Expression, purification and characterization of proteins with unknown function homologous to a novel carbohydrate esterase

Abstract

Recently, FjoAcXE, a novel acetyl xylan esterase was characterised to deacetylate internal xylose residues of glucuronoxylan at all positions displaying low susceptibility to steric hindrance of the substrates. In this thesis eight unknown proteins homologous to FjoAcXE that shared from 47 to 98% sequence similarity to FjoAcXE were studied (K392DRAFT_2214, FF52_18088, EW79DRAFT_2436, T426DRAFT_00687, WP_035683315.1, AlkimDRAFT_0871, ZPR_3026 and CHSO_3300). Six of selected proteins (K392DRAFT_2214, FF52_18088, EW79DRAFT_2436, T426DRAFT_00687, WP_035683315.1, AlkimDRAFT_0871) were successfully expressed and purified. Additionally, pH optimum and temperature stability were determined for the studied proteins. In this work FjoAcXE- homologues showed ability to deacetylate simple synthetic substrates (pNP-acetate and 4MU-acetate). Further characterisation demonstrated that the enzymes were capable of deacetylating per-acetylated xylo-oligosaccharide mixture and branched feruloylated xylo-oligosaccharides from corn fibre and thereby revealed acetyl xylan esterase activity. Following comparison of FjoAcXE and six homologous to it proteins showed analogous level of acetic acid release from xylo-oligosaccharide mixtures.