Alkali-resistant cellulases and microorganisms capable of producing same

Claim 2

A substantially pure alkali-resistant cellulase enzyme (K-534) having the following enzymatic properties: (1) action: acting well on cellulosic materials including carboxymethyl cellulose, cellulose, filter paper and microcrystalline cellulose causing them to be destroyed, thereby forming reducing sugars; (2) substrate specificity; having activity on carboxymethyl cellulose, cellulose powder, microcrystalline cellulose, filter paper and p-nitrophenyl cellobioside; (3) working pH and optimum pH: a working pH range from 3 to 12.5 and an optimum pH of 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: working temperature in a wide range of 15° to 85° C and an optimum temperature of 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 gel filtration).

Claim 3

A substantially pure alkali-resistant cellulase enzyme (K-344) having the following enzymatic properties: (1) action: acting on cellulosic materials including carboxymethyl cellulose, cellulose, filter paper and microcrystalline cellulose causing them to dissolved, thereby forming reducing sugars; (2) substrate specificity: having activity on carboxymethyl cellulose, cellulose powder, microcrystalline cellulose, filter paper and p-nitrophenyl cellobioside; (3) working pH and optimum pH: working pH from 3-12.5 and an optimum pH of 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 inactivate 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: working temperature in a wide range of 15° to 80° C and an optimum temperature of 70° C, in the range of 40° -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 gel filtration).

Claim 4

A substantially pure alkali-resistant cellulase enzyme (K-344) having the following enzymatic properties: (1) action: acting on cellulosic materials including carboxymethyl cellulose, cellulose, filter paper and microcrystalline cellulose causing them to dissolved, thereby forming reducing sugars; (2) substrate specificity: having activity on carboxymethyl cellulose, cellulose powder, microcrystalline cellulose, filter paper and p-nitrophenyl cellobioside; (3) working pH and optimum pH: working pH from 3-12.5 and an optimum pH of 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 inactivate 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: working temperature in a wide range of 15° to 80° C and an optimum temperature of 70° C, in the range of 40° -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 gel filtration).

Claim 5

A substantially pure alkali-resistant cellulose enzyme (K-539) having the following enzymatic properties: (1) action; acting well on cellulosic materials including carboxymethyl cellulose, cellulose, filter paper and microcrystalline cellulose causing them to be dissolved, thereby forming reducing sugars; (2) substrate specificity: having activity on carboxymethyl cellulose, cellulose powder, microcrystalline cellulose, filter paper and p-nitrophenyl cellobioside; (3) working pH and optimum pH: a working pH range of from 3 to 12.5 and an optimum pH of 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: a working temperature in a wide range of 15° to 85° C and an optimum temperature of 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 19,000, respectively (when determined by gel filtration); (7) influences of metal ions: inhibited by Hg 2+ and activated by Ca 2+ , Cd 2+ and Ba 2+ ; (8) influences of surface active agents: little inhibition of activity by sodium linear alkylbenzensulfonates, sodium alkylsulfates, sodium polyoxyethylene alkylsulfates, sodium α- olefin sulfonates, sodium α- sulfonated aliphatic acid esters, sodium alkylsulfonates, soaps and polyoxyethylene secondary alkyl ethers; (9) proteinase resistance: resistant to proteinases; and (10) influences of chelating agents: ethylenediamine tetraacetic acid, ethylene glycol-bis-(β-aminoethylether)-N,N,N', N"-tetraacetic acid, sodium tripolyphosphate, zeolite and citric acid do not inhibit activity.

Claim 6

A substantially pure alkali-resistant cellulose enzyme (K-539) having the following enzymatic properties: (1) action; acting well on cellulosic materials including carboxymethyl cellulose, cellulose, filter paper and microcrystalline cellulose causing them to be dissolved, thereby forming reducing sugars; (2) substrate specificity: having activity on carboxymethyl cellulose, cellulose powder, microcrystalline cellulose, filter paper and p-nitrophenyl cellobioside; (3) working pH and optimum pH: a working pH range of from 3 to 12.5 and an optimum pH of 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: a working temperature in a wide range of 15° to 85° C and an optimum temperature of 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 19,000, respectively (when determined by gel filtration); (7) influences of metal ions: inhibited by Hg 2+ and activated by Ca 2+ , Cd 2+ and Ba 2+ ; (8) influences of surface active agents: little inhibition of activity by sodium linear alkylbenzensulfonates, sodium alkylsulfates, sodium polyoxyethylene alkylsulfates, sodium α- olefin sulfonates, sodium α- sulfonated aliphatic acid esters, sodium alkylsulfonates, soaps and polyoxyethylene secondary alkyl ethers; (9) proteinase resistance: resistant to proteinases; and (10) influences of chelating agents: ethylenediamine tetraacetic acid, ethylene glycol-bis-(β-aminoethylether)-N,N,N', N"-tetraacetic acid, sodium tripolyphosphate, zeolite and citric acid do not inhibit activity.

