2012 lam

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Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE

Characterization of an ethanol-tolerant 1,4-b-xylosidase produced by Pichia membranifaciens A.M. Romero, J.J. Mateo and S. Maicas Departament de Microbiologia i Ecologia, Universitat de Vale`ncia, Burjassot, Spain

Keywords 1,4-b-xylosidase, characterization, ethanol tolerant, Pichia membranifaciens, purification. Correspondence Sergi Maicas, Departament de Microbiologia i Ecologia, Universitat de Vale`ncia, Dr. Moliner 50, Burjassot E-46100, Spain. E-mail: sergi.maicas@uv.es

2012 ⁄ 0470: received 14 March 2012, revised 7 August 2012 and accepted 7 August 2012 doi:10.1111/j.1472-765X.2012.03297.x

Abstract Aims: The purification and biochemical properties of the 1,4-b-xylosidase of an oenological yeast were investigated. Methods and Results: An ethanol-tolerant 1,4-b-xylosidase was purified from cultures of a strain of Pichia membranifaciens grown on xylan at 28 C. The enzyme was purified by sequential chromatography on DEAE cellulose and Sephadex G-100. The relative molecular mass of the enzyme was determined to be 50 kDa by SDS-PAGE. The activity of 1,4-b-xylosidase was optimum at pH 6Æ0 and at 35 C. The activity had a Km of 0Æ48 ± 0Æ06 mmol l)1 and a Vmax of 7Æ4 ± 0Æ1 lmol min)1 mg)1 protein for p-nitrophenyl-b-d-xylopyranoside. Conclusions: The enzyme characteristics (pH and thermal stability, low inhibition rate by glucose and ethanol tolerance) make this enzyme a good candidate to be used in enzymatic production of xylose and improvement of hemicellulose saccharification for production of bioethanol. Significance and Impact of the Study: This study may be useful for assessing the ability of the 1,4-b-xylosidase from P. membranifaciens to be used in the bioethanol production process.

Introduction Pichia membranifaciens is a film-forming yeast causing off-aroma and flavour in table wine (Esteve et al. 2001). Other members of this genus have also been isolated from diverse sources: flowering plants, fruit skins, insect intestinal tracts, human tissue and faeces, dairy and baked food products, contaminated oil, wastewaters, tree exudates, salted foods and from the marine environment (El-Sharoud et al. 2009; Sinigaglia et al. 2010). This represents a wider range of habitats in nature compared with other common yeasts as Saccharomyces cerevisiae. Furthermore, Pichia sp. exhibits great diversity with regard to its natural habitat, growth morphology, metabolism, stress tolerance and antimicrobial properties (Walker 2011). Other potential biotechnological applications of Pichia isolates include environmental bioremediation, biopharmaceuticals and biofuels. b-1,4-Xylan is a heteroglycan with a backbone of b-(1 fi 4)-linked d-xylopyranose residues that can be substituted with l-arabinofuranose, d-glucuronic acid and ⁄ or 4-O-methyl-d-glucuronic acid (Puls and Schuseil 354

1993; Bhat and Hazlewood 2001). It constitutes the major component of hemicelluloses found in the cell walls of monocots and hard woods and represents one of the most abundant biomass resources. Recently, xylanolytic enzymes of microbial origin have received great attention because of their possible industrial applications for sustainable fuel ethanol production from xylan. Two key reactions proceed during hydrolysis of the xylan backbone; endo-1,4-b-xylanases (1,4-b-d-xylan xylanohydrolase) hydrolyse internal b-(1 fi 4)-xylosidic linkages in the insoluble xylan backbone to yield soluble xylooligosaccharides, while 1,4-b-xylosidases are exoglycosidases that cleave terminal xylose monomers from the nonreducing end of short-chain xylooligosaccharides (Polizeli et al. 2005). Additional enzyme activities, such as a-l-arabinofuranosidase, a-d-glucuronidase and acetyl xylan esterase, remove side-chain substituents. 1,4-b-Xylosidase is important in xylan degradation, considering that xylans are not completely hydrolysed by xylanases alone. Many micro-organisms can produce 1,4-b-xylosidase; however, only few yeast species and limited number of strains can produce it (Linden and Hahn-Hagerdal 1989). The use of

ª 2012 The Authors Letters in Applied Microbiology 55, 354–361 ª 2012 The Society for Applied Microbiology


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