黑曲霉耐酸性α-半乳糖苷酶的分离及其酶学性质  

Purification and Characterization of Acid-Tolerant α-Galactosidase from Aspergillus niger L.

作　　者：王剑锋[1] 王璋[2] 李江[1] 饶军[1] 

机构地区：[1]东华理工大学化学生物与材料科学学院生物系,江西抚州344000 [2]中国食品发酵工业研究院,北京100027

出　　处：《食品科学》2012年第21期267-270,共4页Food Science

摘　　要：采用耐酸性黑曲霉(Aspergillus niger L.)发酵豆渣产α-半乳糖苷酶,粗酶液依次经过聚乙二醇6000-KH2PO4双水相萃取、Sephadex G-100凝胶过滤,获得了电泳纯的α-半乳糖苷酶,纯化倍数为36.4,总酶活力回收率达到70%。凝胶过滤表明:该酶表观分子质量为125kD,SDS-PAGE显示其分子质量为58.5kD。该酶水解对硝基苯-α-D-吡喃半乳糖苷的最适pH值为4.0,最适温度为65℃,表观Km值为0.915mmol/L,表观kcat/Km值为1.07×105L/(mol.s);水解蜜二糖的表观Km、kcat/Km值分别是21.0mmol/L、9.96×103L/(mol.s);对棉子糖只有微弱的水解作用。水解活性受多种金属离子的普遍抑制,其中Fe3+、Fe2+、Mn2+、Hg+等强烈抑制酶活力。该酶活力在pH1.5～8.2保持稳定,在60℃时保温60min,剩余酶活力达到了60%,是一种热稳定酸性α-半乳糖苷酶。After the end of the fermentation of soybean dregs by Aspergillus niger L.for α-galactosidase production,the fermentation broth supernatant was harvested as crude enzyme solution.Electrophoretically pure α-galactosidase was obtained from the crude enzyme solution by aqueous two-phase extraction in polyethylene glycol-6000-KH2PO4 system and Sephadex G-100 gel filtration,resulting in a purification factor of 36.4 and a total activity recovery of 70%.The molecular weight of the purified enzyme was approximately 125 kD as determined by Sephadex G-100 gel filtration chromatography and 58.5 kD as determined by SDS-PAGE,suggesting that the native enzyme was a homodimer.The optimum pH and temperature for the hydrolysis of p-nitrophenyl-α-D-galactopyranoside（pNPGal） by the enzyme were 4.0 and 65 ℃,respectively,and the apparent Km and kcat/Km were 0.915 mmol/L and 1.07 × 105 L/（mol？s）,respectively,whereas those for melibiose were 21.0 mmol/L and 9.96 × 103 L/（mol？s）,respectively.The enzyme activity was inhibited by some metal ions,especially Fe2＋,Mn2＋ and Hg＋,but was stable in the pH range of 1.5 to 8.2.After 60 min of exposure to 60 ℃,60% of the original activity was retained.Therefore,the enzyme has good acid and thermal stability.

关 键 词：双水相萃取 蜜二糖 对硝基苯-α-D-吡喃半乳糖苷 同二聚体 Α-半乳糖苷酶 

分 类 号：Q939.97[生物学—微生物学]