Claim 7

A substantially pure alkali-resistant cellulase enzyme (K-577) having the following enzymatic properties: (1) action: acting well on cellulosic materials including carboxymethyl cellulose, cellulose, filter paper and microcrystalline cellulose causing them to be dissolved, thereby forming reducing sugars; (2) substrate specificity: having activity on carboxymethyl cellulose, cellulose powder, microcrystalline cellulose and filter paper; (3) working pH and optimum pH: a working pH range of from 3 to 12 and an optimum pH of 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 about 50% being maintained at a pH of 4.5 to 12.5; (5) optimum temperature: a working temperature in a wide range of from 15° to 75° C and an optimum temperature of 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 gel filtration; (7) influences of metal ions: inhibited by Hg 2+ and activated by Ba 2+ , Ca 2+ , Cd 2+ and Co 2+ ; (8) influences of surface active agents: little inhibition of activity by sodium linear alkylbenzensulfonates, sodium alkylsulfates, sodium polyoxyethylene alkylsulfates, sodium α- olefin sulfonates, sodium α- sulfonated aliphatic acid esters, sodium alkylsulfonates, soaps and polyoxyethylene secondary alkyl ethers; (9) proteinase resistance: resistant to proteinases; and (10) influences of chelating agents: ethylenediamine tetraacetic acid, ethylene glycol-bis-(β-aminoethylether)-N,N,N', N"- tetraacetic acid, sodium tripolyphosphate, zeolite and citric acid do not inhibit activity.

Claim 8

A substantially pure alkali-resistant cellulase enzyme (K-577) having the following enzymatic properties: (1) action: acting well on cellulosic materials including carboxymethyl cellulose, cellulose, filter paper and microcrystalline cellulose causing them to be dissolved, thereby forming reducing sugars; (2) substrate specificity: having activity on carboxymethyl cellulose, cellulose powder, microcrystalline cellulose and filter paper; (3) working pH and optimum pH: a working pH range of from 3 to 12 and an optimum pH of 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 about 50% being maintained at a pH of 4.5 to 12.5; (5) optimum temperature: a working temperature in a wide range of from 15° to 75° C and an optimum temperature of 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 gel filtration; (7) influences of metal ions: inhibited by Hg 2+ and activated by Ba 2+ , Ca 2+ , Cd 2+ and Co 2+ ; (8) influences of surface active agents: little inhibition of activity by sodium linear alkylbenzensulfonates, sodium alkylsulfates, sodium polyoxyethylene alkylsulfates, sodium α- olefin sulfonates, sodium α- sulfonated aliphatic acid esters, sodium alkylsulfonates, soaps and polyoxyethylene secondary alkyl ethers; (9) proteinase resistance: resistant to proteinases; and (10) influences of chelating agents: ethylenediamine tetraacetic acid, ethylene glycol-bis-(β-aminoethylether)-N,N,N', N"- tetraacetic acid, sodium tripolyphosphate, zeolite and citric acid do not inhibit activity.

Claim 9

A substantially pure alkali-resistant cellulase enzyme (K-588) having the following enzymatic properties: (1) action: acting well on cellulosic materials including carboxymethyl cellulose and filter paper and causing them to be dissolved, thereby forming reducing sugars; (2) substrate specificity: having activity on carboxymethyl cellulose, phosphoric acid-swollen cellulose, filter paper and p-nitrophenyl cellobioside; (3) working pH and optimum pH: a working pH in the range from 3 to 13 and an optimum pH of 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 about 50% being maintained at a pH of 4.5 to 12.5; (5) optimum temperature: a working temperature in a wide range of from 15° to 80° C and an optimum temperature of 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 (when determined by gel filtration; (7) influences of metal ions: inhibited by Hg 2+ ; (8) influences of surface active agents: little inhibition of activity by sodium linear alkylbenzensulfonates, sodium alkylsulfates, sodium polyoxyethylene alkylsulfates, sodium α- olefin sulfonates, sodium α- sulfonated aliphatic acid esters, sodium alkylsulfonates, soaps and polyoxyethylene secondary alkyl ethers; (9) proteinase resistance: resistant to proteinases; and (10) influences of chelating agents: ethylenediaminetetraacetic acid and ethyleneglycol-bis-(β-aminoethylether)-N,N,N', N"- tetraacetic acid, sodium tripolyphosphate, zeolite and citric acid do not inhibit activity.

