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A novel process using immobilized recombinant Escherichia coli cells with D-hydantoinase and D-N-carbamoylase activities as biocatalyst for the production of D-Valine
  • Lixi Niu,
  • Lulu Xie
Lixi Niu
Shanxi University

Corresponding Author:[email protected]

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Lulu Xie
Shanxi University
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Abstract

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 putia YZ-26 and D-N-carbamoylase (cab) gene from Sinorhizobium sp. SS-ori in Escherichia coli BL 21 (DE3) was developed. The expression conditions of D-hydantoinase (HYD) and D-N-carbmoylase (CAB) for recombinant cells were optimized. The cells showed the highest HYD and CAB activities (4.650 and 0.75 U/ml-borth, respectively) after 20-h fermentation. The cells of engineered strain HC01 were immobilized in the form of Ca2+-alginate beads, and the conditions for immobilization were investigated. The optimal gel concentration and cell concentration were found to be 2.5% and 0.029 g/mL in the presence of 3% CaCl2. The thermo-stability of immobilized cells was 5 ℃ higher than that of free cells in the same condition. Divalent metal ions, such as Mn2+, Mg2+, Cu2+, Co2+ and Ni2+ did not affect significantly the enzymatic activity of HYD and CAB in immobilized cells. Conversion of about 91.4% was reached after 42-h reaction when the substrate concentration was 50 mmol/L with the initial pH of 9.0 under the protection of nitrogen. With the optimized amino acid purification process, the overall yield of D-Valine was 72.2% with the optical purity of 96.7%.