Cloning And Expression Of The Polysaccharide Deacetylase Gene From Caulobacter Crescentus And Its Bio-activity Analysis

The bioadhesive has good biocompatibility and degradability,it can replace the traditional suture technology,and has been widely concerned in the field of biomedicine.Caulobacter crescentus can secrete the underwater natural viscous substance-holdfast which has the strongest adhesion ability to date.The synthesis and secretion pathway were complex process,in which the polysaccharide deacetylase encoded by hfsH gene affects the viscosity key factors.Therefore,in this paper,the hfsH gene of Caulobacter crescentus was used as the research object,optimizes its codons and analyzes bioinformatics,and performs cloning and soluble expression.Based on the analysis and purification of the recombinant enzyme by nickel column affinity chromatography,a preliminary evaluation of its biological activity was carried out.The main research contents are as follows:Part ?: Cloning of hfsH gene from Caulobacter crescentus and construction vector.We performed codon optimization on the original sequence of the hfsH gene,and used RNAfold online software to analyze the free energy and GC content of the mRNA secondary structure of the 5' and 3' ends of the hfsH gene sequence before and after optimization.It was found that the free energy of the optimized sequence was reduced and the GC content was reduced from 71% to 68%,which was more conducive to later expression.Bioinformatics analysis was found that there were five relatively conserved amino acid functional domains: Motif 1(T(F/Y)DD),Motif 2(H(S/T)xxH),Motif 3(RxPY),Motif 4(DxxD(W/Y)and Motif 5(I(V/I)LxHD).The relative molecular weight is about 29.2 kDa,the isoelectric point is 6.26,and it is a hydrophilic unstable protein with no signal peptide and transmembrane domain,and the secondary structure is mainly ?-helix and irregular coils.The hfsH gene was cloned by PCR amplification using the genomic DNA of Caulobacter crescentus as template,ligating the purified PCR product with pET-28a(+)vector,and then transferred into E.coli DH5? competent cells.Finally,after double digestion identification,sequencing verification was performed.The recombinant plasmid was named pET-28a(+)-hfsH.The sequencing results were correct,indicating that the recombinant plasmid pET-28a(+)-hfsH was successfully constructed.Part ?: Expression of hfsH gene form Caulobacter crescentus and purification of recombinant enzyme.The correctly identified recombinant plasmid pET-28a(+)-hfsH was transformed into E.coli BL21(DE3)for expression.The HfsH protein was analyzed as soluble protein by SDS-PAGE electrophoresis.In order to efficiently express the hfsH gene,the expression conditions were optimized and the optimal expression conditions were determined: the IPTG concentration was 1 mmol/L,and induction at 37°C for 4 h.The HfsH protein was purified by nickel column affinity chromatography,the recovery yield is 4.65g/L and the purity reaches 85%.Part ?: Establishment of bioassay method and preliminary bioactivity analysis of recombinase HfsH.Since acetylxylan esterase belongs to the same family as the polysaccharide deacetylase of Caulobacter crescentus,acetylxylan esterase was selected as the control for the enzyme activity measurement,and p-nitrophenyl acetate(PNPA)was used as the substrate at 415 nm.It was degraded to p-nitrophenol,and the specific activity of polysaccharide deacetylase was 61.8 U/mg.The optimum temperature and pH of the recombinant enzyme were 35? and 7.5.This thesis lays the foundation for the further research on the structure and function of polysaccharide deacetylase from Caulobacter crescentus.