Molecular Insights into the Enhanced Activity and/or Thermostability of PET Hydrolase by D186 Mutations.

PETase exhibits a high degradation activity for polyethylene terephthalate (PET) plastic under moderate temperatures. However, the effect of non-active site residues in the second shell of PETase on the catalytic performance remains unclear. Herein, we proposed a crystal structure- and sequence-based strategy to identify the key non-active site residue. D186 in the second shell of PETase was found to be capable of modulating the enzyme activity and stability. The most active PETaseD186N improved both the activity and thermostability with an increase in Tm by 8.89 °C. The PET degradation product concentrations were 1.86 and 3.69 times higher than those obtained with PETaseWT at 30 and 40 °C, respectively. The most stable PETaseD186V showed an increase in Tm of 12.91 °C over PETaseWT. Molecular dynamics (MD) simulations revealed that the D186 mutations could elevate the substrate binding free energy and change substrate binding mode, and/or rigidify the flexible Loop 10, and lock Loop 10 and Helix 6 by hydrogen bonding, leading to the enhanced activity and/or thermostability of PETase variants. This work unraveled the contribution of the key second-shell residue in PETase in influencing the enzyme activity and stability, which would benefit in the rational design of efficient and thermostable PETase.

New combined absorption/1H NMR method for qualitative and quantitative analysis of PET degradation products.

Poly(ethylene terephthalate) (PET) is a very valuable and beneficial material for industrial purposes, with various different applications. Due to the high annual production volume of over 50 million tons worldwide and the indiscriminate disposal by consumers, the polymers accumulate in the environment, causing negative effects on various ecosystems. Biodegradation via suitable enzymes represents a promising approach to combat the plastic waste issue so validated methods are required to measure the efficiency and efficacy of these enzymes. PETase and MHETase from Ideonella sakaiensis are suitable enzymes needed in combination to completely degrade PET into its environmentally friendly monomers. In this project, we compare and combine a previously described bulk absorbance measurement method with a newly established 1H NMR analysis method of the PET degradation products mono(2-hydroxyethyl) terephthalic acid, bis(2-hydroxyethyl) terephthalic acid and terephthalic acid. Both were optimized regarding different solvents, pH values and drying processes. The accuracy of the measurements can be confirmed with sensitivity limits of 2.5-5 µM for the absorption method and 5-10 µM for the 1H NMR analysis. The combination of the described methods therefore allows a quantitative analysis by using bulk absorption coupled with a qualitative analysis through 1H NMR. The methods established in our work can potentially contribute to the development of suitable recycling strategies of PET using recombinant enzymes.

Analysis of Poly(ethylene terephthalate) degradation kinetics of evolved IsPETase variants using a surface crowding model.

Poly(ethylene terephthalate) (PET) is a major plastic polymer utilized in the single-use and textile industries. The discovery of PET-degrading enzymes (PETases) has led to an increased interest in the biological recycling of PET in addition to mechanical recycling. IsPETase from Ideonella sakaiensis is a candidate catalyst, but little is understood about its structure-function relationships with regards to PET degradation. To understand the effects of mutations on IsPETase productivity, we develop a directed evolution assay to identify mutations beneficial to PET film degradation at 30 °C. IsPETase also displays enzyme concentration-dependent inhibition effects, and surface crowding has been proposed as a causal phenomenon. Based on total internal reflectance fluorescence microscopy and adsorption experiments, IsPETase is likely experiencing crowded conditions on PET films. Molecular dynamics simulations of IsPETase variants reveal a decrease in active site flexibility in free enzymes and reduced probability of productive active site formation in substrate-bound enzymes under crowding. Hence, we develop a surface crowding model to analyze the biochemical effects of three hit mutations (T116P, S238N, S290P) that enhanced ambient temperature activity and/or thermostability. We find that T116P decreases susceptibility to crowding, resulting in higher PET degradation product accumulation despite no change in intrinsic catalytic rate. In conclusion, we show that a macromolecular crowding-based biochemical model can be used to analyze the effects of mutations on properties of PETases and that crowding behavior is a major property to be targeted for enzyme engineering for improved PET degradation.

