The gene encoding an ␣-L-arabinofuranosidase from Thermobacillus xylanilyticus D3, AbfD3, was isolated. Characterization of the purified recombinant ␣-L-arabinofuranosidase produced in Escherichia coli revealed that it is highly stable with respect to both temperature (up to 90°C) and pH (stable in the pH range 4 to 12). On the basis of amino acid sequence similarities, this 56,071-Da enzyme could be assigned to family 51 of the glycosyl hydrolase classification system. However, substrate specificity analysis revealed that AbfD3, unlike the majority of F51 members, displays high activity in the presence of polysaccharides.Microorganisms employ a wide variety of enzymes to degrade hemicellulosic material. Backbone-degrading endoxylanases and ␤-xylosidases are the principal enzymes, but numerous side chain-cleaving enzymes, such as ␣-L-arabinofuranosidases (8, 12, 33), ␣-glucuronidases, acetylxylan esterases, and phenolic acid esterases, are also important. One substituent of xylan, L-arabinose, is present in significant amounts in wheat bran and straw in the form of arabinoxylans. Hydrolysis of such important agricultural by-products using a endo-␤(1,4)-xylanase has identified substituting L-arabinose as a potential barrier for xylanase action (20), and indeed, a synergistic effect between the activities of an ␣-L-arabinofuranosidase and a xylanase has been previously reported (1). Despite the obvious potential role for ␣-L-arabinofuranosidases in the industrial bioconversion of plant material, most of the known enzymes would be unsuitable. Indeed, in addition to their lack of robustness (thermostability and pH tolerance), most ␣-L-arabinofuranosidases exhibit a narrow substrate specificity range (2, 10), which limits their action towards either oligomeric substrates (13,16,19,21) or polymeric substrates (11,12). Our work on a novel thermophilic bacterium, Thermobacillus xylanilyticus, has led to the identification of several hemicellulase-encoding genes (3, 4, 7), including one for an ␣-L-arabinofuranosidase, the products of which may be suitable as biological catalysts for industrial processes (24,25).Isolation and characterization of the ␣-L-arabinofuranosidase-encoding gene, abfD3. Genetic analysis of ϳ9-kb genomic DNA segment revealed the presence of three open reading frames in the same strand (EMBL database accession number Y16849). Of these, one (1,488 bp) encodes a 56-kDa (496-amino-acid) protein which was identified by a database enquiry as a putative family 51 ␣-L-arabinofuranosidase. Comparison of the sequence of this protein, AbfD3, with members of family 51 (F-51) of the glycosyl hydrolase classification system (9) and the creation of a phylogenetic tree revealed that this enzyme is localized within a distinct phylogenetic cluster which contains three other ␣-L-arabinofuranosidases from taxonomically related bacterial sources (Bacillus subtilis [14], Clostridium stercorarium [28], and Bacillus stearothermophilus [6]) (Fig. 1).Expression and purification of recombinant AbfD3. The insertion of the abf...

The alpha-L-arabinofuranosidase D3 from Thermobacillus xylanilyticus is an arabinoxylan-debranching enzyme which belongs to family 51 of the glycosyl hydrolase classification. Previous studies have indicated that members of this family are retaining enzymes and may form part of the 4/7 superfamily of glycosyl hydrolases. To investigate the active site of alpha-L-arabinofuranosidase D3, we have used sequence alignment, site-directed mutagenesis and kinetic analyses. Likewise, we have shown that Glu(28), Glu(176) and Glu(298) are important for catalytic activity. Kinetic data obtained for the mutant Glu(176)-->Gln, combined with the results of chemical rescue using the mutant Glu(176)-->Ala, have shown that Glu(176) is the acid-base residue. Moreover, NMR analysis of the arabinosyl-azide adduct, which was produced by chemical rescue of the mutant Glu(176)-->Ala, indicated that alpha-L-arabinofuranosidase D3 hydrolyses glycosidic bonds with retention of the anomeric configuration. The results of similar chemical rescue studies using other mutant enzymes suggest that Glu(298) might be the catalytic nucleophile and that Glu(28) is a third member of a catalytic triad which may be responsible for modulating the ionization state of the acid-base and implicated in substrate fixation. Overall, these findings support the hypothesis that alpha-L-arabinofuranosidase D3 belongs to the 4/7 superfamily and provide the first experimental evidence concerning the catalytic apparatus of a family 51 arabinofuranosidase.

Starting from gold chips, we have tailor-made three surfaces by the self-assembly monolayer technique: one entirely hydrophobic, one hydrophobic with dispersed carboxyl groups, and one hydrophilic, containing hydroxyl groups. Rhizomucor miehei lipase has been adsorbed to the hydrophobic and the hydrophilic surfaces and covalently bound to the surface containing carboxyl groups. The adsorption of two substrates-capric acid (decanoic acid) and monocaprin-on the lipase-covered surfaces was monitored by the surface plasmon resonance (SPR) technique. Biocatalysis was also performed in the SPR instrument by circulating a solution of the substrate, dissolved in an 85:15 water-glycerol mixture at a(w) = 0.81, through the instrument, thus exposing the capric acid or the monocaprin to the lipase-covered surfaces. The product composition was found to depend on the type of surface used. Lipase adsorbed at the hydrophilic surface favored hydrolysis, and capric acid was the main product formed when monocaprin was used as substrate. Lipase adsorbed at a hydrophobic surface and, in particular, lipase covalently bound to a hydrophobic surface favored condensation. More dicaprin than capric acid was formed in experiments with monocaprin as the substrate. Reactions performed outside the SPR instrument showed that small amounts of triglyceride were also formed under these conditions. We believe that this work constitutes the first example of the SPR instrument being used for in-situ biotransformation.

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