| Vibrio parahaemolyticus is a sort of gram-negative halophilic bacteria, widespread across the sea water, the sediment and the marine products. It was one of intestinal diseases in human after consuming seafood. Hydrogen peroxidase, also called catalase(CAT) is a kind of oxidases, found in almost all animals, plants and microorganisms, was widely used in medical diagnosis, clinical analysis, food sterilization as well as paper making and pulp making industries.In the research, catalase in VP110 were named catVPF1 and the isoenzyme catVPR2, expressed and purified in prokaryotic expression system, enzyme activities were detected by reagent kit. To investigate the function of catalase in VP110, genes were knockout and three mutants were constructed by the means of over-lap PCR. Each mutant antioxidant capacity was detected by kit. Gene expression changes in the culture medium with hydrogen peroxide were detected by RT-PCR.After purified and expressed, catalase in prokaryotic expression system showed strong enzyme activities, catVPF1 was 242.02 IU/mg, catVPR2 was 197.962 IU/mg. It showed that strains with catalase could resisted with oxidant environment. Wild type strain had a stronger antioxidant capacity than mutant strains. Single-mutant strains had a stronger antioxidant ability than that of double-mutant strain. RT-PCR results showed that with the increase concentration of hydrogen peroxide, the catalase enzyme gene expression was down-regulated under anaerobic conditions. But in aerobic conditions, with the increase concentration of hydrogen peroxide oxidant, the catalase enzyme gene expression was up-regulated. But when the concentration of oxidant was increased, hydrogen peroxide enzyme gene expression was restrained. All of those results indicated that catalase had a certain influence on the growth of VP110, it may help the strain lived well under oxidizing environment. |
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