2,5-diketo-D-gluconic acid reductase

Claim 2

Substantially pure 2,5-Diketo-D-gluconic acid reductase having the following physio-chemical properties: (a) enzyme action: catalyzes, in the presence of NADPH as a coenzyme, (i) reduction of 2,5-diketo-D-gluconic acid or its salts to 2-keto-L-gulonic acid or the corresponding salts thereof, and (ii) reduction of 5-keto-D-fructose to L-sorbose; (b) substrate specificity to: 2,5-diketo-D-gluconic acid and 5-keto-D-fructose; (c) optimum pH: pH 6-7; (d) pH stability: pH 5-7; (e) molecular weight: 29,000±2,000; and (f) isoelectric point: pH 4.4±0.3.

Claim 3

A process for the production of 2,5-diketo-D-gluconic acid reductase, which comprises: culturing a microorganism belonging to the genus Corynebacterium in a nutrient medium containing carbon and nitrogen sources and inorganic salt, and isolating the thus produced 2,5-diketo-D-gluconic acid reductase from disrupted cells of the microorganism as a single component which can reduce 5-keto-D-fructose to L-sorbose.

Claim 4

A process for the production of 2,5-diketo-D-gluconic acid reductase, which comprises: culturing a microorganism belonging to the genus Corynebacterium in a nutrient medium containing carbon and nitrogen sources and inorganic salt, and isolating the thus produced 2,5-diketo-D-gluconic acid reductase from disrupted cells of the microorganism as a single component which can reduce 5-keto-D-fructose to L-sorbose.

Claim 5

A composition of matter comprising 2,5-Diketo-D-gluconic acid reductase as a single component which exhibits the following physio-chemical properties: (a) enzyme: action: catalyzes, in the presence of NADPH as a coenzyme, (i) reduction of 2,5-diketo-D-gluconic acid or its salts to 2-keto-L-gulonic acid or the corresponding salts thereof, and (ii) reduction of 5-D-fructose to L-sorbose; (b) substrate specificity to: 2,5-diketo-D-gluconic acid and 5-keto-D-fructose; (c) an optimum pH of from 6 to 7; (d) a pH stability of from 5 to 7; (e) a molecular weight of 29,000±2,000; and (f) an isoelectric point at a pH of 4.4±0.3.

Claim 6

A composition of matter comprising 2,5-Diketo-D-gluconic acid reductase as a single component which exhibits the following physio-chemical properties: (a) enzyme: action: catalyzes, in the presence of NADPH as a coenzyme, (i) reduction of 2,5-diketo-D-gluconic acid or its salts to 2-keto-L-gulonic acid or the corresponding salts thereof, and (ii) reduction of 5-D-fructose to L-sorbose; (b) substrate specificity to: 2,5-diketo-D-gluconic acid and 5-keto-D-fructose; (c) an optimum pH of from 6 to 7; (d) a pH stability of from 5 to 7; (e) a molecular weight of 29,000±2,000; and (f) an isoelectric point at a pH of 4.4±0.3.

Claim 7

A process for converting 2,5-diketo-D-gluconic acid into 2-keto-L-gulonic acid comprising contacting 2,5-diketo-D-gluconic acid with an effective amount of the enzyme composition of claim 3.

Claim 8

A process for converting 5-keto-D-fructose into L-sorbose which comprises contacting 5-keto-D-fructose with a purified 2,5-Diketo-D-gluconic acid reductase enzyme composition which exhibits the following physio-chemical properties: (a) enzyme: action: catalyzes, in the presence of NADPH as a coenzyme, (i) reduction of 2,5-diketo-D-gluconic acid or its salts to 2-keto-L-gulonic acid or the corresponding salts thereof, and (ii) reduction of 5-D-fructose to L-sorbose; (b) substrate specificity to: 2,5-diketo-D-gluconic acid and 5-keto-D-fructose; (c) an optimum pH of from 6 to 7; (d) a pH stability of from 5 to 7; (e) a molecular weight of 29,000±2,000; and (f) an isoelectric point at a pH of 4.4±0.3.

Claim 9

A process for converting 5-keto-D-fructose into L-sorbose which comprises contacting 5-keto-D-fructose with a purified 2,5-Diketo-D-gluconic acid reductase enzyme composition which exhibits the following physio-chemical properties: (a) enzyme: action: catalyzes, in the presence of NADPH as a coenzyme, (i) reduction of 2,5-diketo-D-gluconic acid or its salts to 2-keto-L-gulonic acid or the corresponding salts thereof, and (ii) reduction of 5-D-fructose to L-sorbose; (b) substrate specificity to: 2,5-diketo-D-gluconic acid and 5-keto-D-fructose; (c) an optimum pH of from 6 to 7; (d) a pH stability of from 5 to 7; (e) a molecular weight of 29,000±2,000; and (f) an isoelectric point at a pH of 4.4±0.3.

Claim 10

The composition of matter according to claim 3, wherein the enzyme is an intracellular enzyme extracted from a microorganism belonging to the genus Corynebacterium.

