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(ethyleneterephthalate) (PET), opening up new options for enzymatic routes for polymer recycling and surface modification reactions. Cutinase from Apsergillus 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 i.e. 70 °C. Consequently, in this study we worked towards 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.

Small cause, large effect: Structural characterization of cutinases from Thermobifida cellulosilytica

“…Generally, the structural information of these Thermobifida cutinases was important in engineering towards improved thermostability (Then et al, ; Thumarat et al, ). In addition to Thermobifida cutinases, the structural basis for thermostability of other cutinases like from another compost organisms, namely Saccharomonospora viridis or from Aspergillus oryzae was recently discussed (Miyakawa et al, ; Shirke et al, ).…”

“…Despite the potential of these enzymes, especially processing of cheap bulk polymers will require more efficient enzymes. Recently, various strategies for the improvement of such enzymes hydrolysing polyethylene terephthalate (PET) related to thermostability (Miyakawa et al, ; Shirke et al, ; Then et al, ), ultrasound activation (Pellis et al, ), and active site architecture (Araujo et al, ; Silva et al, ; Wei et al, ) were reported. In addition, we have demonstrated that the attachment of hydrophobic binding modules (Gamerith et al, ; Ribitsch et al, ) or hydrophobins to cutinases (Ribitsch et al, ) or enzyme surface engineering (Herrero Acero et al, ) can tune sorption properties and thereby improve PET hydrolysis .…”

Microbial Degradation and Valorization of Plastic Wastes

“…As a result, it suggests that efficient enzymatic degradation of PET requires thermostable PET depolymerases. Approaches of glycosylation (Shirke et al, 2018) and rational protein engineering, such as the optimization of surface salt bridge (Shirke et al, 2016), mutation of Ca 2+ and Mg 2+ binding sites , introduction of disulfide bridge , stabilization of a β6-β7 connecting loop, and extension of subsite IIc (Son et al, 2019), have been applied to improve the thermostability of these PET hydrolases. Notwithstanding, there is room for increasing the half-life of PET hydrolases above 65 • C.…”

A Middle-Aged Enzyme Still in Its Prime: Recent Advances in the Field of Cutinases

“…However, most variants lost their activity at temperatures well below the thermal unfolding temperature, due to an earlier active site denaturation. Consequently, further research has to be performed in order to design thermostable variants that maintain their activities at elevated temperatures [61].…”