嗜热栖热菌HB 8耐热α-葡萄糖苷酶的提纯和性质

PURIFICATION AND CHARACTERIZATION OF α-GLUCOSIDASE FROM AN EXTREME THERMOPHILE,THERMUS THERMOPHILUS HB 8

嗜热栖热菌 HB 8(Thermus thermophilus HB 8)的耐热α-葡萄糖苷酶(α-glucosidaseEC.3.2.1.20)经硫酸铵分步沉淀、DEAE-纤维素柱层析和垂直板制备凝胶电泳提纯,经盘状凝胶电泳鉴定为单一区带,比活提高17倍。酶作用最适温度80℃,最适 pH5.8,分子量67000,等电点 pI 为4.5。该酶能够水解对硝基酚-α-D-葡萄糖苷(PNPG)、蔗糖和麦芽糖,但不水解纤维二糖、蜜二糖、可溶性淀粉。酶作用于 PNPG 的米氏常数(K_m)为0.4mmol/L,最大反应速度 V_(max)为0.29umol·min^(-1)·mg^(-1)。金属离子 Mg^(2+)、Mn^(2+)、Ca^(2+)和 Ba^(2+)对酶有激活作用,Hg^(2+)和 Cu^(2+)有强烈抑制作用。酶表现出极好的热稳定性,在90℃保温10小时后,仍保留90%的原始酶活力,在95℃的失活半寿期(t_(1/2)为108分钟,经蛋白质侧链化学修饰研究表明,羧基和组氨酸残基为其表现活力所必需。

A highly thermostable α-glucosidase(EC.3.2.1.20)from an extreme thermophile,Thermus thermophilus HB 8,was purifiedto homogeneous by ammonium sulfate frac-tionation,DEAE-cellulose chromatographyand preparative slab gel electrophoresis.Theenzyne was purified 17 fold with 21% recoveryof activity.The enzyme had a molecular wei-ght of 67000 by SDS-PAGE.The isoelectricpoint was pH4.5 by IEF on PAG.The enzymehydrolized p-nitr ophenyl-α-glucoside(PN-PG),sucrose and maltose,but not cellobiose,melibiose and soluble starch.The k_m valuefor PNPG was 0.4mmol/L,the V_(max) was 0.29μmol.min^(-1).mg^(-1).The enzyme exhibited hi-gh thermostability.After incubation at 90℃for 10 h in 50 mmol/L acetate buffer pH5.8,the enzyme ratained 90% of its originalactivity.The half-live(t_(1/2))at 95℃ was108 min.The enzyme was activated by Mg^(2+),Mn^(2+),Ca(2+),Ba(2+)and strongly inhibited byHg^(2+), Cu^(2+).Modification of the enzyme byEDC or DEPC led to complete loss of activity,which suggests that carboxyl group(s)and hi-stidine residue(s)are essential for activity ofα-glucosidase.