Entrapment of a Trigonopsis variabilis D-amino acid oxidase variant F54Y for oxidative deamination of cephalosporin C

Abstract

Trigonopsis variabilis D-amino acid oxidase (TvDAAO) is an enzyme used in the industrial bioconversion of cephalosporin C (CPC) into 7-aminocephalosporanic acid, a crucial biosynthetic nucleus for a wide spectrum of semi-synthetic cephem antibiotics. Using homology modeling and site-directed mutagenesis, we have previously shown that the TvDAAO variant F54Y possesses improved catalytic activity and thermostability. To further explore its industrial application, the conditions for immobilization of the enzyme were examined in the present investigation. The results showed that entrapment in a calcium alginate (Ca-alginate) matrix using 2% alginate, 500mM CaCl 2, and 15min stabilization appeared to be optimal for the immobilization of F54Y. The entrapped enzyme allowed complete CPC conversion. The entrapped enzyme also showed good operational stability and retained at least 90% of its original activity after 20 reaction cycles. To conclude, the entrapment of F54Y in Ca-alginate appeared to be a simple and efficient biocatalysis system with potential application in the antibiotics industry. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.