Alkali-resistant cellulases and microorganisms capable of producing same

Claim 2

An alkali-resistant cellulase which is produced by a microorganism belonging to the genus Bacillus and growing in a neutral medium.

Claim 3

An alkali-resistant cellulase according to Claim 1, wherein said cellulase has the following enzymatic properties: (1) a wide optimum pH range of from 5 to 7; (2) very stable and not inactivated at a pH of from 5 to 10 with a residual activity, at a pH of. 4.5 to 11, of not less than 50% of a maximum activity; (3) its activity being inhibited by the presence of H g 2+ ; and (4) the activity being rarely inhibited with proteinases, surface active agents and chelating agents.

Claim 4

An alkali-resistant cellulase according to Claim 1, wherein said cellulase has the following enzymatic properties and is named K-534. (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 as glucose. (2) Substrate specificity Having activity not only on CMC, but also on cellulose powder, Avicel, filter paper and p-nitrophenyl cellobioside (PNPC). (3) Working pH and optimum pH The working pH range is from 3 to 12.5 and the optimum pH is about 6.5 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 11 with 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 85°C and an optimum temperature is about 70°C. In the range of 45 to 75°C, the activity is not less than 50% of the activity at the optimum temperature. (6) Molecular weight Peaks for the molecular weight are at about 15,000 and about 30,000 (when determined by a gel filtration method using Sephadex G-100). (7) Influences of metal ions Activated by Ba2+ and 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, STPP, zeolite and citric acid do not inhibit the activity.

Claim 5

An alkali-resistant cellulase according to Claim 1, wherein said alkaline cellulase has the following enzymatic properties and is named K-344. (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, and PNPC. (3) Working pH and optimum pH The working pH range is from 3 to 12.5 and the optimum pH is 6 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 11. (4) pH stability Very stable and not inactivated at a pH of 5 to 10 with an activity of not less than about 50% being maintained at a pH of 4.5 to 12. (5) Optimum temperature The working temperature is in a wide range of 15 to 80°C and an optimum temperature is 70°C. In the range of 40 to 75°C, the activity is not less than 50% of the activity at the optimum temperature. (6) Molecular weight A peak for the molecular weight appears at about 16,000 (when determined by a gel filtration method using Sephadex G-100). (7) Influences of metal ions Inhibited by Hg 2+ and Pb 2+ 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, sodium tripolyphosphate, zeolite and citric acid do not inhibit the activity.

Claim 6

An alkali-resistant cellulase according to Claim 1, wherein said alkaline cellulase has the following enzymatic properties and is named K-539. (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 and PNPC. (3) Working pH and optimum pH The working pH range is from 3 to 12.5 and the optimum pH is 7 with a relative activity of not less than 50% of the'activity at the optimum pH being shown in the range of 4 to 11. (4) pH stability Very stable and not inactivated at a pH of 5 to 12 with 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 85°C and an optimum temperature is 60°C. In the range of 45 to 75°C, the activity is not less than 50% of the activity at the optimum temperature. (6) Molecular weight Peaks for the molecular weight appear at about 18,000 and about 29,000, respectively, (when determined by a gel filtration method using Sephadex G-100). (7) Influences of metal ions Inhibited by Hg 2+ and activated by Ca2+, Cd 2+ and Ba2+. (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, sodium tripolyphosphate, zeolite and citric acid do not inhibit the activity.

Claim 7

An alkali-resistant cellulase according to Claim 1, wherein said cellulase has the following enzymatic properties and is named K-577. (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 and filter paper. (3) Working pH and optimum pH The working pH range is from 3 to 12 and the optimum pH is 7 with a relative activity of not less than 50% of the activity at the 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 with 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 75°C and the optimum temperature is 60°C. In the range of 40 to 65°C, the activity is not less than 50% of the activity at the optimum temperature. (6) Molecular weight Peaks for the molecular weight appear at about 17,000 and about 30,000 (when determined by a gel filtration method using Sephadex G-100). (7) Influences of metal ions Inhibited by Hg 2+ and activated by Ba2+, Ca2+, Cd 2+ and 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, sodium tripolyphosphate, zeolite and citric acid do not inhibit the activity.

