刺芹侧耳芳基醇氧化酶的分离纯化及其酶学性质

分离纯化刺芹侧耳Pleurotus eryngii芳基醇氧化酶,并探究其酶学性质。通过硫酸铵盐沉、DEAE-Sepharose Fast Flow弱阴离子交换层析、Sephacryl S-200 High Resolution凝胶过滤层析和Source 15Q强阴离子交换层析,得到纯化的单一酶。经肽指纹图谱鉴定,确定其为芳基醇氧化酶,酶活回收率25.5%,纯化倍数38.2。结合SDS-PAGE和IEF-PAGE分析,确定其分子量和等电点分别为70kDa和4.2。以藜芦醇为底物,该酶最适反应pH为6.0,最适反应温度为70℃,金属离子Zn²⁺、Fe²⁺和Cu²⁺对芳基醇氧化酶的活性抑制作用明显,K_m和V_max分别为0.921mmol/L和80U/mg。

Purification and characterization of aryl-alcohol oxidase from Pleurotus eryngii

Isolation, purification and characterization of aryl-alcohol oxidase from Pleurotus eryngii were studied. The single enzyme was purified by ammonium sulfate precipitation, DEAE-Sepharose Fast Flow weak negative ion-exchange chromatography, Sephacryl S-200 High Resolution gel filtration chromatography and Source 15Q strong negative ion-exchange chromatography. This enzyme was identified by peptide mapping analysis, and it was purified about 38.2-fold with a yield of 25.5%. The molecular mass and isoelectric point of aryl-alcohol oxidase were estimated by SDS-PAGE and IEF-PAGE at 70kDa and 4.2, respectively. The optimum reaction pH value and temperature were 6.0 and 70℃, respectively. The metal ions, Zn²⁺, Fe²⁺ and Cu²⁺, inhibited the enzyme activity significantly. The K_m and V_max of purified aryl-alcohol oxidase toward veratryl alcohol were 0.921mmol/L and 80U/mg, respectively.