Engineering a hybrid chitinase-sonorensin fusion protein for enhanced antibacterial and antifungal activity.

The main objective of the study was to attain improved effectiveness against various bacterial and fungal infections, tackling the widespread problem of multidrug resistance. The study presents the development and evaluation of a hybrid antimicrobial protein created by combining chitinase and sonorensin (HyChiSono). The hybrid protein HyChiSono, a 49.3 kDa chitinase-sonorensin fusion protein, was engineered, expressed in E. coli BL21(DE3), and purified to > 95 % homogeneity. Independent folding of each domain linked by a flexible (GGGGS) spacer was revealed by AlphaFold modelling; a highly ordered α/β structure was confirmed by far-UV CD. HyChiSono exhibited bactericidal activity, as assessed through well diffusion assays, membrane damage assays, and transmission electron microscopy (TEM). Quantitatively, HyChiSono demonstrated robust antibacterial activity, with recorded zones of inhibition (ZOIs) against Gram-positive Staphylococcus aureus (MIC 8 μg/mL; ZOI 17.0 ± 0.3 mm), Listeria monocytogenes (MIC 16 μg/mL; ZOI 19.3 ± 0.5 mm), and Gram-negative Escherichia coli (MIC 4 μg/mL; ZOI 18.5 ± 0.5 mm) and Salmonella enterica serovar Typhi (MIC 2 μg/mL; ZOI 18.0 ± 0.7 mm), outperforming native sonorensin by a factor of 2-4. SYTOX-green uptake and TEM demonstrated rapid membrane permeabilization and peptidoglycan thinning in both cell envelopes. Cell-wall targeting was rationalized by docking scores of -6.7 and -6.4 kcal/mol against N-acetyl-glucosamine and chitin. In addition to its antibacterial properties, the antifungal assays of hybrid protein exhibited 64.7 % growth inhibition of Fusarium oxysporum and 45.9 % against Alternaria solani, superior to standalone chitinase (41 % and 37 %, respectively). The bifunctional HyChiSono construct is offered as a single-molecule platform to combat multidrug-resistant bacteria and phytopathogenic fungi.