New non-naturally occurring variant of glutaryl amidase comprising histidine or glutamate in its substrate binding pocket, binds cephalosporin C (CPC) as substrate and catalyzes conversion of CPC to 7-aminocephalosporanic acid

摘要

Non-naturally occurring variant of a glutaryl amidase (GA) (I) which binds cephalosporin C (CPC) as a substrate and catalyzes conversion of the CPC to 7-aminocephalosporanic acid (7-ACA), where the variant comprises a histidine or glutamate in the substrate binding pocket that binds the alpha -amino adipyl moiety of the CPC, is new. A non-naturally occurring variant of a glutaryl amidase (GA) (I) which binds cephalosporin C (CPC) as a substrate and catalyzes conversion of the CPC to 7-aminocephalosporanic acid (7-ACA), where the variant comprises a histidine or glutamate in the substrate binding pocket that binds the alpha -amino adipyl moiety of the CPC. (I) comprises a combination of mutations chosen from PheB177His, LeuB24Arg, ArgB57His; PheB177His, LeuB24Arg, ArgB57His, GlnB50Val; PheB177His, LeuB24Arg, ArgB57His, GlnB50Ala; PheB177His, LeuB24Arg, ArgB57His, GlnB50Val, ValB70His; PheB177His, LeuB24Arg, ArgB57His, GlnB50Ala, ValB70His; PheB177His, LeuB24Arg, ArgB57His, GlnB50Val, ValB70His, TyrB153Leu; PheB177His, LeuB24Arg, ArgB57His, GlnB50Ala, ValB70His, TyrB153Leu; PheB177His, ThrB176Asp; PheB177His, ThrB176Asp, TyrA150Ala; PheB177His, ThrB176Asp, LeuB24Arg, ArgB57His; PheB177His, ThrB176Asp, LeuB24Arg, ArgB57His, TyrA150Ala; PheB177His, ThrB176Asp, GlnB50Arg, ArgB57His; PheB177His, ThrB176Asp, GlnB50Arg, ArgB57His, TyrA150Ala; TyrA150Glu; TyrA150Glu, PheB177His; TyrA150Glu, LeuB24Arg, ArgB57His; TyrA150Glu, LeuB24Arg, ArgB57His, PheB177His; TyrA150Glu, GlnB50Arg, ArgB57His; TyrA150Glu, GlnB50Arg, ArgB57His, PheB177His; PheB177His, ThrB176Asp, TyrA150Ala, TyrB33Ser; PheB177His, ThrB176Asp, LeuB24Arg, ArgB57His, TyrA150Ala, TyrB33Ser; PheB177His, ThrB176Asp, GlnB50Arg, ArgB57His, TyrA150Ala, TyrB33Ser; TyrA150Glu, PheB177His, TyrB33Ser; TyrA150Glu, LeuB24Arg, ArgB57His, TyrB33Ser; PheB177His, LeuB24Arg, ArgB57His, TyrB33Ser; and PheB177His, LeuB24Arg, ArgB57His, TyrB33Ser, ValB70His. Independent claims are also included for: (1) a polynucleotide (II) encoding (I); (2) a nucleic acid vector (III) comprising (II); (3) a host cell (IV) comprising (II) operably linked to regulatory control sequence which direct expression of the variant GA in the host cell; (4) production of (I); (5) production of 7-aminocephalosporanic acid; and (6) modifying (M1) a GA enzyme to enhance the CPC acylase activity of the enzyme, which involves by introducing a non-naturally occurring histidine or glutamate amino acid residue in the substrate binding pocket of the enzyme which histidine or glutamate binds the alpha -amino adipyl moiety of a CPC substrate molecule.