Production, purification, and properties of an endo-1,3-beta-glucanase from the basidiomycete Agaricus bisporus

Agaricus bisporus H 25 produced extracellular endo-1,3-beta-glucanase when grown in a static culture at 25 degrees C in a minimal synthetic medium sup plemented with A. bisporus cell walls plus fructose. Endo-1,3-beta-glucanas e was purified 17.85-fold from 20-day-old culture filtrates by precipitatio n at 80% ammonium sulfate saturation, Sephadex G-75 gel filtration, and pre parative PAGE followed by electroelution. The purified enzyme yielded a sin gle band in both native and SDS-polyacrylamide gels with a molecular mass o f 32 kDa (SDS-PAGE) and 33.7 kDa (MALDI-MS), showing an isoelectric point o f 3.7. The enzyme was active against beta-1,3- linkages and, to a lesser ex tent, against beta-1,6-, exhibiting an endohydrolytic mode of action and a glycoprotein nature. Significant activities of the endo-glucanase against l aminarin and pustulan were observed between pH 4 and 5.5, and between 40 de grees and 50 degrees C for laminarin, and between 30 degrees and 50 degrees C for pustulan. The optimum pH and temperature were 4.5 and 45 degrees C f or both substrates.