Cloning and Overexpression of Thermostable D-Hydantoinase from Thermophile in E. coli and its Application to the Synthesis of Optically Active D-Amino Acids
Dong-Cheol Lee, Seung-Goo Lee, Seung-Pyo Hong, Moon-Hee Sung, and Hak-Sung Kim
In the 1980s, a simple chemoenzymatic method for the production of various D-amino acids including D-p-hydroxyphenylglycine (D-HPG) was developed by employing asymmetric biotransformation. In this process, a chemically synthesized racemic hydation derivative is asymmetrically hydrolyzed to the N-carbamoyl-D-amino acid by D-stereospecific hydantoinase. The resulting N-carbamoyl-D-amino acid is further converted to the corresponding D-amino acid by nitrous acid treatment or carbamoylse. The residual L-form substrate is spontaneously racemized to the D-form under alkaline conditions, leading to 100% theoretical yield.From a practical standpoint, several problems have been found in the production process of D-HPG from 5-(4-hydroxyphenyl)hydantoin(HPH). On e of them is that the substrate is slightly soluble in water at room terperature, which might be a barrier to a high production rate. Thus, the enzyme reaction at high temperature is more advantageous because the enzyme activity and substrate solubility increase, given that the enzyme is stable at elevated temperature. In this context, the use of a thermostable enzyme is highly required.Recently, we isolated Bacillus stearothermophilus SD-1 from soil that produces a thermostable D-hydantoinase. The enzyme was purified to homogeneity and its biochemical properties were investigated. Here, we report the cloning and overexpression of the thermostable D-hydantoinase in E. coli. Culture conditions of recombinant E. coli for the production fo the enzyme were optimized, and the systhesis of optically active D-HPG using free or immobilized enzyme was conducted.