Christel Thea Jørgensen

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.

ENZYME CATALYSED SYNTHESIS IN AMBIENT TEMPERATURE IONIC LIQUIDS

The activity of three different lipases, a glycosidase and a protease in ionic liquids has been studied. Ambient temperature ionic liquids are a new class of solvents that are nonvolatile and nonflammable and thus an interesting alternative to classical organic solvents. Monitoring the synthesis of a simple ester, all lipases were found to exhibit both excellent activity and stability in the non-polar ionic liquid 1-butyl-3-methylimidazohum hexaflurophosphate ([bmin][PF6], 1). Furthermore, β-galactosidase from E. coli and the Subtilisin protease SavinaseTM were both found to exhibit a hydrolytic activity in a 50% aqueous solution of the water-miscible ionic liquid 1-butyl-3-methyhmidazoUum tetra-fluoroborate ([bmin][BF4], 2) comparable to the activity observed in 50% aqueous solutions of ethanol and acetonitrile.

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.

Investigating the binding of beta-1,4-galactan to Bacillus licheniformis beta-1,4-galactanase by crystallography and computational modeling.

Microbial β‐1,4‐galactanases are glycoside hydrolases belonging to family 53, which degrade galactan and arabinogalactan side chains in the hairy regions of pectin, a major plant cell wall component. They belong to the larger clan GH‐A of glycoside hydrolases, which cover many different poly‐ and oligosaccharidase specificities. Crystallographic complexes of Bacillus licheniformis β‐1,4‐galactanase and its inactive nucleophile mutant have been obtained with methyl‐β(1→4)‐galactotetraoside, providing, for the first time, information on substrate binding to the aglycone side of the β‐1,4‐galactanase substrate binding groove. Using the experimentally determined subsites as a starting point, a β(1→4)‐galactononaose was built into the structure and subjected to molecular dynamics simulations giving further insight into the residues involved in the binding of the polysaccharide from subsite −4 to +5. In particular, this analysis newly identified a conserved β‐turn, which contributes to subsites −2 to +3. This β‐turn is unique to family 53 β‐1,4‐galactanases among all clan GH‐A families that have been structurally characterized and thus might be a structural signature for endo‐β‐1,4‐galactanase specificity. Proteins 2009.