A novel GH43 α-l-arabinofuranosidase from Humicola insolens: mode of action and synergy with GH51 α-l-arabinofuranosidases on wheat arabinoxylan

Abstract

A novel alpha-L-arabinofuranosidase (alpha-AraF) belonging to glycoside hydrolase (GH) family 43 was cloned from Humicola insolens and expressed in Aspergillus oryzae. (1)H-NMR analysis revealed that the novel GH43 enzyme selectively hydrolysed (1-->3)-alpha-L-arabinofuranosyl residues of doubly substituted xylopyranosyl residues in arabinoxylan and in arabinoxylan-derived oligosaccharides. The optimal activity of the cloned enzyme was at pH 6.7 and 53 degrees C. Two other novel alpha-L-arabinofuranosidases (alpha-AraFs), both belonging to GH family 51, were cloned from H. insolens and from the white-rot basidiomycete Meripilus giganteus. Both GH51 enzymes catalysed removal of (1-->2) and (1-->3)-alpha-L-arabinofuranosyl residues from singly substituted xylopyranosyls in arabinoxylan; the highest arabinose yields were obtained with the M. giganteus enzyme. Combinations (50:50) of the GH43 alpha-AraF from H. insolens and the GH51 alpha-AraFs from either M. giganteus or H. insolens resulted in a synergistic increase in arabinose release from water-soluble wheat arabinoxylan in extended reactions at pH 6 and 40 degrees C. This synergistic interaction between GH43 and GH51 alpha-AraFs was also evident when a GH43 alpha-AraF from a Bifidobacterium sp. was supplemented in combination with either of the GH51 enzymes. The synergistic effect is presumed to be a result of the GH51 alpha-AraFs being able to catalyse the removal of single-sitting (1-->2)-alpha-L- arabinofuranosyls that resulted after the GH43 enzyme had catalysed the removal of (1-->3)-alpha-L-arabinofuranosyl residues on doubly substituted xylopyranosyls in the wheat arabinoxylan.

Introducing endo-xylanase activity into an exo-acting arabinofuranosidase that targets side chains

“…By contrast, backbone sugars, decorated at both O2 and O3 with Araf units, are particularly recalcitrant to enzymatic attack; currently only two arabinofuranosidases, defined as AXHd3s, are known to cleave these linkages (3,4) (Fig. 1).…”

“…Single Araf substitutions that decorate Xylp residues are removed by a myriad of arabinofuranosidases (defined as AXHm2, 3). By contrast, backbone sugars, decorated at both O2 and O3 with Araf units, are particularly recalcitrant to enzymatic attack; currently only two arabinofuranosidases, defined as AXHd3s, are known to cleave these linkages (3,4) (Fig.…”

“…The activity displayed by AXHd3s is of biotechnological significance. Indeed, the AXHd3 from Humicola insolens, designated hereafter as HiAXHd3, is a component of the Novozyme product Ultraflo L, which is used in the brewing industry (3).…”

The Biochemistry and Structural Biology of Plant Cell Wall Deconstruction

“…Each of these families contains at least one structural representative, with the exception of GH62 and GH115 (http://www.cazy.org). GH43 enzymes may display the highest level of substrate diversity, exemplified by the activity of two arabinofuranosidases from this family that remove the O3 side chain from Xyl residues that are decorated at both O2 and O3 with Araf (van den Broek et al, 2005;Sorensen et al, 2006). The crystal structure of this enzyme (H.J.…”

Crystallization and preliminary X-ray crystallographic analysis of a novel α-L-arabinofuranosidase (CtGH43) fromClostridium thermocellumATCC 27405

“…The latest classification based on amino-acid sequences, primary-structure similarities and hydrophobic cluster analysis classifies -l-arabinofuranosidases ( -l-Araf) into five glycoside hydrolase (GH) families, i.e. GH30, GH43, GH51, GH54 and GH62 (Zhou et al, 2012;Cartmell et al, 2011;Sørensen et al, 2006;Guais et al, 2010;Hashimoto et al, 2011). Although all GH43 family members belong to clan GH-F and have a fivefoldpropeller core architecture, they have different activities (see the CAZy website, http://www.cazy.org; Lombard et al, 2013).…”