Restriction of arginine induces antibiotic tolerance in Staphylococcus aureus.

Staphylococcus aureus is responsible for a substantial number of invasive infections globally each year. These infections are problematic because they are frequently recalcitrant to antibiotic treatment. Antibiotic tolerance, the ability of bacteria to persist despite normally lethal doses of antibiotics, contributes to antibiotic treatment failure in S. aureus infections. To understand how antibiotic tolerance is induced, S. aureus biofilms exposed to multiple anti-staphylococcal antibiotics are examined using both quantitative proteomics and transposon sequencing. These screens indicate that arginine metabolism is involved in antibiotic tolerance within a biofilm and support the hypothesis that depletion of arginine within S. aureus communities can induce antibiotic tolerance. Consistent with this hypothesis, inactivation of argH, the final gene in the arginine synthesis pathway, induces antibiotic tolerance. Arginine restriction induces antibiotic tolerance via inhibition of protein synthesis. In murine skin and bone infection models, an argH mutant has enhanced ability to survive antibiotic treatment with vancomycin, highlighting the relationship between arginine metabolism and antibiotic tolerance during S. aureus infection. Uncovering this link between arginine metabolism and antibiotic tolerance has the potential to open new therapeutic avenues targeting previously recalcitrant S. aureus infections.

A customized self-assembled synergistic biocatalyst for plastic depolymerization.

The enzymatic degradation of plastic offers a green, sustainable strategy and scalable circular carbon route for solving polyester waste. Among the earlies discovered plastic-degrading enzymes are PET hydrolase (PETase) and MHET hydrolase (MHETase), which act synergistically. To promote the adsorption of enzymes on PET surfaces, increase their robustness, and enable directly depolymerization, we designed hydrophobin HFBI fused-PETase and MHETase. A customized self-assembled synergistic biocatalyst (MC@CaZn-MOF) was further developed to promote the two-step depolymerization process. The tailored catalysts showed better adhesion to the PET surface and desirable durability, retaining over 70% relative activity after incubation at pH 8.0 and 60 °C for 120 h. Importantly, MC@CaZn-MOF could directly decompose untreated AGf-PET to generate 9.5 mM TPA with weight loss over 90%. The successful implementation of a bifunctional customized catalyst makes the large-scale biocatalytic degradation of PET feasible, contributing to polymer upcycling and environmental sustainability.

Identify Regioselective Residues of Ginsenoside Hydrolases by Graph-Based Active Learning from Molecular Dynamics.

Identifying the catalytic regioselectivity of enzymes remains a challenge. Compared to experimental trial-and-error approaches, computational methods like molecular dynamics simulations provide valuable insights into enzyme characteristics. However, the massive data generated by these simulations hinder the extraction of knowledge about enzyme catalytic mechanisms without adequate modeling techniques. Here, we propose a computational framework utilizing graph-based active learning from molecular dynamics to identify the regioselectivity of ginsenoside hydrolases (GHs), which selectively catalyze C6 or C20 positions to obtain rare deglycosylated bioactive compounds from Panax plants. Experimental results reveal that the dynamic-aware graph model can excellently distinguish GH regioselectivity with accuracy as high as 96-98% even when different enzyme-substrate systems exhibit similar dynamic behaviors. The active learning strategy equips our model to work robustly while reducing the reliance on dynamic data, indicating its capacity to mine sufficient knowledge from short multi-replica simulations. Moreover, the model's interpretability identified crucial residues and features associated with regioselectivity. Our findings contribute to the understanding of GH catalytic mechanisms and provide direct assistance for rational design to improve regioselectivity. We presented a general computational framework for modeling enzyme catalytic specificity from simulation data, paving the way for further integration of experimental and computational approaches in enzyme optimization and design.

Biochemical Characterisation of Sis: A Distinct Thermophilic PETase with Enhanced NanoPET Substrate Hydrolysis and Thermal Stability.

Polyethylene terephthalate (PET) degradation by enzymatic hydrolysis is significant for addressing plastic pollution and fostering sustainable waste management practices. Identifying thermophilic and thermostable PET hydrolases is particularly crucial for industrial bioprocesses, where elevated temperatures may enhance enzymatic efficiency and process kinetics. In this study, we present the discovery of a novel thermophilic and thermostable PETase enzyme named Sis, obtained through metagenomic sequence-based analysis. Sis exhibits robust activity on nanoPET substrates, demonstrating effectiveness at temperatures up to 70 °C and displaying exceptional thermal stability with a melting temperature (Tm) of 82 °C. Phylogenetically distinct from previously characterised PET hydrolases, Sis represents a valuable addition to the repertoire of enzymes suitable for PET degradation.

Comprehensive review on Haloalkane dehalogenase (LinB): a ß-hexachlorocyclohexane (HCH) degrading enzyme.

