Alkaline cellulases and microorganisms capable of producing same

Claim 2

An alkaline cellulase which is produced by a microorganism belonging to the genus Bacillus and growing in a neutral medium and which has a working pH in an alkaline range.

Claim 3

An alkaline cellulase according to Claim 1, wherein said cellulase has the following enzymatic properties: (1) a broad optimum pH range of from 8 to 10 with a maximum activity at pH of approximately 10; (2) the activity being inhibited by the presence of Hg²⁺; (3) the activity being rarely inhibited with proteinases, surface active agents and chelating agents; (4) a CMCase being a main activity with additional filter paper-disintegrating activity and Avicelase activity (C₁ activity).

Claim 4

An alkaline cellulase according to Claim 1, wherein said alkaline cellulase has the following enzymatic properties and is named K-580. (1) Action       Acting well on cellulosic materials including carboxymethyl cellulose (CMC), cellulose, filter paper and Avicel and causing them to be dissolved, thereby forming reducing sugars such as glucose. (2) Substrate specificity       Having activity not only on CMC, but also on cellulose powder, Avicel and filter paper. (3) Working pH and optimum pH       The working pH range is from 3 to 12.5 and the optimum pH is from 7 to 10 with a relative activity of not less than 50% of the activity at an optimum pH being shown in the range of 4.5 to 10.5. (4) pH stability       Very stable and not inactivated at a pH of 4.5 to 12 and an activity of not less than about 50% being maintained at a pH of 3.5 to 12.5. (5) Optimum temperature       The working temperature is in a wide range of 15 to 80°C and an optimum temperature is 65°C. In the range of 50 to 75°C, the activity is not less than 50% of the activity at the optimum temperature. (6) Molecular weight       Peaks of the molecular weight are at about 18,000 and about 50,000 (when determined by a gel filtration method using Sephadex G100). (7) Influences of metal ions       Inhibited by Hg²⁺ and activated by Ba²⁺, Ca²⁺, Co²⁺ and Cd²⁺. (8) Influences of surface active agents       LAS, AS, ES, AOS, alpha-SFE, SAS and soaps and polyoxyethylene secondary alkyl ethers do rarely inhibit the activity. (9) Proteinase resistance       Resistant to proteinases. (10) Influences of chelating agents       EDTA, EGTA, citric acid, sodium tripoly­phosphate and zeolite do not inhibit the activity. 4. An alkaline cellulase according to Claim 1, wherein said alkaline cellulase has the following enzymatic properties and is named K-425. (1) Action       Acting well on cellulosic materials including CMC, cellulose, filter paper and Avicel and causing them to be dissolved, thereby forming reducing sugars such as glucose. (2) Substrate specificity       Having activity not only on CMC, but also on cellulose powder, Avicel, filter paper, p-nitrophenyl cellobioside (PNPC) and cellobiose. (3) Working pH and optimum pH       The working pH range is from 3.5 to 12.5 and the optimum pH is from 8 to 10 with a relative activity of not less than 50% of the activity at an optimum pH being shown in the range of 5.5 to 10.5. (4) pH stability       Very stable and not inactivated at a pH of 5 to 11 and an activity of not less than about 50% being maintained at a pH of 3 to 12. (5) Optimum temperature       The working temperature is in a wide range of 15 to 75°C and an optimum temperature is 50°C. In the range of 35 to 55°C, the activity is not less than 50% of the activity at the optimum temperature. (6) Molecular weight       About 35,000 (when determined by a gel filtration method using Sephadex G100). (7) Influences of metal ions       Inhibited by Hg²⁺ and Ba²⁺ and activated by Co²⁺. (8) Influences of surface active agents       LAS, AS, ES, AOS, alpha-SFE, SAS and soap and polyoxyethylene secondary alkyl ethers do rarely inhibit the activity. (9) Proteinase resistance       Resistant to proteinases. (10) Influences of chelating agents       EDTA, EGTA, citric acid, sodium tripoly­phosphate and zeolite do not inhibit the activity. 5. An alkaline cellulase according to Claim 1, wherein said alkaline cellulase has the following enzymatic properties and is named K-521. (1) Action       Acting well on cellulosic materials including CMC, cellulose, filter paper and Avicel and causing them to be dissolved, thereby forming reducing sugars such as glucose. (2) Substrate specificity       Having activity not only on CMC, but also on cellulose powder, Avicel, filter paper, PNPC and cellobiose. (3) Working pH and optimum pH       The working pH range is from 3 to 12.5 and the optimum pH is from 7 to 10 with a relative activity of not less than 50% of the activity at an optimum pH being shown in the range of 4.5 to 10.5. (4) pH stability       Very stable and not inactivated at a pH of 5 to 12 and an activity of not less than about 50% being maintained at a pH of 4.5 to 12.5. (5) Optimum temperature       The working temperature is in a wide range of 15 to 80°C and an optimum temperature is 60°C. In the range of 45 to 65°C, the activity is not less than 50% of the activity at the optimum temperature. (6) Molecular weight       About 31,000 (when determined by a gel filtration method using Sephadex G100). (7) Influences of metal ions       Inhibited by Hg²⁺ and activated by Ca²⁺. (8) Influences of surface active agents       LAS, AS, ES, AOS, alpha-SFE, SAS and soaps and polyoxyethylene secondary alkyl ethers do rarely inhibit the activity. (9) Proteinase resistance       Resistant to proteinases. (10) Influences of chelating agents       EDTA, EGTA, citric acid, sodium tripoly­phosphate and zeolite do not inhibit the activity. 