High-Speed Atomic Force Microscopy Reveals Factors Affecting the Processivity of Chitinases during Interfacial Enzymatic Hydrolysis of Crystalline Chitin

Enzymatic hydrolysis of crystalline chitin is a solid-liquid interfacial catalysis process that is highly dependent on the enzymes' processivity, namely, the number of catalytic cycles consecutively repeated per enzyme-substrate complexation event without the release of the substrate. However, the factors affecting an enzyme's processivity are not fully understood, as processivity in a solid-liquid interface can be hardly determined biochemically. Using high-speed atomic force microscopy (HS-AFM), here we investigated an insect chitinase cocktail containing one exochitinase (OfChi-h) and two endochitinases (OfChtI and OfChtII), the indispensable enzymes for shedding old cuticle. At the single-molecule level, we have revealed endochitinases as a significant factor to elevate the processivity of OfChi-h from 25 to 176 reactions in crystalline chitin hydrolysis, to dramatically increase the number of OfChi-h newly bound onto a chitin surface area unit per chitinase concentration, and to increase the duration time of processive running OfChi-h molecules on the chitin surface (1/k(off)) from 0.48 to 3.13 s. In the presence of endochitinases, OfChi-h molecules were observed to crowd at the ends of chitin fibrils and form clusters that quickly ablate chitin fibrils. The processive initiation including initial OfChi-h-substrate binding and complexation is a factor that affects processive hydrolysis. The time for processive initiation was deduced to be 0.22 +/- 0.07 s according to an aligning behavior of OfChi-h molecules on chitin fibrils. This work improves our knowledge of exo-endo chitinase synergism during the solid-liquid interfacial catalysis of crystalline chitin hydrolysis, highlighting the role of endochitinases in boosting processive hydrolysis.