Influence of surface charge, binding site residues and glycosylation on Thielavia terrestris cutinase biochemical characteristics

Abstract

Cutinases are esterases of industrial importance for applications in recycling and surface modification of polyesters. The cutinase from Thielavia terrestris (TtC) is distinct in terms of its ability to retain its stability and activity in acidic pH. Stability and activity in acidic pHs are desirable for esterases as the pH of the reaction tends to go down with the generation of acid. The pH stability and activity are governed by the charged state of the residues involved in catalysis or in substrate binding. In this study, we performed the detailed structural and biochemical characterization of TtC coupled with surface charge analysis to understand its acidic tolerance. The stability of TtC in acidic pH was rationalized by evaluating the contribution of charge interactions to the Gibbs free energy of unfolding at varying pHs. The activity of TtC was found to be limited by substrate binding affinity, which is a function of the surface charge. Additionally, the presence of glycosylation affects the biochemical characteristics of TtC owing to steric interactions with residues involved in substrate binding.

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

“…TtC-G and TtC-NG were successfully expressed in Pichia pastoris and Escherichia coli, respectively. As we previously reported, analysis of CD and intrinsic tryptophan fluorescence spectra led us to conclude that TtC-G and TtC-NG display similar secondary and tertiary structures (Shirke et al, 2016).…”

“…Thermal Inactivation of Thiellavia terrestris Cutinase Thiellavia terrestris cutinase (TtC) is a fungal enzyme and, owing to the presence of two glycosylation sites (N195 and N123), it naturally exists in a glycosylated form (TtC-G) (Shirke et al, 2016). To evaluate the role of glycosylation on TtC-G thermostability, comparative thermal inactivation analyses of TtC-G and TtC-NG were performed.…”

“…S10). Thus the observed activity loss may be due to steric effects given the proximity of the introduced glycan to the active site (Shirke et al, 2016) (see Fig. S8A and B).…”

Screening of commercial enzymes for poly(ethylene terephthalate) (PET) hydrolysis and synergy studies on different substrate sources

“…As shown in Figs. Moreover, glycosylation patterns have been reported in the literature to influence hydrolysis rates due to steric hindering that difficult the access of the catalytic site to the PET chains [27]. On the other hand, BHET concentrations in the reaction media were very similar for all biocatalysts, including HiC.…”

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

“…In the case of G. cingulata cutinase, a 2.7-fold increase of stability at 35 • C was observed when it was expressed in a eukaryotic host like P. pastoris [50], instead of E. coli Origami B [71]. P. pastoris has been indeed proven to be a proper host for the expression of fungal cutinases, such as in the case of T. terrestris, which showed a 3 • C higher T m compared to when it was expressed in E. coli DH5α [72]. The higher thermotolerance was attributed to the glycosylation modification occurring in the eukaryotic host.…”