Abstract
BackgroundConceptually, multi-functional enzymes are attractive because in the case of complex polymer hydrolysis having two or more activities defined by a single enzyme offers the possibility of synergy and reduced enzyme cocktail complexity. Nevertheless, multi-functional enzymes are quite rare and are generally multi-domain assemblies with each activity being defined by a separate protein module. However, a recent report described a GH51 arabinofuranosidase from Alicyclobacillus sp. A4 that displays both α-l-arabinofuranosidase and β-d-xylanase activities, which are defined by a single active site. Following on from this, we describe in detail another multi-functional GH51 arabinofuranosidase and discuss the molecular basis of multifunctionality.ResultsTHSAbf is a GH51 α-l-arabinofuranosidase. Characterization revealed that THSAbf is active up to 75 °C, stable at 60 °C and active over a broad pH range (4–7). THSAbf preferentially releases para-nitrophenyl from the l-arabinofuranoside (kcat/KM = 1050 s−1 mM−1) and to some extent from d-galactofuranoside and d-xyloside. THSAbf is active on 4-O-methylglucuronoxylans from birch and beechwood (10.8 and 14.4 U mg−1, respectively) and on sugar beet branched and linear arabinans (1.1 ± 0.24 and 1.8 ± 0.1 U mg−1). Further investigation revealed that like the Alicyclobacillus sp. A4 α-l-arabinofuranosidase, THSAbf also displays endo-xylanase activity, cleaving β-1,4 bonds in heteroxylans. The optimum pH for THASAbf activity is substrate dependent, but ablation of the catalytic nucleophile caused a general loss of activity, indicating the involvement of a single active center. Combining the α-l-arabinofuranosidase with a GH11 endoxylanase did not procure synergy. The molecular modeling of THSAbf revealed a wide active site cleft and clues to explain multi-functionality.ConclusionThe discovery of single active site, multifunctional enzymes such as THSAbf opens up exciting avenues for enzyme engineering and the development of new biomass-degrading cocktails that could considerably reduce enzyme production costs.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0550-x) contains supplementary material, which is available to authorized users.
Highlights
Multi-functional enzymes are attractive because in the case of complex polymer hydrolysis having two or more activities defined by a single enzyme offers the possibility of synergy and reduced enzyme cocktail complexity
The core enzyme activities produced by xylanolytic microorganisms are endo-β1,4-d-xylanases (EC 3.2.1.8) and β-1,4-d-xylosidases (EC 3.2.1.37), which act on β-1,4 bonds that link d-xylosyl moieties in polysaccharides and oligosaccharides, and α-l-arabinofuranosidases (EC 3.2.1.55) that hydrolyze the 1,2 and 1,3 glycosidic that link α-l-arabinofuranoyl side chains to the main chain, the actual list of enzyme activities is much longer
THSAbf was most active on pNP-Araf, with its specific activity on this substrate being 27- and 2300-fold higher than that on pNP-Galf and pNP-Xylp, respectively (Table 2). These results reveal that like the majority of GH51 glycoside hydrolases [27], THSAbf can best be described as an Abf and that the subsite −1 has a clear preference for the furanose conformation
Summary
Multi-functional enzymes are attractive because in the case of complex polymer hydrolysis having two or more activities defined by a single enzyme offers the possibility of synergy and reduced enzyme cocktail complexity. Heteroxylans (commonly referred to as xylans) composed of β-1,4-linked d-xylosyl subunits constitute an important class of polysaccharides that are widespread throughout the plant kingdom, notably in flowering plants [1]. In both perennial and annual grassy species, the main chains of xylans are mainly decorated with l-arabinofuranosyl moieties. One consequence of the structural complexity of heteroxylans is the diversity of the enzymes that are required to break them down into sugars This is illustrated by the complex enzymatic arsenals deployed by microbial plant pathogens, plant saprophytes and gut microbiota [3]. The core enzyme activities produced by xylanolytic microorganisms are endo-β1,4-d-xylanases (EC 3.2.1.8) and β-1,4-d-xylosidases (EC 3.2.1.37), which act on β-1,4 bonds that link d-xylosyl moieties in polysaccharides and oligosaccharides, and α-l-arabinofuranosidases (EC 3.2.1.55) that hydrolyze the 1,2 and 1,3 glycosidic that link α-l-arabinofuranoyl side chains to the main chain, the actual list of enzyme activities is much longer
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