对枯草芽孢杆菌BEM01菌株发酵液中的弹性蛋白酶进行了分离纯化,并研究了酶学性质。先后采用了硫酸铵分级沉淀、离子交换层析和分子筛层析等方法,弹性蛋白酶的回收率为7.88%,纯化倍数为13.79,结合SDS-PAGE和分子筛层析确定该弹性蛋白酶是一单链蛋白,分子量约为31ku。对酶学性质的研究表明,该弹性蛋白酶属于含有Ca2+的金属蛋白酶类;其最适作用温度为50℃,具有良好的热稳定性;在硼砂-硼酸缓冲体系、Tris-HCl缓冲体系中酶最适反应pH分别为7.4和8.6,在pH8.0～11.0酶活力趋于稳定;10mmol/L的Ca2+有激活和稳定酶活作用,SDS和吐温-80也有激活酶活作用,而Li+、Na+、Zn+、K+、Ba2+、Mg2+、Mn2+和EDTA对酶活均有不同程度的抑制作用;其催化动力学方程为:y=0.186x+32.493;V=0.0308U/mL,K=0.0057g/mL。

The purification of elastase was achieved by combination methods of ammonium sulfate precipitation,ion-exchange chromatography and gel filtration chromatography.The elastase was purified 13.79-fold to apparent homogeneity with 7.88% overall recovery.It was a single-chain protein with a molecular mass of 31ku,and estimated by SDS-PAGE and gel filtration.Studies on enzymatic properties showed as follows:the elastase belonged to metalloproteinase containing Ca2+.The elastase activity had a temperature optimum of 50℃ along with good thermal stability.Meanwhile it was optimal at pH 7.4 in borax-boric acid buffer or pH 8.6 in Tris-HCl,and tended stability between 8.0 and 11.0.10mmol/L Ca2+ had stabilizing and activation on the elastase activity,whereas SDS and Tween-80 only had activation.Moreover,the elastase activity was inhibited varying degrees by many irons(Li+,Na+,Zn+,K+,Ba2+,Mg2+,Mn2+)and EDTA.The catalyzes dynamics equation was:y=0.186x+ 32.493,Vmax =0.0308 U/mL,Km=0.005 7g/mL.