The Discovery, Enzymatic Characterization and Functional Analysis of a Newly Isolated Chitinase from Marine-Derived Fungus Aspergillus fumigatus df347.

In order to discover a broad-specificity and high stability chitinase, a marine fungus, Aspergillus fumigatus df347, was identified in the sediments of mangrove wetlands in Qinzhou Bay, China. The chitinase gene ( Af Chi28) from A. fumigatus df347 was cloned and heterologously expressed in Escherichia coli , and the recombinant enzyme Af Chi28 was purified and characterized. Af Chi28 is an acido-halotolerant- and temperature-resistant bifunctional enzyme with both endo- and exo-cleavage functions. Its enzymatic products are mainly GlcNAc, (GlcNAc) 2 , (GlcNAc) 3 and (GlcNAc) 4 . Na + , Mg 2+ , K + , Ca 2+ and Tris at a concentration of 50 mM had a strong stimulatory effect on Af 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 β 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 Af Chi28 in subsites -4 to +2 in the deep groove substrate-binding pocket. This study demonstrates that Af 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.