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

<jats:title>Abstract</jats:title> <jats:p> 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‐ <jats:sc>l</jats:sc> ‐arabinofuranosidase activities: (i) one GH51 enzyme from <jats:italic>Meripilus giganteus</jats:italic> and (ii) one GH51 enzyme from <jats:italic>Humicola insolens</jats:italic> , both able to catalyze arabinose release from singly substituted xylose; and (iii) one GH43 enzyme from <jats:italic>H. insolens</jats:italic> 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‐ <jats:sc>l</jats:sc> ‐arabinofuranosidase from <jats:italic>H. insolens</jats:italic> (Abf II), the GH51 α‐ <jats:sc>l</jats:sc> ‐arabinofuranosidase from <jats:italic>M. giganteus</jats:italic> (Abf III), a GH10 <jats:italic>endo</jats:italic> ‐1,4‐β‐xylanase from <jats:italic>H. insolens</jats:italic> (Xyl III), and a GH3 β‐xylosidase from <jats:italic>Trichoderma reesei</jats:italic> (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 <jats:italic>H. insolens</jats:italic> and <jats:italic>T. reesei</jats:italic> . </jats:p>