Secretion, purification and characterisation of a recombinant Volvariella volvacea endoglucanase expressed in the yeast Pichia pastoris

• Published in: Enzyme and microbial technology 2002-10, Vol.31 (5), p.621-626
• Main Authors: Ding, Shao-jun, Ge, Wei, Buswell, John A
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Biology of microorganisms of confirmed or potential industrial interest; Biotechnology; Endoglucanase; Fundamental and applied biological sciences. Psychology; Miscellaneous; Mission oriented research; Pichia pastoris; Recombinant enzyme; Volvariella volvacea
• ISSN: 0141-0229; 1879-0909
• DOI: 10.1016/S0141-0229(02)00168-0

An endo-β-1,4-glucanase (EG1) from the edible straw mushroom, Volvariella volvacea, has been expressed in Pichia pastoris and the catalytic activity of the recombinant enzyme confirmed. Secretion was obtained using Saccharomyces cerevisiae α-factor, and a two-step purification protocol yielded EG1 in pure form. Recombinant EG1 had a molecular mass (45 kDa) slightly higher than the native enzyme (42 kDa) and an isoelectric point of 6.2. Recombinant and native EG1 also exhibited similar properties with respect to pH (pH 7.5) and temperature optima (55 °C), temperature and pH stability, substrate specificity, specific activities towards carboxymethylcellulose (344 and 324 U mg −1, respectively) and sensitivity to various metal ions, chelating agents and detergents. The P. pastoris system represents an attractive means of generating large quantities of endoglucanase for both research and industrial purposes.