Claim 10

A substantially pure alkali-resistant cellulase enzyme (K-588) having the following enzymatic properties: (1) action: acting well on cellulosic materials including carboxymethyl cellulose and filter paper and causing them to be dissolved, thereby forming reducing sugars; (2) substrate specificity: having activity on carboxymethyl cellulose, phosphoric acid-swollen cellulose, filter paper and p-nitrophenyl cellobioside; (3) working pH and optimum pH: a working pH in the range from 3 to 13 and an optimum pH of 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 about 50% being maintained at a pH of 4.5 to 12.5; (5) optimum temperature: a working temperature in a wide range of from 15° to 80° C and an optimum temperature of 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 (when determined by gel filtration; (7) influences of metal ions: inhibited by Hg 2+ ; (8) influences of surface active agents: little inhibition of activity by sodium linear alkylbenzensulfonates, sodium alkylsulfates, sodium polyoxyethylene alkylsulfates, sodium α- olefin sulfonates, sodium α- sulfonated aliphatic acid esters, sodium alkylsulfonates, soaps and polyoxyethylene secondary alkyl ethers; (9) proteinase resistance: resistant to proteinases; and (10) influences of chelating agents: ethylenediaminetetraacetic acid and ethyleneglycol-bis-(β-aminoethylether)-N,N,N', N"- tetraacetic acid, sodium tripolyphosphate, zeolite and citric acid do not inhibit activity.

Claim 11

A substnatially pure alkali-resistant cellulase enzyme (K-597) having the following enzymatic properties: (1) action: acting well on cellulosic material including carboxymethyl cellulose, cellulose, filter paper and microcrystalline cellulose causing them to be dissolved, thereby forming reducing sugars; (2) substrate specificity: having activity on carboxymethyl cellulose, phosphoric acid-swollen cellulose, cellulose powder, microcrystalline cellulose, filter paper and p-nitrophenyl cellobioside; (3) working pH and optimum pH: a working pH range of from 3 to 13 and an optimum pH of 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 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 50% being maintained at a pH of 4.5 to 12.5; (5) optimum temperature: a working temperature in a wide range of from 15° to 80° C and an optimum temperature of 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: a peak for the molecular weight appears at about 40,000 (when determined by gel filtration); (7) influences of metal ions: inhibited by Hg 2+ and activated by Co 2+ ; (8) influences of surface active agents: little inhibition of activity by sodium linear alkylbenzensulfonates, sodium alkylsulfates, sodium polyoxyethylene alkylsulfates, sodium α- olefin sulfonates, sodium α- sulfonated aliphatic acid esters, sodium alkylsulfonates, soaps and polyoxyethylene secondary alkyl ethers; (9) proteinase resistance: resistant to proteinases; and (10) influences of chelating agents: ethylenediamine tetraacetic acid, and ethylene glycol-bis-(β-aminoethylether)-N,N,N', N"- tetraacetic acid, sodium tripolyphosphate, zeolite and citric acid do not inhibit activity.

Claim 12

A substnatially pure alkali-resistant cellulase enzyme (K-597) having the following enzymatic properties: (1) action: acting well on cellulosic material including carboxymethyl cellulose, cellulose, filter paper and microcrystalline cellulose causing them to be dissolved, thereby forming reducing sugars; (2) substrate specificity: having activity on carboxymethyl cellulose, phosphoric acid-swollen cellulose, cellulose powder, microcrystalline cellulose, filter paper and p-nitrophenyl cellobioside; (3) working pH and optimum pH: a working pH range of from 3 to 13 and an optimum pH of 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 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 50% being maintained at a pH of 4.5 to 12.5; (5) optimum temperature: a working temperature in a wide range of from 15° to 80° C and an optimum temperature of 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: a peak for the molecular weight appears at about 40,000 (when determined by gel filtration); (7) influences of metal ions: inhibited by Hg 2+ and activated by Co 2+ ; (8) influences of surface active agents: little inhibition of activity by sodium linear alkylbenzensulfonates, sodium alkylsulfates, sodium polyoxyethylene alkylsulfates, sodium α- olefin sulfonates, sodium α- sulfonated aliphatic acid esters, sodium alkylsulfonates, soaps and polyoxyethylene secondary alkyl ethers; (9) proteinase resistance: resistant to proteinases; and (10) influences of chelating agents: ethylenediamine tetraacetic acid, and ethylene glycol-bis-(β-aminoethylether)-N,N,N', N"- tetraacetic acid, sodium tripolyphosphate, zeolite and citric acid do not inhibit activity.