6. An alkaline cellulase according to Claim 1, wherein said alkaline cellulase has the following enzymatic properties and is named K-522. (1) Action       Acting well on cellulosic materials including CMC, cellulose, filter paper and Avicel and causing them to be dissolved, thereby forming reducing sugars such as glucose. (2) Substrate specificity       Having activity not only on CMC, but also on cellulose powder, phosphoric acid-swollen cellulose, alkali-swollen cellulose, Avicel, filter paper, and PNPC. (3) Working pH and optimum pH       The working pH range is from 3 to 12.5 and the optimum pH is from 7 to 10 with a relative activity of not less than 50% of the activity at an optimum pH being shown in the range of 4.5 to 10.5. (4) pH stability       Very stable and not inactivated at a pH of 5 to 12 and an activity of not less than about 50% being maintained at a pH of 4.5 to 12.5. (5) Optimum temperature       The working temperature is in a wide range of 15 to 80°C and an optimum temperature is 60°C. In the range of 45 to 65°C, the activity is not less than 50% of the activity at the optimum temperature. (6) Molecular weight       About 35,000 (when determined by a gel filtration method using Bio-Gel P-150). (7) Influences of metal ions       Inhibited by Hg²⁺. (8) Influences of surface active agents       LAS, AS, ES, AOS, alpha-SFE, SAS, soaps and polyoxyethylene secondary alkyl ethers do rarely inhibit the activity. (9) Proteinase resistance       Resistant to proteinases. (10) Influences of chelating agents       EDTA, EGTA, citric acid, sodium tripoly­phosphate and zeolite do not inhibit the activity. 7. An alkaline cellulase E-II having the following physicochemical properties. (1) Action       Acting effectively on cellulosic materials including CMC and phosphoric acid-swollen cellulose and causing them to be dissolved, thereby forming reducing sugars such as glucose. (2) Substrate specificity       Having not only activity on CMC and phosphoric acid-swollen cellulose, but also slight activity on xylan, inulin and lichenan. (3) Working pH and optimum pH       The working pH range is from 4 to 12.5 and the optimum pH is from 7 to 10. (4) pH stability       When maintained at 5°C for 24 hours, it is not inactivated at a pH of 6 to 10. (5) Optimum temperature       The working temperature is in a wide range of 10 to 80°C and an optimum temperature is 50°C. (6) Molecular weight       About 34,000 (when determined by a gel filtration method using Bio-Gel P-100). (7) Influences of metal ions       Inhibited by Hg²⁺ and activated by Co²⁺. (8) Influences of surface active agents       LAS, AS, ES, AOS, alpha-SFE, SAS, soaps and polyoxyethylene secondary alkyl ethers do rarely inhibit the activity. (9) Proteinase resistance       Resistant to proteinases. (10) Influences of chelating agents       EDTA, EGTA, citric acid, sodium tripoly­phosphate and zeolite do not inhibit the activity. 8. An alkaline cellulase E-III having the following physicochemical properties. (1) Action       Acting well on cellulosic materials including CMC and phosphoric acid-swollen cellulose and causing them to be dissolved, thereby forming reducing sugars such as glucose. (2) Substrate specificity       Having not only activity on CMC and phosphoric acid-swollen cellulose, but also slight activity on xylan and lichenan. (3) Working pH and optimum pH       The working pH range is from 4 to 12.5 and the optimum pH is from 7 to 9. (4) pH stability       When maintained at 5°C for 24 hours, it is not inactivated at a pH of 6 to 10. (5) Working temperature and optimum temperature       The working temperature is in the range of 10 to 80°C and an optimum temperature is 50°C. (6) Molecular weight       About 35,000 (when determined by a gel filtration method using Bio-Gel P-100). (7) Influences of metal ions       Inhibited by Hg²⁺ and activated by Co²⁺. (8) Influences of surface active agents       LAS, AS, ES, AOS, alpha-SFE, SAS, soaps and polyoxyethylene secondary alkyl ethers do rarely inhibit the activity. (9) Proteinase resistance       Resistant to proteinases. (10) Influences of chelating agents       EDTA, EGTA, citric acid, sodium tripoly­phosphate and zeolite do not inhibit the activity. 9. A microorganism which is capable of producing an alkaline cellulase having an optimum pH at an alkaline side, belongs to the genus Bacillus , and grows up in a neutral medium. 10. A microorganism according to Claim 9, wherein the produced alkaline cellulase has the following enzymatic properties. (1) Having a broad optimum pH range of 8 to 10 and a maximum activity at pH of approximately 10. (2) The activity is inhibited by the presence of Hg²⁺. (3) The activity is rarely inhibited with proteinases, surface active agents and chelating agents. (4) The CMC decomposing activity (Cx activity) is a main activity with additional filter paper disintegrating activity and Avicelase activity (C₁ activity). 11. A microorganism according to Claim 9, wherein said microorganism is an alkaline cellulase K-­580-producing bacterium which is named Bacillus sp. KSM-580 and is deposited as FERM BP-1511. 12. A microorganism according to Claim 9, wherein said microorganism is an alkaline cellulase K-­425-producing bacterium which is named Bacillus sp. KSM-425 and is deposited as FERM BP-1505. 13. A microorganism according to Claim 9, wherein said microorganism is an alkaline cellulase K-­ 521-producing bacterium which is named Bacillus sp. KSM-521 and is deposited as FERM BP-1507. 14. A microorganism according to Claim 9, wherein said microorganism is an alkaline cellulase K-­522 producing bacterium which is named Bacillus sp. KSM-522 and is deposited as FERM BP-1512.