Purification and characterization of L-arginine deiminase from Penicillium chrysogenum.

L-arginine deiminase (ADI, EC 3.5.3.6) hydrolyzes arginine to ammonia and citrulline which is a natural supplement in health care. ADI was purified from Penicillium chrysogenum using 85% ammonium sulfate, DEAE-cellulose and Sephadex G200. ADI was purified 17.2-fold and 4.6% yield with a specific activity of 50 Umg- 1 protein. The molecular weight was 49 kDa. ADI expressed maximum activity at 40oC and an optimum pH of 6.0. ADI thermostability was investigated and the values of both t0.5 and D were determined. Kd increased by temperature and the Z value was 38oC. ATP, ADP and AMP activated ADI up to 0.6 mM. Cysteine and dithiothreitol activated ADI up to 60 µmol whereas the activation by thioglycolate and reduced glutathione (GSH) prolonged to 80 µmol. EDTA, α,α-dipyridyl, and o-phenanthroline inactivated ADI indicating that ADI is a metalloenzyme. N-ethylmaleimide (NEM), N-bromosuccinimide (NBS), butanedione (BD), dansyl chloride (DC), diethylpyrocarbonate (DEPC) and N-acetyl-imidazole (NAI) inhibited ADI activity indicating the necessity of sulfhydryl, tryptophanyl, arginyl, lysyl, histidyl and tyrosyl groups, respectively for ADI catalysis. The obtained results show that ADI from P. chrysogenum could be a potential candidate for industrial and biotechnological applications.

Deciphering the stereo-specific catalytic mechanisms of cis-epoxysuccinate hydrolases producing L(+)-tartaric acid.

Microbial epoxide hydrolases, cis-epoxysuccinate hydrolases (CESHs), have been utilized for commercial production of enantiomerically pure L(+)- and D(-)-tartaric acids for decades. However, the stereo-catalytic mechanism of CESH producing L(+)-tartaric acid (CESH[L]) remains unclear. Herein, the crystal structures of two CESH[L]s in ligand-free, product-complexed, and catalytic intermediate forms were determined. These structures revealed the unique specific binding mode for the mirror-symmetric substrate, an active catalytic triad consisting of Asp-His-Glu, and an arginine providing a proton to the oxirane oxygen to facilitate the epoxide ring-opening reaction, which has been pursued for decades. These results provide the structural basis for the rational engineering of these industrial biocatalysts.

Complete degradation of PET waste using a thermophilic microbe-enzyme system.

Enzymatic degradation has been proposed as a suitable solution for addressing PET pollution, but approximately 10 % of PET is left as nonbiodegradable. Microbes can completely degrade PET at the gram level per year. Based on the complementary benefits of microbes and enzymes, a microbe-enzyme system was created to completely degrade PET. Here, a thermophilic microbe-enzyme (TME) system composed of Bacillus thermoamylovorans JQ3 and leaf-branch compost cutinase variant (ICCG) was used to demonstrate the synergistic degradation of PET, enabling 100 % degradation of PET waste at a high PET loading level (360 g/L). Six endogenous PET hydrolases of strain JQ3 were discovered by employing an ester bond hydrolysis function-first genome mining (EGM) strategy and first successfully expressed in E. coli. These hydrolases could release TPA as the final product from PET and preferentially degraded BHET instead of MHET. Of these, carboxylesterase 39_5 and ICCG could degrade PET in a synergistic manner to generate 50 µM of TPA, which was greater than the sum of the individual treatments. Finally, the degradation pathway of the TME system was speculated to include biofilm formation, PET degradation and utilization. The successful implementation of this study rendered a scale-up degradation feasible of PET at a lower cost.

