Studies On Relevant Enzymes In The Bioconversion Of D-amino Acid By Using Engineered Strains

D-amino acid, as a kind of chiral compound, has been paid attention to the field ofthe industrial biochemistry on the application of the medicine, agriculture and food etc. Asa non-natural product, it is hard directly to be obtained from proteins or other naturalresources. Although it can be greatly synthesized by chemical methods, the complicatedprocess, high pollution and expensive price hinder its development and application.Therefore, the bioconversion is regarded as the most efficient and safe process, e.g.5'-substituted hydantoin as the substrate to the relevant D-amino acid catalyzed byD-hydantoinase (HYD) and D-carbamoylase (CAB). In recent decades, the bioconversionprocess of D-amino acid has become a hot focus in the industrial biochemistry both indomestic and abroad.In this thesis, the random mutation, site-directed mutagenesis and deletion mutationof the HYD gene from Pseudomonas putia YZ26 were conducted and some properties ofits mutants were studied as well. In addition, a CAB gene from Sinorhizobium sp. SS-oriwas cloned and expressed in E. coli in the soluble form. At the same time, two recombinantplasmids involving HYD gene and CAB gene respectively with the different antibioticresistance were constructed, and then, co-transformed into a host cell, E. coli BL21 (DE3).The engineered strain can simultaneously express HYD and CAB. By using this specialengineered strain, an optical activity D-valine was achieved with 5'-isopropyl hydantoinas the substrate. Meanwhile, some parameters of the product were examined, comparedwith the commercial D-valine.HYD mutation and enzymatic activityTo explore the relationship between the structure and function of HYD and toconstruct D-hydantoinase-producing engineered strain with the high activity, somemutation methods containing random mutation, site-directed mutagenesis and deletionmutation in vitro were undertaken. The random mutation of D-hydantoinase gene wasconducted by Error-prone PCR. Based on conditional tests, three pairs of ions werechosen, i.e. 1.5mmol/L Mn²⁺+3mmol/L Mg²⁺; 0.3mmol/L Mn²⁺+3mmol/L Mg²⁺; 0.5mmol/L Mn²⁺+2.5mmol/L Mg²⁺. Each PCR product was separately cloned into vectorpET-3a and the relevant construct was transformed into E.coli BL21 (DE3). The positivemutant was screened on a 96-well microtiter plate by using enzyme activity. Finally, twomutants with the higher activity, pE-hyd₄₁/E.coli BL21(DE3) (A) and pE-hyd₄₅/E.coliBL21(DE3) (B), were selected in about 1000 colony. The enzymatic activity shows thatthe strain A is 2.7-fold and 20-fold, while the strain (B) is 2.0-fold and 15-fold, comparedwith the starting engineered strain (pE-hyd/E.coli BL21(DE3)) and wild-type strain(Pseudomonas putida YZ-26) with p-hydroxyophenyl hydantoin as the substraterespectively. DNA sequence analysis indicates that the enzyme gene in the strain Abelongs to the internal mutation by changing the Glu codon (GAA→GAG) and the Aspcodon (GAT→GAC), whilst the gene in the strain B produces a residue mutation,Ala449Gly.In addition, the site-directed mutagenesis of the gene was also conducted. Firstly, themutation site was selected at His239 (H239Y, H239L) and Asp316 (D316E).Unfortunately, there was no any positive result for three mutated strains described above.Subsequently, when His409 and His410 of HYD were changed to H409T and H410A, twomutated strains retained about 40% and 1.3% activity respectively, compared with thestarting strain, predicting that the function of two close Histidines is quite different inmaintaining the conformation and activity of the enzyme. It was also shown that thepurified D-hydantoinase from the mutated strain pE-hyd_H409T/E, coli BL21(DE3) was still adimer, as determined by native-PAGE.Two variants of D-hydantoinase (HYD), created by one amino acid deletion of eitherthe N- or C-terminus, were expressed in Escherichia coli and purified by two-stepchromatography. Compared with HYD, HYDc1 with the C-terminal Arg deletion retained43% activity, while HYDn1 with the N-terminal