日本鳗鲡肠道蛋白酶的分离纯化及其酶学性质研究
Purification of protease from intestine of Anguilla japonica and its enzymatic characteristics

【目的】从日本鳗鲡(Anguilla japonica)肠道中分离纯化蛋白酶并分析其酶学性质，为日本鳗鲡饲料的科学研制提供理论依据。【方法】通过硫酸铵沉淀分级分离及Sephadex G-100和Sephadex G-75两级凝胶柱层析纯化日本鳗鲡肠道蛋白酶，经聚丙烯酰胺凝胶电泳(PAGE)和SDS-PAGE鉴定蛋白酶纯度和亚基分子质量，利用凝胶层析法测定酶的分子质量，用等电聚焦电泳法测定酶的等电点，并研究以酪蛋白为底物时蛋白酶催化反应的动力学参数及pH、温度、金属离子和修饰剂对蛋白酶活力的影响。【结果】日本鳗鲡肠道蛋白酶亚基分子质量为65.3 ku，酶分子质量为260.3 ku，等电点为8.23；该蛋白酶的最适pH为8.2，最适温度为55 ℃，米氏常数（Km）为4.832 mg/mL，最大反应速度（Vmax）为0.269 U/min。日本鳗鲡肠道蛋白酶在pH 为6.6～9.0时表现稳定，在20～55 ℃时具有较好的热稳定性，在60 ℃以上稳定性迅速下降。Mg2+、Ca2+、Co2+、Ba2+、Mn2+、Cu2+、Zn2+和Hg2+对日本鳗鲡肠道蛋白酶的活力表现出不同程度的抑制作用，其中以Hg2+的抑制作用最强，1 mmol/L Hg2+可使酶活力丧失 87.63%；而Fe2+有激活作用，5 mmol/L Fe2+可使酶活力提高134.53%。修饰剂EDTA、对氯汞苯甲酸(pCMB)和Cys对酶活力没有影响，苯甲酰基磺酰氟(PMSF)和二硫苏糖醇(DTT)对酶活力则有不同程度的抑制作用。【结论】日本鳗鲡肠道蛋白酶由4条相同肽链组成，属于丝氨酸蛋白酶类，二硫键是维持其活性所必需的，酶活力易受环境中酸碱度、温度和金属离子调控。
【Objective】This study purified protease from intestine of Anguilla japonica and investigated its enzymatic characteristics to provide theoretical basis in scientific development of the diet for Anguilla japonica.【Method】The protease was purified by ammonium sulfate fractionation and Sephadex G-100 and Sephadex G-75 columns.The purity was determined by polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS)PAGE.Enzyme molecular weight was determined by gel chromatography,and isoelectric point was determined by isoelectric focusing method.The kinetic parameters of protease for hydrolysis of casein (enzyme substrate) and effects of 9 ions and modifiers were also determined.【Result】The molecular weight of enzyme was 260.3 ku and the molecular of enzyme subunit was 65.3 ku.The isoelectric point value was 8.23.The optimum pH and temperature were 8.2 and 55 ℃,respectively.The Km value was 4.832 mg/mL and the Vmax value was 0.269 U/min,respectively.The enzyme was stable with pH of 6.6 to 9.0 and temperature of 20-55 ℃.The stability of enzyme dropped rapidly when temperature was >60 ℃.Fe2+ activated the enzyme,and the activity was increased by 134.53% when the concentration of Fe2+ was 5 mol/L.Mg2+,Ca2+,Co2+,Ba2+,Mn2+,Cu2+,Zn2+ and Hg2+ showed various degrees of inhibitory effects on the enzyme.Hg2+ inhibited the enzyme the most,and the enzyme activity decreased by 87.63% when its concentration reached 1 mmol/L.EDTA,p-chloromercuribenzoate and cysteine had no influence on the enzyme,while benzoyl sulfonyl fluoride and dithiothreitol inhibited the enzyme.【Conclusion】The protease from intestine of Anguilla japonica contained four peptide chains with same mass.The enzyme was a serine proteinase and the disulfide bonds were essential for the active site of protease.The activity of protease was affected easily by acidity