Characterization of haloacid dehalogenase superfamily acid phosphatase from Staphylococcus lugdunensis.

The haloacid dehalogenase superfamily implicated in bacterial pathogenesis comprises different enzymes having roles in many metabolic pathways. Staphylococcus lugdunensis, a Gram-positive bacterium, is an opportunistic human pathogen causing infections in the central nervous system, urinary tract, bones, peritoneum, systemic conditions and cutaneous infection. The haloacid dehalogenase superfamily proteins play a significant role in the pathogenicity of certain bacteria, facilitating invasion, survival, and proliferation within host cells. The genome of S. lugdunensis encodes more than ten proteins belonging to this superfamily. However, none of them have been characterized. The present work reports the characterization of one of the haloacid dehalogenase superfamily proteins (SLHAD1) from Staphylococcus lugdunensis. The functional analysis revealed that SLHAD1 is a metal-dependent acid phosphatase, which catalyzes the dephosphorylation of phosphorylated metabolites of cellular pathways, including glycolysis, gluconeogenesis, nucleotides, and thiamine metabolism. Based on the substrate specificity and genomic analysis, the physiological function of SLHAD1 in thiamine metabolism has been tentatively assigned. The crystal structure of SLHAD1, lacking 49 residues at the C-terminal, was determined at 1.7 Å resolution with a homodimer in the asymmetric unit. It was observed that SLHAD1 exhibited time-dependent cleavage at a specific point, occurring through a self-initiated process. A combination of bioinformatics, biochemical, biophysical, and structural studies explored unique features of SLHAD1. Overall, the study revealed a detailed characterization of a critical enzyme of the human pathogen Staphylococcus lugdunensis, associated with several life-threatening infections.

Seek and you shall find-news on the quest for novel PET-degrading enzymes.

Plastic-degrading enzymes hold immense potential for eco-friendly recycling methods. However, the catalytic rates of current enzymes do not stack up against the mammoth task of degrading millions of tons of plastic waste per year. In the quest for more efficient polyethylene terephthalate (PET)-degrading enzymes, Zhang et al. report the discovery and characterization of PET40, a versatile PET-hydrolyzing esterase that is divergent from most characterized PETases. While PET40 has comparably low hydrolytic activity on PET, Zhang et al. demonstrate its broad activity on an expanded substrate pool. This sheds light on the potential ecological role of these esterases and suggests that PET might be only a recent addition to their substrate spectrum.

The metagenome-derived esterase PET40 is highly promiscuous and hydrolyses polyethylene terephthalate (PET).

Polyethylene terephthalate (PET) is a widely used synthetic polymer and known to contaminate marine and terrestrial ecosystems. Only few PET-active microorganisms and enzymes (PETases) are currently known, and it is debated whether degradation activity for PET originates from promiscuous enzymes with broad substrate spectra that primarily act on natural polymers or other bulky substrates, or whether microorganisms evolved their genetic makeup to accepting PET as a carbon source. Here, we present a predicted diene lactone hydrolase designated PET40, which acts on a broad spectrum of substrates, including PET. It is the first esterase with activity on PET from a GC-rich Gram-positive Amycolatopsis species belonging to the Pseudonocardiaceae (Actinobacteria). It is highly conserved within the genera Amycolatopsis and Streptomyces. PET40 was identified by sequence-based metagenome search using a PETase-specific hidden Markov model. Besides acting on PET, PET40 has a versatile substrate spectrum, hydrolyzing δ-lactones, ß-lactam antibiotics, the polyester-polyurethane Impranil® DLN, and various para-nitrophenyl ester substrates. Molecular docking suggests that the PET degradative activity is likely a result of the promiscuity of PET40, as potential binding modes were found for substrates encompassing mono(2-hydroxyethyl) terephthalate, bis(2-hydroxyethyl) terephthalate, and a PET trimer. We also solved the crystal structure of the inactive PET40 variant S178A to 1.60 Å resolution. PET40 is active throughout a wide pH (pH 4-10) and temperature range (4-65 °C) and remarkably stable in the presence of 5% SDS, making it a promising enzyme as a starting point for further investigations and optimization approaches.

