In situ microscopic observation of chitin and fungal cells with chitinous cell walls in hydrothermal conditions

Deguchi, Shigeru; Tsujii, Kaoru; Horikoshi, Koki

doi:10.1038/srep11907

Cited by 29 publications

(27 citation statements)

References 42 publications

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“…In addition to the processive movements, single-molecule imaging has revealed that chitinase moves and, accordingly, produces disaccharides ten times faster than cellulase does 4 , 5 , although the physical and chemical stabilities of crystalline cellulose and chitin are similar 24 , 25 . Therefore, the mechanism underlying fast unidirectional movement of chitinase is gaining attention.…”

Section: Introductionmentioning

confidence: 99%

Processive chitinase is Brownian monorail operated by fast catalysis after peeling rail from crystalline chitin

et al. 2018

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Processive chitinase is a linear molecular motor which moves on the surface of crystalline chitin driven by processive hydrolysis of single chitin chain. Here, we analyse the mechanism underlying unidirectional movement of Serratia marcescens chitinase A (SmChiA) using high-precision single-molecule imaging, X-ray crystallography, and all-atom molecular dynamics simulation. SmChiA shows fast unidirectional movement of ~50 nm s−1 with 1 nm forward and backward steps, consistent with the length of reaction product chitobiose. Analysis of the kinetic isotope effect reveals fast substrate-assisted catalysis with time constant of ~3 ms. Decrystallization of the single chitin chain from crystal surface is the rate-limiting step of movement with time constant of ~17 ms, achieved by binding free energy at the product-binding site of SmChiA. Our results demonstrate that SmChiA operates as a burnt-bridge Brownian ratchet wherein the Brownian motion along the single chitin chain is rectified forward by substrate-assisted catalysis.

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Section: Introductionmentioning

confidence: 99%

Processive chitinase is Brownian monorail operated by fast catalysis after peeling rail from crystalline chitin

et al. 2018

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“…Other frequently used biopolymers that also widely occur, often as waste, are chitin, chitosan, and cellulose. Chitin is a biopolymer with relatively high thermal stability, even at 380 ∘ C [23][24][25]. However, the thermal degradation of chitin is dependent on several factors including acetylation, degree of crystallinity, and size of crystallites, and it is also strongly related to the origin of the material.…”

Section: Introductionmentioning

confidence: 99%

Influence of Processing Conditions on the Thermal Stability and Mechanical Properties of PP/Silica-Lignin Composites

Kłapiszewski

Bula

Sobczak

et al. 2016

International Journal of Polymer Science

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Functional silica-lignin dual fillers were obtained via mechanical grinding of the components (Syloid 244 silica and kraft lignin). Of particular importance here is the fact that lignin is a natural polymer and particularly that it is a waste by-product of paper production, whose recycling is highly desirable. The product underwent comprehensive dispersive-morphological and thermal analysis. SiO2-lignin hybrid fillers were also used in polypropylene-based composites, extruded via corotating a twin screw machine with different screw speeds. The thermogravimetric data obtained for the extrudates confirmed that the application of the lignin into PP produces a significant char residue. Addition of silica to lignin via this new hybrid formulation has a positive effect on the thermal stability of PP/silica-lignin composites, which can be seen even when observing the temperature for the maximum rate of weight reduction. Tensile test results show that the addition of silica by means of dual filler incorporation improves the mechanical parameters in comparison with pure PP and PP/lignin composite.

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“…Chitin degradation is important not only in nature, but also in industrial applications such as biomass conversion, and has potential applications in agriculture, biotechnology, and the pharmaceutical industry (4,5). Due to its stable crystalline structure, chitin is very durable, only decomposing at very high temperatures and under high pressure (6).…”

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confidence: 99%

Single-molecule imaging analysis reveals the mechanism of a high-catalytic-activity mutant of chitinase A from Serratia marcescens

Visootsat

Nakamura

Vignon

et al. 2020

Journal of Biological Chemistry

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Chitin degradation is important for biomass conversion and has potential applications for agriculture, biotechnology, and the pharmaceutical industry. Chitinase A from the Gram-negative bacterium Serratia marcescens (SmChiA) is a processive enzyme that hydrolyzes crystalline chitin as it moves linearly along the substrate surface. In a previous study, the catalytic activity of SmChiA against crystalline chitin was found to increase after the tryptophan substitution of two phenylalanine residues (F232W and F396W), located at the entrance and exit of the substrate binding cleft of the catalytic domain, respectively. However, the mechanism underlying this high catalytic activity remains elusive. In this study, single-molecule fluorescence imaging and high-speed atomic force microscopy were applied to understand the mechanism of this high-catalytic-activity mutant. A reaction scheme including processive catalysis was used to reproduce the properties of SmChiA WT and F232W/F396W, in which all of the kinetic parameters were experimentally determined. High activity of F232W/F396W mutant was caused by a high processivity and a low dissociation rate constant after productive binding. The turnover numbers for both WT and F232W/F396W, determined by the biochemical analysis, were well-replicated using the kinetic parameters obtained from single-molecule imaging analysis, indicating the validity of the reaction scheme. Furthermore, alignment of amino acid sequences of 258 SmChiA-like proteins revealed that tryptophan, not phenylalanine, is the predominant amino acid at the corresponding positions (Phe-232 and Phe-396 for SmChiA). Our study will be helpful for understanding the kinetic mechanisms and further improvement of crystalline chitin hydrolytic activity of SmChiA mutants.

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In situ microscopic observation of chitin and fungal cells with chitinous cell walls in hydrothermal conditions

Cited by 29 publications

References 42 publications

Processive chitinase is Brownian monorail operated by fast catalysis after peeling rail from crystalline chitin

Processive chitinase is Brownian monorail operated by fast catalysis after peeling rail from crystalline chitin

Influence of Processing Conditions on the Thermal Stability and Mechanical Properties of PP/Silica-Lignin Composites

Single-molecule imaging analysis reveals the mechanism of a high-catalytic-activity mutant of chitinase A from Serratia marcescens

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