| Summary: | In order to discover a broad-specificity and high stability chitinase, a marine fungus, <i>Aspergillus fumigatus</i> df347, was identified in the sediments of mangrove wetlands in Qinzhou Bay, China. The chitinase gene (<i>Af</i>Chi28) from <i>A. fumigatus</i> df347 was cloned and heterologously expressed in <i>Escherichia coli</i>, and the recombinant enzyme <i>Af</i>Chi28 was purified and characterized. <i>Af</i>Chi28 is an acido-halotolerant- and temperature-resistant bifunctional enzyme with both endo- and exo-cleavage functions. Its enzymatic products are mainly GlcNAc, (GlcNAc)<sub>2</sub>, (GlcNAc)<sub>3</sub> and (GlcNAc)<sub>4</sub>. Na<sup>+</sup>, Mg<sup>2+</sup>, K<sup>+</sup>, Ca<sup>2+</sup> and Tris at a concentration of 50 mM had a strong stimulatory effect on <i>Af</i>Chi28. The crude enzyme and pure enzyme exhibited the highest specific activity of 0.737 mU/mg and 52.414 mU/mg towards colloidal chitin. The DxDxE motif at the end of strand <i>β</i>5 and with Glu154 as the catalytic residue was verified by the AlphaFold2 prediction and sequence alignment of homologous proteins. Moreover, the results of molecular docking showed that molecular modeling of chitohexaose was shown to bind to <i>Af</i>Chi28 in subsites −4 to +2 in the deep groove substrate-binding pocket. This study demonstrates that <i>Af</i>Chi28 is a promising chitinase for the preparation of desirable chitin oligosaccharides, and provides a foundation for elucidating the catalytic mechanism of chitinases from marine fungi.
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