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Thermobifida fusca Cellulases Exhibit Increased Endo–Exo Synergistic Activity, but Lower Exocellulase Activity, on Cellulose-III

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    Research Article

    Thermobifida fusca Cellulases Exhibit Increased Endo–Exo Synergistic Activity, but Lower Exocellulase Activity, on Cellulose-III
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    • Yuxin Liu
      Yuxin Liu
      Department of Chemical and Biochemical Engineering, Rutgers, The State University of New Jersey, 98 Brett Road, Piscataway, New Jersey 08854, United States
      More by Yuxin Liu
    • Bhargava Nemmaru
      Bhargava Nemmaru
      Department of Chemical and Biochemical Engineering, Rutgers, The State University of New Jersey, 98 Brett Road, Piscataway, New Jersey 08854, United States
      More by Bhargava Nemmaru
    • Shishir P. S. Chundawat*
      Shishir P. S. Chundawat
      Department of Chemical and Biochemical Engineering, Rutgers, The State University of New Jersey, 98 Brett Road, Piscataway, New Jersey 08854, United States
      *Email: [email protected]. Tel: 848-445-3678.
      More by Shishir P. S. Chundawat
      Orcidhttp://orcid.org/0000-0003-3677-6735
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    ACS Sustainable Chemistry & Engineering

    Cite this: ACS Sustainable Chem. Eng. 2020, 8, 13, 5028–5039
    Click to copy citationCitation copied!
    https://doi.org/10.1021/acssuschemeng.9b06792
    Published March 16, 2020
    Copyright © 2020 American Chemical Society
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    Abstract

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    Cellulose recalcitrance toward saccharification is a barrier for low-cost biofuels production. Ammonia-based pretreatments can alter the native cellulose-I allomorphic state to form an unnatural cellulose-III allomorph that is less recalcitrant toward enzymatic hydrolysis. Here, we characterize the hydrolytic activity of a thermophilic cellulolytic microbe, Thermobifida fusca, derived cellulase on cellulose-III. Up to 2-fold improved activity was observed for homologously expressed T. fusca cellulase enzymes on cellulose-III. Surprisingly, T. fusca exocellulases like Cel6B alone had lower activity on cellulose-III. We hypothesized that increased activity of T. fusca cellulases on cellulose-III arises mostly due to enhanced endocellulase activity and improved synergism between endo/exocellulases. Representative T. fusca endocellulase (Cel5A) and exocellulase (Cel6B) were heterologously expressed in Escherichia coli, purified, and systematically characterized for synergistic activity on cellulose-III. Hydrolytic activity assays confirmed increased activity of Cel5A on cellulose-III and improved endo/exo synergistic activity for various combinations of Cel6B/Cel5A. We finally conducted a two-step restart hydrolysis assay to also confirm if increased endoactivity results in a endo-treated cellulose-III that is amenable toward increased Cel6B activity. This work provides a mechanistic basis for increased synergistic cellulase activity on cellulose-III and provides a rationale for focusing future T. fusca enzyme engineering efforts toward potentially rate-limiting exocellulases like Cel6B.

    Copyright © 2020 American Chemical Society

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    Supporting Information

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssuschemeng.9b06792.

    • Additional methods, optimization of T. fusca cell culture growth media pH (Figure S1), SDS-PAGE for ammonium sulfate precipitate (Figure S2), chromatograms/SDS-PAGE/activity assays for purification steps employed to isolate Cel6B from T. fusca broth (Figures S3–S6), LC-MS proteomics results for bands observed on SDS-PAGE (Figures S7 and S8), impact of pH and temperature on PASC activity of T. fusca broth (Figure S9), SDS-PAGE for surface-bound endocellulases in restart assay (Figure S10), purification of heterologously expressed Cel6B from E. coli cell lysate (Figure S11), SDS-PAGE for heterologously expressed pure Cel6B, Cel5A, and ß-glucosidase (Figure S12), activity of Cellic C.Tec2 on insoluble cellulose substrates CI/CIII/PASC (Figure S13), chromatographic steps employed during purification of Cel6B from T. fusca cell culture broth (Table T1), Optimization of ammonium sulfate precipitation protocol (Table T2), pH dependence of native T. fusca Cel6B (Table T3) (PDF)

    • List of primers used for SLIC and the three insert gene nucleotide sequences (XLSX)

    • List of full vector maps/sequences for all vectors used or generated in this work (TXT)

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    Cited By

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    This article is cited by 10 publications.

