Application of a thermostable glutamate racemase from Bacillus sp. SK-1 for the production of D-phenylalanine in a multi-enzyme system

Abstract

A gene encoding glutamate racemase (GluRA) was found in a thermophilic Bacillus strain named SK-1. The gene was cloned and expressed in Escherichia coli WM335, a D-glutamate auxotroph. It consists of 792bp with a start codon, TTG. The amino acid sequence deduced from the gene indicates that the GluRA has two cysteines and their surrounding regions are well conserved. The GluRA produced in the recombinant E. coli was purified to homogeneity by heat-treatment and Resource Q and Phenyl sepharose column chromatographies. The enzyme, which was determined to be a monomeric protein with a molecular weight of 29,000, did not require a cofactor such as pyridoxal 5′-phosphate, nicotinamide, or flavin for its activity. The enzyme was stable after incubation at 55°C and retained 60% of its original activity after incubation at 60°C. It was found to be stable in the region of pH 6.0-11.5. The thermostable GluRA was used as a catalyst in a multi-enzyme system composed of four enzyme reactions for the production of D-phenylalanine. By running the multi-enzyme system for 35h, 58gl-1 of D-phenylalanine was produced with 100% of optical purity from equimolar amount of phenylpyruvate.