【摘要】 以斑玉蕈为材料分别从菌盖和菌柄中提取一种酸性磷酸酯酶(ACPase,EC.3.1.3.2),进一步用硫酸铵沉淀分离,Sephadex G-200柱纯化,从菌盖中分离到3个酶组分,从菌柄中分离到4个酶组分,分别对菌盖和菌柄的酶Ⅰ和酶Ⅰ′进行聚丙烯酰胺凝胶(PAGE)电泳纯度鉴定,均呈现单一酶蛋白带。SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)测定酶Ⅰ和酶Ⅰ′的相对分子量均为65kDa,SDS-聚丙烯酰胺凝胶电泳及Sephadex G-75凝胶过滤测定分析,酶Ⅰ和酶Ⅰ′均为单亚基蛋白。紫外吸收光谱(UV)测定结果表明,酶Ⅰ和酶Ⅰ′均呈现两个吸收峰,分别在220nm和280nm处;酶Ⅰ和酶Ⅰ′等电点(pI)分别为5.4和5.0;水解对硝基苯磷酸二钠(PNPP)最适pH值分别为5.0和4.6;酶促反应的最适温度均为50℃,并均有热激活现象,热激活温度均为60℃;耐热实验表明,酶Ⅰ和酶Ⅰ′在最适温度下温育45min后,酶活力分别为最高酶活力的60.2%和57.3%,酶Ⅰ在温育20min时表现最大活性,酶Ⅰ′在温育25min时表现最大活性;酶Ⅰ和酶Ⅰ′在60℃保温25min活力分别下降至最大活力的19.33%和15.25%,且保温时间越短酶活性越大。酶Ⅰ和酶Ⅰ′的酶促反应初速度(υ)分别为17.1×10-3μmol/min和11.3×10-3μmol/min;最大反应速度(Vmax)分别为27.6×10-3μmol/min和85.0×10-3μmol/min,米氏常数(Km)值分别为4.095×10-3mol/L和36.3×10-3mol/L,酶Ⅰ的专一性常数Kcat/Km=3.846×105(mol/L)-1S-1,酶Ⅰ′的Kcat/Km=1.11×105(mol/L)-1S-1,酶Ⅰ的Kcat/Km比酶Ⅰ′的Kcat/Km大3.46倍。重金属离子Hg2+、Ag+、Cd2+和Pb2+对酶均有抑制作用。更多还原

【Abstract】 To investigate the characteristics of acid phosphatase(ACPase,E.C.3.1.3.2) of Hypsizygus marmoreus,the crude acid phosphatase was extracted from the pileus and stipe of H.marmoreus,and purified by ammonium sulfate fraction precipitation and chromatography on Sephadex G-200.Three enzyme components and four enzyme components were obtained from pileus and stipe of this fungus respectively.Among the all obtained enzyme components of ACPase from pileus or stipe,both enzymeⅠand enzymeⅠ’ showed single electrophoretic bands in PAGE,which indicated the same molecular weight with 65kDa determined by SephadexG-75gelatin filtration.Both enzymeⅠand enzymeⅠ’ showed the maximum UV absorbance at 220nm and 280nm,while their isoelectric point was at 5.4 and 5.0,respectively.The enzymeⅠand enzymeⅠ’ showed its optimum pH of 5.0 and 4.6 with p-nitrophenyl-phosphate(PNPP) as substrate,respectively.Both enzymes showed the same optimal catalytic reaction temperature of 50℃,and could be activated by the dispose of 60℃.The thermoduric experiments indicated that 60.2% of the highest activity of the enzymeⅠand 57.3% of the enzymeⅠ’ was remained respectively,after being heated at the optimum temperature for 45mins,and moreover,enzymeⅠshowed its highest activity when incubated for 20mins while enzymeⅠ’ for 25mins.When incubated at 60℃ for 25mins,enzymeⅠand enzymeⅠ’ just remained 19.33% and 15.25% of its highest enzyme activity,respectively,and incubated for much less time,they remained much higher activity.The initial reaction velocity of enzymeⅠand enzymeⅠ’ was 17.1×10-3μmol/min and 11.3×10-3μmol/min,respectively,and their maximum reaction velocity was 27.6×10*3μmol/min and 85.0×10-3μmol/min,respectively.The Michaelis constant(Km) of enzymeⅠand enzymeⅠ’ was 4.095×10-3mol/L and 36.3×10-3mol/L,respectively.The specificity constant of enzymeⅠand enzymeⅠ’ was Kcat/Km=3.846×105(mol/L)*1S-1 and Kcat/Km=1.11×105(mol/L)*1S-1,respectively,and the activities of the both enzymes were inhibited by heavy metal ions including Hg2+,Pb2+,Ag+ and Cd2+.更多还原