Fierobe H P, Stoffer B B, Frandsen T P, Svensson B
Department of Chemistry, Carlsberg Laboratory, Copenhagen Valby, Denmark.
Biochemistry. 1996 Jul 2;35(26):8696-704. doi: 10.1021/bi960241c.
Rational protein engineering based on three-dimensional structure, sequence alignment, and previous mutational analysis served to increase thermostability and modulate bond-type specificity in glucoamylase from Aspergillus awamori. The single free cysteine, Cys320, became disulfide bonded in the Ala246 --> Cys mutant, thus enhancing T50 by 4 degrees C to 73 degrees C. Compared to wild-type, Ala246 --> Cys was roughly twice as active at 66 degrees C, but half as active at 45 degrees C. The alternative, elimination of the thiol group in Cys320 --> Ala, barely improved thermostability or altered activity. Secondly, to acquire exceptionally high specificity toward alpha-1,6 glucosidic linkages, characteristic of Hormoconis resinae glucoamylase, two short sequential mutants, Val181 --> Thr/Asn182 --> Tyr/Gly183 --> Ala(L3 glucoamylase) and Pro307 --> Ala/Thr310 --> Val/Tyr312 --> Met/Asn313 --> Gly (L5 glucoamylase), were made. These homologue mutants are located in the (alpha/alpha)6-fold of the catalytic domain in segments that connect alpha-helices 5 and 6 and alpha-helices 9 and 10, respectively. The kinetics of malto- and isomaltooligosaccharides hydrolysis clearly demonstrated that combination of the mutations in L3L5 compensated adverse effects of the single replacements in L3 or L5 glucoamylases to yield wild-type or higher activity. On alpha-1,4-linked substrates, typically Km increased 2-fold for L3, and Kcat decreased up to 15-fold for L5 glucoamylase. In contrast, on alpha-1,6-linked substrates L3 showed both a 2-fold increase in Km and a 3-fold decrease in kcat, while L5 GA caused a similar kcat reduction, but up to 9-fold increase in Km. L3L5 glucoamylase had remarkably low Km for isomaltotriose through isomaltoheptaose and elevated kcat on isomaltose, resulting in an approximately 2-fold improved catalytic efficiency (kcat/Km). Rational loop replacement thus proved powerful in achieving variants with enhanced properties of a highly evolved enzyme.
基于三维结构、序列比对和先前的突变分析进行合理的蛋白质工程改造，以提高泡盛曲霉葡萄糖淀粉酶的热稳定性并调节键型特异性。单个游离半胱氨酸Cys320在Ala246→Cys突变体中形成二硫键，从而使T50提高4℃至73℃。与野生型相比，Ala246→Cys在66℃时的活性约为野生型的两倍，但在45℃时的活性仅为野生型的一半。另一种方法，即消除Cys320→Ala中的巯基，几乎没有提高热稳定性或改变活性。其次，为了获得对树脂状 Hormoconis 葡萄糖淀粉酶特有的α-1,6糖苷键具有极高的特异性，构建了两个短序列突变体，Val181→Thr/Asn182→Tyr/Gly183→Ala（L3葡萄糖淀粉酶）和Pro307→Ala/Thr310→Val/Tyr312→Met/Asn313→Gly（L5葡萄糖淀粉酶）。这些同源突变体分别位于催化结构域的(α/α)6折叠中连接α螺旋5和6以及α螺旋9和10的片段中。麦芽寡糖和异麦芽寡糖水解的动力学清楚地表明，L3L5中的突变组合补偿了L3或L5葡萄糖淀粉酶中单个替换的不利影响，从而产生野生型或更高的活性。对于α-1,4连接的底物，通常L3的Km增加2倍，L5葡萄糖淀粉酶的Kcat降低高达15倍。相反，对于α-1,6连接的底物，L3的Km增加2倍，kcat降低3倍，而L5葡萄糖淀粉酶导致类似的kcat降低，但Km增加高达9倍。L3L5葡萄糖淀粉酶对异麦芽三糖至异麦芽七糖的Km非常低，对异麦芽糖的kcat升高，导致催化效率（kcat/Km）提高约2倍。因此，合理的环替换在实现具有高度进化酶增强特性的变体方面被证明是有效的。
