title: Development of an improved variant of GH51 α-l-arabinofuranosidase from Pleurotus ostreatus by directed evolution.
abstract: In this study, the α-l-arabinofuranosidase from Pleurotus ostreatus was subjected to directed evolution by expressing a library of around 7000 randomly mutated variants by error prone Polymerase Chain Reaction. High-throughput screening of the library for the most active variants was performed by assaying activity towards p-nitrophenyl α-l-arabinofuranoside, and a variant with higher activity than the wild type was selected, purified and characterised. It exhibited a kcat of 7.3 ×1 0¹ ± 0.3 min⁻¹, around 3-fold higher than that of the wild type (2.2 × 10³ ± 0.2 min⁻¹), and a KM (0.54 ± 0.0 6mM) 30% lower than that of the wild type (0.70 ± 0.05 mM) towards this substrate. The mutant also showed improved catalytic properties towards pNP-β-d-glucopyranoside (kcat of 50.85 ± 0.21 min⁻¹ versus 11.0 ± 0.6 min⁻¹) and it was shown able to hydrolyse larch arabinogalactan which is not recognised by the wild type. The mutant was also more active than the wild type towards arabinoxylan and was able to hydrolyse arabinan, which was not transformed by the wild type. The ability of rPoAbf F435Y/Y446F to hydrolyse these insoluble substrates expands its potential for application also to hemicelluloses, which in some types of pretreatment are recovered in solid fractions.
authors.affiliation: Department of Chemical Sciences, University of Naples "Federico II", Complesso Universitario Monte S. Angelo, via Cintia, 4 80126 Naples, Italy.
authors.forename: Simona
authors.initials: S
authors.lastname: Giacobbe
authors.affiliation: CNRS, UMR7257, Aix-Marseille Universite, 163 Avenue de Luminy, Case 932, 13288 Marseille cedex 09, France.
authors.forename: Florence
authors.initials: F
authors.lastname: Vincent
authors.affiliation: Department of Chemical Sciences, University of Naples "Federico II", Complesso Universitario Monte S. Angelo, via Cintia, 4 80126 Naples, Italy. Electronic address: vfaraco@unina.it.
authors.forename: Vincenza
authors.initials: V
authors.lastname: Faraco
mesh_terms: D001089:Arabinose / Q000378:metabolism; D019020:Directed Molecular Evolution / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D015723:Gene Library; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D020076:Pleurotus / Q000201:enzymology*; D013696:Temperature
chemical_list: D050505:Mutant Proteins; D001089:Arabinose; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase