The Research On Biotransformation Of Cis-4-L-hydroxyproline

cis-4-Hydroxy proline(cis-4-Hyp) produced by hydroxylation of the amino acid proline does not belong to the 20 common amino acids. cis-4-Hyp was clinically evaluated as an anticancer drug in a regio- and stereoslective manner. So the production and application are paid attention by the medicine field. At present, microbe transformation method is the most effective method for the cis-4-Hyp,which use the resting cells as enzyme source and L-proline as substrate and the cis-4-Hyp was produced in the medium of the Fe2+ and α-ketoglutarate.In this study, the DNA sequence encoding cis-4-proline hydroxylase(cis-P4H)was adjusted based on the codon bias of E. coli and m RNA secondary structure.The gene linked to p ET-M-3C plasmid was transformed to E. coli BL21-Codon Plus and the engineered strain expressing the optimized cis-P4 H gene was constructed.The cis-P4 H was purified by Ni-NTA column and its activity and stability were analysed. Whole-cell catalysis was used for the production of cis-4-Hyp, then the related transformation condition was optimized by single factor experiment and orthogonal experiment. We optimized the fermentation conditions of recombinant strain BL21(p ET-cis-P4H) producing cis-P4 H. Single factor experiment and orthogonal experiment were used to determined the optimal medium and the main fermentation conditons(such as the concentration of inducer, the inoculum, the initial p H and the tempretation of fermentation).Finally we used a 5 L fermentor to culture the recombinant strain.The main findings are as follows:1、The DNA sequence encoding cis-4-proline hydroxylase(cis-P4H) from Sinorhizobium meliloti was synthesized after optimization. The gene was linked to p ET-M-3C plasmid and transformed to E. coli BL21-Codon Plus, then the ngineered strain expressing the optimized cis-P4 H gene was constructed.2、The cis-P4 H was induced by IPTG and purified by Ni-NTA column, then itsactivity and stability were analysed. The specific activity of cis-P4 H was2.65 U/mg and the half-life period of cis-P4 H was 2.32 h. 3、Whole-cell catalysis was used for the production of cis-4-Hyp, when theoptical density(OD600) of the cells reached up to 0.9, IPTG was added for nducing protein expression, the cells were induced 5 h at 28°C. then the cells ere used for transformation system. At the condition(p H 6.5, 31°C, 60 h),the transformation rate from L-proline to cis-4-Hyp was more than 83.33%.4、The optimal medium compositions were composed of glucose 20 g/L, yeast xtract powder 5 g/L,(NH4)2SO4 3 g/L, KH2PO4 1 g/L, Na Cl 1.5 g/L, Mg SO4 .1 g/L, Fe SO4 0.1 g/L. The optimal fermentation codition was adding lactose g/L, initial p H 7.0,inoculum amount was 10%, fermentation tempreture was 0°C,fermention time was 28 h. The specific activity was 4.89 U/m L and the ield of cis-4-Hyp was 6.23 g/L.