) Nitrate reduction and denitrification (Medium 5) Nitrate reduction: positive Denitrification: negative (b) MR test (Medium 6) Undeterminable (c) VP test (Medium 6) Positive (d) Production of indole (Medium 7) Negative (e) Production of hydrogen sulfide (Medium 8) Negative (f) Hydrolysis of starch (Medium 9) Positive (g) Utilization of citric acid (Medium 10, Medium 11) Positive in Koser's medium and undeterminable in Christensents medium (h) Utilization of inorganic nitrogen sources (Medium 12) Nitrate and ammonium salts are both utilized.

(j) Urease (Medium 15) Negative (k) Oxidase (Medium 16) Positive (1) Catalase (Medium 17) Positive (m) Growth range (Medium 18) Growth temperature: 15 - 400C, Optimum growth temperature: 30 - 370C Growth pH range: 7 - 11 Optimum pH range: 10.5 (n) Behavior on oxygen Aerobic (o) O-F test (Medium 19) Undeterminable because the medium is an alkaline medium. Cells can grow only in an aerobic condition.

(p) Sugar utilization (Medium 20, +: cells can grow, -: cells do not grow) (i) L-arabinose + (ii) D-xylose + (iii) D-glucose + (iv) D-mannose + (v) D-fructose + (vi) D-galactose + (vii) Maltose + (viii) Sucrose + (ix) Lactose + (x) Trehalose + (xi) D-sorbitol + (xii) D-mannitol + (xiii) Inositol (xiv) Glycerol + (xv) Starch + (q) Growth in a medium containing sodium salt (a modification of Medium 1) Cells can grow in the presence of a 7% of sodium chloride, but can not grow in the presence of a 10% sodium chloride.

(r) Hydrolysis of casein ( Medium 21) Positive (s) G + C content (Tm method) About 37.6 mol% for the DNA of the microorganism of the present invention.

Based on the above mycological characteristics, the strains of the present invention were examined referring to Bergey's Manual of Systematic Bacteriology, Vol. 2 and "The Genus Bacillus" Ruth, E. Gordon, Agriculture Handbook No.

427, Agricultural Research Service, U. S. Department of Agriculture Washington D. C., (1973), and determined as a sort of microorganisms belonging to the genus Bacillus which is an ascospore Bacillus. The strains do not grow in a neutral range, but can grow mostly in an alkaline range.

From this fact, the strains of the present invention are classified as an alkalophilic microorganism which has recently been demonstrated by Horikoshi and Akiba, "Alkalophilic Microorganism", Japan Scientific Society Press (Tokyo), 1982. The strains of the present invention are distinguished from a group of microorganisms belonging to the genus Bacillus which grows in an neutral range.

The strains of the present invention, other than the above-mentioned characteristics, have mycologically different characteristics from those of any conventionally known alkalophilic Bacillus. Accordingly, the strains of the present invention were determined as novel strains and named Bacillus sp. KSM-64, Bacillus sp. KSM-19, Bacillus sp.

KSM-520, which were deposited with Fermentation Research Institute, Agency of Industrial Science and Technology as FERM BP-2886, FERM BP-2885, and FERM BP-2887 respectively.

The production of alkaline cellulase of the present invention can be carried out, for example, by the method of measuring CMC-ase activity to select the alkaline cellulase-producing microorganisms and culturing the microorganisms to obtain said alkaline cellulase from the cultured cells.

The alkaline cellulase of the present invention can also be produced, for example, by the fermentation processes using alkalophilic Bacillus sp. KSM-64, Bacillus sp. KSM-19, or Bacillus sp. KSM-520 or the mutants induced from these strains which have an alkaline cellulase-producing capability with high enzyme titre.

It has also been clarified by the present inventors that Bacillus sp. KSM-64 (FERM BP-2886) is capable of producing alkaline cellulase K-64 which possesses high ss-1,3-; 1,4-glucan- and p-1,3-glucan-decomposable activities. Accordingly, alkaline cellulase K-64 can be produced using Bacillus sp. KSM-64 other than Bacillus sp.

KSM-19, as well as the mutants induced from these strains.

