In aquatic environments, insoluble polymers such as chitin are spatially heterogeneous and rapidly diluted by diffusion, posing a challenge for efficient microbial degradation. A key unresolved question is how extracellular enzymes remain associated with such substrates long enough to sustain activity. Here, we show that dual binding domains in a marine Chitinase from Vibrio parahemolyticus prolong enzyme residence time on chitin surfaces. Compared with a binding-domain–truncated variant, the full-length enzyme exhibits a significantly lower apparent Michaelis constant at low substrate concentrations, indicating enhanced efficiency, while displaying substrate inhibition at higher concentrations. Single-molecule fluorescence imaging reveals that an additional C-terminal carbohydrate-binding module selectively stabilizes binding to specific crystalline chitin surfaces and increases binding duration by over an order of magnitude. Kinetic simulations based on measured dissociation rates indicate that this prolonged residence time arises from frequent rebinding of the catalytic domain, facilitated by the additional binding module. High-speed atomic force microscopy further shows that this extra domain does not affect the enzyme’s movement velocity or processive run length along chitin fibers. Together, these findings demonstrate that selective surface binding and extended binding time, rather than catalytic turnover or motility, are critical determinants of enzymatic efficiency under dilute conditions. Notably, this additional binding domain is common in GH18 chitinases from marine bacteria but absent in those from terrestrial organisms and insects, which operate in high-substrate or low-moisture environments. These results suggest that Chitinase domain architecture has evolved to match environmental conditions, offering new insights into solid polymer degradation.
Tanimoto et al. (Mon,) studied this question.