BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS
EcXyl43 β-xylosidase: molecular modeling, activity on natural
and artificial substrates, and synergism with endoxylanases
for lignocellulose deconstruction
Ornella M. Ontañon
Rubén Marrero Díaz de Villegas
Florencia E. Piccinni
Paola M. Talia
María L. Cerutti
Received: 14 March 2018 /Revised: 10 May 2018 / Accepted: 15 May 2018
Springer-Verlag GmbH Germany, part of Springer Nature 2018
Biomass hydrolysis constitutes a bottleneck for the biotransformation of lignocellulosic residues into bioethanol and high-value
products. The efficient deconstruction of polysaccharides to fermentable sugars requires multiple enzymes acting concertedly.
GH43 β-xylosidases are among the most interesting enzymes involved in hemicellulose deconstruction into xylose. In this work,
the structural and functional properties of β-xylosidase EcXyl43 from Enterobacter sp. were thoroughly characterized.
Molecular modeling suggested a 3D structure formed by a conserved N-terminal catalytic domain linked to an ancillary C-
terminal domain. Both domains resulted essential for enzymatic activity, and the role of critical residues, from the catalytic and
the ancillary modules, was confirmed by mutagenesis. EcXyl43 presented β-xylosidase activity towards natural and artificial
substrates while arabinofuranosidase activity was only detected on nitrophenyl α-L-arabinofuranoside (pNPA). It hydrolyzed
xylobiose and purified xylooligosaccharides (XOS), up to degree of polymerization 6, with higher activity towards longer XOS.
Low levels of activity on commercial xylan were also observed, mainly on the soluble fraction. The addition of EcXyl43 to GH10
and GH11 endoxylanases increased the release of xylose from xylan and pre-treated wheat straw. Additionally, EcXyl43
exhibited high efficiency and thermal stability under its optimal conditions (40 °C, pH 6.5), with a half-life of 58 h. Therefore,
this enzyme could be a suitable additive for hemicellulases in long-term hydrolysis reactions. Because of its moderate inhibition
by monomeric sugars but its high inhibition by ethanol, EcXyl43 could be particularly more useful in separate hydrolysis and
fermentation (SHF) than in simultaneous saccharification and co-fermentation (SSCF) or consolidated bioprocessing (CBP).
The search for sustainable replacements of fossil fuels has
focused the attention on the conversion of non-food lignocel-
lulosic biomass into second-generation fuels along with
higher value compounds, emerging the concept of biorefinery.
Lignocellulosic feedstocks for these biorefineries include res-
idues from agriculture, agro-industries, or wood and pulp and
paper industries, rich in carbohydrate polymers (cellulose and
hemicellulose) and polyphenols (lignin) together with other
less abundant compounds, such as pectins (Jørgensen and
Pinelo 2017). The complex arrangement of these molecules
makes biomass highly recalcitrant, and therefore, the efficient
enzymatic hydrolysis of polymers into monomeric sugars still
remains the major challenge for current bioconversion tech-
nologies (Diogo et al. 2014).
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00253-018-9138-7) contains supplementary
material, which is available to authorized users.
* Eleonora Campos
Instituto de Biotecnología, CICVyA, Instituto Nacional de
Tecnología Agropecuaria (INTA), Dr. N. Repetto y Los Reseros s/n,
Hurlingham (1686), Buenos Aires, Argentina
Consejo Nacional de Investigaciones Científicas y Técnicas
(CONICET), Buenos Aires, Argentina
Instituto de Suelos, CIRN, Instituto Nacional de Tecnología
Agropecuaria (INTA), Buenos Aires, Argentina
Fundación Instituto Leloir, IIBBA-CONICET, Avenida Patricias
Argentinas 435, C1405BWE Buenos Aires, Argentina
Applied Microbiology and Biotechnology