Optimizing The Fermentation Conditions, Mutant Breeding, Gene Cloning, Expression And Purification Of Catalase A From Micrococcus Luteus

Objective:The fermentation conditions of catalase A from Micrococcus luteus was optimized to improve Catalase A productivity.M.luteus were physically or chemically mutated,and were screened forward mutations.In this research,the gene of catalase A from M.luteus was cloned into the expression plasmid pProExHTa-CAT.The recombinant CAT fusion protein was expressed in E.coli with high efficiency.Then the fusion protein was purified by metal-chelating affinity chromatography and the purified CAT protein was studied.These results not only provide foundation for further studies on the anti-peroxidatve mechanism of M.luteus and the defensive system of antioxidant enzyme,but also do for further development and application of CAT expression plasmid.Methods:The research included five parts:1.Optimize the fermentation conditions of catalase A from M.luteus.Improve Catalase A productivity and activity.2.In order to improve the catalase activity,M.luteus were mutated by NTG or UV,then forward mutants were screened.Three mutants were obtained,whose catalase abilities were two or three times as high as the activities of wild strains.3.Clone the gene of catalse A from M.luteus and construct the recombinant plasmid pProExHTa-CAT.CAT gene was amplified by PCR from genomic DNA of M.luteus and was inserted into the cloning plasmid pMD18-T to construct the recombinant plasmid pMD18-CAT. The recombinant plasmid was transformed into the host strain E.coli DH5α.The positive clones were selected by using Ampicillin resistance,and recombinant plasmids were identified by using EcoRâ… +Hindâ…¢double digestion and sequence analysis.After the same double enzyme digestion,the products containing the CAT gene were ligated to the same products of the vector pProEx-HTa.The recombinant plasmid pProExHTa-CAT was transformed into the host strain E.coli DH5α.The positive clones were also selected by using Ampicillin resistance,and the recombinant plasmid was identified by using EcoRâ… +Hindâ…¢double digestion and sequence analysis.4.Expression and purification of the recombinant CAT fusion protein.The recombinant plasmid pProExHTa-CAT was transformed into the host strain E.coli DH5α.The recombinant protein was expressed by the induction of isopropyl-O-D-thiogalactopyranoside(IPTG) and was analyzed by SDS-PAGE.Then the CAT fusion protein was purified by metal-chelating affinity chromatography and the purified product was assessed by SDS-PAGE.5.Study on the properties of the purified CAT fusion protein.The purified CAT was analyzed by determining total protein and enzyme activity,and testing activity stability under different conditions.Results:1.Optimize the fermentation conditions of Catalase A from M.luteus IFO 3064:beef extract 2g/dL,MgCl₂ 0.25g/dL,peptone 1g/dL,pH 6.5,the fermentation time 44h.2.Three forward mutants were screened.3.The recombinant cloning plasmid pMD18-CAT was constructed.After double enzyme digestion,the plasmid vector fragment(2.7kb) and the target gene fragment(1.5 kb) were shown respectively.Sequence analysis indicated that the target CAT gene was 1518 bp;there is a three-base difference between the obtained sequence and the previously published sequence in GenBank.The homology was 99.8%.After EcoRâ… +Hindâ…¢double enzyme digestion of pMD 18-CAT and pProEx-HTa,the products containing CAT were ligated to the same products of the vector pProEx-HTa.The recombinant expression plasmid pProExHTa-CAT was confirmed by double restriction enzymes digestion and DNA sequencing.The plasmid vector fragment(4.7Kb) and target gene fragment(1.5 kb) were shown respectively.Sequence analysis indicated that the target CAT gene was 1 518 bp;there is a six-base difference between the obtained sequence and the previously published sequence in GenBank.The homology was also 99.6%.The coding sequence of CAT fusion protein is 1 509bp,coding 503 amino acid residues.There is a two-codon difference between the obtained sequence and the previously published sequence in GenBank.The homology was 99.6%.4.The expression CAT protein was induced by IPTG at 37℃.The 60.3 kD protein was found after SDS-PAGE electrophoresis gels.After purified by the affinity chromatography,the CAT fusion protein showed almost a single band after SDS-PAGE electrophoresis gels. 5.The purified CAT was identified to be catalase.Its specific activity reaches 166U /(mg·protein).It can maintain high-activity in a wide pH range between 5～8,and it is also stable to the wide temperature range between 25～50℃.Conclusion:The recombinant expression plasmid pProExHTa-CAT was constructed successfully. The CAT fusion protein was expressed effectively and detected by SDS-PAGE electrophoresis gels.By affinity chromatography,the purified product could be obtained conveniently and fast with high purity,high enzyme activity and good stability.These results will provide theoretical and experimental foundation for further studies on the anti-peroxidatve mechanism of M.luteus and on establishing a convenient and efficient way to product catalase A.This will be helpful for the development and application of CAT.