Production of thermostable endo-1,5-α-l-arabinanase in Pichia pastoris for enzymatically releasing functional oligosaccharides from sugar beet pulp

Sugar beet pulp is an agricultural processing residue that is a rich source of the cell wall polysaccharide arabinan. Functional oligosaccharides, specifically feruloylated arabino-oligosaccharides (FAOs), can be isolated from sugar beet pulp through selective action by endo-arabinanase (glycoside hydrolase family 43). This study aimed to develop yeast (Pichia pastoris) as an efficient, eukaryotic platform to produce a thermophilic endo-1,5-α-L-arabinanase (TS-ABN) for extracting FAOs from sugar beet pulp. Recombinant TS-ABN was secreted into yeast culture medium at a yield of ~ 80 mg/L, and the protein exhibited specific enzyme activity, pH and temperature optimum, and thermostability comparable to those of the native enzyme. Treatment of sugar beet pulp with Pichia-secreted TS-ABN released FAOs recovered by hydrophobic chromatography at 1.52% (w/w). The isolated FAOs averaged seven arabinose residues per ferulic acid, and treatment of T84 human colon epithelial cells significantly increased expression of two key tight junction-related proteins-zonula occludens-1 and occludin-in a dose-dependent manner. This research establishes a biochemical platform for utilizing sugar beet pulp to produce value-added bioproducts with potential nutraceutical applications.