嗜热脂肪芽孢杆菌HY-69耐热金属蛋白酶基因表达产物的纯化及性质研究

Purification and Properties of Product of the Thermostable Protease Gene from Bacillus stearothermophilus HY 69 *

利用ＣＭ纤维素离子交换层析法，从宿主细胞枯草芽孢杆菌ＭＩ１１３中纯化了嗜热脂肪芽孢杆菌ＨＹ６９的耐热金属蛋白酶基因的表达产物，达电泳纯。该酶的最适反应温度为７０℃，有着较好的热稳定性和极高的盐酸胍抗性。７０℃的半寿期为４５ｍｉｎ。在３ｍｏｌ／Ｌ的盐酸胍中变性２０ｍｉｎ，仍残余近４０％的酶活。利用凝胶过滤和ＳＤＳＰＡＧＥ，测定其分子量均为２７０００±１０００。通过ＣＤ光谱得知，该酶含有６６％的α螺旋，２８％的β转角，６％的无规则卷曲，无β折叠。利用ＣＤ光谱和荧光光谱研究了该酶在盐酸胍变性过程中的构象变化，推测其主要是通过增加包装效率，减少无规则卷曲来提高酶的稳定性。

The thermostable metal protesase gene from Bacillus stearothermophilus HY 69 had been cloned and expressed in Bacillus subtilis MI113.Its product was purified from host bacteria by CM cellulose chromatography.The product showed homogeneity in PAGE.Its molecular weight is 27 000 ±1 000,measured by SDS PAGE and sephadex G 100 gel filtration,respectively.The protease is made up of 66% of α helix,28% of β turn,6% of random and no β sheet by calculations from CD dada.The protease shows rather resistance to heat.Its optimum temperature is 70℃.It also shows high resistance to Gdn HCl.Denatured in 3 mol/L Gdn HCl for 20 minutes the enzyme remaims about 40% of original activity.In order to explore the thermostable mechanisms of the enzyme,its conformational variety in the course of denaturation by Gdn HCl was investigated by CD spectra and fluorescence spectra.It is concluded that the enzyme increases its stability by increasing packing efficiency and decreasing mount of the random.