News & Notes Production, Purification, and Properties of an Endo-1,3--Glucanase from the Basidiomycete Agaricus bisporus Beatriz Gala ´n, Concepcio ´ n Garcı ´a Mendoza, Myriam Calonje, Monique Novaes-Ledieu Centro de Investigaciones Biolo ´gicas, CSIC, Vela ´zquez 144, 28006 Madrid, Spain Received: 17 June 1998 / Accepted: 24 September 1998 Abstract. Agaricus bisporus H 25 produced extracellular endo-1,3--glucanase when grown in a static culture at 25°C in a minimal synthetic medium supplemented with A. bisporus cell walls plus fructose. Endo-1,3--glucanase was purified 17.85-fold from 20-day-old culture filtrates by precipitation at 80% ammonium sulfate saturation, Sephadex G-75 gel filtration, and preparative PAGE followed by electroelu- tion. The purified enzyme yielded a single band in both native and SDS-polyacrylamide gels with a molecular mass of 32 kDa (SDS-PAGE) and 33.7 kDa (MALDI-MS), showing an isoelectric point of 3.7. The enzyme was active against -1,3- linkages and, to a lesser extent, against -1,6-, exhibiting an endohydrolytic mode of action and a glycoprotein nature. Significant activities of the endo-glucanase against laminarin and pustulan were observed between pH 4 and 5.5, and between 40° and 50°C for laminarin, and between 30° and 50°C for pustulan. The optimum pH and temperature were 4.5 and 45°C for both substrates. -D-Glucans are the main structural components of the cell walls of most fungi and are primarily responsible for the shape and rigidity of the walls. The production of -glucanases by fungi represents the cell wall autolytic potential for processes such as morphogenesis, turnover of cell wall components, and the survival of the cell under conditions of nutrient deprivation. Numerous fungi have been described as producers of -glucanases: yeasts [4], filamentous fungi [20, 22, 23, 25–29], and also the basidiomycete Agaricus bisporus, which has been reported as the producer of an extracellu- lar 1,4--glucanase [14, 21]. In the present study we describe the production, purification, and properties of a -glucanase from A. bisporus that digests laminarin and also pustulan, but to a lesser extent in an endohydrolytic mode of action. Organism, culture conditions, and enzyme production and evaluation. Fragments of commercial Agaricus bisporus fruiting bodies (strain H25) harvested at the button stage were cultivated on compost-agar (5% dehy- drated compost) for isolating vegetative mycelium. On the other hand, cell walls of A. bisporus fruiting bodies H25 (free of lamellae and basidiospores) were prepared by the method of Avella ´n et al. [1]. Agaricus bisporus vegetative mycelium was grown on Hanseler medium [9] modified by adding 0.1% (wt/vol) fructose plus 0.1% (wt/vol) A. bisporus cell walls as carbon source. Roux flasks containing 100 ml of culture medium were inocu- lated with A. bisporus mycelium and incubated statically at 25°C for 20 days. Culture fluids were filtered through Whatman 3MM paper in Millipore filtration apparatus, and the filtrate was extensively dialysed against water at 4°C. Protein concentrations were determined according to Bradford [3]. 1,3-- and 1,6--Glucanase activities were determined by measuring the amount of reducing sugars [16, 24] in 50 mM sodium acetate buffer pH 5.2 with 5 mg/ml of either laminarin (Koch-Light), periodate- oxidized laminarin [8], or pustulan (Calbiochem) at 40°C for 16 h. Activities (U) are expressed as μmol of glucose released h -1 , and specific activity as units of enzyme activity (mg protein) -1 . Both -glucosidase and exo-1,3- glucanase activities were determined by measuring the rate of hydrolysis of p-nitrophenyl--glucopyranoside (Sigma, 0.5 mg/ml) in sodium acetate buffer pH 5.2 at Correspondence to: C.G. Mendoza CURRENT MICROBIOLOGY Vol. 38 (1999), pp. 190–193 An International Journal  Springer-Verlag New York Inc. 1999