Purification And Characterization Of The Elastase From Bacillus Subtilis EL32

Elastase is a kind of protease that specially degrades elastin. Due to its special enzymaticcharacteristics, it has broad applications in food, pharmaceutical and cosmetics industries. It hadthe advantages of high production ability and low cost for using microorganism fermentation toproduce elastase, which had attracted reasearchers more and more interest in domestic andforeign. The reasearchers mainly concentrated on isolation and screening of elastase-production,the optimization of fermentation process, but seldomly care about the characterization of elastase.In this study, the strain EL32which was isolated to excrete elastase was identified, and theelastase was purified and characterized. The results were listed below:The16S rDNA of strain EL32had99%homology with Bacillus subtilis. The colonies ofstrain EL32were milky which could produce spores. Its cell gram-stained and V-P reaction werepositive; the srain EL32produced acid but not gas in the glucose culture. It can hydrolyze gelatinas well as starch. The strain EL32was identified as Bacillus subtilis.Elastase from Bacillius subtilis EL32was purified by the procedure of ammonium sulfateprecipitation, DEAE-Sepharose and Sepecryl-S HR100, having the molecular mass of31kDa bySDS-PAGE analysis. The elastase was identified to be substilisin by LTQ-MS, the nucleotidesequence of elastase gene cloned shared high homology of99%with substilisin. The threedimensional structure of the elastase was simulated, which indicated that His, Asp and Ser werethe key groups of active center.The elastase had optimal pH value of7.4and temperature of50°C, was stable in pH rangefrom6.0to8.5. The enzyme was inhibited by Zn²⁺, Al³⁺, Mn²⁺, Cu²⁺, Ca²⁺, Co²⁺and Ba²⁺ionsand EDTA. It was active and stable in25%（v/v） solutions of methanol, ethanol, isopropanol anddimethyl sulfoxide （DMSO）. The K_ms were14.3mg/mL towards elastin congo red and3.4mg/mL towards casein. The V_maxs were estimated at35.7U/mg and1428.6U/mg for elastincongo red and casein, respectively. The elastase could effectively hydrolyze beef, beef tendonand bullflog’s skin. The elastase is easy to prepare and has desirable characteristics for practicaluse.In conslusion, the elastase-producing strain EL32was identified to be Bacillus subtilis, andthe elastase was identified to be subtilisin. It was revealed that the enzyme could hydrolyze manykinds of protein in food materials with a wide pH and termperature range. All these results wereexpected to set up the foundation for the application of elastase in food industry.