Claim 11

The composition of matter according to claim 6, wherein the Corynebacterium microorganism is selected from the group consisting of ATCC 31,090; FERM-BP 108; ATCC 31,098; ATCC 31,088 and any mutant derived therefrom.

Claim 12

The composition according to claim 3, wherein said enzyme is catalytically active at temperatures ranging from 25° to 45° C.

Claim 13

The composition according to claim 3 further comprising a stabilizer selected from the group consisting of glycerol and sucrose.

Claim 14

The process according to claim 2, wherein the culturing is at 25°-35° C. for 15-30 hours during microbial growth.

Claim 15

A process for the production of 2,5-diketo-D-gluconic acid reductase, which comprises: culturing a microorganism belonging to genus Corynebacterium in a nutrient medium containing carbon and nitrogen sources and inorganic salt during the growth phase of said microorganism; harvesting the microorganism by centrifugation; disrupting the harvested cells by sonification, french press treatment or enzyme treatment; separating unbroken cells and cell debris from a supernatant enzyme solution; salting-out crude enzyme solution from the supernatant enzyme solution with a salting-out solution; dissolving the salted-out crude enzyme solution in a buffer so that the enzyme is not inactivated; dialyzing the enzyme solution to remove the salting-out solution; and purifying the enzyme solution by ion-exhange chromatography and affinity, chromatography to produce purified 2,5-diketo-D-gluconic acid reductase as a single component.

Claim 16

A process for the production of 2,5-diketo-D-gluconic acid reductase, which comprises: culturing a microorganism belonging to genus Corynebacterium in a nutrient medium containing carbon and nitrogen sources and inorganic salt during the growth phase of said microorganism; harvesting the microorganism by centrifugation; disrupting the harvested cells by sonification, french press treatment or enzyme treatment; separating unbroken cells and cell debris from a supernatant enzyme solution; salting-out crude enzyme solution from the supernatant enzyme solution with a salting-out solution; dissolving the salted-out crude enzyme solution in a buffer so that the enzyme is not inactivated; dialyzing the enzyme solution to remove the salting-out solution; and purifying the enzyme solution by ion-exhange chromatography and affinity, chromatography to produce purified 2,5-diketo-D-gluconic acid reductase as a single component.

Claim 17

The process according to claim 4, wherein the contacting occurs at 24°-45° C. and pH 6-7.

Claim 18

The process according to claim 4, wherein the molar concentration of 2,5-diketo-D-gluconic acid is equal to that of the coenzyme.

Claim 19

The process according to claim 13, wherein the molar concentration of said 2,5-diketo-D-gluconic acid is in a concentration of 20 to 200 mM.

Claim 20

The process according to claim 5, wherein the enzyme reacts with the 5-keto-D-fructose in a concentration ranging from 0.5 to 5.0 units/ml.

Claim 21

The process according to claim 5, wherein the enzyme is immobilized onto a carrier.

Claim 22

The process according to claim 4, wherein at least 90% of 2,5-diketo-D-gluconic acid is reduced to 2-keto-L-gulonic acid in 60 to 120 minutes at 30° C.

Claim 23

The process according to claim 11, wherein the salting out solution is ammonium sulfate.

Claim 24

The composition according to claim 3, wherein the Km value is 1.8±1.0 mM.

Claim 25

The enzyme composition according to claim 3 having an enzyme activity for 2,5-diketo-D-gluconic acid between 22 and 59 unit/ml and an enzyme activity for 5-keto-L-fructose between 33 and 96 unit/ml at a pH ranging from 5 to 7.

Claim 26

A composition of matter comprising 2,5-diketo-D-gluconic acid reductase as a single component which exhibits the following physio-chemical properties: (a) enzyme catalyzing action, in the presence of a hydrogen donor as a coenzyme, (i) reduction of 2,5-diketo-D-gluconic acid or its salts to 2-keto-L-gulonic acid or the corresponding salts thereof, and (ii) reduction of 5-D-fructose to L-sorbose; (b) substrate specificity to: 2,5-diketo-D-gluconic acid and 5-keto-D-fructose; (c) an optimum pH of from 6 to 7; (d) a pH stability of from 5 to 7; (e) a molecular weight of 29,000±2,000; and (f) an isoelectric point at a pH of 4.4±0.3.

Claim 27

A composition of matter comprising 2,5-diketo-D-gluconic acid reductase as a single component which exhibits the following physio-chemical properties: (a) enzyme catalyzing action, in the presence of a hydrogen donor as a coenzyme, (i) reduction of 2,5-diketo-D-gluconic acid or its salts to 2-keto-L-gulonic acid or the corresponding salts thereof, and (ii) reduction of 5-D-fructose to L-sorbose; (b) substrate specificity to: 2,5-diketo-D-gluconic acid and 5-keto-D-fructose; (c) an optimum pH of from 6 to 7; (d) a pH stability of from 5 to 7; (e) a molecular weight of 29,000±2,000; and (f) an isoelectric point at a pH of 4.4±0.3.

Claim 28

A product produced by the process according to claim 11.

Claim 29

A product produced by the process according to claim 2.