Designed enzyme preparations for the hydrolysis of corn silage polysaccharides

This thesis describes the design of hemicellulolytic enzyme preparations with high activity towards the rather recalcitrant xylan present in corn silage, a major biogas feedstock. Also, recalcitrance factors towards the enzymatic conversion of xylans, varying in type and level of substitution, are addressed

Acetyl Xylan Esterase Axe1 (T. reesei, Carbohydrate Esterase Family 5) Supplemented to a (Hemi)cellulolytic Preparation Enhances Degradation of Recalcitrant Corn Silage Polysaccharides

The increase in hydrolytic activity towards corn silage water unextractable solids by supplementation of acetyl xylan esterase 1 (T. reesei, TrAxe1), belonging to arbohydrate esterase (CE) family 5, to an A. niger / T. emersonii enzyme preparation is presented. TrAxe1 was cloned and expressed in A niger. The hydrolytic activity of TrAxe1 was analysed with a p-nitrophenyl-acetate standard assay and by the measurement of the acetic acid released from neutral and acidic eucalyptus xylo-oligosaccharides. The activity obtained for TrAxe1 was compared to acetyl xylan esterases belonging to CE families 1, 5 and 16 from A. niger. High activities were obtained for the CE5 classified acetyl xylan esterases from T. reesei and A. niger. The data obtained confirm the suggested increase in hydrolytic activity by supplementation of CE5 classified acetyl xylan esterases to the (hemi)cellulolytic A. niger / T. emersonii preparation (Neumüller et al. 2014b

Trichoderma longibrachiatum acetyl xylan esterase 1 enhances hemicellulolytic preparations to degrade corn silage polysaccharides

Supplementation of a Trichoderma longibrachiatum preparation to an industrial Aspergillus niger/Talaromyces emersonii enzyme mixture demonstrated synergy for the saccharification of corn silage water-unextractable solids (WUS). Sub-fractions of the crude T. longibrachiatum preparation obtained after chromatography were analyzed regarding their hydrolytic activity. An acetyl xylan esterase 1 [Axe1, carbohydrate esterase (CE) family 5]-enriched sub-fraction closely mimicked the hydrolytic gain as obtained by supplementation of the complete, crude enzyme mixture (increase of 50%, 62% and 29% for Xyl, Ara and Glc, respectively). The acetic acid released from model polysaccharides (WUS) and oligosaccharides [neutral (AcXOS) and acidic (AcUXOS) xylo-oligosaccharides] by Axe1 was two and up to six times higher compared to the acetic acid released by acetyl xylan esterase A (AxeA, CE 1). Characterization of Axe1 treated AcXOS and AcUXOS revealed deacetylation of oligosaccharides that were not deacetylated by AxeA or the A. niger/T. emersonii preparation

Synergistic action of enzyme preparations towards recalcitrant corn silage polysaccharides

Corn silage, its water unextractable solids (WUS) and enzyme recalcitrant solids (ErCS) and an industrial corn silage-based anaerobic fermentation residue (AFR) represent corn substrates with different levels of recalcitrance. Compositional analysis reveals different levels of arabinoxylan substitution for WUS, ErCS and AFR, being most pronounced regarding acetic acid, glucuronic acid- and arabinose content. By screening for enzymatic degradation of WUS, ErCS and AFR, enzyme preparations exhibiting high conversion rates were identified. Furthermore significant synergistic effects were detected by blending Aspergillus niger/Talaromyces emersonii culture filtrates with various enzymes. These findings clearly highlight a necessity for a combinatorial use of enzyme preparations towards substrates with high recalcitrance characteristics to reach high degrees of degradation. Enzyme blends were identified, outperforming the individual commercial preparations. These enzyme preparations provide a basis for new, designed enzyme mixtures for corn polysaccharide degradation as a source of necessary, accessory enzyme activities

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 1H NMR, using common conditions for biofuel production (pH 5.0, 50°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°C. The data allow the design of optimal, deacetylating hemicellulolytic enzyme mixtures for the hydrolysis of non-alkaline-pretreated bioenergy feedstocks

Fast and Robust Method To Determine Phenoyl and Acetyl Esters of Polysaccharides by Quantitative 1H NMR

The acetyl (AcE), feruloyl (FE), and p-coumaroyl (pCE) ester contents of different cereal and grass polysaccharides were determined by a quantitative 1H NMR-based method. The repeatability and the robustness of the method were demonstrated by analyzing different plant polysaccharide preparations. Good sensitivity and selectivity for AcE, FE, and pCE were observed. Moreover, an optimized and easy sample preparation allowed for simultaneous quantification of AcE, FE, and pCE. The method is suitable for high-throughput analysis, and it is a good alternative for currently used analytical procedures. A comparison of the method presented to a conventional HPLC-based method showed that the results obtained are in good agreement, whereas the combination of the optimized sample preparation and analysis by the 1H NMR-based methodology results in significantly reduced analysis time