蓝圆鲹肝脏酯酶的分离纯化与性质分析

Purification and Characterization of Esterase from Liver of Blue Round Scads(Decapterus maruadsi)

以蓝圆鲹肝脏为对象,通过硫酸铵盐析、Q-Sepharose阴离子交换柱层析、Q-HP阴离子交换柱层析、Phenyl Fast Flow疏水柱层析和Superdex G75凝胶过滤柱层析技术分离纯化得到一种酯酶。该酶的回收率为0.69%,纯化倍数为1150倍。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳与Superdex G75凝胶过滤柱层析结果显示纯化得到的酯酶分子质量为61.18 kDa。肽指纹图谱显示其肽段与红鳍东方鲀假定脂酰辅酶A水解酶一致。该酶的最适条件为pH 8.0、50℃;在pH 6.0~10.0、温度4~40℃条件下稳定性较高;金属离子K^(+)、Zn^(2+)、Cu^(2+)、Al^(3+)和Fe^(2+)会使酯酶活性显著下降,而Ba^(2+)、Ca^(2+)、Mn^(2+)、Co^(2+)和Mg^(2+)则会促进酶活性;该酶对甲醇、乙醇、丙酮和异丙醇等有机溶剂与盐具有较强的耐受性。底物特异性表明该酶在水溶液中偏向于水解中短链酯,对长链酯无水解活性,而在乳化体系中对长链酯也有较高的水解活性,显示出其具有酯酶与脂肪酶的双重活性。

In this study,a novel esterase was purified from the liver of blue round scads via ammonium sulfate fractionation followed by a series of column chromatographies on Q-Sepharose,Q-HP,Phenyl Fast Flow and Superdex G75 with a recovery of 0.69%and 1150-fold purification factor.The esterase was a monomer with a molecular mass of 61.18 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and Superdex G75 column chromatography.Peptide mass fingerprint analysis showed that the peptides showed a similarity of 100%to the predicted fatty acyl-CoA hydrolase precursor from Takifugu rubripes with 14 amino acid residues.The esterase showed an optimal activity at pH 8.0 and 50℃,and was comparably stable at pH 6.0–10.0 and 4–40℃.The metal ions K^(+),Zn^(2+),Cu^(2+),Al^(3+) and Fe^(2+)strongly suppressed the esterase activity while Ba^(2+),Ca^(2+),Mn^(2+),Co^(2+)and Mg^(2+)showed obvious activation effect on the enzyme’s activity.Moreover,the esterase exhibited strong tolerance against methanol,ethanol,acetone and isopropanol,as well as salt.Interestingly,the esterase preferably hydrolyzed esters with short carbon chain in aqueous solution,and exhibited hydrolytic activity toward long-chain esters in an emulsion system but not in aqueous solution.The results indicated that the esterase has both esterase and lipase activity.