The strains of the present invention are inoculated into an appropriate medium and cultured according to a conventional culture method to obtain the alkaline cellulase of the present invention. Inclusion of a suitable amount of carbon and nitrogen sources which the microorganism can utilize in the medium is desirable. There are no specific limitations as to the carbon and nitrogen sources.

Enumerated as nitrogen sources are ammonium nitrate, ammonium sulfate, ammonium phosphate, sodium nitrate, corn gluten meal, soybean flour, corn steep liquor, casamino acid, yeast extract, pharma media, sardine meal, meat extract, peptone, hypro, ajipower, corn-soybean meal, coffee meal, cotton seed meal, cultivator, ajipron, zest, and the like. Given as examples of carbon sources are fibrous substances such as chaff, hull, filter paper, general paper, sawdust; waste molasses, invert sugar, CMC, Avicel, cellulose cotton, xylan, pectin, ribose, arabinose, xylose, glucose, mannose, fructose, galactose, maltose, sucrose, lactose, trehalose, mannitol, inositol, glycerol, starch, acetic acid, citric acid, and the like.In addition to these carbon and nitrogen sources, phosphoric acid, salts of metal such as Mg2+, Ca2+, Mn2+, Zn2+, Co2+, Na+, K+, and the like, as required, other micro-nutritious organic or inorganic substances can be added into the culture medium.

The alkaline cellulase of the present invention can be prepared from the culture broth by means of conventional collection and purification methods adapted for general enzymes. Specifically, the cells are separated from the culture broth by means of conventional solid-liquid separation methods, such as, centrifugation, filtration, or the like, to obtain a crude enzyme liquid. While it is possible to use the crude enzyme liquid per se thus obtained, it can be served as a purified enzyme, as required, after separating the crude enzyme by means of conventional separation methods, e.g. salting out, precipitation, ultrafiltration, and the like, and then purifying and crystallizing the crude enzyme by conventional methods.

Enzymological characteristics of a novel enzyme, alkaline cellulases K-64, K-19, and K-520, of this invention are now discussed. Enzymatic activities were measured according to the following methods.

(1) CMC-ase activity To the base solution comprised of 0.4 ml of 2.5% CMC, (Sunrose AOIL, manufactured by Sanyo-Kokusaku Pulp Co., Ltd.), 0.2 ml of 500 mM glycine buffer (pH 9), and 0.3 ml of deionized water was added 0.1 ml of enzyme solution, and the mixture was reacted for 20 minutes at 300C. On completion of the reaction, reducing sugar was quantitatively analyzed by the 3,5-dinitro-salicylic acid (DNS) method.

Specifically, 1 ml of DNS reagent was added to 1.0 ml of reaction liquid, and mixture was caused to color by heating at 1000C for 5 minutes. After cooling, the colored mixture was diluted with 4.0 ml of deionized water and subjected to colorimetric quantitative analysis at 535 nm. One (1) unit of enzyme titer is defined as the amount of the enzyme producing reducing sugar corresponding to 1 mol of glucose per minute.

The buffer solutions used are as follows: pH 3 - 8: MacIlvaine's buffer pH 8 - 11: glycine-NaOH buffer pH 12 - 13: KCl-NaOH buffer (2) P-nitrophenylglucoside- or pnitrophenylcellobioside-resolving activity To 1.0 ml of reaction liquid comprised of 0.8 mol of p-nitrophenylcellobioside (manufactured by Sigma Co.) or pnitrophenylglycoside (manufactured by Sigma Co.) and 50 mol of phosphoric acid buffer (pH 7.0) or 100 jimol of glycine buffer (pH 9) was effected a suitable amount of enzyme liquid at 300C. 0.4 ml of 1 M Na2CO3 was added to the mixture. Free p-nitrophenol was subjected to colorimetric quantitative analysis at 410 nm. One (1) unit of enzyme titer is defined as the amount of the enzyme producing 1 pinol of free p-nitrophenol per minute.

(3) Avicel-, cellulose powder-, phosphoric acid swollen cellulose-, and filter paper-resolving activity To 1.0 mg of reaction liquid comprised of 10 mg of avicel and 100 pinol of glycine buffer (pH 9) was added an appropriate amount of enzyme and the mixture was reacted at 300C with shaking at 280 rpm. On completion of the reaction, reducing sugar was quantitatively determined by means of the DNS method. One (1) unit of enzyme titer is defined as the amount of the enzyme producing reducing sugar corresponding to 1 pinol of glucose per minute.

