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, 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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