尼罗罗非鱼肝脏N-乙酰-β-D-氨基葡萄糖苷酶的分离纯化及酶学性质分析

Purification and enzymatic properties analyses of N-acetyl-β-D-glucosaminidase from the liver of Oreochromis niloticus

N-乙酰-β-D-氨基葡萄糖苷酶(NAGase)是生物体内主要的溶酶体水解酶之一,广泛存在于动植物和微生物体内.以尼罗罗非鱼(Oreochromis niloticus)肝脏作为材料,采用硫酸铵(饱和度30%~70%)分级沉淀、DEAE Sepharose Fast Flow离子交换柱层析、Sephadex G-200分子筛凝胶过滤柱层析的方法分离纯化NAGase,得到比活力为2 988 U/mg的酶制剂.经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测呈现单一条带,说明所得酶为PAGE单一纯.通过Sephadex G-200柱层析法测得NAGase的总分子质量为61.8 ku,结合SDS-PAGE结果,说明该酶仅有一个亚基.进一步测定了NAGase的酶学性质:NAGase水解底物对硝基苯-N-乙酰-β-D-氨基葡萄糖苷(pNP-NAG)的最适温度为55℃,温度稳定范围为0~55℃,超过75℃时则完全丧失活性;NAGase水解底物的最适pH为5.8,pH稳定范围为4.0~9.0,pH大于9.0时几乎完全失活;该酶水解底物的动力学参数K m和v m分别为0.229 mmol/L和9.346μmol/(L·min);运用化学修饰法分析发现二硫键、氨基、组氨酸咪唑基和色氨酸吲哚基均是NAGase的活性必需基团,而精氨酸胍基则不是其活性必需基团.

N-Acetyl-β-D-glucosaminidase(NAGase)is one of the main lysosomal hydrolases in organisms,which widely exists in animals,plants and microorganisms.In this study,NAGase was purified from the liver of Oreochromis niloticus using the following techniques:(NH4)2SO4 fractionation(saturation degree of 30%to 70%),DEAE Sepharose Fast Flow ion exchange column chromatography and Sephadex G-200 molecular sieve gelfiltration column chromatography.The specific activity of the purified enzyme was 2 988 U/mg.The purity of the enzyme was determined by SDS-PAGE,which showed one single band suggesting its PAGE-pure level.The molecular mass of NAGase was estimated to be 61.8 ku by Sephadex G-200 chromatography,which combined with results from SDS-PAGE,indicates that NAGase is composed of only one subunit.In addition,the enzymatic properties of NAGase were further determined.The enzyme was stable in the range of 0 to 55℃,and its activity was completely lost above 75℃,and the optimum temperature for hydrolysis of pNP-NAG was 55℃.NAGase was stable in the pH range of 4.0 to 9.0 with an optimal pH of 5.8,and the enzyme was almost totally inactivated when pH was higher than 9.0.The kinetic parameters,K m and v m,were determined to be 0.229 mmol/L and 9.346μmol/(L·min),respectively.The essential active groups of NAGase were determined using chemical modification method.The results demonstrated that essential active groups of NAGase included disulfide bond,amidogen,imidazole of histidine,and indolyl of tryptophan,while guanidyl of arginine was not an essential active group of the enzyme.