Production and purification of an extracellular β-galactosidase from the Dutch elm disease fungusOphiostoma novo-ulmi

Abstract

The causal agents of Dutch elm disease, Ophiostoma ulmi (isolate H200) and Ophiostoma novo-ulmi (isolate CKT-11), secreted similar amounts of β-galactosidase in liquid shake cultures when grown on galacturonic acid or sodium pectate (1.45 ± 0.16 and 1.03 ± 0.24 nkat∙mL−1for O. ulmi, respectively, and 1.30 ± 0.08 and 1.28 ± 0.26 nkat∙mL−1for O. novo-ulmi, respectively). Rhamnose and pectin also stimulated secretion but to a lesser extent, whereas on glucose, enzyme activity was barely detectable (≤0.01 nkat∙mL−1). Ophiostoma novo-ulmi was shown by Q-Sepharose chromatography to form two β-galactosidases, named β-galactosidases I and II. In cultures grown on galacturonic acid β-galactosidase I accounted for approximately 75% of the total activity in the culture filtrate. β-Galactosidase I was further purified to apparent electrophoretic homogeneity by means of Sephacryl gel filtration chromatography, chromatofocusing, and Superdex75 gel filtration. The molecular mass of the enzyme was 135 kDa by SDS–PAGE and 123 kDa by gel filtration. Its isoelectric point, determined by chromatofocusing, was 4.9. The optimal pH for enzyme activity was 5.8 and the optimal temperature was 50 °C. The Kmvalues for p-nitrophenyl β-D-galactopyranoside and lactose were 7.52 and 14.23 mM, respectively, and the maximum velocities for these substrates were 1733 and 355 nkat∙mg protein−1, respectively. The Kivalue for D(−)-galactonic acid γ-lactone was 2.29 mM.Key words: Dutch elm disease, β-galactosidase, Ophiostoma ulmi, Ophiostoma novo-ulmi.