Thermal unfolding and refolding of a lytic polysaccharide monooxygenase from Thermoascus aurantiacus |
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| Authors: | Raushan K. Singh Benedikt M. Blossom D. A. Russo B. van Oort R. Croce P. E. Jensen C. Felby M. J. Bjerrum |
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| Affiliation: | Department of Chemistry, University of Copenhagen, Copenhagen Denmark.; Department of Geosciences and Natural Resource Management, University of Copenhagen, Frederiksberg C Denmark ; Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg C Denmark ; Biophysics of Photosynthesis, Vrije Universiteit Amsterdam, Amsterdam Netherlands |
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| Abstract: | Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes which promote the degradation of recalcitrant polysaccharides like cellulose or chitin. Here, we have investigated the thermostability of an LPMO from Thermoascus aurantiacus (TaLPMO9A). TaLPMO9A was found to retain most of its initial activity after incubating at 100 °C while its apparent melting temperature (Tm) is 69 °C at neutral pH. Interestingly, our studies show that holoTaLPMO9A, apoTaLPMO9A and deglycosylated TaLPMO9A can fold back to their original conformation upon lowering the temperature. In the presence of β-mercaptoethanol the protein does not refold. Activity of TaLPMO9A and refolded TaLPMO9A was studied by an Amplex® Red assay as well as by TaLPMO9A catalysed oxidation of phosphoric acid swollen cellulose (PASC). These studies confirm the functional regain of TaLPMO9A activity upon going through one cycle of unfolding and refolding. The thermal unfolding and refolding of TaLPMO9A was measured spectroscopically. Utilizing the two-state model, detailed thermodynamic parameters were obtained for holoTaLPMO. Furthermore, we have investigated the kinetics of TaLPMO9A unfolding and refolding. Our results have implications in understanding LPMO stability, which is crucial for the efficient application of LPMOs as biocatalysts during biomass degradation.TaLPMO9A regains its catalytic power after a thermal unfolding and refolding cycle. |
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