Haloalkane dehalogenase, LinB, is a member of the α/ß hydrolase family of enzymes. It has a wide range of halogenated substrates, but, has been mostly studied in context of degradation of hexachlorocyclohexane (HCH) isomers, especially ß-HCH (5-12% of total HCH isomers), which is the most recalcitrant and persistent among all the HCH isomers. LinB was identified to directly act on ß-HCH in a one or two step transformation which decreases its toxicity manifold. Thereafter, many studies focused on LinB including its structure determination using X-ray crystallographic studies, structure comparison with other haloalkane dehalogenases, substrate specificity and kinetic studies, protein engineering and site-directed mutagenesis studies in search of better catalytic activity of the enzyme. LinB was mainly identified and characterized in bacteria belonging to sphingomonads. Detailed sequence comparison of LinB from different sphingomonads further revealed the residues critical for its activity and ability to catalyze either one or two step transformation of ß-HCH. Association of LinB with IS6100 elements is also being discussed in detail in sphingomonads. In this review, we summarized vigorous efforts done by different research groups on LinB for developing better bioremediation strategies against HCH contamination. Also, kinetic studies, protein engineering and site directed mutagenesis studies discussed here forms the basis of further exploration of LinB's role as an efficient enzyme in bioremediation projects.

Mechanistic conformational and substrate selectivity profiles emerging in the evolution of enzymes via parallel trajectories.

Laboratory evolution studies have demonstrated that parallel evolutionary trajectories can lead to genetically distinct enzymes with high activity towards a non-preferred substrate. However, it is unknown whether such enzymes have convergent conformational dynamics and mechanistic features. To address this question, we use as a model the wild-type Homo sapiens kynureninase (HsKYNase), which is of great interest for cancer immunotherapy. Earlier, we isolated HsKYNase_66 through an unusual evolutionary trajectory, having a 410-fold increase in the kcat/KM for kynurenine (KYN) and reverse substrate selectivity relative to HsKYNase. Here, by following a different evolutionary trajectory we generate a genetically distinct variant, HsKYNase_93D9, that exhibits KYN catalytic activity comparable to that of HsKYNase_66, but instead it is a "generalist" that accepts 3'-hydroxykynurenine (OH-KYN) with the same proficiency. Pre-steady-state kinetic analysis reveals that while the evolution of HsKYNase_66 is accompanied by a change in the rate-determining step of the reactions, HsKYNase_93D9 retains the same catalytic mechanism as HsKYNase. HDX-MS shows that the conformational dynamics of the two enzymes are markedly different and distinct from ortholog prokaryotic enzymes with high KYN activity. Our work provides a mechanistic framework for understanding the relationship between evolutionary mechanisms and phenotypic traits of evolved generalist and specialist enzyme species.

Modeling HBV Infection and Therapy in Immunodeficient NOD-Rag1-/-IL2RgammaC-null (NRG) Fumarylacetoacetate Hydrolase (FAH) Knockout Mice with Human Chimeric Liver.

Chimeric mouse models with a humanized liver (Hu-HEP mice) provide a unique tool to study human hepatotropic virus diseases, including viral infection, viral pathogenesis, and anti-viral therapy. Here, we describe a detailed protocol for studying hepatitis B infection in NRG-derived fumarylacetoacetate hydrolase (FAH) knockout mice repopulated with human hepatocytes (FRG-Hu HEP mice). The procedures include (1) maintenance and genotyping of the FRG mice, (2) intrasplenic injection of primary human hepatocytes (PHH), (3) 2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) drug reduction cycling to improve human hepatocyte repopulation, (4) human albumin detection, and (5) HBV infection and detection. The method is simple and allows for highly reproducible generation of FRG-Hu HEP mice for HBV infection and therapy investigations.

Shifting Mycobacterial Serine Hydrolase Activity Visualized Using Multi-Layer In-Gel Activity Assays.

The ability of Mycobacterium tuberculosis to derive lipids from the host, store them intracellularly, and then break them down into energy requires a battery of serine hydrolases. Serine hydrolases are a large, diverse enzyme family with functional roles in dormant, active, and reactivating mycobacterial cultures. To rapidly measure substrate-dependent shifts in mycobacterial serine hydrolase activity, we combined a robust mycobacterial growth system of nitrogen limitation and variable carbon availability with nimble in-gel fluorogenic enzyme measurements. Using this methodology, we rapidly analyzed a range of ester substrates, identified multiple hydrolases concurrently, observed functional enzyme shifts, and measured global substrate preferences. Within every growth condition, mycobacterial hydrolases displayed the full, dynamic range of upregulated, downregulated, and constitutively active hydrolases independent of the ester substrate. Increasing the alkyl chain length of the ester substrate also allowed visualization of distinct hydrolase activity likely corresponding with lipases most responsible for lipid breakdown. The most robust expression of hydrolase activity was observed under the highest stress growth conditions, reflecting the induction of multiple metabolic pathways scavenging for energy to survive under this high stress. The unique hydrolases present under these high-stress conditions could represent novel drug targets for combination treatment with current front-line therapeutics. Combining diverse fluorogenic esters with in-gel activity measurements provides a rapid, customizable, and sensitive detection method for mycobacterial serine hydrolase activity.

