Screening, Cloning And Expression For C2/c3 Haloacid Dehalogenases

Dehalogenases catalyze the breakdown of the carbon-halid bondings. They have significant applications in biosynthesis of critical medical and agriculture intermediates, such as D-lactic acid, S-2-chloropropionic acid (S-2-CPA), and D-2, 3-dichloropropionic acid. In this study, we took the preparation of valuable S-2-CPA, hydroxyacetic acid, 3-hydroxypropionic acid for examples, and investigated the catalytic properties and regularity of haloacid dehalogenases. This work is valuable in fundamental research and potential application. In our contents, based on the high-throughput screening method, we first screened strains which could produce R-2-haloacid dehalogenase, chloroacetic acid dehalogenase, and 3-chloropropionic acid dehalogenase, respectively. The encoding genes of above three dehalogenases were identified by degenerate PCR and genome walking, or genome library. The encoding genes of the dehalogenases were heterologous expressed. Finally, the catalytic properties were investigated by molecular modeling. The main contents of this work were introduced as follows:1) A Pseudomonas sp. ZJU26, with priority dehalogenation of R-2-chloropropionic acid (R-2-CPA), was screened based on the high-throughput screening method. The N-dificient gene of the R-2-haloacid dehalogenase (dehDIV) from Pseudomonas sp. ZJU26 was identified by degenerate PCR and genome walking. A revised dehalogenase gene (dehDIV-R) was constructed by fusing an N-terminal fragment (24aa) of an R-2-haloacid dehalogenase gene (hadD) from Pseudomonas putida AJ1.The dehDIV-R was subcloned to pET30a(+) and heterologous expressed in E. coli BL21(DE3). The maximum dehalogenase activity of the recombinant enzyme (rDehDIV-R) occurred when it was incubated in pH 9.0 at 60℃. The Km and specific activity of the rDehDIV-R were 2.245 mM and 20.56 U/mg. Finally, the purified rDehDIV-R was successfully used to resolute 100 mM recemic 2-CPA to obtain S-2-CPA with ees above 99% within 4 h.With an RS-2-haloacid dehalogenase (PDB code 3BJX) as a template, a three dimensional structure of DehDIV-R was constructed by homology modeling. The docking results displayed that the R-2-CPA could bind to the active site of the DehDIV-R with slower binding energy. The critical residue dictating the enantioselectivity of the DehDIV-R was the Asn203. The Asn203 was mutated to Ala, Gly, and Gin, respectively, by amino acid mutation in Sybyl. The mutants Asn203/Gly or Ala with smaller steric space, have no enantioselectivity, while the mutant Asn203/Gln, decreased in the enantioselectivity because of the hydrogen bonding from the Gln. Overall, the results proved that the steric hindrance of Asn203 caused the enantioselectivity of the DehDIV-R.2) A high chloroacetic acid dehalogenase producing strain Pseudomonas sp. CMGCC 3267 was screened using high-throughput screening method. Native polyacerylamide gel electrophoresis displayed that the chloroacetic acid dehalogenase was one kind of S-2-haloacid dehalogenases. Thus, based on the conserve sequence of dehlls from Psudomonas sp., the dehll-S was identified.The dehⅡ-S was subcloned to pET30a(+) and expressed in E. coli BL21(DE3), and the recombinant enzyme (rDehⅡ-S) was obtained. The rDehⅡ-S had maximum dehalogenase activity in pH 9.5 and at 50℃. Its Km and specific activity were 0.97 mM and 10.3 U/mg, respectively.Magnesium-aluminum oxide was used to resolve the chloride ion inhibition. The yield achived 100% within 15 h when the dehalogenation was incubated in 200 mM chloroacetate with 0.02 g/L magnesium-aluminum oxide addition. The purity of the product could get to above 99%.Results of homology modeling for DehⅡ-S suggested that the catalytic pockage was exposed to bulk solvents, indicating that the DehⅡ-S had wide susbtrates range, stronge dehalogenase activity. Results of molecular docking explicated the reason of the substrate enantioselectivity for DehⅡ-S.3) An isolate Bacillus sp. CGMCC 4196 had an ability of dehalogenating high-concentration 3-chloropropionic acid (3-CPA) and 3-chlorobutyric acid (3-CBA). Results of metabolic product analysis in 3-CPA biodegradation proved that the dehalogenation mechanism was hydrolytic. The gene (465bp) encoding the 3-chloropropionic acid dehalogenase (DehⅢ) was identified from genome library.The dehⅢwas subcloned to pET30a(+) and expressed in E. coli BL21 codon plus(DE3), and the recombinant 3-chloropropionic acid dehalogenase (rDehⅢ) was obtained. The optimum temperature and pH for rDehⅢwere 30℃and pH 7.0. The Km and specific activity of the rDehⅢwere 3.213 mM and 19.28 U/g, respectively. With the purified rDehⅢas biocatalyst,3-hydroxypropionic acid was prepared from 3-CPA, and the yield could achieve above 99%.A three dimensional structure of DehⅢwas built. The catalytic residues, including Asp55, Arg77, and Arg78, were predicted by SiteID and Surflex-docking.