Please cite this article in press as: L.C. Cintra, et al., Int. J. Biol. Macromol. (2017), http://dx.doi.org/10.1016/j.ijbiomac.2017.07.039 ARTICLE IN PRESS G Model BIOMAC-7847; No. of Pages 10 International Journal of Biological Macromolecules xxx (2017) xxx–xxx Contents lists available at ScienceDirect International Journal of Biological Macromolecules j ourna l h o mepa ge: www.elsevier.com/locate/ijbiomac Characterization of a recombinant xylose tolerant -xylosidase from Humicola grisea var. thermoidea and its use in sugarcane bagasse hydrolysis Lorena Cardoso Cintra a,c , Amanda Gregorim Fernandes a , Izadora Cristina Moreira de Oliveira b , Saulo J. Linhares Siqueira b,d , Ilítia Ganaê Oliveira Costa b , Francieli Colussi b , Rosália Santos Amorim Jesuíno b , Cirano José Ulhoa a,b,∗ , Fabrícia Paula de Faria b a Department of Cellular Biology, University of Brasília, Brasília, DF, Brazil b Department of Biochemistry and Molecular Biology, Federal University of Goiás, Goiânia, Goiás, Brazil c Department of Veterinary Pathology, School of Veterinary and Animal Science, Federal University of Goiás, Goiânia, Goiás, Brazil d State University of Goiás, Palmeiras de Goiás, Goiás, Brazil a r t i c l e i n f o Article history: Received 8 December 2016 Received in revised form 7 April 2017 Accepted 6 July 2017 Available online xxx Keywords: Humicola grisea var. thermoidea -xylosidase Xylose tolerant Sinergism Sugarcane bagasse a b s t r a c t One full-length -xylosidase gene (hxylA) was identified from the Humicola grisea var. thermoidea genome and the cDNA was successfully expressed by Pichia pastoris SMD1168. An optimization of enzyme pro- duction was carried out, and methanol was found to be the most important parameter. The purified enzyme was characterized and showed the optimal conditions for the highest activity at pH 7.0 and 50 ◦ C, being thermostable by maintaining 41% of its activity after 12 h incubated at 50 ◦ C. HXYLA is a bifunctional enzyme; it showed both -xylosidase and -arabinfuranosidase activities. The K m and V max values were 1.3 mM and 39.1 U/mg, respectively, against 4-nitrophenyl -xylopyranoside. HXYLA showed a relatively strong tolerance to xylose with high K i value of 603 mM, with the xylose being a non-competitive inhibitor. HXYLA was successfully used simultaneously and sequentially with an endo- xylanase for analysis of synergism in the degradation of commercial xylans. Furthermore, commercial cellulases supplementation with HXYLA during sugarcane bagasse hydrolysis increased hydrolysis in 29%. HXYLA is distinguished from other -xylosidases by the attractive characteristics for industrial applica- tions such as thermostability, high tolerance xylose and saccharification of biomass by convert xylan into fementable monosaccharides and improve cellulose hydrolysis. © 2017 Elsevier B.V. All rights reserved. 1. Introduction Plant biomass represents the most abundant renewable energy source found in nature and it is mainly composed by lignocellulosic materials. Lignocellulose is composed of cellulose, hemicellulose, pectin, proteins and lignin. Xylan is the main component of hemi- cellulose and is the second most abundant biomass resource in nature after cellulose [1]. Xylan is composed of a backbone of -(1,4)-linked d-xylopyranose residues with the presence of var- ious substitutions in the side chains such as l-arabinofuranose, 4-O-methyl-glucuronic acids and acetyl groups. Xylan-rich agricul- ∗ Corresponding author at: Department of Biochemistry and Molecular Biology, Federal University of Goiás, Goiânia, Go, Brazil. E-mail address: ulhoa@ufg.br (C.J. Ulhoa). turals waste can be converted into xylose by enzymatic hydrolysis and xylose has been used for bioethanol and xylitol production. Additionally, xylan can be converted into food additives and other economically important products [2]. Due its heterogeneity and complexity, the complete degrada- tion of xylan requires the synergistic action of several enzymes termed the xylanolytic system [3]. The xylanolytic system consist of: endoxylanases (endo-1,4--xylanases), that release xylo-oligossacharides since catalyse random cleavage of internal bonds of the xylan chain; and -xylosidases, that release xylose monomers from these xylo-oligosaccharides and xylobiose. - xylosidases and endoxylanases act in synergy, since endoxylanases generate more reducing ends for -xylosidases to act upon. Also, -xylosidases play an important role in the degradation of xylan by removing the final product that can inhibit xylanses, thereby limiting the hydrolysis of xylan [3]. Moreover, other accessory http://dx.doi.org/10.1016/j.ijbiomac.2017.07.039 0141-8130/© 2017 Elsevier B.V. All rights reserved.