Intracellular removal of acetyl, feruloyl and p-coumaroyl decorations on arabinoxylo-oligosaccharides imported from lignocellulosic biomass degradation by Ruminiclostridium cellulolyticum.

BACKGROUND: Xylans are polysaccharides that are naturally abundant in agricultural by-products, such as cereal brans and straws. Microbial degradation of arabinoxylan is facilitated by extracellular esterases that remove acetyl, feruloyl, and p-coumaroyl decorations. The bacterium Ruminiclostridium cellulolyticum possesses the Xua (xylan utilization associated) system, which is responsible for importing and intracellularly degrading arabinoxylodextrins. This system includes an arabinoxylodextrins importer, four intracellular glycosyl hydrolases, and two intracellular esterases, XuaH and XuaJ which are encoded at the end of the gene cluster. RESULTS: Genetic studies demonstrate that the genes xuaH and xuaJ are part of the xua operon, which covers xuaABCDD'EFGHIJ. This operon forms a functional unit regulated by the two-component system XuaSR. The esterases encoded at the end of the cluster have been further characterized: XuaJ is an acetyl esterase active on model substrates, while XuaH is a xylan feruloyl- and p-coumaryl-esterase. This latter is active on oligosaccharides derived from wheat bran and wheat straw. Modelling studies indicate that XuaH has the potential to interact with arabinoxylobiose acylated with mono- or diferulate. The intracellular esterases XuaH and XuaJ are believed to allow the cell to fully utilize the complex acylated arabinoxylo-dextrins imported into the cytoplasm during growth on wheat bran or straw. CONCLUSIONS: This study reports for the first time that a cytosolic feruloyl esterase is part of an intracellular arabinoxylo-dextrin import and degradation system, completing its cytosolic enzymatic arsenal. This system represents a new pathway for processing highly-decorated arabinoxylo-dextrins, which could provide a competitive advantage to the cell and may have interesting biotechnological applications.

Biochemical characterization of an esterase from Thermobifida fusca YX with acetyl xylan esterase activity.

BACKGROUND: Esterases (EC 3.1.1.X) are enzymes that catalyze the hydrolysis ester bonds. These enzymes have large potential for diverse applications in fine industries, particularly in pharmaceuticals, cosmetics, and bioethanol production. METHODS AND RESULTS: In this study, a gene encoding an esterase from Thermobifida fusca YX (TfEst) was successfully cloned, and its product was overexpressed in Escherichia coli and purified using affinity chromatography. The TfEst kinetic assay revealed catalytic efficiencies of 0.58 s-1 mM-1, 1.09 s-1 mM-1, and 0.062 s-1 mM-1 against p-Nitrophenyl acetate, p-Nitrophenyl butyrate, and 1-naphthyl acetate substrates, respectively. Furthermore, TfEst also exhibited activity in a pH range from 6.0 to 10.0, with maximum activity at pH 8.0. The enzyme demonstrated a half-life of 20 min at 70 °C. Notably, TfEst displayed acetyl xylan esterase activity as evidenced by the acetylated xylan assay. The structural prediction of TfEst using AlphaFold indicated that has an α/ß-hydrolase fold, which is consistent with other esterases. CONCLUSIONS: The enzyme stability over a broad pH range and its activity at elevated temperatures make it an appealing candidate for industrial processes. Overall, TfEst emerges as a promising enzymatic tool with significant implications for the advancement of biotechnology and biofuels industries.