A new tool for studying the molecular architecture of the fungal cell wall: one-step purification of recombinant Trichoderma β-(1-6)-glucanase expressed in Pichia pastoris

• Published in: Biochimica et biophysica acta Vol. 1425; no. 2; pp. 419 - 424
• Main Authors: Bom, I.J., Dielbandhoesing, S.K., Harvey, K.N., Oomes, S.J.C.M., Klis, F.M., Brul, S.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 23.10.1998
• Subjects: beta-glucanase; biosynthesis; Cell Membrane; Cell Membrane - chemistry; cell wall components; chemistry; Enzyme purification; Enzyme Stability; enzymology; Fungal cell wall; genetics; Glycoside Hydrolases; Glycoside Hydrolases - biosynthesis; Glycoside Hydrolases - chemistry; Glycoside Hydrolases - isolation & purification; Heterogeneous protein expression; Hydrogen-Ion Concentration; isolation & purification; Mannoprotein; Pichia; Pichia - enzymology; Pichia - genetics; Recombinant Proteins; Recombinant Proteins - isolation & purification; Trichoderma; Trichoderma - enzymology; Trichoderma - genetics; β-(1-6)-Glucanase; Enzyme purification; β-(1-6)-Glucanase; Mannoprotein; Heterogeneous protein expression; Fungal cell wall
• ISSN: 0304-4165; 0006-3002; 1872-8006; 1878-2434
• DOI: 10.1016/S0304-4165(98)00096-8

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 β-(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 β-(1-6)-glucan is an important component of fungal cell walls, the easy availability of pure β-(1-6)-glucanase will highly facilitate studies of the molecular organization of the fungal cell wall.