BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS Molecular characterization and heterologous expression of a Xanthophyllomyces dendrorhous α-glucosidase with potential for prebiotics production Patricia Gutiérrez-Alonso 1 & María Gimeno-Pérez 1 & Mercedes Ramírez-Escudero 2 & Francisco J. Plou 3 & Julia Sanz-Aparicio 2 & María Fernández-Lobato 1 Received: 4 August 2015 /Revised: 9 November 2015 /Accepted: 11 November 2015 /Published online: 28 November 2015 # Springer-Verlag Berlin Heidelberg 2015 Abstract Basidiomycetous yeast Xanthophyllomyces dendrorhous expresses an α-glucosidase with strong transglycosylation activity producing prebiotic sugars such as panose and an unusual tetrasaccharides mixture including α– (1 –6) bonds as major products, which makes it of biotechnological interest. Initial analysis pointed to a homodimeric protein of 60 kDa subunit as responsible for this activity. In this study, the gene Xd-AlphaGlu was characterized. The 4131-bp-long gene is interrupted by 13 short introns and encodes a protein of 990 amino acids (Xd-AlphaGlu). The N-terminal se- quence of the previously detected 60 kDa protein resides in this larger protein at residues 583–602. Functionality of the gene was proved in Saccharomyces cerevisiae, which produced a protein of about 130 kDa containing Xd-AlphaGlu sequences. All properties of the heterolo- gously expressed protein, including thermal and pH profiles, activity on different substrates, and ability to produce prebiotic sugars were similar to that of the α-glucosidase produced in X. dendrorhous. No activity was detected in S. cerevisiae containing exclusively the 1256-bp from gene Xd-AlphaGlu that would encode syn- thesis of the 60 kDa protein previously detected. Data were compatible with an active monomeric α-glucosidase of 990 amino acids and an inactive hydrolysis product of 60 kDa. Protein Xd-AlphaGlu contained most of the elements charac- teristic of α-glucosidases included in the glycoside hydrolases family GH31 and its structural model based on the homolo- gous human maltase-glucoamylase was obtained. Remark- ably, the Xd-AlphaGlu C-terminal domain presents an unusu- ally long 115-residue insertion that could be involved in this enzyme’s activity against long-size substrates such as maltoheptaose and soluble starch. Keywords Xanthophyllomyces dendrorhous . Alpha-glucosidase . GH31 family . Maltooligosaccharides . Panose Introduction Glycosidases are widely used as biocatalysts in the biotechno- logical industries using oligo- and polysaccharides as raw ma- terial. α-Glucosidases (EC. 3.2.1.20) are a particularly impor- tant subset of these enzymes that hydrolyze α-glycosidic link- ages from the non-reducing end of different size substrates. They show major roles in biology ranging from breakdown of polysaccharides to biosynthesis of glycoproteins (Chiba 1997; Melo et al. 2006). According to the Carbohydrate-Active En- zymes database (CAZy) glycoside hydrolase (GH) classifica- tion, the α-glucosidases are included into two major families, GH13 and GH31, and, to a lesser extent, in families GH4, GH63, and GH97 (Lovering et al. 2005). Family GH13 in- cludes important enzymes such as α -amylases and Electronic supplementary material The online version of this article (doi:10.1007/s00253-015-7171-3) contains supplementary material, which is available to authorized users. * María Fernández-Lobato mfernandez@cbm.csic.es 1 Centro de Biología Molecular Severo Ochoa, Departamento de Biología Molecular (CSIC-UAM), Nicolás Cabrera 1. Universidad Autónoma Madrid, 28049 Madrid, Spain 2 Departamento de Cristalografía y Biología Estructural, Instituto de Química-Física Rocasolano, CSIC, Serrano 119, 28006 Madrid, Spain 3 Instituto de Catálisis y Petroleoquímica, CSIC, Marie Curie 2, 28049 Madrid, Spain Appl Microbiol Biotechnol (2016) 100:3125–3135 DOI 10.1007/s00253-015-7171-3