Gene Cloning,Expression and Enzymatic Properties of a Thermotolerant and Alkalophilic Protease Gene

Alkaline protease is one of the most important industrial proteases. To further improve its activity under high temperature alkaline conditions and enhance its industrial application values,the alkaline protease-encoding gene aprE539 was obtained by PCR amplification from Bacillus licheniformis CTCCC M2018539 and cloned into the expression vector pND-113 to express alkaline protease ApaE539 in Bacillus subtilis WB600 in this study. The recombinant AprE539 was purified by ammonium sulfate precipitation(30%~70%),dialysis,DEAE anion sepharose anion-exchange chromatography separating to obtain pur enzyme. And the molecular weight of the purified AprE539 was determined by SDS-PAGE. The experimental results were as follows:The molecular weight of the purified AprE539 was 30 kDa. The recombinant AprE539 had the maximum activity at pH11.0 and 65~70℃. It was stable at pH6.0~12.0 and remained about 70% of the activity after incubation at 60℃ for 1 h. Its activity was significantly enhanced with the existence of Cu²⁺,Mn²⁺,Ca²⁺ and Mg²⁺. Two amino acid residues difference were found between AprE539 and alkaline protease 2709. However their properties were different remarkably,which would lay a foundation for further exploring the differences of their enzymatic properties.