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The synergy between LPMOs and cellulases in enzymatic saccharification of cellulose is both enzyme- and substrate-dependent

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Abstract

Objectives

The synergistic effects between cellulases and lytic polysaccharide monooxygenases (LPMOs) were investigated systematically in terms of their degree of synergy (DS) on amorphous and crystalline cellulose. Synergy curves were obtained for enzyme pairs containing a cellulase from Trichoderma reesei (Cel6A and Cel7A) and three LPMOs from Thermoascus aurantiacus (TaAA9A), Lentinus similis (LsAA9A) and Thielavia terrestris (TtAA9E).

Results

The synergistic experiments showed that the three LPMOs significantly improved the hydrolytic efficiency of Cel6A, on both cellulosic substrates; a more pronounced effect being seen for TtAA9E on amorphous cellulose at low cellulase:LPMO ratios. In contrast, the highly processive, reducing-end acting Cel7A synergised with the C1-C4 oxidising LPMOs, TaAA9A and LsAA9A, but was inhibited by the presence of C1-oxidizing TtAA9E.

Conclusions

The degree of synergy exhibited by the cellulase-LPMO mixtures was enzyme- and substrate-specific. The observed Cel7A inhibition, rather than synergy, by the C1-oxidizing LPMO, TtAA9E, warrants further investigations.

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Acknowledgements

This work was supported by the Novo Nordisk Foundation, Grant NO. NNF17SA0027704 to Katja Salomon Johansen.

Supporting information: Supplementary Fig. 1 – Comparison of cellulose degradation fingerprint of TaAA9A, LsAA9A and TtAA9E.

Supplementary material

Supplementary experiments were performed, in order to confirm the purity of the three LPMO enzyme preparations, as well as to compare directly their activity. Supplementary Fig. 1 shows a direct comparison of the activity of the three tested LPMOs – TaAA9A, LsAA9A and TtAA9E. PASC degradation experiments were carried out in 50 mM Citrate/Phosphate buffer (pH 6), with copper-loaded LPMO at a concentration of 4 µM, 0.4 g/L PASC and 4 mM ascorbic acid. Samples were incubated at 50 °C for 24 h in an Eppendorf Thermomixer at 1100 rpm. The reaction mixture was centrifuged through filter plates with 0.22 µm pore size (Millipore MultiScreenHTS, 3 min, 3500 rpm) and the supernatant was analysed by HPAEC-PAD.

TtAA9E’s strict C1-oxidation mechanism is confirmed by the lack of C4-oxidised oligosaccharides on the chromatogram (after 25 min). The proposed substrate-specific regioselectivity of LsAA9A can be observed by the presence of C4- and absence of C1-oxidised sugars. LsAA9A’s ability to cleave short-chain oligosaccharides is also evident by the presence of a larger glucose peak, compared to the other two LPMOs.

Author information

Authors and Affiliations

  1. Department of Plant and Environmental Sciences, University of Copenhagen, 1871, Frederiksberg, Denmark

    Radina Tokin, Johan Ørskov Ipsen, Peter Westh & Katja Salomon Johansen

  2. Department of Biotechnology and Biomedicine, Technical University of Denmark, 2800, Kgs Lyngby, Denmark

    Peter Westh

  3. Department of Geoscience and Natural Resource Management, University of Copenhagen, 1958, Frederiksberg, Denmark

    Katja Salomon Johansen

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  1. Radina Tokin
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  2. Johan Ørskov Ipsen
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  3. Peter Westh
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  4. Katja Salomon Johansen
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Correspondence to Katja Salomon Johansen.

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The authors declare that they have no conflict of interest. This article does not describe any studies with animals performed by any of the authors.

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Tokin, R., Ipsen, J.Ø., Westh, P. et al. The synergy between LPMOs and cellulases in enzymatic saccharification of cellulose is both enzyme- and substrate-dependent. Biotechnol Lett 42, 1975–1984 (2020). https://doi.org/10.1007/s10529-020-02922-0

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  • DOI: https://doi.org/10.1007/s10529-020-02922-0

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