Thermostable Glutamate Racemase from a Thermophilic Bacillus sp. SK-1, Symbiont of thermophilic Symbiobacterium sp. SC-1' Gene Cloning, Overproduction, and Characterization
Hee-Sung BAE*, Jin-Seo PARK, Seung-Pyo HONG, Seung-Goo LEE, Dae-Heoun BAEK, and Moon-Hee SUNG
Microbial Conversion Research Unit, Korea Research Institute of Bioscience and Biotechnology (KRIBB), P.O.BOX 115, Yusong, Taejon 305-600, Korea
A thermostable glutamate racemase was found in a thermophilic Bacillus sp. SK- 1, the symbiont of a thermophilic Symbiobacterium sp. SC-1, and cloned into Escherichia coli WM335, a D-glutamate auxotroph, by the genetic complement method. The thermostable glutamate racemase was expressed efficiently in Escherichia coli, the production level being about 10% of the total soluble proteins of the recombinant Escherichia coli extract. The recombinant glutamate racemase was purified to homogeneity by heat treatment and two column chromatography steps (Resource Q and Phenyl sepharose column chromatography) with a final yield of 40%. The molecular weight of the enzyme was determined to be 29.9 kDa on gel filteration column chromatography and 29 kDa on SDS-PAGE, indicating the enzyme is a monomer protein. The enzyme was most active at 70℃, and had higher thermostability than the enzyme from Escherichia coli.
