重组大肠杆菌共表达D-海因酶和N-氨甲酰基-D-氨基酸酰胺水解酶

通过PCR分别扩增得到N-carbamoylase基因和上游带有RBS区的D-hydantoinase基因，并将其依次克隆入pBAD/His A质粒中，得到呈多顺反子结构的双酶表达质粒pBAD-CRH，转化E. coli Top10F￠得到重组菌TaraCRH. 实验证明，E. coli TaraCRH对外源蛋白的表达控制严紧，而加入阿拉伯糖诱导后可以成功表达两酶基因. 诱导条件优化结果表明，诱导温度采用29℃，培养基中阿拉伯糖浓度0.05 g/L可达到最高酶活. 进一步尝试采用玉米浆培养基代替LB培养基，在经过优化的玉米浆培养基中培养TaraCRH菌株，经阿拉伯糖诱导，海因酶和水解酶酶活分别达到3.52和4.21 U/mL.

Co-expression of D-Hydantoinase and N-Carbamoylase in Recombinant Escherichia coli

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.