Engineering transglycosidase activity into a GH51 α-L-arabinofuranosidase
Engineering transglycosidase activity into a GH51 α-L-arabinofuranosidase
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DOI：
10.1016/j.nbt.2013.04.002
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发表时间：
2013-06-25
影响因子：
5.4
通讯作者：
O'Donohue, Michael J.
中科院分区：
文献类型：
--
作者：
Arab-Jaziri, Faten;Bissaro, Bastien;O'Donohue, Michael J.
将定向进化方法应用于木霉α-L-阿拉伯呋喃糖苷酶的糖基化反应，特别是以对硝基苯基-α-L-阿拉伯呋喃糖苷(供体)和α-D-二甲基吡喃糖苷(受体)为起始原料合成了苄基alpha-L-arabinofuranosyl-(1，2)-alpha-D-xylopyranoside，。目的是获得同时具有较低的水解性和较高的转糖基化活性的突变体，以利于目标二糖的稳定生产。简单显色筛选的实施最终提供了三种突变酶，它们的性质与所寻找的酶相对应。这些都表现出较低的水解性和保守或略有改善的转移活性，其中一个也表现出较低的转糖基化产物的二次水解度。对3~7个突变体的DNA序列分析和生化分析结合STD-核磁共振实验表明，3个突变体之间存在着不同的分子机制。
Directed evolution was applied to the alpha-L-arabinofuranosidase from Thermobacillus xylanilyticus to confer better transglycosylation ability, particularly for the synthesis of benzyl alpha-L-arabinofuranosyl-(1,2)-alpha-D-xylopyranoside, starting from p-nitrophenyl alpha-L-arabinofuranoside (donor) and benzyl alpha-D-xylopyranoside (acceptor). The aim was to obtain mutants displaying both lower hydrolytic and greater transglycosylation activities to favour the stable production of the target disaccharide. The implementation of a simple chromogenic screen ultimately provided three mutant enzymes whose properties correspond to those sought after. These all displayed lowered hydrolytic activity and conserved or slightly improved transfer activity, while one of them also displayed lowered secondary hydrolysis of the transglycosylation product. DNA sequence analysis of the mutants revealed between three and seven point mutations and biochemical analysis combined with STD-NMR experiments indicated that distinct molecular mechanisms were active among the three mutants.