Purification and Characterization of a Newly Identified GH18 Chitinase BhGH18 from Bacillus haynesii and Its Ultrasound-Assisted Hydrolysis of Chitin

A previously uncharacterized GH18 Chitinase from Bacillus haynesii (BhGH18) was heterologously expressed and characterized. BhGH18 displayed maximal activity at pH 7.5 and 40 ° C, and activity was enhanced by Mg 2+ /Zn 2+ but inhibited by Cu 2+ /Fe 2+ /Ni 2+ . After further purification, the enzyme exhibited markedly improved kinetics toward pNP-(GlcNAc) 2 ( K m = 0.103 mM, V max = 0.217 μmol/min). Importantly, on the natural substrate colloidal chitin, BhGH18 gave an apparent K m of 3.51 mg/mL and V max of 1.69 μmol/min (for colloidal chitin). The enzyme displayed an endohydrolytic preference, converting ultrasonically pretreated chitin predominantly into chitobiose (DP2). Ultrasonication at 500 W increased the hydrolysis yield nearly 6-fold (23.86% conversion). Molecular docking identified Trp157, Glu197, Tyr198 and Arg333 as key substrate-binding residues. This work demonstrates an integrated ultrasound-enzyme strategy using BhGH18 for sustainable production of DP2-enriched chitin oligosaccharides.