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Title: Identification of the nucleophile catalytic residue of GH51 α-l-arabinofuranosidase from Pleurotus ostreatus

In this paper, the recombinant α-l-arabinofuranosidase from the fungus Pleurotus ostreatus (rPoAbf) was subjected to site-directed mutagenesis in order to identify the catalytic nucleophile residue. Based on bioinformatics and homology modelling analyses, E449 was revealed to be the potential nucleophilic residue. Thus, the mutant E449G of PoAbf was recombinantly expressed in Pichia pastoris and its recombinant expression level and reactivity were investigated in comparison to the wild-type. The design of a suitable set of hydrolysis experiments in the presence or absence of alcoholic arabinosyl acceptors and/or formate salts allowed to unambiguously identify the residue E449 as the nucleophile residue involved in the retaining mechanism of this GH51 arabinofuranosidase. 1H NMR analysis was applied for the identification of the products and the assignement of their anomeric configuration.
Authors:
 [1] ;  [2] ;  [3] ;  [2]
  1. Univ. of Naples Federico II (Italy); National Renewable Energy Lab. (NREL), Golden, CO (United States)
  2. Univ. of Naples Federico II (Italy)
  3. Aix-Marseille Univ. (France)
Publication Date:
OSTI Identifier:
1236038
Report Number(s):
NREL/JA--2700-65692
Journal ID: ISSN 2191-0855
Grant/Contract Number:
AC36-08GO28308
Type:
Accepted Manuscript
Journal Name:
AMB Express
Additional Journal Information:
Journal Volume: 5; Journal Issue: 1; Related Information: AMB Express; Journal ID: ISSN 2191-0855
Research Org:
NREL (National Renewable Energy Laboratory (NREL), Golden, CO (United States))
Sponsoring Org:
Ministero dell'Universita e della Ricerca Scientifica-Industrial Research Project
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANAYLYTICAL CHEMISTRY arabinofuranosidase; lignocellulose; site-directed mutagenesis