Other enzymatic activities were measured in the same manner as above. Cellulose powder (manufactured by Toyo Roshi Co.), cellulase activity test filter paper (Toyo No.

51-Toku manufactured by Toyo Roshi Co.), and alkali swollen cellulose and phosphoric acid swollen cellulose which were treated according to the method by Tomita et al (Tomita, Y.

et al.,: j. Ferment. Technol., 52, 235, 1974).

(4) ss-1,3-; 1,4-glucanase- and ss-1,3-glucanase-activity The activity was measured in the same manner as in (3) using lichenan (from Usnera barubata, manufactured by Sigma Co.) and laminarin (from Laminaria digitata, manufactured by Sigma Co.) as substrates.

Enzvmoioaical characteristics of alkaline cellulase K-64 (1) Activity The enzyme of the present invention acts well on cellulosic materials such as CMC, cellulose powder, avicel, and the like dissolves them, and produces reducing sugars.

(2) Substrate specificity The substrate specificity of the enzyme of the present invention is exemplified in Table 1. The enzyme of this invention possesses a high hydrolytic activity against lichenan which is ss-1,3-; 1,4-glucan as well as CMC; acts on laminarin which is ss-1,3-glucan; and slightly acts on filter paper, p-nitrophenylglucoside, and p-nitrophenylcellobioside.

Table 1 Substrate Concentration Relative Activity (%) CMC 1% 100 Avicel 1% 2 Filter Paper 1% 4 Phosphoric acid swollen 18 34 cellulose PNPC * 8 mM 1 PNPG ** 8 mM 1 Lichenan 1% 154 Laminarin 1% 25 * p-nitrophenylcellobioside ** p-nitrophenylglucoside (3) Active pH range and optimum pH The active pH range on CMC is broad, i.e. 3 - 13.0 with an optimum pH range of 8.5 - 10. It shows a relative activity more than 50% of the activity of the optimum pH range even in pHs ranging 7.0 - 12.0 (see Fig. 1).

(4) pH stability After the enzyme adjusted to various pHs was left stand at 300C for one hour, and the residual activity was measured to evaluate pH stability. The enzyme is quite stable in pHs ranging 5.0 to 10.5 and has the activity of more than 50% even in pHs ranging 3.0 to 11 (see Fig. 2).

(5) Active temperature and optimum temperature The enzyme of the present invention acts at wide temperatures ranging 10 to 700C with an optimum temperature of 500C and has more than 50% of the activity of the optimum temperature even in temperatures ranging 35 to 600C (see Fig. 3).

(6) Temperature stability Residual activities after heat-treatment in glycine-NaOH buffer (pH 9.0) at various temperatures for 10 minutes were measured. The enzyme of this invention scarcely loses its activity at 500C, and has the residual activity of about 50% even at 550C (see Fig. 4).

(7) Molecular weight The molecular weight of the enzyme of the present invention was measured by means of a gel filtration method using Bio-Gel A 0.5m (manufactured by Biorad Co.). The enzyme of this invention has peaks at approximately 180,000 + 10,000 and 80,000 + 2,000, when CMC is a substrate.

(8) Effect of metal ions The effect of metal ions was investigated, at the same time when the activities of the enzyme were measured, by incorporating a metal ion such as Na+, K+, Ca2+, Cu2+, Co2+, Cd2+, Mn2+, Mg2+, Ba2+, Ni2+, Hg2+ Fe2+ pub2+ z 2+ Al3+, and Fe3+. Addition of Hg2+ ions (1 mM each) gives an adverse effect and addition of Co2+ and Mn2+ ions (1 mM each) gives an activated effect.

(9) Effect of surfactants 0.05% by weight of a surfactant such as LAS, AS, ES, AOS, a-SFE, SAS, soap, and polyoxyethylene secondary alkylether (SDS) was tested and confirmed no inhibitive effects on enzymatic activities.