    1. Zhe Dong, Peng Zhang, Slavko Kralj, Yu Ji, Ulrich Schwaneberg. Synergism of Endo and Exo-α-1,3-Glucanases in α-1,3-Glucan Degradation: A Kinetic Study. ACS Sustainable Chemistry & Engineering 2024, 12 (24) , 9123-9132. https://doi.org/10.1021/acssuschemeng.4c01469
    2. Antonio DeChellis, Bhargava Nemmaru, Deanne Sammond, Jenna Douglass, Nivedita Patil, Olivia Reste, Shishir P. S. Chundawat. Supercharging Carbohydrate Binding Module Alone Enhances Endocellulase Thermostability, Binding, and Activity on Cellulosic Biomass. ACS Sustainable Chemistry & Engineering 2024, 12 (9) , 3500-3516. https://doi.org/10.1021/acssuschemeng.3c06266
    3. Paula Cavion Costa, Eduardo Echer dos Reis, Laura Salgueiro de Carvalho, Roselei Claudete Fontana, Willian Daniel Hahn Schneider, Marli Camassola. Making the Process of Enzyme Production in Solid-State Cultivation Cleaner and More Sustainable—Reuse of Raw Materials and a Syringe-Type Bioreactor Enter in the Scene. ACS Sustainable Chemistry & Engineering 2021, 9 (42) , 14134-14142. https://doi.org/10.1021/acssuschemeng.1c04257
    4. Bhargava Nemmaru, Jenna Douglass, John M. Yarbrough, Antonio DeChellis, Srivatsan Shankar, Alina Thokkadam, Allan Wang, Shishir P. S. Chundawat. Supercharged cellulases show superior thermal stability and enhanced activity towards pretreated biomass and cellulose. Frontiers in Energy Research 2024, 12 https://doi.org/10.3389/fenrg.2024.1372916
    5. Liwei Yin, Chuncheng Yang, Duoqi Zhou, Zhu Huang, Yu Zhu, Lin Wu. Synergistic study of cellulose enzymatic factors and cellulase adsorption properties under bioinformatics. Applied Mathematics and Nonlinear Sciences 2024, 9 (1) https://doi.org/10.2478/amns.2023.2.00042
    6. Bhargava Nemmaru, Antonio DeChellis, Nivedita Patil, Shishir P. S. Chundawat. CAZyme Characterization and Engineering for Biofuels Applications. 2023, 1-34. https://doi.org/10.1007/978-94-007-6724-9_32-1
    7. Hangyu Luo, Xiaofang Liu, Dayong Yu, Junfa Yuan, Jinyu Tan, Hu Li. Research Progress on Lignocellulosic Biomass Degradation Catalyzed by Enzymatic Nanomaterials. Chemistry – An Asian Journal 2022, 17 (18) https://doi.org/10.1002/asia.202200566
    8. Mpho S. Mafa, Brett I. Pletschke, Samkelo Malgas. Defining the Frontiers of Synergism between Cellulolytic Enzymes for Improved Hydrolysis of Lignocellulosic Feedstocks. Catalysts 2021, 11 (11) , 1343. https://doi.org/10.3390/catal11111343
    9. Bhargava Nemmaru, Nicholas Ramirez, Cindy J. Farino, John M. Yarbrough, Nicholas Kravchenko, Shishir P. S. Chundawat. Reduced type‐A carbohydrate‐binding module interactions to cellulose I leads to improved endocellulase activity. Biotechnology and Bioengineering 2021, 118 (3) , 1141-1151. https://doi.org/10.1002/bit.27637
    10. Merja H. Kontro, Jayachandra S. Yaradoddi, Nagaraj R. Banapurmath, Sharanabasava V. Ganachari, Basavaraj S. Hungund. Biotechnological Importance of Actinomycetes. 2021, 271-290. https://doi.org/10.1007/978-981-16-3353-9_15
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    ACS Sustainable Chemistry & Engineering

    Cite this: ACS Sustainable Chem. Eng. 2020, 8, 13, 5028–5039
    Click to copy citationCitation copied!
    https://doi.org/10.1021/acssuschemeng.9b06792
    Published March 16, 2020
    Copyright © 2020 American Chemical Society
    Request reuse permissions

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