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, 500 mM CaCl2, and 15 min 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.

Effects of an extra Trp113Tyr substitution on yeast D-amino acid oxidase variant

“…Purity of the enzyme was analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and its concentration was determined by Bradford assay. The catalytic activity of the purified enzyme was determined by measuring the formation of GL-7-ACA at 22°C with agitation [ 3 ]. One mL of the reaction mixture contained 20 μg TvDAAO F54Y/W113Y variant, 5 mM cephalosporin C, and 50 mM sodium phosphate (pH 7.5).…”

“…A single amino acid substitution at the 54th position of TvDAAO from phenylalanine (F) to tyrosine (Y) resulted in a 6-fold improvement in catalytic activity towards CPC and in thermostability after heat treatment at 55°C [ 2 ]. The potential application of the TvDAAO F54Y variant in the antibiotics industry as a simple and efficient biocatalyst was evaluated [ 3 ]. To further elucidate the structural basis of the catalytic activity and thermostability of TvDAAO F54Y, we investigated the role of the 113th amino acid residue (tryptophan, W113) that locates near the substrate binding cleft.…”

Principles, techniques, and applications of biocatalyst immobilization for industrial application

“…It can be concluded that entrapment in Ca-alginate matrix appeared to be an efficient method for antibiotics industry. Because of the availability of Ca-alginate and simplicity of the immobilization technique, this production can be considered economically viable (Wong et al 2011).…”

Synthesis of peroxidase‐encapsulated sodium cellulose sulphate/poly‐dimethyl‐diallyl‐ammonium chloride biopolymer via polyelectrolyte complexation for enhanced removal of phenol

“…Encapsulation using polyelectrolyte complexation has found wide applications in various industries such as food industry, pharmaceutical, enzyme production, and pulp and paper . Encapsulation via calcium alginate beads has been studied extensively due to simple preparation, inexpensive process, and good biocompatibility.…”