Hanne R. Sørensen

Enzymatic Hydrolysis of Wheat Arabinoxylan by a Recombinant “Minimal” Enzyme Cocktail Containing β‐Xylosidase and Novel endo‐1,4‐β‐Xylanase and α‐l‐Arabinofuranosidase Activities

This study describes the identification of the key enzyme activities required in a “minimal” enzyme cocktail able to catalyze hydrolysis of water‐soluble and water‐insoluble wheat arabinoxylan and whole vinasse, a fermentation effluent resulting from industrial ethanol manufacture from wheat. The optimal arabinose‐releasing and xylan‐depolymerizing enzyme activities were identified from data obtained when selected, recombinant enzymes were systematically supplemented to the different arabinoxylan substrates in mixtures; this examination revealed three novel α‐l‐arabinofuranosidase activities: (i) one GH51 enzyme from Meripilus giganteus and (ii) one GH51 enzyme from Humicola insolens, both able to catalyze arabinose release from singly substituted xylose; and (iii) one GH43 enzyme from H. insolens able to catalyze the release of arabinose from doubly substituted xylose. Treatment of water‐soluble and water‐insoluble wheat arabinoxylan with an enzyme cocktail containing a 20%:20%:20%:40% mixture and a 25%:25%:25%:25% mixture, respectively, of the GH43 α‐l‐arabinofuranosidase from H. insolens (Abf II), the GH51 α‐l‐arabinofuranosidase from M. giganteus (Abf III), a GH10 endo‐1,4‐β‐xylanase from H. insolens (Xyl III), and a GH3 β‐xylosidase from Trichoderma reesei (β‐xyl) released 322 mg of arabinose and 512 mg of xylose per gram of water‐soluble wheat arabinoxylan dry matter and 150 mg of arabinose and 266 mg of xylose per gram of water‐insoluble wheat arabinoxylan dry matter after 24 h at pH 5, 50 °C. A 10%:40%:50% mixture of Abf II, Abf III, and β‐xyl released 56 mg of arabinose and 91 mg of xylose per gram of vinasse dry matter after 24 h at pH 5, 50 °C. The optimal dosages of the “minimal” enzyme cocktails were determined to be 0.4, 0.3, and 0.2 g enzyme protein per kilogram of substrate dry matter for the water‐soluble wheat arabinoxylan, the water‐insoluble wheat arabinoxylan, and the vinasse, respectively. These enzyme protein dosage levels were ∼14, ∼18, and ∼27 times lower than the dosages used previously, when the same wheat arabinoxylan substrates were hydrolyzed with a combination of Ultraflo L and Celluclast 1.5 L, two commercially available enzyme preparations produced by H. insolens and T. reesei.

Optimization of Reaction Conditions for Enzymatic Viscosity Reduction and Hydrolysis of Wheat Arabinoxylan in an Industrial Ethanol Fermentation Residue

This study examined enzyme‐catalyzed viscosity reduction and evaluated the effects of substrate dry matter concentration on enzymatic degradation of arabinoxylan in a fermentation residue, “vinasse”, resulting from industrial ethanol manufacture on wheat. Enzymatic catalysis was accomplished with a 50:50 mixture of an enzyme preparation from Humicola insolens, Ultraflo L, and a cellulolytic enzyme preparation from Trichoderma reesei, Celluclast 1.5 L. This enzyme mixture was previously shown to exhibit a synergistic action on arabinoxylan degradation. The viscosity of vinasse decreased with increased enzyme dosage and treatment time at pH 5, 50 °C, 5 wt % vinasse dry matter. After 24 h of enzymatic treatment, 76–84%, 75–80%, and 43–47%, respectively, of the theoretically maximal arabinose, xylose, and glucose releases were achieved, indicating that the viscosity decrease was a result of enzyme‐catalyzed hydrolysis of arabinoxylan, β‐glucan, and cellulose. In designed response surface experiments, the optimal enzyme reaction conditions with respect to pH and temperature of the vinasse, the vinasse supernatant (mainly soluble material), and the vinasse sediment (mainly insoluble substances) varied from pH 5.2–6.4 and 41–49 °C for arabinose release and from pH 4.9–5.3 and 42–46 °C for xylose release. Even though only limited hydrolysis of the arabinoxylan in the vinasse sediment fraction was obtained, the results indicated that the same enzyme activities acted on the arabinoxylan in the different vinasse fractions irrespective of the state of solubility of the substrate material. The levels of liberated arabinose and xylose increased with increased dry matter concentration during enzymatic hydrolysis in the vinasse and the vinasse supernatant, but at the same time, increased substrate dry matter concentrations gave corresponding linear decreases in the hydrolytic efficiency as evaluated from levels of monosaccharide release per weight unit dry matter. The study thus documents that enzymatic arabinoxylan hydrolysis of the vinasse significantly decreases the vinasse viscosity and that a compromise in the dry matter must be found if enzymatic efficiency must be balanced with monosaccharide yields.

Characterization of oligosaccharides from industrial fermentation residues by matrix-assisted laser desorption/ionization, electro spray mass spectrometry, and gas chromatography mass spectrometry

We report here the preliminary characterization of oligosaccharides present in an enzyme-treated industrial fermentation residue using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), electrospray ion trap mass spectrometry (ESI-ITMS), and gas chromatography mass spectrometry (GC-MS). After sample cleaning with carbon graphite columns, analysis of oligosaccharides present in the sample using MALDI-TOF-MS resulted in identification of molecular ions representing sodiated hexose and pentose oligo/polysaccharides. The GC-MS analyses revealed that the signals observed in the mass spectrum for hexose oligomers represent linear structures, whereas the pentose oligomers were identified as arabinoxylans with a (1»4) linked Xylp backbone where the Xylp residues were either not substituted or singly substituted with Araf branching residues at positions C-2 or C-3 of the Xylp ring. Analyses by ESI-ITMS of the signals corresponding to arabinoxylan oligosaccharides with four and five monosaccharide residues showed the presence of isomeric structure differing in degree of branching and localization of the branched residue along the Xylp backbone.