title: Comparative thermal inactivation analysis of Aspergillus oryzae and Thiellavia terrestris cutinase: Role of glycosylation.
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. Biotechnol. Bioeng. 2017;114: 63-73. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, New York.|Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York.
authors.forename: Abhijit N
authors.initials: AN
authors.lastname: Shirke
authors.affiliation: Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, New York.|Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York.
authors.forename: An
authors.initials: A
authors.lastname: Su
authors.affiliation: Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York.|Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York.
authors.forename: J Andrew
authors.initials: JA
authors.lastname: Jones
authors.affiliation: Center for Genomics and Systems Biology, New York University Abu Dhabi, Abu Dhabi, UAE.
authors.forename: Glenn L
authors.initials: GL
authors.lastname: Butterfoss
authors.affiliation: Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York.|Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York.
authors.forename: Mattheos A G
authors.initials: MA
authors.lastname: Koffas
authors.affiliation: Department of Chemical and Biomolecular Engineering, New York University Tandon School of Engineering, Brooklyn, New York.
authors.forename: Jin Ryoun
authors.initials: JR
authors.lastname: Kim
authors.affiliation: Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, New York.|Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Gross
mesh_terms: D001236:Aspergillus oryzae / Q000201:enzymology* / Q000235:genetics; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006358:Hot Temperature; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020037:Sordariales / Q000201:enzymology* / Q000235:genetics
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases; C019952:cutinase
keywords: aggregation; cutinase; glycosylation; thermal inactivation