Catalytic function of a newly purified exo-β-d-glucosaminidase from the entomopathogenic fungus Paecilomyces lilacinus

An entomopathogenic fungus, Paecilomyces lilacinus, was found to grow on chitosanase-detecting plates. Besides an endo-chitosanase, an exo-β-d-glucosaminidase was purified by cation-exchange chromatography from this microorganism cultivated in M9 minimal media containing 0.5% chitosan as the sole carbon source. The molecular weight of the enzyme is 95 kDa; the optimum pH and temperature for activity are 6.0 and 45 °C, respectively. The purified exo-β-d-GlcNase promotes the hydrolysis of 95% deacetylated chitosan from its non-reducing end and liberates 2-amino-2-deoxy-d-glucopyranose (GlcN) as the sole product; however, 2-acetamido-2-deoxy-d-glucopyranose (GlcNAc) was not detected when chitin was used as the substrate. The cleavage pattern confirmed using real-time mass spectrometry shows that exo-β-d-glucosaminidase cleaves the glycosidic bonds between GlcN–GlcN and GlcN–GlcNAc but not between GlcNAc–GlcN or GlcNAc–GlcNAc. In the presence of a 10% solution of various alcohols, many alkyl-β-d-glucosaminides were obtained, indicating that exo-β-d-glucosaminidase is a retaining enzyme.

Exo-β-d-glucosaminidase of Paecilomyces lilacinus was induced, purified and characterized. To clarify its cleavage ability, several compounds including GlcN–GlcNAc–butyl and a mixture of partially acetylated chitooligosaccharides were used as the substrate and analyzed by real-time mass spectrometry. Based on the anomeric configuration of the transglycosylation product by 1H NMR, exo-β-d-glucosaminidase was identified as a retaining enzyme.Figure optionsDownload as PowerPoint slideHighlights
► β-d-Glucosaminidase was first found in Paecilomyces lilacinus.
► Paecilomyces was engineered to secrete more β-d-glucosaminidase.
► A retaining β-d-glucosaminidase was purified and characterized.
► This enzyme hydrolyzes 95% deacetylated chitosan but not chitin.
► The cleavage pattern was identified by real-time mass spectrometry.