Abstract

Rhamnogalacturonan acetylesterases (RAEs) catalyze the deacetylation of rhamnogalacturonan. Penicillium purpurogenum is a fungus which grows with sugar beet pulp (SBP) wich is composed, in part, by esterified rhamnogalacturonan. The aim of this work is the heterologous expression of a RAE from Penicillium purpurogenum and to characterize the recombinant enzyme. Penicillium purpurogenum was grown with SBP. Partially purified culture supernatant was loaded on a zymogram. An area active on methylumbelliferyl-acetate (MUA) was analyzed by mass spectrometry. The peptides obtained matched a hypothetical RAE (RAEA). RAEA coding sequence was heterologously expressed in Pichia pastoris. The recombinant enzyme was purified. SDS-PAGE showed a molecular mass of 30kDa. RAEA is active toward: MUA, indoxyl acetate. fluorescein diacetate and p-nitrophenyl acetate (Km= 1.63 mM). It is not active toward p-nitrophenyl derivatives of ferulate, decanoate, palmitate and dodecanoate. Bioinformatic analysis allowed prediction of the typical catalytic triad of serine esterases (ser-his-asp). Distinctive motifs of the SGNH-hydrolase family were also detected. In conclusion, RAEA is a rhamnogalacturonan acetylesterase secreted by Penicillium purpurogenum during sugar beet pulp degradation. It belongs to the SGNH-hydrolases family. It is the second fungal RAEA biochemically characterized, thus highlighting the novelty of this work.