Chemical synthesis of a 28 kDa full-length PET degrading enzyme ICCG by the removable backbone modification strategy.

Chemical protein synthesis offers a powerful way to access otherwise-difficult-to-obtain proteins such as mirror-image proteins. Although a large number of proteins have been chemically synthesized to date, the acquisition to proteins containing hydrophobic peptide fragments has proven challenging. Here, we describe an approach that combines the removable backbone modification strategy and the peptide hydrazide-based native chemical ligation for the chemical synthesis of a 28 kDa full-length PET degrading enzyme IGGC (a higher depolymerization efficiency of variant leaf-branch compost cutinase (LCC)) containing hydrophobic peptide segments. The synthetic ICCG exhibits the enzymatic activity and will be useful in establishing the corresponding mirror-image version of ICCG.

A Hydrolase Produced by Rhodococcus erythropolis HQ Is Responsible for the Detoxification of Zearalenone.

Zearalenone (ZEN), an estrogenic mycotoxin, is one of the prevalent contaminants found in food and feed, posing risks to human and animal health. In this study, we isolated a ZEN-degrading strain from soil and identified it as Rhodococcus erythropolis HQ. Analysis of degradation products clarified the mechanism by which R. erythropolis HQ degrades ZEN. The gene zenR responsible for degrading ZEN was identified from strain HQ, in which zenR is the key gene for R. erythropolis HQ to degrade ZEN, and its expression product is a hydrolase named ZenR. ZenR shared 58% sequence identity with the hydrolase ZenH from Aeromicrobium sp. HA, but their enzymatic properties were significantly different. ZenR exhibited maximal enzymatic activity at pH 8.0-9.0 and 55 °C, with a Michaelis constant of 21.14 µM, and its enzymatic activity is 2.8 times that of ZenH. The catalytic triad was identified as S132-D157-H307 via molecular docking and site-directed mutagenesis. Furthermore, the fermentation broth of recombinant Bacillus containing ZenR can be effectively applied to liquefied corn samples, with the residual amount of ZEN decreased to 0.21 µg/g, resulting in a remarkable ZEN removal rate of 93%. Thus, ZenR may serve as a new template for the modification of ZEN hydrolases and a new resource for the industrial application of biological detoxification. Consequently, ZenR could potentially be regarded as a novel blueprint for modifying ZEN hydrolases and as a fresh resource for the industrial implementation of biological detoxification.

First Display of Haloalkane Dehalogenase LinB on the Surface of Bacillus subtilis Spore.

BACKGROUND: LinB, as a Haloalkane dehalogenase, has good catalytic activity for many highly toxic and recalcitrant compounds, and can realize the elimination of chemical weapons HD in a green non-toxic mode. OBJECTIVES: In order to display Haloalkane dehalogenase LinB on the surface of Bacillus subtilis spore. METHODS: We have constituted the B. subtilis spore surface display system of halogenated alkanes dehalogenase LinB by gene recombination. RESULTS: Data revealed that LinB can display on spore surface successfully. The hydrolyzing HD analogue 2-chloroethyl ethylsulfide (2-CEES) activity of displayed LinB spores was 4.30±0.09 U/mL, and its specific activity was 0.78±0.03U/mg. Meanwhile, LinB spores showed a stronger stress resistance activity on 2-CEES than free LinB. This study obtained B. subtilis spores of LinB (phingobium japonicum UT26) with enzyme activity that was not reported before. CONCLUSION: Spore surface display technology uses resistance spore as the carrier to guarantee LinB activity, enhances its stability, and reduces the production cost, thus expanding the range of its application.

Computational design of highly efficient thermostable MHET hydrolases and dual enzyme system for PET recycling.

