短芽孢杆菌低温弹性蛋白酶酶学特性分析

Characterization of Cold-active Elastase from Bacillus brevis

旨在从短芽孢杆菌(Bacillus brevis)XZE116发酵液中分离纯化低温弹性蛋白酶,并对酶学性质进行研究。利用硫酸铵分级盐析、DEAE-Sepharose阴离子交换层析和Sephadex G-75分子筛凝胶过滤层析等方法进行纯化。结果显示,分离纯化到了均一的酶蛋白,酶纯度提高了37.21倍,回收率为35.3%。SDS-PAGE及Sephadex G-75分子筛凝胶过滤层析显示酶蛋白为单亚基蛋白,分子量是32.6 kD。最适作用温度25℃。在pH7.5-10.5范围内酶活性及稳定性较高,最适作用pH9.0,Mg2+对酶有明显激活作用。丝氨酸蛋白酶特异性抑制剂强烈抑制酶活性,表明所纯化到的弹性蛋白酶属于丝氨酸蛋白酶。酶对阴离子表面活性剂(0.1%SDS)、阳离子表面活性剂(0.1%CTAB)和非离子型表面活性剂(1%Tween80)均具有很强的稳定性。鉴于短芽孢杆菌XZE116弹性蛋白酶具有以上优良酶特性,它在肉品嫩化领域具有潜在的应用价值。

It was to purify a cold-active elastase from Bacillus brevis XZE116 and to characterize the enzyme. We purified the elastase from strain XZE116 using precipitation with ammonium sulfate, DEAE-Sepharose ion exchange chromatography and Sephadex G-75 gel filtration chromatography and examined the characterization of the elastase. Results showed the elastase from strain XZE 116 was purified to homogeneity with a 37.21-fold increase in specific activity and 35.3% recovery. Elastase purified is composed of a single polypeptide chain having an apparent molecular mass of 32.6 kD as determined by SDS-PAGE and gel filtration through Sephadex G-75 column. The enzyme was highly active and stable in the pH range of 7.5-10.5, with an optimum at pH9.0. It exhibited maximal activity at 25℃. The elastase was obviously activated by Mg2＋. Its activity was strongly inhibited by serine-protease inhibitors, suggesting that the purified enzyme is a serine- protease. The enzyme showed extreme stability towards anionic （ 0.1% SDS ）, cationic （ 0.1% CTAB ） non-ionic surfactants （ 1% Tween80 ） . Considering its promising properties, the elastase from Bacillus brevis XZE116 may find potential application in meat tenderization field.