Enzymatic production of glucaric acid from glucuronic acid
View Patent ↗Described herein are processes and enzymes for the enzymatic production of glucaric acid from glucuronic acid, and more specifically the enzymatic production of substituted glucaric acid from substituted glucuronic acid. Advantageously, the process and enzymes described herein make preferential use substituted glucuronic acid obtained from natural sources, such as underutilized hemicellulose stream from wood and agricultural biorefineries, to produce substantially enantiomerically pure substituted D-glucaric acid.
1 . A process for producing 4-O-methyl glucaric acid, comprising:
providing a solution comprising dissolved 4-O-methyl glucuronic acid;
providing a recombinant gluco-oligosaccharide oxidase (GOOX) having at least 99% sequence identity to SEQ ID NO: 1; and
contacting the dissolved 4-O-methyl glucuronic acid with said recombinant GOOX under conditions that catalyze enzymatic conversion of the 4-O-methyl glucuronic acid to 4-O-methyl glucaric acid.
2 . The process of claim 1 , wherein the recombinant GOOX comprises 300A, 314A, or 388S, relative to the amino acid positioning of SEQ ID NO: 1.
3 . The process of claim 1 , wherein the recombinant GOOX is immobilized to a solid support, particle, or matrix.
4 . The process of claim 1 , wherein the enzymatic conversion of the 4-O-methyl glucuronic acid to the 4-O-methyl glucaric acid by the recombinant GOOX is performed at a pH of 7.5 to 11.
5 . The process of claim 1 , wherein the enzymatic conversion of the 4-O-methyl glucuronic acid to the 4-O-methyl glucaric acid by the recombinant GOOX is performed in a buffer having an ionic strength of at least 100 mM.
6 . The process of claim 1 , wherein the enzymatic conversion of the 4-O-methyl glucuronic acid to the 4-O-methyl glucaric acid by the recombinant GOOX is performed in the absence of exogenous cofactor supplementation.
7 . The process of claim 1 , wherein the enzymatic conversion of the 4-O-methyl glucuronic acid to the 4-O-methyl glucaric acid by the recombinant GOOX is performed at a temperature between 38° C. and 42° C.
8 . The process of claim 1 , further comprising enzymatic treatment of a polysaccharide comprising 4-O-methyl glucuronic acid moieties with a glycoside hydrolase to release the 4-O-methyl glucuronic acid prior to enzymatic conversion of the 4-O-methyl glucuronic acid to the 4-O-methyl glucaric acid by the recombinant GOOX.
9 . The process of claim 8 , wherein the enzymatic treatment of the polysaccharide to release the 4-O-methyl glucuronic acid and the enzymatic conversion of the 4-O-methyl glucuronic acid to the 4-O-methyl glucaric acid by the recombinant GOOX are performed in the same reaction vessel.
10 . The process of claim 8 , further comprising the use of a catalase to catalyze the breakdown of hydrogen peroxide generated by the recombinant GOOX.
11 . A composition comprising a recombinant gluco-oligosaccharide oxidase (GOOX) having at least 99% sequence identity to SEQ ID NO: 1, O-methyl glucuronic acid as a GOOX substrate, and an alkaline reaction buffer enabling enzymatic conversion of the 4-O-methyl glucuronic acid to 4-O-methyl glucaric acid.
12 . The composition of claim 11 , wherein the recombinant GOOX is immobilized to a solid support, particle, or matrix.
13 . The composition of claim 11 , wherein the alkaline reaction buffer has a pH of 7.5 to 11.
14 . The composition of claim 11 , wherein the alkaline reaction buffer has an ionic strength of at least 100 mM.
15 . The composition of claim 11 , wherein the composition is free of exogenous cofactors.
16 . The composition of claim 11 , further comprising a catalase to catalyze the breakdown of hydrogen peroxide generated by the recombinant GOOX.
17 . The composition of claim 11 , wherein the recombinant GOOX comprises 300A, 314A, or 388S, relative to the amino acid positioning of SEQ ID NO: 1.