Claim 13

A biologically pure culture of Bacillus species KSM-534 deposited as FERM BP-1508 which is capable of producing the alkali-resistant cellulase of claim 1.

Claim 14

A biologically pure culture of Bacillus species KSM- 534 deposited as FERM BP-1506 which is capable of producing the alkali-resistant cellulase K-344 of claim 2.

Claim 15

A biologically pure culture of Bacillus species KSM- 539 deposited as FERM BP-1509 which is capable of producing the alkali-resistant cellulase K-539 of claim 3.

Claim 16

A biologically pure culture of Bacillus species KSM- 577 deposited as FERM BP-1510 which is capable of producing the alkali-resistant cellulase K-577 of claim 4.

Claim 17

A biologically pure culture of Bacillus species KSM- 588 deposited as FERM BP-1513 which is capable of producing the alkali-resistant cellulase K-588 of claim 5.

Claim 18

A biologically pure culture of Bacillus species KSM- 597 deposited as FERM BP-1514 which is capable of producing the alkali-resistant cellulase K-597 of claim 6.

Claim 19

A process of producing an alkali-resistant cellulase which is stable and not inactivated at a pH of 5 to 10 with a residual activity, at a pH of 4.5 to 11, of not less than 50% of a maximum activity, comprising: cultivating an alkali-resistant cellulase-producing bacterium selected from the group consisting of Bacillus species KSM-534 deposited as FERM BP-1508, Bacillus species KSM-344 deposited as FERM BP-1506, Bacillus species KSM 539 deposited as FERM BP -1509, Bacillus species KSM-577 deposited as FERM BP-1510, Bacillus species KSM-588 deposited as FERM BP- 1513 and Bacillus species KSM - 597 deposited as FERM BP-1514, which is capable of growing in a neutral medium, in a neutral medium, and collecting said alkali-resistant cellulase from the resulting culture product.

Claim 20

A process of producing an alkali-resistant cellulase which is stable and not inactivated at a pH of 5 to 10 with a residual activity, at a pH of 4.5 to 11, of not less than 50% of a maximum activity, comprising: cultivating an alkali-resistant cellulase-producing bacterium selected from the group consisting of Bacillus species KSM-534 deposited as FERM BP-1508, Bacillus species KSM-344 deposited as FERM BP-1506, Bacillus species KSM 539 deposited as FERM BP -1509, Bacillus species KSM-577 deposited as FERM BP-1510, Bacillus species KSM-588 deposited as FERM BP- 1513 and Bacillus species KSM - 597 deposited as FERM BP-1514, which is capable of growing in a neutral medium, in a neutral medium, and collecting said alkali-resistant cellulase from the resulting culture product.

Claim 21

The process of claim 13, wherein the produced alkali-resistant 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 the maximum activity; (3) activity being inhibited by the presence of Hg 2+ ; and (4) activity being not inhibited by proteinases, surface acting agents and chelating agents.

Claim 22

The process of claim 13, wherein said bacterium is an alkali-resistant cellulase K-534-producing bacterium named Bacillus species KSM-534 deposited as FERM BP-1508.

Claim 23

The process of claim 13, wherein said bacterium is an alkali-resistant cellulase K-344-producing bacterium named Bacillus species KSM-344 deposited as FERM BP-1506.

Claim 24

The process of claim 13, wherein said bacterium is an alkali-resistant cellulase K-539-producing bacterium named Bacillus species KSM-539 deposited as FERM BP-1509.

Claim 25

The process of claim 13, wherein said bacterium is an alkali-resistant cellulase K-577-producing bacterium named Bacillus species KSM-577 deposited as FERM BP-1510.

Claim 26

The process of claim 13, wherein said bacterium is an alkali-resistant cellulase K-588-producing bacterium named Bacillus species KSM-588 deposited as FERM BP-1513.

Claim 27

The process of claim 13, wherein said bacterium is an alkali-resistant cellulase K-597-producing bacterium named Bacillus species KSM-597 deposited as FERM BP-1514.