Sabatier principle for rationalizing enzymatic hydrolysis of a synthetic polyester

Abstract

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.

Concentration‐Dependent Inhibition of Mesophilic PETases on Poly(ethylene terephthalate) Can Be Eliminated by Enzyme Engineering

“…In such interfacial catalysis, the reaction rate is hyperbolic with respect to enzyme concentration, due to the saturation of accessible scissile bonds on the substrate. [15,17] In contrast to this, here we have shown that for IsPETase and numerous mesophilic homologues, the extent of reaction is not hyperbolic in profile; instead, it reaches a maximum then falls as the enzyme concentration rises. [2,15,20] In this study, we analyzed concentration-dependent inhibition in multiple natural IsPETase homologues and engineered variants of IsPETase itself, and we demonstrated that the extent of inhibition on PET is related to the dynamic properties of the enzyme, including regions around the active site; less thermostable enzymes are often more susceptible to inhibition at high enzyme concentration.…”

Unveiling PET Hydrolase Surface Dynamics through Fluorescence Microscopy

“…The results of the TIRF experiments suggest that PET hydrolases spend enough time on a PET surface to hydrolyse a number of bonds, given that the enzyme turnover number (k cat ) of LCC ICCG is approximately 0.3 s À 1 under relevant conditions. [14] However, the non-specific binding model suggested based on the TIRF data in this study would support the conclusion that the majority of enzymes bound to the surface of PET do not exhibit processive behavior. Furthermore, the results from the FRAP experiments indicate negligible lateral diffusion over the PET surface, as discussed below.…”

“…This has also been suggested previously when comparing LCC ICCG to other PET hydrolases, in accordance with the Sabatier principle, which states that the optimum activity of a protein often occurs when an enzyme has an intermediate affinity, being neither adsorption or desorption limited. [14] It has also been recently shown that the two enzymes produce markedly different degradation profiles on a PET surface, [27] with PHL7 producing a crater-like effect on the surface, whereas LCC ICCG produces a smoother surface upon degradation. This observation could also be due to the longer residence time of PHL7 compared to LCC ICCG , as the longer an enzyme spends in one position, the more likely it is to bore into the surface.…”

“…[12] It has also been shown that a lower affinity can actually have a positive effect on the activity of the enzymes, with LCC showcasing lower affinity yet higher activity than other cutinases. [13,14] However, increased affinity originating from fusion of carbohydrate-binding modules (CBMs) to PET hydrolases can improve activity, [15] at least at low substrate loadings. These results suggest that the dynamics of adsorption and desorption to the insoluble surface are key to high activity, and warrant further investigation.…”

Relationships of crystallinity and reaction rates for enzymatic degradation of poly (ethylene terephthalate), PET

“…We note that DMSO has previously been shown to either modestly suppress or enhance the enzymatic efficiency of PET-hydrolases for bulk reactions on PET. [23,45,46] Duplicates and substrate blanks (for quantification of autohydrolysis) were included, and all reactions were quenched and analyzed by RP-HPLC as described above.…”