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

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

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
JournalBiotechnology and Bioengineering
DOIs
StateAccepted/In press - 2016

Fingerprint

Aspergillus oryzae
Glycosylation
Aspergillus
Thermoanalysis
Hot Temperature
Agglomeration
cutinase
Enzyme activity
Glass transition
Enzymes
Trehalose
Pichia
Proteins

Keywords

  • Aggregation
  • Cutinase
  • Glycosylation
  • Thermal inactivation

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

Cite this

Comparative thermal inactivation analysis of Aspergillus oryzae and Thiellavia terrestris cutinase : Role of glycosylation. / Shirke, Abhijit N.; Su, An; Jones, J. Andrew; Butterfoss, Glenn; Koffas, Mattheos A G; Kim, Jin; Gross, Richard A.

In: Biotechnology and Bioengineering, 2016.

Research output: Contribution to journalArticle

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AU - Shirke, Abhijit N.

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AU - Jones, J. Andrew

AU - Butterfoss, Glenn

AU - Koffas, Mattheos A G

AU - Kim, Jin

AU - Gross, Richard A.

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