New enzymatic process for production of modified hop products

Abstract

The present invention relates to a process for producing a beer bittering agent via enzyme catalyzed bioconversion of hop-derived isoalpha acids to dihydro-(rho)-isoalpha acids and to the novel enzyme catalysts which may be employed in such a process.

1 . A process for the preparation of dihydro-(rho)-isoalpha acids, comprising treating isoalpha acids with a ketoreductase enzyme or a microorganism expressing a gene that encodes the ketoreductase. 
     
     
         2 . The process according to  claim 1 , wherein the process is carried out in an aqueous system. 
     
     
         3 . The process according to  claim 2 , wherein the process is carried out under mild temperature and pH conditions. 
     
     
         4 . The process according to  claim 1 , comprising adding the ketoreductase enzyme and NADPH or NADP to a mixture of isoalpha acids followed by incubation. 
     
     
         5 . The process according to  claim 1 , comprising adding the ketoreductase enzyme and NADPH or NADP to a mixture of isoalpha acids in the presence of isopropanol for cofactor recycling, followed by incubation. 
     
     
         6 . The process according to  claim 5 , wherein the concentration of isoalpha acids, i.e. the substrate, is maximized to increase the volumetric productivity of the bioconversion. 
     
     
         7 . The process according to  claim 5 , wherein the concentration of the cofactor NADPH or NADP in the mixture is minimized to improve the economics of the bioconversion. 
     
     
         8 . The process according to  claim 1 , comprising adding the ketoreductase enzyme and NADPH or NADP to a mixture of isoalpha acids in the presence of another enzyme for cofactor recycling, followed by incubation. 
     
     
         9 . The process according to  claim 1 , comprising adding a whole cell biocatalyst, wherein the whole cell biocatalyst is an immobilized microorganism expressing the gene which encodes a ketoreductase, to a mixture of isoalpha acids followed by incubation. 
     
     
         10 . The process according to  claim 1 , comprising culturing a microorganism expressing the gene which encodes the ketoreductase and adding isoalpha acids to the culture. 
     
     
         11 . The process according to  claim 1 , comprising adding the ketoreductase enzyme, wherein the ketoreductase is thermostable, to an extract of isoalpha acids wherein heat is applied, and the mixture is incubated. 
     
     
         12 . The process according to  claim 1 , wherein the ketoreductase specifically reduces cis-isohumulone, cis-isocohumulone, and cis-isoadhumulone. 
     
     
         13 . The process according to  claim 1 , wherein the ketoreductase specifically reduces trans-isohumulone, trans-isocohumulone, and trans-isoadhumulone. 
     
     
         14 . The process according to  claim 1 , comprising adding a mixture of 2 or more ketoreductase enzymes in an amount effective to reduce a mixture of cis- and trans-isoalpha acids, to their respective dihydroisoalpha acids. 
     
     
         15 . The process according to  claim 14 , wherein the mixture of 2 or more ketoreductase enzymes produces a unique mixture of dihydroisoalpha acids that is distinct from that produced by chemical reducing agents, such as sodium borohydride. 
     
     
         16 . (canceled) 
     
     
         17 . (canceled) 
     
     
         18 . (canceled) 
     
     
         19 . A ketoreductase enzyme which comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:16 SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:22. 
     
     
         20 . The ketoreductase enzyme according to  claim 19 , wherein the reductase enzyme or microorganism expressing a gene which encodes the reductase can optionally have one or more differences at amino acid residues as compared to the ketoreductase of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO;8, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:22, 
     
     
         21 . The ketoreductase enzyme according to  claim 20 , wherein the ketoreductase is 99, 95, 90, 85, 80, 75 or 70 percent homologous to the ketoreductase of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:22. 
     
     
         22 . The process according to  claim 1 , wherein the ketoreductase enzyme comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:22. 
     
     
         23 . The process according to  claim 22 , wherein the ketoreductase enzyme or microorganism expressing a gene which encodes the ketoreductase can optionally have one or more differences at amino acid residues as compared to the ketoreductase of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:22. 
     
     
         24 . The process according to  claim 23 , wherein the ketoreductase is 99, 95, 90, 85, 80, 75 or 70 percent homologous to the ketoreductase of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:22.