根霉ZM-10脂肪酶经过硫酸铵盐析、DEAE-Sepharose FF阴离子交换层析、SephardexG100凝胶过滤层析得到电泳纯脂肪酶,分子量约为42 ku,纯化倍数15.3倍,酶活回收率22.2%。采用NBS、EDC、DEPC、CH-T、PMSF、DTNB等6种化学修饰剂对根霉ZM-10脂肪酶进行化学修饰和底物保护实验,研究了其分子中氨基酸侧链基团与酶活性中心的关系。实验结果表明,酸性氨基酸(天冬氨酸?谷氨酸)残基、组氨酸残基、丝氨酸残基、色氨酸残基为根霉ZM-10脂肪酶活性的必需基团。酶分子中酸性氨基酸(天冬氨酸?谷氨酸)残基、组氨酸残基和丝氨酸残基位于根霉ZM-10脂肪酶的活性中心部位,而色氨酸残基在保持脂肪酶活性中起到重要作用,但其不位于根霉ZM-10脂肪酶的活性中心部位。

A lipase from Rhizopus sp.ZM-10 was purified to homogeneity using ammonium sulfate precipitation,dialysis,DEAE-Sepharose Fast Flow anion exchange chromatography and Sephadex G-100 gel filtration chromatography.This purification protocol resulted in a 15.3-fold purification of lipase with 22.2% final yield,and the relative molecular weight of the enzyme was determined to be approximately 42 ku using SDS-PAGE.Six modifiers(NBS,EDC,DEPC,CH-T,PMSF,DTNB) were used to react with Rhizopus sp.ZM-10 lipase.The result indicated that aspartate(glutamate),histidine,serine and tryptophan residues were essential groups for the catalytic activity;aspartate(glutamate),histidine and serine residue were in the substrate binding site,and tryptophan residue was important in maintaining the enzyme activity,but not in the substrate binding site of the enzyme.