| In this study, Bacillus subtilis K91 was isolated from a hot spring in Tengchong Yunnan Province, China. The strain optimum growth temperature is 50 ℃. Through the analysis of the complete genome sequencing and annotation, it is found that an esterase gene Car EW is contained in its genome sequence. The study researches on the expression and purification of obtaining recombinant esterase Car EW, and studies the characterization of enzymes, the degradation mechanism of pesticide malathion and plasticizer diisobutyl phthalate. This thesis mainly as follows:(1) The esterase gene Car EW is cloned from a thermophilic bacterium Bacillus subtilis K91. The length of Car EW is 1464 bp, which encodes 487 amino acid proteins. The enzyme shows a monomric structure with a molecular mass of approximately 53.76 k Da and p I of 4.88. The sequence analysis showed that the protein contains a motif Gly-X-Ser-X-Gly, and catalytic triad formed by Ser189, Glu289, and His 388. The serine is of the active site. However, the Car EW shared 99% sequences identity to a carboxylesterase of Bacillus pumilus(AAU04567.1) whose function is unknown.(2) Through assay enzyme activity of recombinant esterase Car EW, it is found that the enzyme exhibited maximal activity with p H 7.5 and 45 ℃, which uses p-NP butyrate as the best substrate. The enzyme was fairly stable within the p H range from 6.5 to 9.5, which retains more than 60%. The temperature stability of Car EW was examined by measuring its resisual activity after incubating for 60 min ant 37 and 45 ℃, and retained approximately 54% and 58%. K+, Na+, Cu2+ and Zn2+ activated Car EW. Hg2+, Mn2+, Fe2+ and Ag+ had a strong inhibitory effect. Triton X-100, DTT and SDS, CTAB strongly inhibited Car EW. 2-propanol, methanol and ethanol strongly activated Car EW. The Car EW can hydrolyze the carbon chain whose length is less than or equal to 12 of p-nitrophenol ester. The values of Km and Kcat were 0.8 m M and 1792 s-1. The Car EW also displayed quite high enzyme activity on different carbon chain length of the substrate, and stability differs in organic solvents.(3) The esterase Car EW can hydrolyze malathion and diisobutyl phthalate, and its potential biodegradation pathway was proposed. Let recombinant esterase Car EW make a response with 14 kinds of pesticide and plasticizer diisobutyl phthalate. This study found that 0.3 U recombinant esterase Car EW can degradate 48% malathion within 60 min at 37 ℃, and hydrolyze after malathion dicarboxylic acid and malathion monocarboxylic acid. Car EW also can hydrolyze diisobutyl phthalate. 10 U Car EW can degradate 92% Di BP within 120 min at 37 ℃, and produce of Mi BP to PTH after hydrolysis, and also hydrolyze Mi BP to PTH. The catalytic efficiency of Di BP is higher than Mi BP, and the values of Kcat/Km were 0.109 s-1 m M-1and 0.031 s-1 m M-1.(4) Cell surface displays esterase Car EW in E.coli BL21(DE3). Pseudomonas syringae KCTC1832 ice nucleation protein N’s end as the anchor protein make a fusion with esterase Car EW to construct recombinant plasmid p ET28-N/Car EW and convert it to E. coli BL21(DE3). Through cell surface localization analysis, it demonstrates that esterase Car EW successfully localizes on the cell surface.The optimal temperature and p H of the surface-displayed Car EW are 45 ℃ and 8.0. The surface-displayed Car EW can hydrolyze the carbon chain whose length is less than 10 of p-nitrophenol ester. The surface-displayed Car EW showed long-trem stability than its purified form,which lays a foundation for the application of esterase in bioremediation. |
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