Studies On Bioconversion Of D-Amino Acids

D-amino acids as the synthetic intermediates are widely used in the pharmaceutical fields, with applications such as antimicrobial and Antifical sweeteners, peptide hormone, pesticides and pyrethroids. For an example of these intermediates, D-hydroxyphenylglycine, is an important precursor used for the synthesis of semi-synthetic antibiotics. The production of D-amino acids is usually achieved by chemo-enzymatic or enzymatic Routes. In the enzymatic route, 5' -monosubstituted hydantoin is firstly transferred into intermediate, N-carbamy-D-amino acid by D-hydantoinase, and the resulting N-carbamyl-D-amino acid is further converted to the corresponding D-amino acid by N-carbamyolase. Therefore, D-hydantoinase is one of the crux enzymes in bioconversion of D-amino acids and becomes a hot field in the industrial production of chiral amino acids.l.The culture condition of producing D-hydantoinase from Pseudomonas putida YZ-II6Pseudomonas putida YZ-II6 with the high D-hydantoinase activity was stored in this Laboratry. The optimal culture condition of the strain producing D-hydantoinase is in YCG medium by the induction of 0. 1% hydantoin at 26 C for 20 h. D-hydantoinase activity can reach 1. 3U/mL at 10 OD600 of cells under the condition described above.2. The culture condition and stability of the engineered stainpET-hdase/E. coli BL21The open reading frame encoding D-hydantoinase from strain YZ-II6 was achieved by PCR. Various factors including inducer, temperature, culture time and stability of pET-hdase/E. coli BL21 were studied.. It was shown that the D-hydantoinase activity was 10 folds as high as that of the wild strain YZ-II6 at 30 C for 12-14h in LB medium without adding any inducers.3. The immobilization of pET-hdase/E. coli BL21Artificial cells were immobilized by mixing with calcium alginate.The specific activity of immobilized cells was decreased with increasing the amount of cells. The optimal reaction conditions of immobilized cells were at 50 C in 0.05mol/L Tris-HCl buffer, pH9.0 and D-hydantoinase activity for substrate hydantoin reached 40U/(g)beads. If D-p-HPH as the substrate, the activity of immobilized cells was 6. 8U/(g)beads, which was two folds as high as the same free cells from the engineered strain. The activity of immobilized cells was not any changeable and the beads were not any disrupted after they were repeatedly used 5 times. The immobilized cells were stored in water supplemented with 100uL/mL Amp can keep the activity for two weeks more.4. The bioconversion of the intermediate CpHPGD-p-hydroxyphenylhydantoin was chemically synthesized from glyoxylic as described in reference. Afterwards, D-p-hydroxyphenyl -hydantoin was converted to CpHPG by immobilized cells. It was indicated from the result of HPLC analysis that the product, CpHPG mainly exists in the supernatant. With increasing the Tris-base concentration by reduced pressure evaporation, the extract of CpHPG becomed more and more difficult. Therefore, the further work is in progress.