Asparagine 42 of the conserved endo-inulinase INU2 motif WMNDPN from Aspergillus ficuum plays a role in activity specificity

Autor: Isabelle Housen, Aurélie Mayard, Anne-Michèle Vandamme, Catherine Michaux
Jazyk: angličtina
Rok vydání: 2013
Předmět:
Zdroj: Vandamme, A-M, Michaux, C, Mayard, A & Housen, I 2013, ' Asparagine 42 of the conserved endo-inulinase INU2 motif WMNDPN from Aspergillus ficuum plays a role in activity specificity ', FEBS open bio, vol. 3, pp. 467-72 . https://doi.org/10.1016/j.fob.2013.10.009
FEBS Open Bio
ISSN: 2211-5463
Popis: 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.
Highlights • Endo-inulinase INU2 degrades inulin into fructo oligosaccharides. • 12 residues around the catalytic pockets of INU2 enzyme were determined. • These residues were mutated to either a G or A residue. • The activity has been tested by plate, spectrophotometric and TLC assays. • One mutation, N42G, which changes the specificity of activity, has been identified.
Databáze: OpenAIRE
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