1046-5928/$ - see front matter  2006 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2006.05.011 Expression and puriWcation of dalcochinase, a -glucosidase from Dalbergia cochinchinensis Pierre, in yeast and bacterial hosts Prachumporn Toonkool a , Pornphimon Metheenukul b , Penporn Sujiwattanarat a , Patcharee Paiboon a , Nusra Tongtubtim a , Mariena Ketudat-Cairns c , James Ketudat-Cairns b , Jisnuson Svasti d,¤ a Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand b School of Biochemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand c School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand d Center for Protein Structure and Function and Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand Received 5 December 2005, and in revised form 19 April 2006 Available online 27 May 2006 Abstract The coding sequence of the mature dalcochinase, a -glucosidase from Dalbergia cochinchinensis Pierre, was cloned and expressed in various systems. Expression in Escherichia coli resulted in an insoluble protein, which could be made soluble by co-expression with bacte- rial chaperonin GroESL. However, the enzyme had no activity. Recombinant expression in Pichia pastoris and Saccharomyces cerevisiae yielded an active enzyme. Dalcochinase was expressed under methanol induction in P. pastoris, since this was much more eYcient than constitutive expression in P. pastoris or in S. cerevisiae. Addition of 0.5% casamino acids to the culture medium stabilized the pH of the culture and increased the protein yield by 3- to 5-folds. Insertion of a polyhistidine-tag either after the N-terminal  factor signal sequence or at the C-terminus failed to assist in puriWcation by immobilized metal-ion aYnity chromatography (IMAC) due to post-translational processing at both termini. A new construct of dalcochinase with an N-terminal truncation following the propeptide and eight histidine residues enabled its puriWcation by IMAC, following hydrophobic interaction chromatography. The puriWed recombinant dalcochinase was apparently composed of diVerently post-translationally modiWed forms, but had kinetic properties and pH and temperature optima comparable to natural dalcochinase. The procedures reported here overcome the limitation in enzyme supply from natural sources, and allow further studies on structure–function relationships in this enzyme.  2006 Elsevier Inc. All rights reserved. Keywords: Dalcochinase; -Glucosidase; Thai rosewood; Pichia pastoris; Optimized expression -Glucosidases (E.C. 3.2.1.21) represent a group of ubiquitously expressed, hydrolytic enzymes, which cata- lyze the hydrolysis of -O-glucosidic linkages between - D-glucose and an aglycone or another sugar [1]. -Gluco- sidases exhibit similar speciWcity for a -glucoside sub- strate, but distinct speci Wcities for the aglycone linked to the glucosyl group [1], suggesting their diverse biological functions. In plant physiology, -glucosidases are impli- cated in growth regulation, stress response, cellobiose degradation, ligniWcation, and defence [2]. Human -glu- cosidase hydrolyses glucosylceramide in lysosomes, and its deWciency leads to Gaucher’s disease [3]. In cellulolytic organisms, such as fungi and bacteria, -glucosidase is part of the cellulase complex, which breaks down cellulose to glucose [4]. Previous studies on -glucosidases have focused on the identiWcation of catalytic residues and catalytic mecha- nisms [5–10]. -Glucosidases are classiWed as retaining enzymes since their product (-D-glucose) retains the same anomeric conWguration as the substrate (a -D-glucoside) * Corresponding author. Fax: +66 2 201 5843. E-mail address: scjsv@mahidol.ac.th (J. Svasti).