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

Abstract

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 alpha-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 alpha-l-arabinofuranosidase from H. insolens (Abf II), the GH51 alpha-l-arabinofuranosidase from M. giganteus (Abf III), a GH10 endo-1,4-beta-xylanase from H. insolens (Xyl III), and a GH3 beta-xylosidase from Trichoderma reesei (beta-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 degrees C. A 10%:40%:50% mixture of Abf II, Abf III, and beta-xyl released 56 mg of arabinose and 91 mg of xylose per gram of vinasse dry matter after 24 h at pH 5, 50 degrees 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 approximately 14, approximately 18, and approximately 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.

Multifunctional Properties of Glycoside Hydrolase Family 43 from Paenibacillus curdlanolyticus Strain B-6 Including Exo-β-xylosidase, Endo-xylanase, and α-L-Arabinofuranosidase Activities

“…However, the specificity of the arabinosyl position on AX was not examined. Arabinofuranosidases are categorised by the specificity of the arabinosyl positions, which are specific on α-1,2 and 1,3 monosubstitution (Bourgois et al 2007;Lagaert et al 2010), and only on α-1,3 of diarabinosyl substitution (van den Broek et al 2005, Sørensen et al 2007.…”

Improving Enzymes for Biomass Conversion: A Basic Research Perspective

“…Based on press releases, patents, talks at scientific meetings, and presentations scattered across the Internet, the companies are taking several approaches to reduce the cost of enzymes. These include screening new organisms (mainly fungi) for superior versions of current enzymes and for enzymes that act synergistically with existing commercial enzymes, continued strain improvement by conventional and molecular mutagenesis, enzyme improvement by protein engineering and directed evolution, and improved efficiencies in industrial-scale enzyme production [1,11,40,48,49,56].…”

Biochemical Characterization and Relative Expression Levels of Multiple Carbohydrate Esterases of the Xylanolytic Rumen Bacterium Prevotella ruminicola 23 Grown on an Ester-Enriched Substrate

“…A mixture of hemicellulases with an endoxylanase (Humicola insolens GH10 [49]), ␤-xylosidase (Trichoderma reesei GH3 [49]), and arabinofuranosidases (Meripilus giganteus GH51 and H. insolens GH43 [49]) (Novozymes, Bagsvaerd, Denmark) and the commercially available amyloglucosidase Spirizyme (Novozymes, Bagsvaerd, Denmark) were added to the suspension in order to degrade the most easily accessible starch and hemicellulose components (enzyme or protein [dry matter] in suspension was 0.025 and 0.05% [by weight] for each of the hemicellulases and amyloglucosidase, respectively). Two yeast strains (Nedalco, Bergen op Zoom, Netherlands) were used to consume the resulting arabinose, xylose, and glucose monosaccharides.…”