Development of enzyme cocktails for complete saccharification of chitin using mono-component enzymes from Serratia marcescens

Mekasha, Sophanit; Byman, Ida Roksvåg; Lynch, Catherine; Toupalová, Hana; Anděra, Ladislav; Næs, Tormod; Vaaje‐Kolstad, Gustav; Eijsink, Vincent G. H.

doi:10.1016/j.procbio.2017.02.021

Cited by 39 publications

(28 citation statements)

References 33 publications

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“…Serratia marcescens GH18 chitinases ( Sm Chi18A, Sm Chi18B, and Sm Chi18C), its GH20 chitobiase ( Sm GH20), and its AA10 LPMO ( Sm LPMO10A) were expressed and purified as previously described .…”

Section: Methodsmentioning

confidence: 99%

Characterization and synergistic action of a tetra‐modular lytic polysaccharide monooxygenase from Bacillus cereus

et al. 2018

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Lytic polysaccharide monooxygenases (LPMOs) contribute to enzymatic conversion of recalcitrant polysaccharides such as chitin and cellulose and may also play a role in bacterial infections. Some LPMOs are multimodular, the implications of which remain only partly understood. We have studied the properties of a tetra‐modular LPMO from the food poisoning bacterium Bacillus cereus (named BcLPMO10A). We show that BcLPMO10A, comprising an LPMO domain, two fibronectin‐type III (FnIII)‐like domains, and a carbohydrate‐binding module (CBM5), is a powerful chitin‐active LPMO. While the role of the FnIII domains remains unclear, we show that enzyme functionality strongly depends on the CBM5, which, by promoting substrate binding, protects the enzyme from inactivation. BcLPMO10A enhances the activity of chitinases during the degradation of α‐chitin.

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Section: Methodsmentioning

confidence: 99%

Characterization and synergistic action of a tetra‐modular lytic polysaccharide monooxygenase from Bacillus cereus

et al. 2018

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“…Moreover, the chitinases of the bacterium Serratia marcescens are a well-known model for the study of chitin degradation. S. marcescens chitinase A (SmChiA), 3 an exo-processive enzyme, is the most powerful enzyme among the chitinases of Serratia in the hydrolysis of crystalline chitin (8,9). SmChiA is a linear molecular motor enzyme that hydrolyzes chitin from the reducing end and works in the extracellular environment without the need for ATP.…”

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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

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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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“…While the present results show that the TG activity of different GH18 chitinases may be enhanced by similar types of mutations, and thus reveal general principles of TG activity, our study also reveals conspicuous differences between SpChiD and SmChiA. SmChiA is a well-known powerful chitin-degrading enzyme with an essential role in the chitinolytic machinery of S. marcescens 47,48 . Even the D313N mutant of this enzyme still has so much hydrolytic power that TG products are converted to chitobiose and chitotriose 12 .…”

Section: Discussionmentioning

confidence: 46%

Key Residues Affecting Transglycosylation Activity in Family 18 Chitinases: Insights into Donor and Acceptor Subsites

et al. 2018

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Understanding features that determine transglycosylation (TG) activity in glycoside hydrolases is important because it would allow the construction of enzymes that can catalyze controlled synthesis of oligosaccharides. To increase TG activity in two family 18 chitinases, chitinase D from Serratia proteamaculans ( SpChiD) and chitinase A from Serratia marcescens ( SmChiA), we have mutated residues important for stabilizing the reaction intermediate and substrate binding in both donor and acceptor sites. To help mutant design, the crystal structure of the inactive SpChiD-E153Q mutant in complex with chitobiose was determined. We identified three mutations with a beneficial effect on TG activity: Y28A (affecting the -1 subsite and the intermediate), Y222A (affecting the intermediate), and Y226W (affecting the +2 subsite). Furthermore, exchange of D151, the middle residue in the catalytically important DXDXE motif, to asparagine reduced hydrolytic activity ≤99% with a concomitant increase in apparent TG activity. The combination of mutations yielded even higher degrees of TG activity. Reactions with the best mutant, SpChiD-D151N/Y226W/Y222A, led to rapid accumulation of high levels of TG products that remained stable over time. Importantly, the introduction of analogous mutations at the same positions in SmChiA (Y163A equal to Y28A and Y390F similar to Y222A) had similar effects on TG efficiency. Thus, the combination of the decreasing hydrolytic power, subsite affinity, and stability of intermediate states provides a powerful, general strategy for creating hypertransglycosylating mutants of retaining glycoside hydrolases.

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Development of enzyme cocktails for complete saccharification of chitin using mono-component enzymes from Serratia marcescens

Cited by 39 publications

References 33 publications

Characterization and synergistic action of a tetra‐modular lytic polysaccharide monooxygenase from Bacillus cereus

Characterization and synergistic action of a tetra‐modular lytic polysaccharide monooxygenase from Bacillus cereus

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

Key Residues Affecting Transglycosylation Activity in Family 18 Chitinases: Insights into Donor and Acceptor Subsites

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