Identification, Fermentation And Characterization Of A Catalase-producing Bacterium YS0810

Catalase (whose abbreviation is CAT), widely distributed in nature and found inall aerobic microorganisms, plant and animal cells, can decompose H2O2into H2Oeffectively. Therefore, the catalases are widely used in food, pharmaceutical, chemicalindustries and environmental protection. It was studied that screening, identification,fermentation-conditions optimization and purification of marine bacteria producingcatalase, the properties of the catalase in order to lay the foundation for the industrialproduction of catalase and its application in the environmental protection.1. A strain named YS0810with high catalase activity had been screened out.According to the result of16S rDNA, the morphological, physiological andbiochemical identification, this strain belonged to Acinetobacter sp. and was namedAcinetobacter sp. YS0810.2. The fermentation conditions of marine Acinetobacter sp. YS0810wereoptimized in order to improve the yield of the catalase. Firstly, single factorexperiment was used to screen these eight factors: the nigrogen source, the carbonsource, sodium acetate, inorganic salt, culture volume, inoculation volume, initial pH,fermentation temperature and fermentation time. The result indicated that when theproduction of catalase reached the highest yield, the value of each single factor was asfollows: peptone15g/L, sucrose20g/L, sodium acetate10g/L, MgSO4·7H2O0.9g/L,NaCl5g/L, KH2PO41.5g/L, respectively. Secondly, those variables were valuatedthrough the Plackett-Burman design method. Results showed sodium acetate, peptoneand sucrose that were the significantly affecting variables. Then, the central compositedesign and the analysis by Design-Expert8.05software were adopted to determinethe optimal level of the three main factors. The most suitable variables were identifiedas follows: sodium acetate10.36g/L, peptone16.68g/L, sucrose16.22g/L.Thecatalase activity was predicted to be6814U/mL. The result of verification experimentunder the optimum conditions showed that6578U/mL was the maximum catalaseactivity. Therefore, the yield of catalase was raised obviously by through optimizingthe fermentation conditions. These results suggested that the predicted model was reliable and available for the optimization of catalase fermentation conditions.3. The result of characterization of catalase from strain YS0810show: withaprocedure consisting of ammonium sulfate precipitation, ion exchangechromatography and gel filtration, CAT had been purified from strain YS0810. Thepurification, specific activity and yield were37.5-fold,112466.70U/mg and19.23%respectively.4. The optimum pH of was12.0, with relative activities were all above50%atthe rang of pH6.0to pH10.0. The optimum temperature of was60℃, whose residualactivity retained more than of the initial activity from30℃to60℃. The catalase wasfound with good alkali tolerance and thermal stability. It was stable at the pH rangingfrom6.0to12.0, still possessing81%of its maximal activity at pH11under4℃for3h. It still remained about73%of its activity at pH7.0under60℃for30min. Thesubunit molecular weight was estimated to be50kDa by SDS-PAGE. The molecularmass of CAT was estimated to be201.2kDa by gel filtration chromatography, and it isa dipolymer comprising of four identical subunits. And the isoelectric point of thecatalase was determined to be5.5by using isoelectric focusing electrophoresis.