(10) Effect of proteases In the presence of a protease for detergents, for example, API-21 (manufactured by Showa Denko), Maxatase (manufactured by IBIS), and Sabinase, Alkalase, and Esperase (manufactured by Novo) in an amount of 0.2 AU/l, the activities were measured, to confirm that the enzyme of this invention had an excellent resistance to any proteases.

(11) Effect of chelating agents In the presence of a chelating agent such as EDTA, EGTA, sodium tripolyphosphate, zeolite, citric acid, or the like, the activities were measured and confirmed that any chelating agents had no inhibitive effects on enzymatic activities.

Enzvmoloaical characteristics of alkaline cellulase K-19 (1) Activity The enzyme of the present invention acts well on cellulosic materials such as CMC, cellulose powder, avicel, and the like dissolves them, and produces reducing sugars.

(2) Substrate specificity The substrate specificity of the enzyme of the present invention is exemplified in Table 2. The enzyme of this invention acts on, cellulose powder, phosphoric acid swollen cellulose, avicel, alkali swollen cellulose, filter paper, p-nitrophenylglucoside, and p-nitrophenylcellobioside, and the like, as well as CMC; possesses a high hydrolytic activity against lichenan which is ss-1,3-; 1,4glucan; acts on laminarin which is ss-1,3-glucan.

Table 2 Substrate Concentration Relative Activity (t) CMC 1% 100 Avicel 1% 5 Filter Paper 1% 4 Cellulose powder 1% 3 Phosphoric acid swollen 1% 3 cellulose Alkali swollen cellulose 1% 3 PNPC * 8 mM 1 PNPG ** 8 mM 2 Lichenan 1% 161 Laminarin 1% 37 * p-nitrophenylcellobioside ** p-nitrophenylglucoside (3) Active pH range and optimum pH The active pH range on CMC is broad, i.e. 4.5 12.5 with an optimum pH range of 9.0. It shows a relative activity more than 50% of the activity of the optimum pH range even in pRs ranging 7.0 - 11.0 (see Fig. 5).

(4) pH stability After the enzyme adjusted to various pHs was left stand at 300C for one hour, the residual activity was measured to evaluate pH stability. The enzyme is quite stable in pHs ranging 4.5 to 11.0 and shows more than 50% of the activity even in pHs ranging 4.0 to 12.0 (see Fig. 6).

(5) Active temperature and optimum temperature The enzyme of the present invention acts at wide temperatures ranging 10 to 700C with an optimum temperature of 500C and has more than 50% of the activity of the optimum temperature even in temperatures ranging 35 to 600C (see Fig. 7).

(6) Temperature stability The residual activities after heat-treatment in glycine-NaOH buffer (pH 9.0) at various temperatures for 10 minutes were measured. The enzyme of this invention scarcely loses its activity at 500C, and has the residual activity of about 50% even at 550C (see Fig. 8).

(7) Molecular weight The molecular weight of the enzyme of the present invention was measured by means of a gel filtration method using Bio-Gel A 0.5m (manufactured by Biorad Co.). The enzyme of this invention has peaks at about 120,000 + 20,000 and 34,000 + 2,000 (small peak) when CMC is a substrate.

(8) Effect of metal ions The effect of metal ions was investigated, at the same time when the activities of the enzyme were measured, by incorporating a metal ion such as Na+, K+, Ca2+, Cu2+, Co2+, Cd2+, Mn2+, Mg2+, Boa2+, Ni2+, Hg2+ Foe2+ pub2+ Zn2+ Al3+, and Fe3+. Addition of Hg2+ and Pb2+ ions (1 mM each) gives an adverse effect and addition of Co2+ and Mn2+ ions (1 mM each) gives an activated effect.

(9) Effect of surfactants 0.05% by weight of a surfactant such as LAS, AS, ES, AOS, a-SFE, SAS, soap, and polyoxyethylene secondary alkylether (SDS) was tested to confirm no inhibitive effects on enzymatic activities.

(10) Effect of proteases In the presence of a protease for detergents, for example, API-21 (Showa Denko), Maxatase (IBIS), and Sabinase, Alkalase, and Esperase (Novo) in an amount of 0.2 AU/l, the activities were measured, to confirm that the enzyme of this invention had an excellent resistance to any proteases.

