Comparative thermal inactivation analysis of Aspergillus oryzae and Thiellavia terrestris cutinase: Role of glycosylation

Abhijit N. Shirke, An Su, J. Andrew Jones, Glenn L. Butterfoss, Mattheos A.G. Koffas, Jin Ryoun Kim, Richard A. Gross

Research output: Contribution to journalArticlepeer-review


Cutinase thermostability is important so that the enzymes can function above the glass transition of what are often rigid polymer substrates. A detailed thermal inactivation analysis was performed for two well-characterized cutinases, Aspergillus oryzae Cutinase (AoC) and Thiellavia terrestris Cutinase (TtC). Both AoC and TtC are prone to thermal aggregation upon unfolding at high temperature, which was found to be a major reason for irreversible loss of enzyme activity. Our study demonstrates that glycosylation stabilizes TtC expressed in Pichia pastoris by inhibiting its thermal aggregation. Based on the comparative thermal inactivation analyses of non-glycosylated AoC, glycosylated (TtC-G), and non-glycosylated TtC (TtC-NG), a unified model for thermal inactivation is proposed that accounts for thermal aggregation and may be applicable to other cutinase homologues. Inspired by glycosylated TtC, we successfully employed glycosylation site engineering to inhibit AoC thermal aggregation. Indeed, the inhibition of thermal aggregation by AoC glycosylation was greater than that achieved by conventional use of trehalose under a typical condition. Collectively, this study demonstrates the excellent potential of implementing glycosylation site engineering for thermal aggregation inhibition, which is one of the most common reasons for the irreversible thermal inactivation of cutinases and many proteins. Biotechnol. Bioeng. 2017;114: 63–73.

Original languageEnglish (US)
Pages (from-to)63-73
Number of pages11
JournalBiotechnology and Bioengineering
Issue number1
StatePublished - Jan 1 2017


  • aggregation
  • cutinase
  • glycosylation
  • thermal inactivation

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology


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