枯草芽孢杆菌弹性蛋白酶的纯化及酶学性质研究  

Purification and Characterization of Elastase from Bacillus subtilis

作　　者：刘书亮[1] 吴琦[2] 詹莉[2] 赖文[1] 

机构地区：[1]四川农业大学食品学院 [2]四川农业大学生命与理学院,四川雅安625014

出　　处：《食品与发酵工业》2010年第6期26-30,共5页Food and Fermentation Industries

基　　金：四川省教育厅重点项目(07ZA054)

摘　　要：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, ionexchange 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 Ca^2＋. 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 Ca^2＋ 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 ^＋ ,Ba^2＋ ,Mg^2＋ ,Mn^2＋ ） and EDTA. The catalyzes dynamics equation was： y =0. 186x ＋ 32.493, Vn,ax =0. 0308 U/mL, Km =0.005 7g/mL.

关 键 词：弹性蛋白酶 枯草芽孢杆菌 纯化 酶学性质 

分 类 号：TQ925.2[轻工技术与工程—发酵工程]