Ser deletion had no any activity usingDL-Hydantoin as substrate. Based on HYD dimer with a molecular weight of 103 kDa,HYDc1 is a monomer of 52 kDa and HYDn1 is a mixture of dimer and monomer asdetermined by SDS-PAGE, Native-PAGE and HPLC respectively. Moreover, besidesHYDc1 displays the higher pH stability, higher anti-SDS ability and lower thermalstability, the secondary and tertiary structures of HYDc1 were not significantly changedand on contrary, the obvious change of HYDn1 conformation is occurred in the comparison of HYD. All data imply that the C-terminal Arg of the HYD is a crucialresidue for homodimeric architecture of the enzyme, but non-essential for catalysis, whilethe N-terminal Ser is required for both conformation and catalysis of the enzyme.Cloning and expression of CAB geneCAB gene was amplified by PCR with genomic DNA from the strain Sinorhizobiumsp. SS-ori as the template, using the forward primer designed by N-terminal amino acidsequence analysis of the purified CAB in this Lab and the reverse primer designed bycomparing C-terminal amino acid sequence of CABs according to the amino acidsequence homology of some strains reported. The PCR product indicates that the openreading frame of CAB gene is 915 bp by DNA sequence analysis.The CAB gene was inserted into various vectors e.g. pUC18,pET3a,pET32M,pRSET,pGEX4T-2,pExSecâ… and pMAL-c2â…©to be different constructs. After beingintroduced into host strains, for instance, E. coli DH5α,E. coli 2426 and E. coli BL21 (DE3),the target product expressed from them was mostly in the form of inclusion bodies exceptpMD/E.coli BL21(DE3) which can produce an active CAB in the soluble form with 20%yield of total soluble proteins in cells by GDS analysis. The activity of cells reaches 0.11U/ml·10OD_600nm with N-carbamoyl-D-valine as the substrate. In addition, the fusionprotein MBP-CAB was also purified by amylose beads affinity and Superose 12 gelfiltration. A 77 kDa band was occurred on SDS-PAGE, with which corresponded the sumof molecular weight for MBP (42 kDa) and CAB (35 kDa). Meanwhile, the pure CABremoved the fusion tag could be obtained by Factor Xa cleavage.Co-expression of HYD and CABConsidering the compatibility and resistance of plasmid, HYD gene was cloned tovector pET-28a with Kan^r and CAB gene to pQE30 with Amp^r. Two recombinant plasmids,pET28a-hyd and pQE30-cab were co-transformed into the host cell E.coli BL21(DE3) togenerate the engineered strain which could simultaneously express HYD and CAB. By theanalysis of Western blot and enzymatic activity, the active HYD and CAB were confirmed. In addition, when employing the strain to convert several 5'-substituted hydatoins, thehydrophobic side chain of substrate is much favorable than the charged side chain.The preparation process of D-Valine on benchThe engineered cells from 1 L culture could convert 91.4% of 5'-isopropyl hydantoin(50 mmol/L, 400 ml) to D-valine at 37℃for 42 h. After several steps, such ascentrifugation, de-coloration, lyophilization and ethanol treatment etc., the product wasroughly purified. The analytic data of the D-valine contain HPLC, IR and ¹H NMR andoptical rotation etc.HPLC analysis of DL-hydroxyphenyl hydantoin and its enzymatic productA quantitative analysis of substrate DL-hydroxyphenyl hydantoin (DL-HPH) and itsproduct N-carbamyl-D-p-hydroxyphenyl-glycine (CpHPG) or D-p-hydroxyphenyl-glycine(D-HPG) bioconversed by an engineered strain pE-hyd/E, coli BL21 (DE3) or native strainSinorhizobium sp. SS-or was conducted by the reverse-phase HPLC. It is more efficient toseparate the mixture of DL-HPH, Cp-HPG and D-HPG on BONDAPAK^TM C₁₈ columnwith the optimal mobile-phase of 50mmol/L acetate buffer (pH 4.2) and methanol at theratio of 90:10(V/V) and at 254nm detection. It is also shown from the linear relation of thepeak area for the above three samples that the relative coefficient reaches over 0.99. ByHPLC analysis reported here, not only the activity of D-Hydantoinase or D-Carbamoylasewith DL-HPH as the substrate can be determined, but also its bioconversion products canbe quantitatively assayed.