A novel process using immobilized engineered strain HC01 cells with co-expressed D-hydantoinase and D-N-carbamoylase as biocatalyst for the production of D-Valine.

The demand for D-Valine increases because of its wide range of use. A whole-cell biocatalyst for the production of D-Valine from 5'-isopropyl hydantoin by co-expression of the D-hydantoinase (hyd) gene from Pseudomonas putida YZ-26 and D-N-carbamoylase (cab) gene from Sinorhizobium sp. SS-ori in Escherichia coli BL 21 (DE3) was developed. The expression condition of the engineered strain HC01 co-expressing D-hydantoinase (HYD) and D-N-carbamoylase (CAB) was optimized. HYD and CAB reached the highest activities (4.65 and 0.75 U/ml-broth) after inducing for 8-12 h. Subsequently, the cells of HC01 were immobilized in the form of Ca2+-alginate beads, and the optimal conditions for immobilizing were obtained as 2.5% gel concentration and 0.029 g/mL cell concentration in the presence of 3% CaCl2. The thermostability of immobilized cells was 5 ℃ higher than that of free cells in the same condition. And the divalent metal ions such as Mn2+, Mg2+, Cu2+, Co2+, and Ni2+ did not significantly affect the enzymatic activity of HYD and CAB in immobilized cells. Bioconversion rate reached to 91% after a 42-h reaction when the substrate concentration was 50 mmol/L with the initial pH of 9.0 under the nitrogen protection. This method provides D-Valine with optical purity of 97% and an overall yield of 72%. Furthermore, the immobilized cells can be reused for more than 7 cycles and maintain their capacity of over 70%. Hence the immobilized cells of engineered strain HC01 could potentially be used to prepare D-Valine.