(11) Effect of chelating agents In the presence of a chelating agent such as EDTA, EGTA, sodium tripolyphosphate, zeolite, and citric acid, the activities were measured, to confirm that any chelating agents had no inhibitive effects on enzymatic activities.

Enzvmoloaical characteristics of alkaline cellulase K-520 (1) Activity The enzyme of the present invention acts well on cellulosic materials such as CMC, cellulose powder, avicel, and the like and dissolves them, to produce, reducing sugars.

(2) Substrate specificity The substrate specificity of the enzyme of the present invention is exemplified in Table 3. The enzyme of this invention slightly acts on laminarin, lichenan, filter paper, p-nitrophenylglucoside, and p-nitrophenylcellobioside.

Table 3 Substrate Concentration Relative Activity (%) CMC 1% 100 Avicel 1% 20 Filter Paper 1% 0.2 Cellulose powder 1% 6 Phosphoric acid swollen 1% 160 cellulose Alkali swollen cellulose 1% 28 PNPC * 8 mM 0.8 PNPG ** 8 mM 0.2 Lichenan 1% 6 Laminarin 1% 2 * p-nitrophenylcellobioside ** p-nitrophenylglucoside (3) Active pH range and optimum pH The active pH range on CMC is broad, i.e. 3 - 13.0 with an optimum pH range of 8.5 - 9.5. It shows a relative activity of more than 50% of the activity of the optimum pH range even in pHs ranging 7.0 - 11.0 (see Fig. 9).

(4) pH stability After the enzyme adjusted to various pHs was left stand at 300C for one hour, the residual activity was measured to evaluate its pH stability. The enzyme is quite stable in pHs ranging 6.0 to 11.0 and has more than 50% of the activity even in pHs ranging 5.0 to 11.5 (see Fig. 10).

(5) Active temperature and optimum temperature The enzyme of the present invention acts at wide temperatures ranging 10 to 750C with an optimum temperature of 500C and has more than 50% of the activity of the optimum temperature even in temperatures ranging 35 to 600C (see Fig. 11).

(6) Temperature stability The residual activities after heat-treatment in glycine-NaOH buffer (pH 9.0) at various temperatures for 10 minutes were measured. The enzyme of this invention scarcely loses its activity at 400C, and has the residual activity of about 50% even at 500C (see Fig. 12).

(7) Molecular weight The molecular weight of the enzyme of the present invention was measured by means of a gel filtration method using Bio-Gel A 0.5m (manufactured by Biorad Co.). The enzyme of this invention has peaks at about 170,000 + 10,000 and 80,000 + 2,000.

(8) Effect of metal ions The effect of metal ions was investigated, at the same time when the activities of the enzyme were measured, by incorporating a metal ion such as Na+, K+, Ca2+, Cu2+, Cho2+ Cud2+ Mn2+, Mug2+ Boa2+, Ni2+, Hg2+, Fe2+, Pb2+, On2+, Al3+, and Fe3+. Addition of Hg2+, Cd2+, and Pb2+ ions (1 mM each) gives an adverse effect and addition of Co2+ and Mn2+ ions (1 mM each) gives an activated effect.

(9) Effect of surfactants 0.05% by weight of a surfactant such as LAS, AS, ES, AOS, SAS, soap, and polyoxyethylene secondary alkylether was tested to confirm no inhibitive effects on enzymatic activities.

(11) Effect of chelating agents In the presence of a chelating agent such as EDTA, EGTA, sodium tripolyphosphate, zeolite, citric acid, or the like, the activities were measured, to confirm that any chelating agents had no inhibitive effects on enzymatic activities.

Other features of the invention will become apparent in the course of the following description of the exemplary embodiments which are given for illustration of the invention and are not intended to be limiting thereof.

