High ionic strength tolerance of pyranose oxidase from Trametes versicolor and its purification

The stability of pyranose oxidase (EC 1.1.3.10) from Trametes versicolor was studied in the presence of high ionic strengths of various salts. With the exception of nickel chloride, none of the other salts studied inhibited pyranose oxidase activity. This unusual high ionic strength tolerance was exploited to develop a high yield purification protocol. Fungal pellets were subjected to osmotic shock by washing in buffer containing 9% NaCl (I= 1.54 M), followed by buffer in the absence of salt. This gave a 2.7-fold increase in the amount of pyranose oxidase released in comparison with that using an isotonic solution. Optimal conditions for the extraction of pyranose oxidase were determined to be a combination of homogenisation together with sonication. The pyranose oxidase was purified using a two step protocol involving hydrophobic interaction chromatography followed by anion exchange chromatography. The improved protocol gave near total recovery of the enzyme at a specific activity of 18.4 U/mg protein. (C) 2002 Elsevier Science Ltd. All rights reserved.