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

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posted on 10.11.2020, 17:41 by Mingbo Qu, Takahiro Watanabe-Nakayama, Shaopeng Sun, Kenichi Umeda, Xiaoxi Guo, Yuansheng Liu, Toshio Ando, Qing Yang
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/koff) 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.

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