凡纳滨对虾蛋白酶的分离纯化及生化特性

Purification and Biochemical Properties of Protease from White Leg Shrimp Litopenaeus vannamei

采用Tris-HCl缓冲液抽提、Sephadex G-100凝胶过滤层析、DEAE-Sepharose F.F.阴离子交换层析和SDS-PAGE等方法,分离和纯化了凡纳滨对虾蛋白酶,研究了其生化特性。试验结果表明,该蛋白酶粗提物经层析后,得到聚丙烯酰胺凝胶电泳纯的一酶组分。该蛋白酶的比活力从128.35 U/mg增至1835.65 U/mg,提高了14.3倍,产率达31.9%。SDS-PAGE显示该蛋白酶只含一条谱带,相对分子量为25 kD。以酪蛋白为底物,该酶的最适pH为7.0,最适温度为60℃,在50℃以下,比较稳定,放置1 h后活性仍超过60%,而超过50℃时蛋白酶活性急剧下降,60℃放置1 h后,酶活性只残留4.7%。10 mmol/L EDTA、Fe2+、Ba2+和Zn2+对该蛋白酶有较强的抑制作用,抑制率分别为84%、53%、43%和38%,10 mmol/L Mg2+和Cu2+对蛋白酶活性有轻微抑制作用,而10 mmol/L Ca2+能显著促进蛋白酶活性,据此推测凡纳滨对虾蛋白酶可能为一种金属蛋白酶。

The protease was extracted and purified from heads of white leg shrimp Litopenaeus vannamei by Tris-HCl buffer solution, gel-filtertion with Sephadex G-100, and ion-exchange with DEAE-Sepharose F.F. The enzymological nature was studied with biochemical and electrophoretic techniques. The enzyme was purified to an electrophoretically homogenous state, reaching 14. g-fold purification with a yield of 31.9%. The extracted protease showed a single band in its relative molecular weight at 25 kD based on SDS-PAGE. The optimum pH was 7.0 for the enzymatic reaction and the optimum temperature was 60 ℃ for casein as the substrate This protease showed a good thermal stability below 50 ℃, and more than 60% of the activity still remained when the protease was cultured at 50 ℃ for 1 h. But the enzyme became inactivated rapidly at the temperature above 60℃, and only 4.7% of protease activity remained at 60℃ for 1 h. The protease activity was found to be inhibited by 10 mmol/L of EDTA（at a rate of 84%）, and Fe2＋ （53%）, Bae＋（43%）and Zne＋（38%）. However, 10 mmol/L of Ca2＋ increased the enzyme activity,while 10 mmol/L of Mg2＋ and Cu2＋ had less effect on the protease activity. It is suggested that the protease from the shrimp be a type of metalloenzyme.