关键词: D-海因酶, N-氨甲酰基-D-氨基酸酰胺水解酶, 多顺反子, 大肠杆菌, 共表达

Abstract: Fully enzymatic synthesis process for the production of D-amino acids involves two enzymes, D-hydantoinase and N-carbamoylase. In an attempt to select more efficient biocatalysts, the hydantoinase and carbamoylase genes from A. radiobacter TH572 were cloned in Escherichia coli. The genes were assembled to give polycistronic structures, and under the control of a strong araBAD promoter, the recombinant strain TaraCRH was obtained. Expression of the two enzymes was tightly controlled by araBAD promoter, high level expression of both hydantoinase and carbamoylase was achieved with the induction of arabinose, but no activity could be detected in the absence of arabinose. The expression conditions were then optimized. The highest level of soluble expression was obtained at an induction temperature of 29℃ and arabinose concentration of 0.05 g/L. Furthermore, we tried to use corn steep liquor medium instead of LB medium for the culture of E. coli TaraCRH, the optimized corn steep liquor medium consisting of corn steep liquor (45 g/L), glycerol (3 g/L), NaCl (3 g/L), KH2PO4 (0.5 g/L), CoCl2 (0.2 g/L) was developed by using orthogonal experiments. After culturing and inducing E. coli TaraCRH in this optimized medium, the activities of hydantoinase and carbamoylase achieved 3.52 and 4.21 U/mL, respectively, which enabled efficient production of D-HPG from DL-HPH.

Key words: D-hydantoinase, N-carbamoylase, polycistron, Escherichia coli, co-expression