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Comparative Biochemistry of Four Polyester (PET) Hydrolases

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Comparative Biochemistry of Four Polyester (PET) Hydrolases bioRxiv preprint doi: https://doi.org/10.1101/2020.11.20.392019; this version posted November 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Comparative biochemistry of four polyester (PET) hydrolases Jenny Arnling Bååth1, Kim Borch2, Kenneth Jensen2, Jesper Brask2 and Peter Westh1* 1Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads, DK- 2800 Kgs. Lyngby, Denmark 2Novozymes A/S, Biologiens Vej 2, DK-2800 Kgs. Lyngby, Denmark *Corresponding author: Peter Westh E-mail: [email protected] Running title: Comparative biochemistry of PET hydrolases Keywords: Enzyme kinetics, Michaelis-Menten, cutinase, PET hydrolase, serine esterase, enzyme turnover, enzyme degradation, biotechnology, interfacial enzymology, heterogeneous catalysis 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.20.392019; this version posted November 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Abstract monomer recovery and re-synthesis of virgin residue. Recent progress suggests that this may be The potential of bioprocessing in a circular plastic achieved through bioprocessing (1,4), and one economy has strongly stimulated research in crucial requirement for this is the development of enzymatic degradation of different synthetic resins. efficient enzymes for PET degradation. 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 Research on enzymatic degradation of PET has framework for comparisons of PET hydrolases (or been ongoing for over 20 years, and enzymes other plastic degrading enzymes) acting on the classified as cutinases have been described as the insoluble substrate, has not been established. Here, most promising PET hydrolases (2,3,5). Cutinases we propose such a framework and test it against are typical serine hydrolases, with an α/β fold and a kinetic measurements on four PET hydrolases. The catalytic triad consisting of serine, histidine and aspartate (6). Compared to lipases and many ester- analysis provided values of kcat and KM, as well as an apparent specificity constant in the conventional active enzymes, cutinases exhibit a flat, surface units of M-1s-1. These parameters, together with exposed active site, and this have been described as experimental values for the number of enzyme essential for their interaction with the PET polymer, attack sites on the PET surface, enabled which is bulkier than their preferred, aliphatic comparative analyses. We found that the PET substrate, cutin (7,8). PET hydrolases have been hydrolase from Ideonella sakaiensis was the most described as enzymes with low to moderate efficient enzyme at ambient conditions, and that turnover rates, reflecting the fact that PET is an unnatural substrate for these enzymes (2). this relied on a high kcat rather than a low KM. Moreover, both soluble and insoluble PET However, in 2016 the bacterium Ideonella fragments were consistently hydrolyzed much sakaiensis was discovered, possessing an faster than intact PET. This suggests that unprecedented capacity to use PET as a source of interactions between polymer strands slow down carbon and energy. This bacterium was isolated PET degradation, while the chemical steps of from a PET-rich environment and secretes a PET catalysis and the low accessibility associated with hydrolase, which is homologous to cutinases, and solid substrate were less important for the overall may represent a short evolutionary adaptation to the rate. Finally, the investigated enzymes showed a synthetic substrate. Due to its superior PET remarkable substrate affinity, and reached half the degrading ability and a significantly lower activity saturation rate on PET, when the concentration of on natural, aliphatic polyesters it has been attack sites in the suspension was only about 50 nM. categorized into a novel family of enzymes, named We propose that this is linked to nonspecific PETases (EC 3.1.1.101) (5). The discovery of the I. adsorption, which promotes the nearness of enzyme sakaiensis PETase has led to several structural and attack sites. studies, which have improved our understanding of the catalytic process and consequently outlined strategies for rational engineering of enzyme variants with improved activity against the Introduction synthetic substrate (9–12). However, this progress Polyethylene terephthalate (PET) is a copolymer of in structural understanding has not been paralleled terephthalic acid (TPA) and ethylene glycol (EG) by biochemical investigations. Thus, while some linked by ester bonds (Fig. 1), and among the most studies have reported kinetic parameters for PET produced plastics. The stable nature of the PET hydrolases (4,8,13–17), most functional molecule has been an advantage for its use in fibers, assessments have used long contact times and films and packaging, but this characteristic is also empirical discussions of either end-point becoming an increasing problem, as it leads to concentrations or progress curves (18–23). Results accumulation of PET in the environment (1–3). from the latter type of work primarily elucidate This causes a growing need for cost-effective time- and dose requirements to achieve a significant means to recycle PET waste for example through degree of polymer conversion, and are hence 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.20.392019; this version posted November 21, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. important for technical applications of PET solid substrates (29–31) including PET (8,14,15). hydrolases. However, in the absence of physically Both convMM plots (rate vs. substrate load) and meaningful kinetic parameters, one can typically invMM plots (rate vs. enzyme concentration) may only make superficial analyses of structure- lead to hyperbolic curves (27), and the benefit of function relationships. This makes it difficult to combining these two approaches may be illustrated compare results across different studies, and by considering the conditions at saturation. For impedes the potential for interpretations regarding convMM, saturation reflects the well-known molecular mechanisms of the catalytic process. situation, where all enzyme are engaged in a complex, and the rate becomes (c.f. Fig. 2) conv Vmax =kcatE0 (1) The scarcity of kinetic parameters can be linked to the interfacial nature of the reaction, which gives For invMM, saturation occurs at high enzyme rise to some complications that are unknown in concentration as all attack sites on the surface conventional (bulk) enzyme kinetics. One become occupied (and additional enzyme particular difficulty arises because the molar accumulates in the bulk). If we assume that the concentration of the substrate is unknown. This conversion of these sites is governed by kcat, the challenges the use of mass-action kinetics and inverse saturation rate may be written hence the conventional Michaelis-Menten inv (convMM) framework (8,24). However, mass-action Vmax =kcatΓattackS0 (2) kinetics is widely used for non-biochemical, where S0 is the (known) mass load of substrate. The interfacial catalysis (25,26), and the normal way to expression in eq. (2) emerges intuitively as handle the solid material in rate equations is to illustrated in Fig. 2, but has been derived rigorously conv inv define a number of sites on the surface, which are elsewhere (27). As Vmax and Vmax can be competent for the process in question. Along these derived from experiments, we can estimate Γattack as lines, we define an attack site on the PET surface as the ratio of the maximal specific rates. Specifically, a locus, where the PET hydrolase is able to form a combining eqs. (1) and (2) yields productive substrate complex (Fig. 2). If the density 푖푛푣푉 of attack sites, Γattack in units of mol sites per gram 푚푎푥 푆0 = Γ PET, can be experimentally established, it is 푐표푛푣푉푚푎푥 attack possible to convert a substrate load (in g/L) into a 퐸0 (3) molar concentration of sites (27). This is only an apparent molar concentration, because the surface sites are not evenly distributed in the suspension, We have used these ideas to characterize a group of but the approach opens up for the use of mass- enzymes consisting of a catalytically improved action kinetics, and hence the derivation of rigorous variant of I. sakaiensis PETase (12), cutinases from kinetic parameters for a heterogeneous enzyme respectively the fungus Humicola insolens and the reaction. bacterium Thermobifida fusca and a carboxyl- esterase from Bacillus subtilis. These enzymes have previously been reported to hydrolyze PET In the current work, we have tested a kinetic (8,14,21) and here we conduct a comparative approach based on these ideas for PET hydrolases. kinetic analysis with respect to their activity on both Specifically, we analyzed rate measurements polymeric PET and smaller (soluble or insoluble) obtained under two different
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