Extraction, Purification And Characterization Of A Catalase From Bacillus Subtilis WSHDZ-01

Catalase can decompose H2O2 into H2O and O2 effectively. Physiologically it acts as a regulator of H2O2 levels in organelles and protect organism from the damage of reactive oxygen species. Catalases from various sources are utilized in several applications, such as diseases diagnosis and removal of hydrogen peroxide in food sterilization. Catalase is used in removal of hydrogen peroxide in the textile pretreatment as a substitute for high-temperature water washing and using chemical reducing agents.In this study, we investigated the methods for extraction, purification and characterization of a catalase from Bacillus subtilis WSHDZ-01. And be intended to improve the thermostability of the catalse by using additives. The main results were given as follows:(1) The Bacillus subtilis WSHDZ-01 cells were permeabilized by the method of chloroform shock. Experimental results showed that, under the biomass condition of 0.01 g DCW, addition of 20μL chloroform, treating 30min, and permeation in water for 60 min at 37℃, the release of catalase was up to 85 % of total.(2) The purification was performed with a three-step procedure consisting of ethanol precipitation, DEAE ion exchange and hydrophobic interaction chromatography, and finally achieved a 6.8-fold-purifying over the crude extract. SDS-PAGE of the purified catalase revealed the final presence of a single band at an apparent molecular mass of 63 kD. The native enzyme showed the typical Sort band appearing at 405 nm, which indicated that the purified catalase is a heme-containing enzyme. The apparent Km value for enzyme activity on H2O2 was calculated to be 26.87 mmol/L. The activity of this catalase was not reduced by dithionite but was strongly inhibited by cyanide, azide and 3-amino-1, 2, 4-triazole (the specific inhibitor to monofunctional catalase). No peroxidase activity of this enzyme was detected when using p-phenylenediamine as an electron donor. Therefore, it suggested that this catalase belonged to monofunctional catalases.(3) Besides, this catalase was thermosensitive, and its activity exhibited pH-independently between pH 5-10 but showed a sharp maximum at pH 11-12. No activity was lost when the enzyme was incubated at 25℃and pH 11 for 60 min. The catalase reacted best at 55℃,and it is quite stable when maintained at below 50℃.(4) Sugars, polyols, metal ions, anions and other chemical materials were added in catalase solution to improve its stability. The remaining activity of CAT when maintained at 60℃for 15 min was increased to 80 % in the presence of 500 mmol/L sodium acetate, 500 mmol/L sodium sulfate, 50 % ethylene glycol and 50 % PEG..(5) According to the study, the deactivation process at different temperatures of the native CAT is followed the first inactivation order. But it is followed second inactivation order in the presence of sodium acetate and sodium sulfate. Through the study of the thermodynamic properties, we found that the thermostability of native catalase was poor at high temperature. It was improved greatly in the presence of the two additives, but it showed great variation at different temperatures as well as in the absence of them.