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Expression, Characterization And The Primary Research Of Catalase From Vitis Vinifera In Pichia Pastoris

Posted on:2013-05-19Degree:MasterType:Thesis
Country:ChinaCandidate:C C JiaFull Text:PDF
GTID:2253330398993066Subject:Pomology
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Catalase(EC1.11.1.6) is a kind of widely exists in animal, plants and microorganisms within the terminal oxidase, with hydrogen peroxide as the substrate, a pair of electron transfer through catalysis and its decomposed into water and oxygen. In plants, CAT eliminated hydrogen peroxide formed during light respiration, mitochondrial electron transport and fatty acid beta oxidation process. A large number of studies showed that,CAT played important roles in plant defense, stress response, delaying senility and controlling of cellular redox balance etc.To construct the grape catalase (VvCAT) expression plasmid, expression in Pichia pastoris and purification of catalase, thus further study of catalase enzyme properties, which can provide a research foundation for the physiological function in the metabolism of plants.In this study, a gene(VvCAT) encoding catalase was cloned by RT-PCR from grape seedlings, and was delated BamH I restriction site of VvCAT gene by overlap extension PCR according to a yeast bias codon. Methanol induction resulted in the production of active catalase. The catalase had a calculated molecular mass of approximately55kD. The main biochemical properties of the catalase, such as thermo dependence and thermo stability of enzyme, pH optimum and pH stability of enzyme, and the effect of metal ions and chemical reagents, were characterized. With H2O2as the substrate, VvCAT displayed pH and temperature optima of9.0and40℃, respectively. The Km value of VvCAT for H2O2was4.806mmol/L,and the Vmax was102.04μmol mg-1min-1. The enzyme was strongly activated byK+Al3+and was obviously inhibited byMn2+. Recombinant VvCAT activity was inhibited by azide, hydroxylamine, and vitaminC, especially by the catalase-specific inhibitor3-amino-1,2,4-triazole.To detect the peroxidase activity of enzyme using guaiacol, but had no obvious color reaction. Therefore, the enzyme was a monofunctional catalase.
Keywords/Search Tags:Catalase, Vitis vinifera, Secretory expression, Protein purification
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