Recently developed enzymes for the depolymerization of polyethylene terephthalate (PET) such as FAST-PETase and LCC-ICCG are inhibited by the intermediate PET product mono(2-hydroxyethyl) terephthalate (MHET). Consequently, the conversion of PET enzymatically into its constituent monomers terephthalic acid (TPA) and ethylene glycol (EG) is inefficient. In this study, a protein scaffold (1TQH) corresponding to a thermophilic carboxylesterase (Est30) was selected from the structural database and redesigned in silico. Among designs, a double variant KL-MHETase (I171K/G130L) with a similar protein melting temperature (67.58 °C) to that of the PET hydrolase FAST-PETase (67.80 °C) exhibited a 67-fold higher activity for MHET hydrolysis than FAST-PETase. A fused dual enzyme system comprising KL-MHETase and FAST-PETase exhibited a 2.6-fold faster PET depolymerization rate than FAST-PETase alone. Synergy increased the yield of TPA by 1.64 fold, and its purity in the released aromatic products reached 99.5%. In large reaction systems with 100 g/L substrate concentrations, the dual enzyme system KL36F achieved over 90% PET depolymerization into monomers, demonstrating its potential applicability in the industrial recycling of PET plastics. Therefore, a dual enzyme system can greatly reduce the reaction and separation cost for sustainable enzymatic PET recycling.

De novo design of a polycarbonate hydrolase.

Enzymatic degradation of plastics is currently limited to the use of engineered natural enzymes. As of yet, all engineering approaches applied to plastic degrading enzymes retain the natural $\alpha /\beta $-fold. While mutations can be used to increase thermostability, an inherent maximum likely exists for the $\alpha /\beta $-fold. It is thus of interest to introduce catalytic activity toward plastics in a different protein fold to escape the sequence space of plastic degrading enzymes. Here, a method for designing highly thermostable enzymes that can degrade plastics is described. With the help of Rosetta an active site catalysing the hydrolysis of polycarbonate is introduced into a set of thermostable scaffolds. Through computational evaluation, a potential PCase was selected and produced recombinantly in Escherichia coli. Thermal analysis suggests that the design has a melting temperature of >95$^{\circ }$C. Activity toward polycarbonate was confirmed using atomic force spectroscopy (AFM), proving the successful design of a PCase.

Atypical homodimerization revealed by the structure of the (S)-enantioselective haloalkane dehalogenase DmmarA from Mycobacterium marinum.

Haloalkane dehalogenases (HLDs) are a family of α/ß-hydrolase fold enzymes that employ SN2 nucleophilic substitution to cleave the carbon-halogen bond in diverse chemical structures, the biological role of which is still poorly understood. Atomic-level knowledge of both the inner organization and supramolecular complexation of HLDs is thus crucial to understand their catalytic and noncatalytic functions. Here, crystallographic structures of the (S)-enantioselective haloalkane dehalogenase DmmarA from the waterborne pathogenic microbe Mycobacterium marinum were determined at 1.6 and 1.85 Šresolution. The structures show a canonical αßα-sandwich HLD fold with several unusual structural features. Mechanistically, the atypical composition of the proton-relay catalytic triad (aspartate-histidine-aspartate) and uncommon active-site pocket reveal the molecular specificities of a catalytic apparatus that exhibits a rare (S)-enantiopreference. Additionally, the structures reveal a previously unobserved mode of symmetric homodimerization, which is predominantly mediated through unusual L5-to-L5 loop interactions. This homodimeric association in solution is confirmed experimentally by data obtained from small-angle X-ray scattering. Utilizing the newly determined structures of DmmarA, molecular modelling techniques were employed to elucidate the underlying mechanism behind its uncommon enantioselectivity. The (S)-preference can be attributed to the presence of a distinct binding pocket and variance in the activation barrier for nucleophilic substitution.

Impact of GSK199 and GSK106 binding on protein arginine deiminase IV stability and flexibility: A computational approach.

