Abstract
BackgroundAcetylation 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 1H NMR, using common conditions for biofuel production (pH 5.0, 50°C).ResultsDifferences 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).ConclusionsDifferent 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°C. The data allow the design of optimal, deacetylating hemicellulolytic enzyme mixtures for the hydrolysis of non-alkaline-pretreated bioenergy feedstocks.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-014-0187-6) contains supplementary material, which is available to authorized users.
Highlights
Acetylation of the xylan backbone restricts the hydrolysis of plant poly- and oligosaccharides by hemicellulolytic enzyme preparations to constituent monosaccharides
The protein bands of 34 kDa, 34 kDa, 26 kDa, and 32 kDa matched with the molecular mass reported previously for CE5 classified acetyl esterase from Trichoderma reesei (TrCE5) (34 kDa) [14] and with the calculated molecular masses based on the protein sequences, which were 33.8 kDa (AnCE16), 25.8 kDa (AnCE5), and 32.5 kDa (TeCE1)
For the commercially available acetyl esterases, protein bands were observed at 37 kDa, 24 kDa, 24 kDa, and 34 kDa, respectively, and these matched with the calculated molecular masses based on the protein sequences, which were 36.4 kDa (CtCE2), 22.8 kDa (CtCE3), 22.8 kDa (CtCE4), and 34.9 kDa (OsCE6)
Summary
Acetylation of the xylan backbone restricts the hydrolysis of plant poly- and oligosaccharides by hemicellulolytic enzyme preparations to constituent monosaccharides. In only a few cases has the specificity towards different acetylated xylopyranosyl units (Xylp) been investigated on Neumüller et al Biotechnology for Biofuels (2015) 8:7 actual plant oligosaccharides [7,8], and even in those cases the deacetylation rates towards the differently acetylated Xylp were not reported. Knowledge of these rates is required to determine the positional preference of the acetyl esterases in the early stage of the reaction when many competing substrates are still present
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