Nonnatural D-amino Acid Bioconversion By Engineered Strains

Hydantoinase (EC 3.5.2) hydrolyzes its substrate 5'-monosubstantial hydantoin to enantiomerical N-carbamyl-amino acids and in turn, the resulting product can be chemically or enzymatically converted into the corresponding optically active amino acids. A number of bacterial D-hydantoinases with the different stereoselectivity and substrate specificity have been used in industrial bioconversion of optically active D-amino acids. D-Amino acids as unnatural chiral products are important intermediates in the synthesis of products, such as β-lactam semisynthetic antibiotics, antiviral agents, artificial sweeteners, pesticide, peptide hormones, and pyrethroids, etc. Though bioconversion of D-amino acids using microbial cells has been realized in industrialization for decades, some limited factors hinder the development of D-amino acid production, and ultimately result in shortage of the raw material in the related industrial fields. By the technology of gene cloning, bioconversion of D-amino acids with engineered cells containing D-hydantoinase and D-carbamoylase would be expected to overcome the drawbacks presented by using the original strains described above.According to the reported amino acid sequence of D-hydantoinases, two primers were designed and synthesized. Two DNA fragments encoding D-hydantoinase gene were amplified by PCR from chromosome DNA of Pseudomonas putida YZ-26 and Sinorhizobium morelense SS-ori, respectively, and confirmed by DNA sequence analysis. D-Hydantoinase from strain YZ-26 contains an open reading frame of 1440 bp corresponding 479 amino acids, which is a new gene as accepted by GenBank (AY387829). Whilst D-hydantoinase from strain SS-ori contains an open reading frame of 1374 bp encoding 457 amino acids.D-Hydantoinase gene from strain YZ-26 was cloned to pET-3a and pGEX-4T-1 vectors, respectively. The engineered strain EHD-YZ26/E.coli BL21(DE3) was incubated at 30 ℃ for 10 hrs without adding any inducers. The results show that the target product is soluble and active, which account for about 20 % compared with the total proteins in cells, and the enzymatic activity reaches 13 U/mL that is about 13 folds compared with the activity of strain YZ-26. The D-hydantoinase was purified by three steps, ammonium sulfate fractionation, Phenyl Sepharose hydrophobic chromatography and Sephacryl S-200 gel-filtration. With the procedures, the overall recovery of enzymatic activity reached 55 % and the specific activity for substrate hydantoin was about 16 U/mg protein. The purification factor was 3.4 folds with the purity about more than 95 % as estimated by SDS-PAGE analysis. The enzyme is homologous dimmer with molecular mass 102600 as determined by gel filtration on HPLC and subunit mass 52042 as determined by MALDI-TOF mass spectrometry. The optimal pH of D-hydantoinase is at near 9.5 and the optimal temperature is at 45 ℃. Based on the effect of divalent metal ions and chelator EDTA on the activity, the enzyme is metal-dependent only. The engineered strain GHD-YZ26/E.coli BL21(DE3) was incubated and induced at 30 ℃ for 8 hrs. The results show that the target fusion protein is soluble and active, which is about 15 % compared with the total proteins in cells, and the enzymatic activity reaches 5 U/mL that is about 8 folds compared with the activity of strain YZ-26. Fusion protein GST-HDT was purified by GSTrap affinity chromatography. With the procedures, the overall recovery of enzymatic activity reached 20 % and the specific activity for substrate hydantoin was about 4 U/mg protein. The purification factor was about 4.7 folds with the purity about more than 95 % as estimated by SDS-PAGE analysis.D-Hydantoinase gene from strain SS-ori was cloned to five different vectors to be five recombinant plasmids and in turn to transfer into five different E.coli strains, respectively. Then the total 25 engineered strains were cultured and induced in the respective conditions. The results show that the target products expressed in 3 strains of them are partly soluble and active, whils...