New polyphosphate-dependent glucokinase and method for producing glucose 6-phosphate using same

Abstract

The present invention relates to a novel thermophilic and thermoresistant polyphosphate-dependent glucokinase having excellent stability, a composition comprising the same, and a method for producing glucose 6-phosphate using the same.

1 . A thermoresistant polyphosphate-dependent glucokinase derived from the genus  Deinococcus.    
     
     
         2 . The thermoresistant polyphosphate-dependent glucokinase according to  claim 1 , wherein the glucokinase is expressed from the DNA sequence set forth in SEQ ID NO. 1. 
     
     
         3 . The thermoresistant polyphosphate-dependent glucokinase according to  claim 1 , wherein the glucokinase has the amino acid sequence set forth in SEQ ID NO. 2. 
     
     
         4 . A composition for the production of glucose 6-phosphate comprising a thermoresistant polyphosphate-dependent glucokinase derived from the genus  Deinococcus , glucose, and polyphosphate. 
     
     
         5 . The composition according to  claim 4 , wherein the composition achieves a conversion yield of at least 60% to glucose 6-phosphate, when the composition comprises 1% to 3% by weight of the glucose, 1% to 10% by weight of the polyphosphate, and 0.1 mg/ml to 2.0 mg/ml of the polyphosphate-dependent glucokinase, based on the total volume of the composition. 
     
     
         6 . The composition according to  claim 4 , wherein the composition achieves a conversion yield of at least 50% to glucose 6-phosphate, when the composition comprises 5% to 20% by weight of the glucose, 5% to 10% by weight of the polyphosphate, and 0.1 mg/ml to 2.0 mg/ml of the polyphosphate-dependent glucokinase, based on the total volume of the composition. 
     
     
         7 . The composition according to  claim 5  or  6 , further comprising magnesium ions. 
     
     
         8 . The composition according to  claim 7 , wherein the magnesium ions are present at a concentration of 1 mM to 20 mM. 
     
     
         9 . The composition according to  claim 4 , wherein glucose 6-phosphate is produced at a temperature of 40° C. to 60° C. and/or a pH of 7 to 9. 
     
     
         10 . A method for producing glucose 6-phosphate from a composition comprising a thermoresistant polyphosphate-dependent glucokinase derived from the genus  Deinococcus , glucose, and polyphosphate. 
     
     
         11 . The method according to  claim 10 , wherein the glucose is prepared by liquefaction or glycosylation of starch or cellulose. 
     
     
         12 . The method according to  claim 10 , wherein the polyphosphate is sodium hexametaphosphate. 
     
     
         13 . The method according to  claim 10 , wherein glucose 6-phosphate is produced at a temperature of 40° C. to 60° C. and/or a pH of 7 to 9. 
     
     
         14 . The method according to  claim 10 , wherein the composition further comprises magnesium ions. 
     
     
         15 . The method according to  claim 10 , wherein the polyphosphate-dependent glucokinase is present in an amount of 0.1 mg/ml to 0.5 mg/ml. 
     
     
         16 . The method according to  claim 10 , wherein the glucose is present in an amount of 1% to 30% by weight, based on the total volume of the composition. 
     
     
         17 . The method according to  claim 10 , wherein the polyphosphate is present in an amount of 1% to 10% by weight, based on the total volume of the composition. 
     
     
         18 . A method for producing glucose 6-phosphate from a composition comprising a thermoresistant polyphosphate-dependent glucokinase derived from the genus  Deinococcus , a diastatic enzyme, starch, and polyphosphate. 
     
     
         19 . The method according to  claim 18 , wherein the diastatic enzyme is selected from alpha-amylases, glucoamylases, alpha-glycosidases, and mixtures thereof. 
     
     
         20 . A recombinant expression vector comprising a gene encoding the polyphosphate-dependent glucokinase according to  claim 3 . 
     
     
         21 .  Escherichia coli  BL21 DE3 CJ pET21a-Dg_ppgk (KCCM11793P) transformed with the recombinant expression vector according to  claim 20 . 
     
     
         22 . A method for producing a compound selected from D-glucose 1-phosphate, D-fructose 6-phosphate, D-fructose 1,6-bisphosphate, myo-inositol 3-phosphate, myo-inositol, D-glucuronate, D-glucosamine 6-phosphate, D-glucosamine, N-acetyl-D-glucosamine 6-phosphate, N-acetyl-D-glucosamine, N-acetyl-D-mannosamine 6-phosphate, N-acetyl-D-mannosamine, N-acetylneuraminic acid (sialic acid), D-mannose 6-phosphate, D-mannose, D-tagatose 6-phosphate, D-tagatose, D-allulose 6-phosphate, D-allulose, D-glyceraldehyde 3-phosphate, and dihydroxyacetone phosphate, the method comprising reacting a thermoresistant polyphosphate-dependent glucokinase derived from the genus  Deinococcus , glucose or starch, polyphosphate, and an additional enzyme.