Nonnatural D-amino Acid Bioconversion By Engineered Strains

Optically active D-amino acids are widely used as intermediates for synthesis of semisynthetic antibiotics, peptide hormones, pyrethroids and pesticides etc. Non-natural D-amino acids are usually produced by enzymatic or chemo-enzymatic method. In the reaction process, the substrate DL-5'- monosubstitued hydantoin is enantioselectively hydrolyzed to the corresponding N-carbamoyl- D-amino acid by D-hydantoinase, and in turn, the intermediate is further converted to D-amino acid by D-Carbamoylase (DCase) or chemical decarbamoylation. Nowadays, D-amino acid production bio-conversed by enzymes has been performed in the industrial scale.DCase gene was amplified by PCR with genomic DNA from the original strain SS-ori as the template, using the forward primer deduced by N-terminal amino acid sequence of DCase purified by this Lab. and the reverse primer designed by C-terminal amino acid sequence of DCase according to the amino acid sequence homology of some strains reported. It was shown from DNA sequence analysis that the open reading frame of DCase is 912bp.DCase gene described above was inserted into vectors e.g. pUC18, pET3a, pET32M, pRSET, pGEX4T-2, pExSecI and pMAL-c2X to be different constructs. After transferred into host strains, for instance, E.coli DH5 a , E.coli 2426 and E.coli BL21, the target product expressed from most engineered strains was in the form of inclusion bodies. Finally, we found that the engineered strain, E.coli BL21/pMD, could produce an active DCase in the soluble form, which reached 20% of total soluble protein in cells by GDS analysis. The cells from 1L culture at the concentration of 10OD600nm could converse N-carbamoyl-DL -valine to about 1.0 g D-valine per an hour in this experimental condition. In addition, the fusion protein MBP-DCase was also purified by amylose beads affinity chromatography and Superose 12 gel filtration. A 77kD band was occurred on SDS-PAGE, which corresponded the sum of molecular weight for MBP and Dcase. Meanwhile, the pure Dcase (35kD) removed from fusion product could be obtained by Factor Xa cleavage.A quantitative analysis of substrate DL-hydroxyphenyl hydantoin (DL-HPH) and its product N-carbamyl-D-p-hydroxyphenyl-glycine (CpHPG) or D-p-hydroxyphenyl-glycine ( D-HPG) bioconversed by an engineered strain E.coli EHD-YZII 6 or native strain SS-ori was conducted by the reverse-phase HPLC. It is more efficient to separate the mixture of DL-HPH, Cp-HPG and D-HPG onBONDAPAK C18 column with the optimal mobile-phase of 50mmol/L acetate buffer(pH 4.2) and methanol at the ratio of 90:10(V/V) as detected at 254nm. It is also shown from the linear relation of the peak area for the above three samples that the relative coefficient reaches over 0.99. By HPLC analysis reported here, not only the activity of D-Hydantoinase or D-Carbamoylase can be determined, but also their bioconversion products can quantitatively assayed.To improve the heat stability and anti-oxidation of DCase, molecule-directed evolution in vitro was undertaken by error prone PCR. The preliminary results show that it is possible to achieve the goal we expected. In order to engage the high throughput screening, we have constructed a modification plasmid, pET32M-CAT, by which soluble mutant of DCase can be easily distinguished in vivo in E. coli. This method is based on that the screen for the mutant pool of Dcase can be underwent by the high solubility of chloramphenicol acetyltransferase(CAT). The further work is in progress.