Development of enzymatic process for the production of D-amino acids from hydantoin derivatives

Abstract

KBEL101 was isolated from soil and used to obtain the whole cell and crude dihydropyrimidinase (E.C.3.5.2.2., hydantoinase). Hydantoinase activity of screened KBEL101 were exhibited about 3 times higher hydantoinase acivity than pseudomonas striata IFO 12996. Cultivation conditions of KBEL 101 are initial pH 5.5, temperature 30$^\circ$C, and culture time 18 hr. In the growth conditions for production of hydantoinase activity, yeaxt extract is best nitrogen source and the formation of enzyme increased with increasing concentrations up to 1\%. Glucose as carbon source increased the total activity, while the specific activity was not increased. Hydantoinase activity was observed regardless of the presence of a hydantion dervative during cultivation. Hydantoinase was severely inhibited by products, N-carbamoylhydroxyphenlyglycine and N-carbamoyl-glycine, etc.. The optimal conditions of KBEL101 for whole cell hydantoinase are pH 9.0 and 40$^\circ$C. However, when considering the stabilities of whole cell hydantoinase and substrate, pH 8.0 and 30$^\circ$C are the best actual reaction condition. Various water-miscible organic solvents were tested with respect to both the activity and the stability of whole cell enzyme. Among the vorious organic solvents, 5\% dimethylfomamide showed increase about 2.7 times than control and 5\% dimethylsulfoxide was chosen to be a proper solvent : hydantoinase acitivity increased two fold in the presence of 5\% dimethylsulfoxide and stability in the presence of 5\% dimethylsulfoxide was like that of control. From the practical stand point, enhancement of substrate solubility is advantageous to obtain highest conversion yield by increase of reaction rate. Enzymatic synthesis fo N-carbamoyl-D-p-hydroxyphenylglycine was carried out in repeated batch operation in the presence of 5\% dimethylsulfoxide. The amount of entrapted whole cell linearly increased with increasing loading of whole cell hydantoinase up to 50 mg dry cell per mL acrylam...