Thermal unfolding and refolding of a lytic polysaccharide monooxygenase from: Thermoascus aurantiacus

Raushan K. Singh, Benedikt M. Blossom, D. A. Russo, B. Van Oort, R. Croce, P. E. Jensen, C. Felby, M. J. Bjerrum

Research output: Contribution to JournalArticleAcademicpeer-review

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.

Original languageEnglish
Pages (from-to)29734-29742
Number of pages9
JournalRSC Advances
Volume9
Issue number51
DOIs
Publication statusE-pub ahead of print - 19 Sep 2019

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Polysaccharides
Mixed Function Oxygenases
Cellulose
Degradation
Chitin
Regain
Biocatalysts
Mercaptoethanol
Phosphoric acid
Enzymes
Melting point
Conformations
Copper
Assays
Biomass
Hot Temperature
Thermodynamics
Proteins
Oxidation
Kinetics

Cite this

Singh, Raushan K. ; Blossom, Benedikt M. ; Russo, D. A. ; Van Oort, B. ; Croce, R. ; Jensen, P. E. ; Felby, C. ; Bjerrum, M. J. / Thermal unfolding and refolding of a lytic polysaccharide monooxygenase from : Thermoascus aurantiacus. In: RSC Advances. 2019 ; Vol. 9, No. 51. pp. 29734-29742.
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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{\circledR} 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.",
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Singh, RK, Blossom, BM, Russo, DA, Van Oort, B, Croce, R, Jensen, PE, Felby, C & Bjerrum, MJ 2019, 'Thermal unfolding and refolding of a lytic polysaccharide monooxygenase from: Thermoascus aurantiacus' RSC Advances, vol. 9, no. 51, pp. 29734-29742. https://doi.org/10.1039/c9ra05920b

Thermal unfolding and refolding of a lytic polysaccharide monooxygenase from : Thermoascus aurantiacus. / Singh, Raushan K.; Blossom, Benedikt M.; Russo, D. A.; Van Oort, B.; Croce, R.; Jensen, P. E.; Felby, C.; Bjerrum, M. J.

In: RSC Advances, Vol. 9, No. 51, 19.09.2019, p. 29734-29742.

Research output: Contribution to JournalArticleAcademicpeer-review

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AU - Singh, Raushan K.

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