EXAMPLES Example 1 A spoonful of soil (about 0.5 g) of Nasu-gun, Tochigiken, Japan was suspended in sterilized saline and the mixture was heat-treated at 800C for 10 minutes. A supernatant of the heat-treated mixture was appropriately diluted, applied onto an isolating agar medium (Medium A), and cultured at 300C for 3 days to grow colonies. The colonies which formed a transparent range in their peripheries due to CMC dissolution were collected to obtain CMC-ase producing strains. These strains were inoculated into the liquid medium B and shake-cultured at 300C for 3 days. After culturing, the cultured broth was centrifuged to separate a supernatant. The CMC-ase activity of the supernatant was measured at pH ranging 3 to 13 to select alkaline cellulase-producing strains. Bacillus sp. KSM-64 (FERM BP-2886) and Bacillus sp. KSM-520 (FERM BP-2887) were thus obtained.

% by weight Medium A CMC 2 Polypeptone 0.5 Yeast extract 0.5 KH2PO4 0.1 MgSO4C7H2O 0.02 Na2CO3 0.8 Agar 0.75 pH: 10 (approximately) Medium B CMC 1 Meat extract 1.5 Yeast extract 0.5 KH2P04 0.1 Na2CO3 0.5 pH: 10 (approximately) Example 2 Bacillus sp. KSM-64 obtained in Example 1 was inoculated into the liquid medium B of Example 1 and shakecultured at 300C for 3 days. After culturing, the cells were removed by means of centrifugation to obtain a crude enzyme liquid. The enzyme liquid was processed according to a conventional method to prepare ethanol-dry powder. As a result, the production of enzyme cellulase was confirmed as shown in Table 4.

Table 4 Strain Amount of enzyme Enzymatic * per 1 of medium (g) activity (U/g) KSM-64 1.1 1,500 * Value at pH 9 Example 3 KSM-64 was inoculated into a medium which had the same composition as the liquid medium B in Example 1 except that 1% of cellobiose and 5% of CSL were added instead of CMC and polypeptone respectively, and shake-cultured at 300C for 2 3 days. CMC-ase activity of a supernatant centrifuged was measured to be 3,000 U/l.

Example 4 Bacillus sp. KSM-520 obtained in Example 1 was inoculated into the liquid medium B of Example 1 and shakecultured at 300C for 3 days. After culturing, the cells were removed by means of centrifugation to obtain a crude enzyme liquid. The enzyme liquid was processed according to a conventional method to prepare ethanol-dry powder. As a result, the production of enzyme cellulase was confirmed as shown in Table 5.

Table 5 Strain Amount of enzyme Enzymatic * per 1 of medium (g) activity (U/g) KSM-520 0.1 2,530 * Value at pH 9 Example 5 KSM-520 was inoculated into a medium which had the same composition as the liquid medium B in Example 1 except that 1% of cellobiose and 5% of CSL were added instead of CMC and polypeptone respectively, and shake-cultured at 300C for 2 3 days. CMC-ase activity of a supernatant centrifuged was measured to be 3,542 U/l.

Example 6 A spoonful of soil (about 0.5 g) of Nasu-gun, Tochigiken, Japan was suspended in sterilized saline and the mixture was heat-treated at 800C for 10 minutes. A supernatant of the heat-treated mixture was appropriately diluted, applied onto an isolating agar medium (Medium A in Example 1), and cultured at 300C for 3 days to grow colonies. The colonies which formed a transparent range in their peripheries due to CMC dissolution were collected to obtain CMC-ase producing strains.

Next, these strains were inoculated into the similar medium as the medium A except that lichenan was used instead of CMC and shake-cultured at 300C for 3 days, to grow colonies. By means of Congo-red method, colonies forming a transparent region in the peripheries of the colonies due to lichenan dissolution were selected to obtain lichenaseproducing strains.

Tïle strains obtained was inoculated into the liquid medium B (used in Example 1) and shake-cultured at 300C for 2 - 3 days. CMC-ase activity and lichenase activity of a supernatant centrifuged was measured at pHs of 3 - 13 to select an alkaline cellulase-producing microorganism.

Example 7 Bacillus sp. KSM-19 obtained in Example 6 was inoculated into the liquid medium B of Example 1 and shakecultured at 300C for 3 days. After culturing, the cells were removed by means of centrifugation to obtain a crude enzyme liquid. The enzyme liquid was processed according to a conventional method to prepare ethanol-dry powder. As a result, the production of enzyme cellulase was confirmed as shown in Table 6.

