Screening, Isolation And Identification Of The Strains With Cholesterol Oxidase And Protein Modification Of Cholesterol Oxidase

Purpose:The strains with cholesterol oxidase were screened from natural environment They were authenticated and genetically modified.In order to their improve enzyme activities,these strains were modified by protein engineering after gene recombination in vitro.Method:?1?Soil samples were collected from natural environment with the help of the primary screening medium using cholesterol as the sole carbon source.These strains were classified and saved by their shapes and colors.The strains were fermented in liquid medium.After the fermentation culture,the culture medium was assayed by silica gel thin-layer chromatography.The culture medium which generated new spots were chosen for further characterization of chromatography mass spectrometry?GC?.The strains with cholesterol oxidase could be screened by using this culture medium.The genome of the strain L71 were extracted and further compared with the 16S rDNA for identification.?2?Pure cholesterol oxidas'e were extracted from the strain by cell disruption,salting out method,weak anion exchange,hydrophobic chromatography and molecular sieve.Their amino acid's sequence were measured by Edman method and carboxypeptidase method.Their coding sequences were obtained by being compared with GenBank.Followed by Polymerase Chain Reaction?PCR?amplification,gene recombination and transformation into E.coli Top 10 receptor and expression.?3?After that,its basic physical and chemical properties such as optimum pH,optimum temperature,organic solvent tolerance and substrate spectrum were measured.?4?3D model of this pure enzyme docked with the substrate molecule was constructed.Mutant site was selected for saturation mutation and its mutant activity was measured.The pure enzyme was extracted from the positive mutant.Their enzymatic activity was measured and compared with the wild-type's.The enzymes which exhibit better enzymatic activity were chosen for further measuring their physical and chemical properties.Result:Sixteen strains containing cholesterol oxidase were obtained.Three strains of them are identified as Cellulosimicrobium strains,Stenotrophomonas sp.and Arthrobacter sp.The strain of Arthrobacter sp was chosen for the further investigation.After pure enzyme gene was obtained,their gene expression were determined.The optimum pH of this strain was 7,the optimum temperature was 40 ? under the above conditions Km was 1.41 mM/L and the enzyme activity was 94.7 U/mg.Pure enzyme showed good organic solvent tolerance for N,N-dimethylformamide,methanol and dimethyl sulfoxide.The conversion of the substrates include?-Sitostero,pregnenolone and chiral compound 3,5,5-trimethyl-2-cyclohexen-l-ol?few chiral selectivity?.The Glu144,Gly160 and Asn164 sites of the substrate-binding pocket were selected for saturation mutation according to the three-dimensional docking model.After the site of Glu 144 was transformed into Tyr,the catalytic performance to the substrate 3,5,5_trimethyl₂-cyclohexen-l-ol was increased by 5 times.And the chiral selectivity was as high as 9.8%;When the site of Glul44 was transformed into Met,its chiral selectivity could be reached to 14.92%;When the site of Gly160 was transformed into Thr,its catalytic activity to substrate cholesterol was increased by 565.77%.Conclusion:In this thesis the strains with cholesterol oxidase were obtained by screening,Pure enzyme and their coding gene were obtained by experiments,its basic physical and chemical properties were measured.Its function a series of experiments of saturation mutation were conducted,which could effectively improve its enzymatic activity to specific substrates.The enzyme showed a selective enhancement in steric structure of the substrate.