A novel, enantioselective, thermostable recombinant hydantoinase to aid the synthesis of industrially valuable non-proteinogenic amino acids

Enzyme and Microbial Technology
Anupama S EngineerPrashant K Dhakephalkar

Abstract

Overexpression of a novel hydantoinase (hyuH) from P. aeruginosa (MCM B-887) in E. coli yielded optically pure carbamoyl amino acids. The use of optically pure carbamoyl amino acids as substrates facilitates the synthesis of non-proteinogenic amino acids. The enzyme hyuH shared a maximum of 92 % homology with proven hydantoinase protein sequences from the GenBank database, highlighting its novelty. Expression of hydantoinase gene was improved by >150 % by overexpressing it as a fusion protein in specialized E. coli CODON + host cells, providing adequate machinery for effective translation of the GC-rich gene. The presence of distinct residues in the substrate binding and active site of MCM B-887 hydantoinase enzyme explained its unique and broad substrate profile desirable for industrial applications. The purified enzyme, with a specific activity of 53U/mg of protein, was optimally active at 42 °C and pH 9.0 with a requirement of 2 mM Mn2+ ions. Supplementation of 500 mM of Na-glutamate enhanced the thermostability of the enzyme by more than 200 %.

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