Table 6 Strain Amount of enzyme Enzymatic * per 1 of medium (g) activity (U/g) KSM-19 0.4 1,600 * Value at pH 9 Example 8 KSM-19 was inoculated into a medium which had the same composition as the liquid medium B in Example 1 except that 1% of cellobiose and 5% of CSL were added instead of CMC and polypeptone respectively, and shake-cultured at 300C for 2 3 days. CMC-ase activity of a supernatant centrifuged was measured to be 769 U/l of CMC-ase activity, 1,238 U/l of lichenan activity, and 285 U/l of laminarin decomposing activity Cellulases of the present invention are the novel alkaline cellulases which have an optimum pH at an alkaline region and are stable in a broad pH range.The enzymes of the present invention are not adversely affected by the addition of detergent components such as surfactants, proteases, chelating agents, and the like, and, accordingly, can effectively be used as a detergent component.

Further, alkaline cellulase K-19 of the present invention possesses ss-1,3; 1,4-glucanase- and ss-1,3- glucanase-activitiy, so that it can dissolve ss-glucan derived from grain, e.g. barley. Accordingly, the alkaline cellulase of the present invention can be utilized for techniques of, for example, shortening time for filtering wheat sap in the process for producing beer and preventing a beer product from becoming turbid.

Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

Claims

WHAT IS CLAIMED IS:

1. An alkaline cellulase K-64 possessing the following physicochemical characteristics: (A) Activity Acts well on cellulosic materials such as carboxymethyl cellulose (CMC), cellulose, filter paper, avicel, and the like, dissolves them and produces reducing sugars.

(B) Substrate specificity Besides CMC, acts on cellulose powder, phosphoric acid swollen cellulose, avicel, filter paper, p-nitrophenyl-ss-D-glucopyranoside, and p-nitrophenyl-ss-D- cellobioside.

(C) Active pH range and optimum pH Has an active pH range on CMC of 3 - 13.0 with an optimum pH range being 8.5 - 10.0 and even in pHs ranging 7.0 - 12.0 has a relative activity of more than 50% of the activity of the optimum pH range (D) pH stability Is quite stable at pHs ranging 5 - 10.5 and even in pHs ranging 3 - 11 has an activity of more than 50% of'the activity of the optimum pH, when CMC is a substrate.

(H) Effect of surfactants Exhibits almost no inhibitive effects on enzymatic activities by linear alkylbenzene sulfonate, alkyl sulfate, polyoxyethylene alkyl sulfonate, a-olefin sulfonate, acarboxymethyl alkylsulfonate, secondary alkyle sulfonate, dodecyl sulfonate, soap, and/or polyoxyethylene secondary alkylether, when CMC is a substrate.

(J) Effect of chelating agents Exhibits no inhibitive effects on enzymatic activities by EDTA, EGTA, citric acid, sodium tripolyphosphate, zeolite, when CXC is a substrate.

2. An alkaline cellulase K-19 possessing the following physicochemical characteristics: (A) Activity Acts well on cellulosic materials such as carboxymethyl cellulose (CMC), cellulose, filter paper, avicel, and the like, dissolves them and produces reducing sugars.

(B) Substrate specificity Besides CXC, acts on cellulose powder, phosphoric acid swollen cellulose, alkali swollen cellulose, avicel, filter paper, p-nitrophenyl-ss-D-glucopyranoside, and p nitrophenyl-ss-D-cellobioside.

(C) Active pH range and optimum pH Has an active pH range on CMC of 4.5 - 12.5 with an optimum pH range being 9 and even in pHs ranging 7.0 - 11.0 has a relative activity of more than 50% of the activity of the optimum pH range (D) pH stability Is quite stable in pHs ranging 4.5 - 11.0 and even in pHs ranging 4.0 - 12.0 has an activity of more than 50% of the activity of the optimum pH, when CMC is a substrate.

(F) Molecular weight Has peaks of activities at about 120,000 + 10,000 and 34,000 i 2,000 (small peak), (measured by means of the gel filtration method using Bio-Gel A 0.5m), when CMC is a substrate.

(G) Effect of metal ions Is adversely affected by the addition of Hg2+, and Pb2+ ions and activated by the addition of Cho2+ and Mn2+ ions, when CXC is a substrate.

