Patent ID: 7932064

Claim:
A method for making (R)-ethyl-4-cyano-3-hydroxybutyrate, comprising (a) providing an epichlorohydrin; (b) providing a polypeptide having an amino acid sequence with at least 80% sequence identity to SEQ ID NO:210 and having nitrilase activity (c) preparing a 3-hydroxyglutaronitrile (HGN) from the epichlorohydrin, followed by the addition of a neutralizing agent, a buffer or a diluent, wherein the epichlorohydrin is converted to 3-hydroxyglutaronitrile (HGN) by cyanide treatment; (d) catalyzing the conversion of the 3-hydroxyglutaronitrile (HGN) to 4-cyano-3-hydroxybutyric acid by contacting the 3-hydroxyglutaronitrile (HGN) with the polypeptide of step (b); (e) isolating the 4-cyano-3-hydroxybutyric acid made in step (d) by crystallization using a protocol comprising: (1) adding concentrated potassium hydroxide or calcium hydroxide to the 4-cyano-3-hydroxybutyric acid, followed by concentration of the solution and crystallization precipitation using a hot alcohol, or (2) converting the 4-cyano-3-hydroxybutyric acid to its potassium salt by reaction with potassium hydroxide in water or an alcohol, followed by crystallization of the 4-cyano-3-hydroxybutyric acid potassium salt; or (3) the protocol of (e)(1) or (e)(2) further comprising a second or additional recrystallization after re-suspension of the crystals formed in (e)(1) or (e)(2); and (f) converting the crystallization precipitate to (R)-Ethyl-4-Cyano-3-Hydroxybutyrate by esterification using an acid catalyst.