Short Communication High-level production of thermotolerant b-xylosidase of Aspergillus sp. BCC125 in Pichia pastoris: Characterization and its application in ethanol production Sriwan Wongwisansri ⇑ , Peerada Promdonkoy, Panida Matetaviparee, Niran Roongsawang, Lily Eurwilaichitr, Sutipa Tanapongpipat Bioresources Technology Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, 113 Thailand Science Park, Phahonyothin Road, Klong Nueng, Klong Luang, Pathum Thani 12120, Thailand highlights " Thermotolerant b-xylosidase from fungal origin was produced to highest yield. " Enzyme efficiency (K cat /K m ) toward p-nitrophenyl-b-D-xylopyranoside was superior. " Simple enzyme production with high yield from Pichia pastoris was described. " Synergism of xylanolytic activity contributed by b-xylosidase was demonstrated. " Application in enzymatic conversion of xylan to ethanol was demonstrated. article info Article history: Received 21 September 2012 Received in revised form 26 November 2012 Accepted 26 November 2012 Available online 5 December 2012 Keywords: b-xylosidase Xylan hydrolysis Pichia pastoris Pichia stipitis Ethanol fermentation abstract A gene coding for thermotolerant b-xylosidase from Aspergillus sp. BCC125 was characterized. The recom- binant enzyme was expressed in methylotrophic yeast Pichia pastoris KM71 and especially high yield of secreted enzyme was obtained. b-xylosidase possessed high enzyme efficiency (K cat /K m = 198.8 mM 1 s 1 ) toward pNP-b-D-xylopyranoside (pNPbX) with optimal temperature and pH for activity of 60 °C and pH 4.0–5.0, respectively. The identified b-xylosidase showed clear synergism with previously identified xylanase for hydrolysis of xylan in vitro as well as simultaneous saccharification and fermen- tation process (SSF) in vivo with Pichia stipitis. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction The conversion of plant biomass, which comprises mostly of cellulose and hemicellulose, into its sugar components by enzy- matic treatment is considered a sustainable technology with lit- tle effects on the environment. Hemicellulose contains b-1,4 xylan as a major component, with its main chain made of xylose monomer. The hydrolysis of xylan requires the cooperative ac- tion of xylanase (EC 3.2.1.8) and b-xylosidase (EC 3.2.1.37). Xylanase catalyzes the endohydrolysis of the 1,4- b-D-xylosidic linkage randomly and gives rise to xylooligosaccharide and xy- lose monomer, while b-xylosidase completes the hydrolysis by cleaving off xylose monomer from the non-reducing end of xylooligosaccharide (Knob et al., 2010). Previously, the produc- tion of recombinant xylanase (XylB) by heterologous expression in the methylotrophic yeast Pichia pastoris was reported (Ruan- glek et al., 2007). To obtain xylan-degrading enzymes available for studying lignocellulosic biomass degradation, b-xylosidase was sought from fungal collections in Thailand’s BIOTEC Culture Collection (BCC), and the Aspergillus sp. BCC125 strain was iden- tified as a strong producer of b-xylosidase. In this study, the re- combinant b-xylosidase from Aspergillus sp. BCC125 was produced to very high yield using P. pastoris expression system and enzyme kinetic properties were characterized. The synergis- tic action of recombinant b-xylosidase and xylanase for xylan hydrolysis was demonstrated, as well as the synergism of both enzymes in ethanol fermentation in the presence of xylose fer- menting yeast, P. stipitis. 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.11.117 ⇑ Corresponding author. Tel.: +66 2 564 6700x3474; fax: +66 2 564 6707. E-mail address: sriwan@biotec.or.th (S. Wongwisansri). Bioresource Technology 132 (2013) 410–413 Contents lists available at SciVerse ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech