碱性蛋白酶是一类重要的工业酶制剂。为进一步提高碱性蛋白酶在高温碱性条件下的活力,增强其工业应用价值,本文从地衣芽胞杆菌CCTCC M2018539中首先通过PCR扩增获得碱性蛋白酶编码基因aprE539并克隆入表达载体pND-113中,在枯草芽胞杆菌WB600中实现碱性蛋白酶AprE539的表达;重组酶AprE539经硫酸铵沉降(30%~70%)、透析、DEAE阴离子交换和超滤获得纯酶,并以SDS-PAGE电泳测定AprE539的分子量大小。结果表明,AprE539的分子量大小为30 kDa。进一步对其酶学性质研究表明:AprE539的最适作用pH为11.0,最适作用温度为65~70℃;在pH6.0~12.0范围内具有较好的稳定性;在60℃保温1 h后酶活剩余70%;Cu2+、Mn2+、Ca2+和Mg2+对酶活有明显促进作用。AprE539在氨基酸序列上仅有2个氨基酸残基与碱性蛋白酶2709存在差异,但其酶学特征差异明显,为后续进一步探究其酶学性质差异性奠定基础。
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.