Protein arginine deiminase IV (PAD4) is a potential target for diseases including rheumatoid arthritis and cancers. Currently, GSK199 is a potent, selective yet reversible PAD4 inhibitor. Its derivative, GSK106, on the other hand, was reported as an inactive compound when tested against PAD4 assay. Although they had similar skeleton, their impact towards PAD4 structural and flexibility is unknown. In order to fill the research gap, the impact of GSK199 and GSK106 binding towards PAD4 stability and flexibility is investigated via a combination of computational methods. Molecular docking indicates that GSK199 and GSK106 are capable to bind at PAD4 pocket by using its back door with -10.6 kcal/mol and -9.6 kcal/mol, respectively. The simulations of both complexes were stable throughout 100 ns. The structure of PAD4 exhibited a tighter packing in the presence of GSK106 compared to GSK199. The RMSF analysis demonstrates significant changes between the PAD4-GSK199 and PAD4-GSK106 simulations in the regions containing residues 136, 160, 220, 438, and 606. The Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) analysis shows a marked difference in binding free energies, with -11.339 kcal/mol for the PAD4-GSK199 complex and 1.063 kcal/mol for the PAD4-GSK106 complex. The hydrogen bond analysis revealed that the GSK199 and GSK106 binding to PAD4 are assisted by six hydrogen bonds and three hydrogen bonds, respectively. The visualisation of the MD simulations revealed that GSK199 remained in the PAD4 pocket, whereas GSK106 shifted away from the catalytic site. Meanwhile, molecular dockings of benzoyl arginine amide (BAEE) substrate have shown that BAEE is able to bind to PAD4 catalytic site when GSK106 was present but not when GSK199 occupied the site. Overall, combination of computational approaches successfully described the behaviour of binding pocket of PAD4 structure in the presence of the active and inactive compounds.

Cationic Polymers Remarkably Boost Haloalkane Dehalogenase Activity in Organic Solvent Solutions and the Molecular Implications.

Applications of haloalkane dehalogenase DhaA in biocatalysis are limited by its unfavorable performance in organic solvents. Our previous work proved that mutations of surface positive-charged residues enhanced the organic solvent resistance of DhaA, which inspired us to explore the effect of cationic polymers on DhaA in organic solvents. Remarkably boosted performance was achieved in different organic solvent solutions by introducing cationic polymers, for example, there was a 6.1-fold activity increase with poly(allylamine hydrochloride) and a 5.5-fold activity increase with poly(ethylene imine) in 40 vol.% dimethylsulfoxide. The presence of cationic polymers protected DhaA from damage by organic solvents and increased the substrate concentration around the enzyme-polymer complex. Fluorescence spectroscopy and molecular dynamics simulations revealed that the binding of cationic polymers onto DhaA weakened the interactions between organic solvents and DhaA, decreased the organic solvent solvation level around DhaA, and enhanced the structural stability of DhaA in organic solvents. This comprehensive understanding of the effect of cationic polymers on DhaA can help to broaden the applications of DhaA in organic solvent-involved biocatalysis.

Functional annotation of haloacid dehalogenase superfamily structural genomics proteins.

