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Synthetic carbohydrate-binding module-endogalacturonase chimeras increase catalytic efficiency and saccharification of lignocellulose residues

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Abstract

Primary cell walls and middle lamella of higher plants include homogalacturonan, the substrate for polygalacturonases. Seeking to enhance lignocellulose saccharification, chimeras between the endopolygalacturonase I from Chondrostereum (Stereum) purpureum (EndoPG-I) and family 3, 44, and 77 carbohydrate-binding modules (CBMs) from Hungateiclostridium thermocellum (Ht) or Ruminococcus flavefaciens (Rf) were constructed, expressed, and characterized. Chimeras presented similar KM values and pH/temperature optima as unfused EndoPG-I against citrus pectin, kcat/KM values 1.6, 1.7, and 1.3-fold higher for the HtCBM3-EndoPG, HtCBM44-EndoPG, and RtCBM77-EndoPG, respectively. Commercial enzyme cocktail supplementation with HtCBM44-EndoPG and RtCBM77-EndoPG increased reducing sugar release from untreated sugarcane bagasse by 35 and 25%, respectively. All chimeras increased reducing sugar release by 20–25% against orange bagasse compared with EndoPG-I or equimolar EndoPG-I/CBM mixtures. These results show that proximity between plant cell wall components in situ influences CBM-enzyme chimera activity and improves saccharification of lignocellulosic materials.

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Acknowledgements

We thank André Justino for technical assistance.

Funding

This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) grants 2017/13734–3 (SC), 2016/17582–0 (LPM), 2019/21989–7 (JCSS), 2016/24139–6 (RJW), CNPq grant 305788/2017–5 (RJW) and the National Institute of Science and Technology of Bioethanol (INCT-Bioethanol) (FAPESP 2011/57908–6 and 2014/50884–5, CNPq 574002/2008–1 and 465319/2014–9).

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  1. Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil

    Sibeli Carli, Luana Parras Meleiro, José Carlos Santos Salgado & Richard John Ward

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  1. Sibeli Carli
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Sibeli Carli, data curation, formal analysis, investigation, methodology, figure preparation, visualization, and writing—original draft; Luana Parras Meleiro, data curation, formal analysis, investigation, methodology, figure preparation, visualization, and writing—review and editing; José Carlos Santos Salgado, data curation, formal analysis, investigation, methodology, figure preparation, visualization, and writing—original draft; and Richard John Ward, conceptualization, funding acquisition, project administration, resource management, team supervision, and writing—review and editing.

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Correspondence to Richard John Ward.

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Carli, S., Parras Meleiro, L., Salgado, J.C.S. et al. Synthetic carbohydrate-binding module-endogalacturonase chimeras increase catalytic efficiency and saccharification of lignocellulose residues. Biomass Conv. Bioref. 14, 6369–6380 (2024). https://doi.org/10.1007/s13399-022-02716-6

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