Arnling Baath, J. | Search bioRxiv

bioRxiv2022

Sabatier principle for rationalizing enzymatic hydrolysis of a synthetic polyester

Interfacial enzyme reactions are common in nature and in industrial settings, including the enzymatic deconstruction of poly(ethylene terephthalate) (PET) waste. Kinetic descriptions of PET hydrolases are necessary for both comparative analyses, discussions of structure-function relations and rational optimization of technical processes. We investigated whether the Sabatier principle could be used for this purpose. Specifically, we compared the kinetics of two well-known PET hydrolases, leaf-branch compost cutinase (LCC) and a cutinase from the bacterium T. fusca (TfC) when adding different concentrations of the surfactant cetyltrimethylammonium bromide (CTAB). We found that CTAB consistently lowered the strength of enzyme-PET interactions, while its effect on enzymatic turnover was strongly biphasic. Thus, at gradually increasing CTAB concentrations, turnover was initially promoted and subsequently suppressed. This correlation with maximal turnover at an intermediate binding strength is in accordance with the Sabatier principle. One consequence of these results is that both enzymes had too strong intrinsic interaction with PET for optimal turnover, especially TfC, which showed a 20-fold improvement of kcat at the maximum. LCC on the other hand had an intrinsic substrate affinity closer to the Sabatier optimum and the turnover rate was 5-fold improved at weakened substrate binding. Our results show that the Sabatier principle may indeed rationalize enzymatic PET degradation and support process optimization. Finally, we suggest that future discovery efforts should consider enzymes with weakened substrate binding, since strong adsorption seems to limit their catalytic performance. ToC graphics O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

biochemistry↗

bioRxiv2020

Comparative biochemistry of four polyester (PET) hydrolases

The potential of bioprocessing in a circular plastic economy has strongly stimulated research in enzymatic degradation of different synthetic resins. Particular interest has been devoted to the commonly used polyester, poly(ethylene terephthalate) (PET), and a number of PET hydrolases have been described. However, a kinetic framework for comparisons of PET hydrolases (or other plastic degrading enzymes) acting on the insoluble substrate, has not been established. Here, we propose such a framework and test it against kinetic measurements on four PET hydrolases. The analysis provided values of kcat and KM, as well as an apparent specificity constant in the conventional units of M-1s-1. These parameters, together with experimental values for the number of enzyme attack sites on the PET surface, enabled comparative analyses. We found that the PET hydrolase from Ideonella sakaiensis was the most efficient enzyme at ambient conditions, and that this relied on a high kcat rather than a low KM. Moreover, both soluble and insoluble PET fragments were consistently hydrolyzed much faster than intact PET. This suggests that interactions between polymer strands slow down PET degradation, while the chemical steps of catalysis and the low accessibility associated with solid substrate were less important for the overall rate. Finally, the investigated enzymes showed a remarkable substrate affinity, and reached half the saturation rate on PET, when the concentration of attack sites in the suspension was only about 50 nM. We propose that this is linked to nonspecific adsorption, which promotes the nearness of enzyme and attack sites.

biochemistry↗