(H) Effect of surfactants Exhibits almost no inhibitive effects on enzymatic activities by linear alkylbenzene sulfonate, alkyl sulfate, polyoxyethylene alkyl sulfonate, a-olefin sulfonate, acarboxymethyl alkylsulfonate, dodecyl sulfonate, soap, and/or polyoxyethylene secondary alkylether, when CMC is a substrate.

3. An alkaline cellulase K-520 possessing the following physicochemical characteristics: (A) Activity Acts well on cellulosic materials such as carboxymethyl cellulose (CXC), cellulose, filter paper, avicel, and the like, dissolves them and produces reducing sugars.

(B) Substrate specificity Besides CMC, acts on cellulose powder, phosphoric acid swollen cellulose, alkali swollen cellulose, avicel, filter paper, p-nitrophenyl-ss-D-glucoside, and p-nitrophenyl-ss-D-cellobioside.

(C) Active pH range and optimum pH Has an active pH range on CXC of 3 - 13.0 with an optimum pH range being 8.5 - 9.5 and even in pHs ranging 7.0 - 11.0 has a relative activity of more than 50% of the activity of the optimum pH range.

(D) pH stability Is quite stable in pHs ranging 6.0 - 11.0 and even in pHs ranging 5 - 11.5 has an activity of more than 50% of the optimum pH, when CXC is a substrate.

(F) Molecular weight About 170,000 t 10,000 and 80,000 + 2,000 (measured by means of the gel filtration method using Bio-Gel A 0.5m), when CXC is a substrate.

(G) Effect of metal ions Is adversely affected by the addition of Hg2+, Cd2+, and Pb2+ ions and activated by the addition of Co2+ and Mn2+ ions, when CXC is a substrate.

(H) Effect of surfactants Exhibits almost no inhibitive effects on enzymatic activities by linear alkylbenzene sulfonate, alkyl sulfate, polyoxyethylene alkyl sulfonate, a-olef in sulfonate, secondary alkane sulfonate, soap, and/or polyoxyethylene secondary alkylether, when CXC is a substrate.

4. An alkaline cellulase K-64 producing microorganism belonging to the genus Bacillus and growing in alkaline media.

5. An alkaline cellulase K-64 producing microorganism which is named as Bacillus sp. KSM-64 and deposited as FERM BP-2886 with Fermentation Research Institute, Agency of Industrial Science and Technology.

6. An alkaline cellulase K-19 producing microorganism belonging to the genus Bacillus and growing in alkaline media.

7. An alkaline cellulase K-19 producing microorganism which is named as Bacillus sp. KSM-19 and deposited as FERM BP-2885 with Fermentation Research Institute, Agency of Industrial Science and Technology.

8. An alkaline cellulase K-520 producing microorganism belonging to the genus Bacillus and growing in alkaline media.

9. An alkaline cellulase K-520 producing microorganism which is named as Bacillus sp. KSM-520 and deposited as FERM BP-2887 with Fermentation Research Institute, Agency of Industrial Science and Technology.

10. A process for producing the alkaline cellulase K64 comprising: culturing an alkaline cellulase K-64 producing microorganism belonging to the genus Bacillus and growing in alkaline media; and collecting the alkaline cellulase K-64 from the cultured medium.

11. A process for producing an alkaline cellulase comprising: culturing a microorganism belonging to the genus Bacillus and capable of producing an alkaline cellulase having ss-1,3-;1,4-glucanase activity and ss-1,3-glucanase activity and collecting the alkaline cellulase from the cultured medium.

12. A process for producing alkaline the cellulase K19 comprising: culturing an alkaline cellulase K-19 producing microorganism belonging to the genus Bacillus and growing in alkaline media; and collecting the alkaline cellulase K-19 from the cultured medium.

13. A process for producing the alkaline cellulase K520 comprising: culturing an alkaline cellulase K-520 producing microorganism belonging to the genus Bacillus and growing in alkaline media; and collecting the alkaline cellulase K-520 from the cultured medium.

14. An alkaline cellulase substantially as hereinbefore described.

15. A process for producing alkaline cellulase substantially as.hereinbefore described.