Unité de Recherche en Biologie des Microorganismes, Biology Department, University of Namur, Belgium.
FEBS Open Bio. 2013 Nov 1;3:467-72. doi: 10.1016/j.fob.2013.10.009. eCollection 2013.
Endo-inulinase INU2 from Aspergillus ficuum belongs to glycosidase hydrolase family 32 (GH32) that degrades inulin into fructo oligosaccharides consisting mainly of inulotriose and inulotetraose. The 3D structure of INU2 was recently obtained (Pouyez et al., 2012, Biochimie, 94, 2423-2430). An enlarged cavity compared to exo-inulinase formed by the conserved motif W-M(I)-N-D(E)-P-N-G, the so-called loop 1 and the loop 4, was identified. In the present study we have characterized the importance of 12 residues situated around the enlarged cavity. These residues were mutated by site-directed mutagenesis. Comparative activity analysis was done by plate, spectrophotometric and thin-layer chromatography assay. Most of the mutants were less active than the wild-type enzyme. Most interestingly, mutant N42G differed in the size distribution of the FOS synthesized.
从曲霉（Aspergillus ficuum）中提取的内切菊粉酶 INU2 属于糖苷水解酶家族 32（GH32），可将菊粉降解为主要由菊三糖和菊四糖组成的低聚果糖。最近获得了 INU2 的三维结构（Pouyez 等人，2012 年，《生物化学》，94，2423-2430）。与由保守基序 W-M（I）-N-D（E）-P-N-G、所谓的环 1 和环 4 形成的外切菊粉酶相比，鉴定出了一个更大的腔。在本研究中，我们研究了位于扩大腔周围的 12 个残基的重要性。这些残基通过定点诱变发生突变。通过平板、分光光度法和薄层层析法进行比较活性分析。大多数突变体的活性均低于野生型酶。最有趣的是，突变体 N42G 在合成的 FOS 分布大小上有所不同。
