[关键词]
[摘要]
利用超滤、硫酸铵盐析、DEAE Sepharose Fast Flow阴离子交换柱层析、Sephadex G-75分子筛柱层析对一株南海深海来源菌苏云金芽孢杆菌(B. thuringiensis)SWJS07所产蛋白酶进行分离纯化,纯化后经SDS-PAGE鉴定达到电泳纯,相对分子质量为37.0 kDa,酶的比活力提高了6.39倍,回收率为37.14%。研究其酶学性质表明,该蛋白酶最适催化温度为55 ℃,在30 ℃~45 ℃下稳定性较高,保温300 min残留酶活在80%以上;最适pH 6.5,在pH 6.0~9.0蛋白酶稳定,4 ℃放置24 h残留酶活在80%以上;2 mM Ca2+、Mn2+对蛋白酶有不同程度的激活作用,而Hg2+、Cd2+、Al3+则强烈地抑制蛋白酶活;当在蛋白酶中添加2 mM Ca2+、Mn2+时,其最适催化温度分别为60 ℃和55 ℃,蛋白酶活分别提高了32.86%和28.35%,60 ℃保温30 min相对酶活基本保持不变,与纯酶(相对酶活残留21.02%)相比蛋白酶的热稳定性显著提高;EDTA-Na2可强烈抑制蛋白酶活,推测该蛋白酶属于金属蛋白酶。
[Key word]
[Abstract]
The protease produced by the marine strain B. thuringiensis SWJS07 was purified to homogeneity by ultrafiltration, ammonium sulfate precipitation, anion-exchange chromatography (DEAE-Sepharose Fast Flow) and gel filtration chromatography (Sephadex G-75), with a 6.39-fold increase in specific activity and 37.14% recovery and the molecular weight was estimated to be 37.0 kDa on SDS-PAGE. The optimal temperature and pH for the purified protease were determined to be 55 ℃ and pH 6.5. The protease was highly stable from 30 ℃ to 45 ℃ and between pH 6.0 and 9.0 and it was activated by Ca2+and Mn2+, while Hg2+, Cd2+, Al3+ had a strong inhibitory effect. The optimal temperature were 60 ℃ and 55 ℃ in the presence of 2 mM Ca2+and Mn2+, and the activity were increase by 32.86%, 28.35%, respectively. Meanwhile, thermostability of the protease was enhanced by Ca2+ and Mn2+. In the presence of 2 mM Ca2+and Mn2+, the activity of the protease were retained unchanged after heating for 30 min at 60 ℃, however, it retained 21.02% of its initial activity in the absence of them. It was strongly inhibited by EDTA-Na2, indicating that the protease may be metalloprotease.
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[基金项目]
国家高技术研究发展计划(863计划)课题(2012AA092104;2013AA102201-1);海洋公益性行业科研专项(201305018-7);广东省海洋经济创新发展区域示范专项(GD2012-D01-002)