Due to the steric effects imposed by bulky polymers, the formation of catalytically competent enzyme and substrate conformations is critical in the biodegradation of plastics. In poly­(ethylene terephthalate) (PET), the backbone adopts different conformations, gauche and trans, coexisting to different extents in amorphous and crystalline regions. However, which conformation is susceptible to biodegradation and the extent of enzyme and substrate conformational changes required for expedient catalysis remain poorly understood. To overcome this obstacle, we utilized molecular dynamics simulations, docking, and enzyme engineering in concert with high-resolution microscopy imaging and solid-state nuclear magnetic resonance (NMR) to demonstrate the importance of conformational selection in biocatalytic plastic hydrolysis. Our results demonstrate how single-amino acid substitutions in Ideonella sakaiensis PETase can alter its conformational landscape, significantly affecting the relative abundance of productive ground-state structures ready to bind discrete substrate conformers. We experimentally show how an enzyme binds to plastic and provide a model for key residues involved in the recognition of gauche and trans conformations supported by in silico simulations. We demonstrate how enzyme engineering can be used to create a trans-selective variant, resulting in higher activity when combined with an all-trans PET-derived oligomeric substrate, stemming from both increased accessibility and conformational preference. Our work cements the importance of matching enzyme and substrate conformations in plastic hydrolysis, and we show that also the noncanonical trans conformation in PET is conducive for degradation. Understanding the contribution of enzyme and substrate conformations to biocatalytic plastic degradation could facilitate the generation of designer enzymes with increased performance.

Certain α/β hydrolases have the ability to hydrolyze synthetic polyesters. While their partial hydrolysis has a potential for surface functionalization, complete hydrolysis allows recycling of valuable building blocks. Although knowledge about biodegradation of these materials is important regarding their fate in the environment, it is currently limited to aerobic organisms. A lipase from the anaerobic groundwater organism Pelosinus fermentans DSM 17108(PfL1) was cloned and expressed in Escherichia coli BL21-Gold (DE3) and purified from the cell extract. Biochemical characterization with small substrates showed thermoalkalophilic properties (Topt=50 °C, pHopt=7.5) and higher activity towards para-nitrophenyl octanoate (12.7 U mg(-1)) compared to longer and shorter chain lengths (C14 0.7 U mg(-1) and C2 4.3 U mg(-1), respectively). Crystallization and determination of the 3-D structure displayed the presence of a lid structure and a zinc ion surrounded by an extra domain. These properties classify the enzyme into the I.5 lipase family. PfL1 is able to hydrolyze poly(1,4-butylene adipate-co-terephthalate) (PBAT) polymeric substrates. The hydrolysis of PBAT showed the release of small building blocks as detected by liquid chromatography mass spectrometry (LC-MS). Protein dynamics seem to be involved with lid opening for the hydrolysis of PBAT by PfL1.

Structural information is crucial for understanding catalytic mechanisms and to guide enzyme engineering efforts of biocatalysts, such as terpene cyclases. However, low sequence similarity can impede homology modeling, and inherent protein instability presents challenges for structural studies. We hypothesized that X-ray crystallography of engineered thermostable ancestral enzymes can enable access to reliable homology models of extant biocatalysts. We have applied this concept in concert with molecular modeling and enzymatic assays to understand the structure activity relationship of spiroviolene synthase, a class I terpene cyclase, aiming to engineer its specificity. Engineering a surface patch in the reconstructed ancestor afforded a template structure for generation of a high-confidence homology model of the extant enzyme. On the basis of structural considerations, we designed and crystallized ancestral variants with single residue exchanges that exhibited tailored substrate specificity and preserved thermostability. We show how the two single amino acid alterations identified in the ancestral scaffold can be transferred to the extant enzyme, conferring a specificity switch that impacts the extant enzyme’s specificity for formation of the diterpene spiroviolene over formation of sesquiterpenes hedycaryol and farnesol by up to 25-fold. This study emphasizes the value of ancestral sequence reconstruction combined with enzyme engineering as a versatile tool in chemical biology.

The utilization of wood-derived building blocks (xylochemicals) to replace fossil-based precursors is an attractive research subject of modern polymer science. Here, we demonstrate that bisguaiacol (BG), a lignin-derived bisphenol analogue, can be used to prepare biobased polyesters with remarkable thermal properties. BG was treated with different activated diacids to investigate the effect of co-monomer structures on the physical properties of the products. Namely, derivatives of adipic acid, succinic acid, and 2,5-furandicarboxylic acid were used. Moreover, a terephthalic acid derivative was used for comparison purposes. The products were characterized by H NMR spectroscopy, attenuated total reflectance FTIR spectroscopy, gel-permeation chromatography, thermogravimetric analysis, and differential scanning calorimetry to assess their structural and thermal properties in detail. The polymers showed glass-transition temperatures ranging up to 160 °C and thermal stabilities in excess of 300 °C. Furthermore, the susceptibility of the polyester to enzymatic hydrolysis was investigated to assess the potential for further surface functionalization and/or recycling and biodegradation. Indeed, hydrolysis with two different enzymes from the bacteria Thermobifida cellulosilytica led to the release of monomers, as quantified by HPLC. The results of this study indicate that our new polyesters represent promising renewable and biodegradable alternatives to petroleum-based polyesters currently employed in the plastics industry, specifically for applications in which high-temperature stability is essential to ensure overall system integrity.