Abstract
In this study, the recombinant α-L-arabinofuranosidase from the fungus Pleurotus ostreatus (rPoAbf) was subjected to site-directed mutagenesis with the aim of elucidating the role of glycosylation on the properties of the enzyme at the level of S160 residue. As a matter of fact, previous mass spectral analyses had led to the localization of a single O-glycosylation at this site. Recombinant expression and characterization of the rPoAbf mutant S160G was therefore performed. It was shown that the catalytic properties are slightly changed by the mutation, with a more evident modification of the Kcat and KM toward the synthetic substrate pN-glucopyranoside. More importantly, the mutation negatively affected the stability of the enzyme at various pHs and temperatures. Circular dichroism (CD) analyses showed a minimum at 210 nm for wild-type (wt) rPoAbf, typical of the beta-sheets structure, whereas this minimum is shifted for rPoAbf S160G, suggesting the presence of an unfolded structure. A similar behavior was revealed when wt rPoAbf was enzymatically deglycosylated. CD structural analyses of both the site-directed mutant and the enzymatically deglycosylated wild-type enzyme indicate a role of the glycosylation at the S160 residue in rPoAbf secondary structure stability.