Claim 8

An alkali-resistant cellulase according to Claim 1, wherein said alkali-resistant cellulase has the following enzymatic properties and is named K-588. (1) Action Acting well on cellulosic materials including CMC and filter paper and causing them to be dissolved, thereby forming reducing sugars such as glucose. (2) Substrate specificity Having not only activity on CMC, but also activity on phosphoric acid-swollen cellulose, filter paper and PNPC. (3) Working pH and optimum pH The working pH range is from 3 to 13 and the optimum pH is 7, with a relative activity of not less than 50% at the 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 11.5 with an activity of not less than about 50% being maintain 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 a range of 45 to 65°C, the activity is not less than 50% of the activity at the optimum temperature. (6) Molecular weight Peaks for the molecular weight appear at about 27,000 and about 30,000, respectively (when determined by a gel filtration method using Biogel P-150). (7) Influences of metal ions Inhibited by H g2 + . (8) Influences of surface active agents LAS, AS, ES, AOS, alpha-SFE, SAS, soap and polyoxyethylene secondary alkyl ethers do rearely inhibit the activity. (9) Proteinase resistance Resistant to proteinases. (10) Influences of chelating agents EDTA, EGTA, sodium tripolyphosphate, zeolite and citric acid do not inhibit the activity.

Claim 9

An alkali-resistant cellulase according to Claim 1, wherein said alkali-resistant cellulase has enzymatic properties and is named K-597. (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 phosphoric acid-swollen cellulose, cellulose powder, Avicel, filter paper and PNPC. (3) Working pH and optimum pH The working pH range is from 3 to 13 and the optimum pH is 7, with a relative activity of not less than 50% of the activity at the optimum pH being shown in the range of from 4.5 to 11.5. (4) pH stability Very stable and not inactivated at a pH of 5 to 11.5 with an activity of not less than about 50% being maintained at a pH of 4.5 to 12.5. (5) Working temperature and optimum temperature The working temperature is in the range of 15 to 80°C and the optimum temperature is 60°C. In the range of from 45 to 75°C, the activity is not less than 50% of the activity at the optimum temperature. (6) Molecular weight A peak for the molecular weight appears at about 40,000 (when determined by a gel filtration method using Bio-Gel P-150). (7) Influences of metal ions Inhibited by Hg 2+ and activated by Co 2+ . (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, sodium-tripolyphosphate, zeolite and citric acid do not inhibit the activity.

Claim 10

A microorganism which is capable of producing an alkali-resistant cellulase, grows up in a neutral medium, and belongs to the genus Bacillus.

Claim 11

A microorganism which is capable of producing an alkali-resistant cellulase, grows up in a neutral medium, and belongs to the genus Bacillus.

Claim 12

A microorganism according to Claim 9, wherein the produced alkali-resistant cellulase has the following enzymatic properties. (1) Having a wide working pH range of from 5 to 7. (2) Very stable and not inactivated at a pH of from 5 to 10 with a residual activity of not less than 50% of the maximum activity being shown in the pH range of from 4.5 to 11. (3) The activity is inhibited by the presence of H g2 + . (4) The activity is rarely inhibited by proteinases, surface active agents and chelating agents.

Claim 13

A microorganism according to Claim 9, wherein said microorganism is named Bacillus sp. KSM-534 and is deposited as FERM BP-1508.

Claim 14

A microorganism according to Claim 9, wherein said microorganism is named Bacillus sp. KSM-344 and is deposited as FERM BP-1506.

Claim 15

A microorganism according to Claim 9, wherein said microorganism is named Bacillus sp. KSM-539 and is deposited as FERM BP-1509.

Claim 16

A microorganism according to Claim 9, wherein said microorganism is named Bacillus sp. KSM-577 and is deposited as FERM BP-1510.

Claim 17

A microorganism according to Claim 9, wherein said microorganism is named Bacillus sp. KSM-588 and is deposited as PERM BP-1513.

Claim 18

A microorganism according to Claim 9, wherein said microorganism is named Bacillus sp. KSM-597 and is deposited as FERM BP-1514.