A new tool for studying the molecular architecture of the fungal cell wall : one-step puri¢cation of recombinant Trichoderma L-(1-6)-glucanase expressed in Pichia pastoris I.J. Bom a , S.K. Dielbandhoesing a , K.N. Harvey b , S.J.C.M. Oomes a , F.M. Klis c , S. Brul a; * a Unilever Research Laboratories, Olivier van Noortlaan 120, 3133 AT Vlaardingen, The Netherlands b Dept. of Molecular and Cell Biology, Institute of Medical Sciences, University of Aberdeen, Forresterhill, Aberdeen, AB25 2ZD, UK c Institute of Molecular Cell Biology, University of Amsterdam BioCenter Amsterdam, Kruislaan 318, 1098 SM Amsterdam, The Netherlands Received 27 March 1998; revised 15 June 1998; accepted 4 August 1998 Abstract The fungal cell wall is a supramolecular network of glycoproteins and polysaccharides. Its analysis is seriously hampered by the lack of easily available hydrolytic enzymes in a pure form. Here we describe a simple and efficient purification procedure of a recombinant L-(1-6)-glucanase from Trichoderma harzianum expressed in Pichia pastoris. Transformed cells efficiently secreted the enzyme into the induction medium. We purified the enzyme using a one-step method based on hydrophobic interaction chromatography. The yield was 80%. SDS-PAGE of the purified enzyme revealed a single band with an apparent molecular mass of 43 kDa. The isoelectric point of the enzyme was 5.8, and it showed maximal enzyme activity and stability at pH 5.0. As L-(1-6)-glucan is an important component of fungal cell walls, the easy availability of pure L-(1-6)- glucanase will highly facilitate studies of the molecular organization of the fungal cell wall. ß 1998 Elsevier Science B.V. All rights reserved. Keywords : Heterogeneous protein expression ; Enzyme puri¢cation ; L-(1-6)-Glucanase ; Fungal cell wall ; Mannoprotein 1. Introduction The cell wall of fungi is a supramolecular structure that o¡ers strength to the cells and protects them against mechanical damage. Although the separate components of the cell wall of fungi have been studied extensively, the way they are interconnected and form a cell wall is still largely unknown [1]. Recently, Brul et al. [2] presented immunological evi- dence that L-(1-6)-glucosylated cell wall proteins are wide-spread in yeast and ¢lamentous fungi, raising the question how these proteins are precisely linked to the cell wall frame work. An important require- ment for elucidating the molecular architecture of the cell wall is the easy availability of pure enzymes that can be used to isolate and purify speci¢c cell wall cross linkages [1,3,4]. Here we focus on a L-(1-6)- glucanase from Trichoderma harzianum [5]. We chose this organism, because it is a mycoparasitic fungus, making it likely that the enzyme will be e¡ective towards fungal cell walls [6^8]. For easy expression 0304-4165 / 98 / $ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved. PII:S0304-4165(98)00096-8 * Corresponding author. Biochimica et Biophysica Acta 1425 (1998) 419^424