Production Of D-HPG By Using Immobilized Cells Of Engineered Strain HC01

Optically pure D-amino acids which have advantages that natural amino acid without are widely used in the production of pharmaceutical chemicals, feed, food and so on. The optical active D-amino acids can be produced in a hydantoin-transforming reaction by starting from DL-5'-monosubstituted hydantoin. In this two-step reaction, D-hydantoinase (HYD) and D-carbamoylase (CAB) catalyze substrates to relative D-amino acids one after another. This method has been generally accepted owing to its gentle reaction condition, high yield, high reaction rate, and pollution-free technology for commercial production of D-amino acids. People cloned and co-expressed of D-hydantoinase gene (hyd) and D-carbamoylase gene (cab) from wild strains in E. coli to gain a suitable engineered strain for commercial use. We have constructed an engineered strain HC01 that coexpressed HYD and CAB, Preliminary work provides research foundation for further experiment.The cells of engineered strain HC01 were immobilized in the form of Ca2+-alginate beads. 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 N-carbamoylase CAB in immobilized cells. By contrast, Mn2+ and Ni2+ could independently enhance the activity of CAB up to 210% and 270% in free cells. The present data showed that the optimal reaction condition of immobilized cells was at pH 7.5 and 40℃as same as that of free cells. The immobilized cells remained 80% activity of original when they were repeatedly used for six times. The immobilized cells can keep the activity for 12 days at 4℃.The immobilized cells were applied to produce D-p-Hydroxyphenylglycine (D-HPG) directly from the substrate DL-Hydroxyphenyl Hydantoin (DL-HPH) in a batch reactor. Conversion of about 97% was reached after 36-h reaction when the substrate concentration was 30 g/L with the initial pH of 9.0 under the protection of nitrogen gas. The overall yield of D-HPG was 85% with the optical purity of 99.7% after purification. The product was characterized by HPLC, IR and 1HNMR.Overproduction of heterologous proteins in Escherichia coli commonly results in the formation of insoluble aggregates. Prior to the purification and immobilization of insoluble aggregates, an indispensable need calls for their renaturation. To complete protein refolding and immobilization in one step, the oleosin gene was fused to the C terminus of HYD and the fusion gene was inserted into vectors e.g. pQE-30,pET-3a and pET-28a to be different constructs. The recombinants were transferred into host strains and the engineering strains were cultured in LB medium by the induction of IPTG. SDS-PAGE analysis indicated the hybrid protein was largely produced in the form of inclusion bodies in pET28a-hydole/BL21, and the protein was harvested by forming AOBs, but the D-hydantoinase activity could not be detected. It may be the condition is not suitable for renaturation of D-hydantoinase. The further research is necessary to explore the conditions of production of AOBs.