title: Toward rational thermostabilization of Aspergillus oryzae cutinase: Insights into catalytic and structural stability.
abstract: Cutinases are powerful hydrolases that can cleave ester bonds of polyesters such as poly(ethylene terephthalate) (PET), opening up new options for enzymatic routes for polymer recycling and surface modification reactions. Cutinase from Aspergillus oryzae (AoC) is promising owing to the presence of an extended groove near the catalytic triad which is important for the orientation of polymeric chains. However, the catalytic efficiency of AoC on rigid polymers like PET is limited by its low thermostability; as it is essential to work at or over the glass transition temperature (Tg) of PET, that is, 70 °C. Consequently, in this study we worked toward the thermostabilization of AoC. Use of Rosetta computational protein design software in conjunction with rational design led to a 6 °C improvement in the thermal unfolding temperature (Tm) and a 10-fold increase in the half-life of the enzyme activity at 60 °C. Surprisingly, thermostabilization did not improve the rate or temperature optimum of enzyme activity. Three notable findings are presented as steps toward designing more thermophilic cutinase: (a) surface salt bridge optimization produced enthalpic stabilization, (b) mutations to proline reduced the entropy loss upon folding, and (c) the lack of a correlative increase in the temperature optimum of catalytic activity with thermodynamic stability suggests that the active site is locally denatured at a temperature below the Tm of the global structure.
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: Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York.|Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, New York.
authors.forename: Danielle
authors.initials: D
authors.lastname: Basore
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 Genomics and Systems Biology, New York University, New York.
authors.forename: Richard
authors.initials: R
authors.lastname: Bonneau
authors.affiliation: Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York.|Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, New York.|Department of Computer Science, Rensselaer Polytechnic Institute, Troy, New York.
authors.forename: Christopher
authors.initials: C
authors.lastname: Bystroff
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 / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D008958:Models, Molecular; D009154:Mutation; D011093:Polyethylene Terephthalates / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering; D013696:Temperature
chemical_list: D011093:Polyethylene Terephthalates; D002265:Carboxylic Ester Hydrolases; C019952:cutinase
keywords: PET; Rosetta; esterase; green chemistry; poly(ethylene terephthalate); protein design; protein engineering; thermophile