Positional preferences of acetyl esterases from different CE families towards acetylated 4-O-methyl glucuronic acid-substituted xylo-oligosaccharides

Background: Acetylation of the xylan backbone restricts the hydrolysis of plant poly-and oligosaccharides by hemicellulolytic enzyme preparations to constituent monosaccharides. The positional preferences and deacetylation efficiencies of acetyl esterases from seven different carbohydrate esterase (CE) families towards different acetylated xylopyranosyl units (Xylp) - as present in 4-O-methyl-glucuronic acid (MeGlcA)-substituted xylo-oligosaccharides (AcUXOS) derived from Eucalyptus globulus - were monitored by H-1 NMR, using common conditions for biofuel production (pH 5.0, 50 degrees C). Results: Differences were observed regarding the hydrolysis of 2-O, 3-O, and 2,3-di-O acetylated Xylp and 3-O acetylated Xylp 2-O substituted with MeGlcA. The acetyl esterases tested could be categorized in three groups having activities towards (i) 2-O and 3-O acetylated Xylp, (ii) 2-O, 3-O, and 2,3-di-O acetylated Xylp, and (iii) 2-O, 3-O, and 2,3-di-O acetylated Xylp, as well as 3-O acetylated Xylp 2-O substituted with MeGlcA at the non-reducing end. A high deacetylation efficiency of up to 83% was observed for CE5 and CE1 acetyl esterases. Positional preferences were observed towards 2,3-di-O acetylated Xylp (TeCE1, AnCE5, and OsCE6) or 3-O acetylated Xylp (CtCE4). Conclusions: Different positional preferences, deacetylation efficiencies, and initial deacetylation rates towards 2-O, 3-O, and 2,3-di-O acetylated Xylp and 3-O acetylated Xylp 2-O substituted with MeGlcA were demonstrated for acetyl esterases from different CE families at pH 5.0 and 50 degrees C. The data allow the design of optimal, deacetylating hemicellulolytic enzyme mixtures for the hydrolysis of non-alkaline-pretreated bioenergy feedstocks.