Summary auto-generated
This study investigated the inducible chitinolytic system of Aspergillus fumigatus, a ubiquitous saprophytic fungus and opportunistic human pathogen. When cultured on chitin as sole carbon source, A. fumigatus secreted extracellular chitinase activity reaching 1.5 pmol GlcNAc released per minute per mg protein. Chitinase induction was repressed by GlcNAc through negative feedback, preventing wasteful enzyme synthesis when the end product was abundant. The researchers identified multiple extracellular chitinolytic enzymes via zymography, with different profiles induced by colloidal versus crystalline chitin preparations. They purified a major 45 kDa chitinase using ammonium sulfate precipitation, chitin affinity chromatography, and novel electroelution from substrate-containing gels. This enzyme is a glycoprotein with endochitinase activity, preferentially cleaving longer chitooligomers. The 45 kDa chitinase showed strong homology to chitinases from other fungi including Coccidioides immitis and Trichoderma harzianum. The chitinase was potently inhibited by allosamidin (IC₅₀ 0.12 μM). Results suggest A. fumigatus contains multiple chitinase genes and can detect different chitin configurations in its environment, enabling utilization of diverse chitinous substrates.
Key findings
- Aspergillus fumigatus produces abundant extracellular chitinase when grown on chitin as sole carbon source, with activity repressed by GlcNAc through negative feedback regulation
- Multiple extracellular chitinolytic enzymes of varying molecular mass (ranging from ~20 to 110 kDa) are induced, with different enzyme profiles produced by colloidal versus crystalline chitin preparations
- A purified 45 kDa extracellular chitinase is a glycoprotein with endochitinase activity that shows homology to chitinases from other fungal pathogens
- The chitinase system is potently inhibited by allosamidin (IC₅₀ 0.12 μM), confirming the activity is from genuine chitinase enzymes
- Evidence suggests A. fumigatus can discriminate between different chitin configurations and likely detects chitin through oligomers generated by constitutive low-level chitinase secretion
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Abstract
Incubation of Aspergillus fumigatus NCPF 2140 in growth medium containing 1% chitin as sole carbon source led to induction of specific extracellular chitinolytic activity of 1.5 mumol GlcNAc released min-1 (mg protein)-1. The effect was repressed by the inclusion of GlcNAc in the medium, indicating regulation by a negative feedback mechanism. Extracellular chitinase activity was inhibited by allosamidin (IC50 0.12 microM). Multiple chitinolytic enzymes were detected on zymograms of extracellular preparations; levels of individual enzymes induced were dependent upon whether cells were incubated with purified colloidal chitin or a crude preparation of crystalline chitin. A major, inducible, 45 kDa chitinase was purified using ammonium sulphate precipitation, chitin affinity chromatography and a novel procedure involving the electroelution of the enzyme from a substrate gel containing glycol chitin. The enzyme is a glycoprotein with endochitinase activity.