Haloacid dehalogenases (HAD) are members of a large superfamily that includes many Structural Genomics proteins with poorly characterized functionality. This superfamily consists of multiple types of enzymes that can act as sugar phosphatases, haloacid dehalogenases, phosphonoacetaldehyde hydrolases, ATPases, or phosphate monoesterases. Here, we report on predicted functional annotations and experimental testing by direct biochemical assay for Structural Genomics proteins from the HAD superfamily. To characterize the functions of HAD superfamily members, nine representative HAD proteins and 21 structural genomics proteins are analyzed. Using techniques based on computed chemical and electrostatic properties of individual amino acids, the functions of five structural genomics proteins from the HAD superfamily are predicted and validated by biochemical assays. A dehalogenase-like hydrolase, RSc1362 (Uniprot Q8XZN3, PDB 3UMB) is predicted to be a dehalogenase and dehalogenase activity is confirmed experimentally. Four proteins predicted to be sugar phosphatases are characterized as follows: a sugar phosphatase from Thermophilus volcanium (Uniprot Q978Y6) with trehalose-6-phosphate phosphatase and fructose-6-phosphate phosphatase activity; haloacid dehalogenase-like hydrolase from Bacteroides thetaiotaomicron (Uniprot Q8A2F3; PDB 3NIW) with fructose-6-phosphate phosphatase and sucrose-6-phosphate phosphatase activity; putative phosphatase from Eubacterium rectale (Uniprot D0VWU2; PDB 3DAO) as a sucrose-6-phosphate phosphatase; and hypothetical protein from Geobacillus kaustophilus (Uniprot Q5L139; PDB 2PQ0) as a fructose-6-phosphate phosphatase. Most of these sugar phosphatases showed some substrate promiscuity.

Multimeric structure of a subfamily III haloalkane dehalogenase-like enzyme solved by combination of cryo-EM and x-ray crystallography.

Haloalkane dehalogenase (HLD) enzymes employ an SN 2 nucleophilic substitution mechanism to erase halogen substituents in diverse organohalogen compounds. Subfamily I and II HLDs are well-characterized enzymes, but the mode and purpose of multimerization of subfamily III HLDs are unknown. Here we probe the structural organization of DhmeA, a subfamily III HLD-like enzyme from the archaeon Haloferax mediterranei, by combining cryo-electron microscopy (cryo-EM) and x-ray crystallography. We show that full-length wild-type DhmeA forms diverse quaternary structures, ranging from small oligomers to large supramolecular ring-like assemblies of various sizes and symmetries. We optimized sample preparation steps, enabling three-dimensional reconstructions of an oligomeric species by single-particle cryo-EM. Moreover, we engineered a crystallizable mutant (DhmeAΔGG ) that provided diffraction-quality crystals. The 3.3 Å crystal structure reveals that DhmeAΔGG forms a ring-like 20-mer structure with outer and inner diameter of ~200 and ~80 Å, respectively. An enzyme homodimer represents a basic repeating building unit of the crystallographic ring. Three assembly interfaces (dimerization, tetramerization, and multimerization) were identified to form the supramolecular ring that displays a negatively charged exterior, while its interior part harboring catalytic sites is positively charged. Localization and exposure of catalytic machineries suggest a possible processing of large negatively charged macromolecular substrates.

Engineering the catalytic activity of an Antarctic PET-degrading enzyme by loop exchange.

Several hydrolases have been described to degrade polyethylene terephthalate (PET) at moderate temperatures ranging from 25°C to 40°C. These mesophilic PET hydrolases (PETases) are less efficient in degrading this plastic polymer than their thermophilic homologs and have, therefore, been the subject of many protein engineering campaigns. However, enhancing their enzymatic activity through rational design or directed evolution poses a formidable challenge due to the need for exploring a large number of mutations. Additionally, evaluating the improvements in both activity and stability requires screening numerous variants, either individually or using high-throughput screening methods. Here, we utilize instead the design of chimeras as a protein engineering strategy to increase the activity and stability of Mors1, an Antarctic PETase active at 25°C. First, we obtained the crystal structure of Mors1 at 1.6 Å resolution, which we used as a scaffold for structure- and sequence-based chimeric design. Then, we designed a Mors1 chimera via loop exchange of a highly divergent active site loop from the thermophilic leaf-branch compost cutinase (LCC) into the equivalent region in Mors1. After restitution of an active site disulfide bond into this chimera, the enzyme exhibited a shift in optimal temperature for activity to 45°C and an increase in fivefold in PET hydrolysis when compared with wild-type Mors1 at 25°C. Our results serve as a proof of concept of the utility of chimeric design to further improve the activity and stability of PETases active at moderate temperatures.