Engineered Humicola insolens cutinase for efficient cellulose acetate deacetylation

Abhijit N. Shirke, Glenn Butterfoss, Rakhi Saikia, Aditya Basu, Leonardo de Maria, Allan Svendsen, Richard A. Gross

    Research output: Contribution to journalArticle

    Abstract

    Cutinases comprise a family of esterases with broad hydrolytic activity for chain and pendant ester groups. This work aimed to identify and improve an efficient cutinase for cellulose acetate (CA) deacetylation. The development of a mild method for CA fiber surface deacetylation will result in improved surface hydrophilicity and reactivity while, when combined with cellulases, a route to the full recycling of CA to acetate and glucose. In this study, the comparative CA deacetylation activity of four homologous wild-type (wt) fungal cutinases from Aspergillus oryzae (AoC), Thiellavia terrestris (TtC), Fusarium solani (FsC), and Humicola insolens (HiC) was determined by analysis of CA deacetylation kinetics. wt-HiC had the highest catalytic efficiency (≈32 [cm2 L-1]-1 h-1). Comparison of wt-cutinase catalytic constants revealed that differences in catalytic efficiency are primarily due to corresponding variations in corresponding substrate binding constants. Docking studies with model tetrameric substrates also revealed structural origins for differential substrate binding amongst these cutinases. Comparative docking studies of HiC point mutations led to the identification of two important rationales for engineering cutinases for CA deacetylation: (i) create a tight but not too closed binding groove, (ii) allow for hydrogen bonding in the extended region around the active site. Rationally designed HiC with amino acid substitutions I36S, predicted to hydrogen bond to CA, combined with F70A, predicted to remove steric constraints, showed a two-fold improvement in catalytic efficiency. Continued cutinase optimization guided by a detailed understanding of structure-activity relationships, as demonstrated here, will be an important tool to developing practical cutinases for commercial green chemistry technologies.

    Original languageEnglish (US)
    Article number1700188
    JournalBiotechnology Journal
    Volume12
    Issue number8
    DOIs
    StatePublished - Aug 1 2017

    Fingerprint

    Green Chemistry Technology
    Aspergillus oryzae
    Cellulases
    cutinase
    acetylcellulose
    Fusarium
    Recycling
    Esterases
    Amino Acid Substitution
    Hydrogen Bonding
    Structure-Activity Relationship
    Hydrophobic and Hydrophilic Interactions
    Point Mutation
    Hydrogen
    Catalytic Domain
    Esters
    Acetates
    Glucose

    Keywords

    • Cellulose acetate deacetylation
    • Cutinase
    • Enzyme kinetics
    • Substrate docking

    ASJC Scopus subject areas

    • Applied Microbiology and Biotechnology
    • Molecular Medicine

    Cite this

    Shirke, A. N., Butterfoss, G., Saikia, R., Basu, A., de Maria, L., Svendsen, A., & Gross, R. A. (2017). Engineered Humicola insolens cutinase for efficient cellulose acetate deacetylation. Biotechnology Journal, 12(8), [1700188]. https://doi.org/10.1002/biot.201700188

    Engineered Humicola insolens cutinase for efficient cellulose acetate deacetylation. / Shirke, Abhijit N.; Butterfoss, Glenn; Saikia, Rakhi; Basu, Aditya; de Maria, Leonardo; Svendsen, Allan; Gross, Richard A.

    In: Biotechnology Journal, Vol. 12, No. 8, 1700188, 01.08.2017.

    Research output: Contribution to journalArticle

    Shirke, AN, Butterfoss, G, Saikia, R, Basu, A, de Maria, L, Svendsen, A & Gross, RA 2017, 'Engineered Humicola insolens cutinase for efficient cellulose acetate deacetylation', Biotechnology Journal, vol. 12, no. 8, 1700188. https://doi.org/10.1002/biot.201700188
    Shirke AN, Butterfoss G, Saikia R, Basu A, de Maria L, Svendsen A et al. Engineered Humicola insolens cutinase for efficient cellulose acetate deacetylation. Biotechnology Journal. 2017 Aug 1;12(8). 1700188. https://doi.org/10.1002/biot.201700188
    Shirke, Abhijit N. ; Butterfoss, Glenn ; Saikia, Rakhi ; Basu, Aditya ; de Maria, Leonardo ; Svendsen, Allan ; Gross, Richard A. / Engineered Humicola insolens cutinase for efficient cellulose acetate deacetylation. In: Biotechnology Journal. 2017 ; Vol. 12, No. 8.
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