Novel DNA-Binding Activity Exhibited by Poly(aspartic acid) Hydrolase-1 Inhibits Poly(aspartic acid) Hydrolase Activity.

Significant attention has been shifted toward the use and development of biodegradable polymeric materials to mitigate environmental accumulation and potential health impacts. One such material, poly(aspartic acid) (PAA), is a biodegradable alternative to superabsorbent poly(carboxylates), like poly(acrylate). Three enzymes are known to hydrolyze PAA: PahZ1KT-1 and PahZ2KT-1 from Sphingomonas sp. KT-1 and PahZ1KP-2 from Pedobacter sp. KP-2. We previously reported the X-ray crystal structure for PahZ1KT-1, which revealed a homodimer complex with a strongly cationic surface spanning one side of each monomer. Here, we report the first characterization of any polymer hydrolase binding to DNA, where modeling data predict binding of the polyanionic DNA near the cationic substrate binding surface. Our data reveal that PahZ1 homologues from Sphingomonas sp. KT-1 and Pedobacter sp. KP-2 bind ssDNA and dsDNA with nanomolar binding affinities. PahZ1KT-1 binds ssDNA and dsDNA with an apparent dissociation constant, KD,app = 81 ± 14 and 19 ± 1 nM, respectively, and these estimates are similar to the same behaviors exhibited by PahZ1KP-2. Gel permeation chromatography data reveal that dsDNA binding promotes inhibition of PahZ1-catalyzed PAA biodegradation for each homologue. We propose a working model wherein binding of PahZ1 to extracellular biofilm DNA aids in the localization of the hydrolase to the environment in which PAA would first be encountered, thereby providing a mechanism to degrade extracellular PAA and potentially harvest aspartic acid for nutritional uptake.

Discovery of a Ni2+-dependent heterohexameric metformin hydrolase.

The biguanide drug metformin is a first-line blood glucose-lowering medication for type 2 diabetes, leading to its presence in the global environment. However, little is known about the fate of metformin by microbial catabolism. Here, we characterize a Ni2+-dependent heterohexameric enzyme (MetCaCb) from the ureohydrolase superfamily, catalyzing the hydrolysis of metformin into guanylurea and dimethylamine. Either subunit alone is catalytically inactive, but together they work as an active enzyme highly specific for metformin. The crystal structure of the MetCaCb complex shows the coordination of the binuclear metal cluster only in MetCa, with MetCb as a protein binder of its active cognate. An in-silico search and functional assay discover a group of MetCaCb-like protein pairs exhibiting metformin hydrolase activity in the environment. Our findings not only establish the genetic and biochemical foundation for metformin catabolism but also provide additional insights into the adaption of the ancient enzymes toward newly occurred substrate.

Incorporating Prior Knowledge in the Seeds of Adaptive Sampling Molecular Dynamics Simulations of Ligand Transport in Enzymes with Buried Active Sites.

Because most proteins have buried active sites, protein tunnels or channels play a crucial role in the transport of small molecules into buried cavities for enzymatic catalysis. Tunnels can critically modulate the biological process of protein-ligand recognition. Various molecular dynamics methods have been developed for exploring and exploiting the protein-ligand conformational space to extract high-resolution details of the binding processes, a recent example being energetically unbiased high-throughput adaptive sampling simulations. The current study systematically contrasted the role of integrating prior knowledge while generating useful initial protein-ligand configurations, called seeds, for these simulations. Using a nontrivial system of a haloalkane dehalogenase mutant with multiple transport tunnels leading to a deeply buried active site, simulations were employed to derive kinetic models describing the process of association and dissociation of the substrate molecule. The most knowledge-based seed generation enabled high-throughput simulations that could more consistently capture the entire transport process, explore the complex network of transport tunnels, and predict equilibrium dissociation constants, koff/kon, on the same order of magnitude as experimental measurements. Overall, the infusion of more knowledge into the initial seeds of adaptive sampling simulations could render analyses of transport mechanisms in enzymes more consistent even for very complex biomolecular systems, thereby promoting drug development efforts and the rational design of enzymes with buried active sites.

Illuminating a biologics development challenge: systematic characterization of CHO cell-derived hydrolases identified in monoclonal antibody formulations.

Monoclonal antibodies (mAb) and other biological drugs are affected by enzymatic polysorbate (PS) degradation that reduces product stability and jeopardizes the supply of innovative medicines. PS represents a critical surfactant stabilizing the active pharmaceutical ingredients, which are produced by recombinant Chinese hamster ovary (CHO) cell lines. While the list of potential PS-degrading CHO host cell proteins (HCPs) has grown over the years, tangible data on industrially relevant HCPs are still scarce. By means of a highly sensitive liquid chromatography-tandem mass spectrometry method, we investigated seven different mAb products, resulting in the identification of 12 potentially PS-degrading hydrolases, including the strongly PS-degrading lipoprotein lipase (LPL). Using an LPL knockout CHO host cell line, we were able to stably overexpress and purify the remaining candidate hydrolases through orthogonal affinity chromatography methods, enabling their detailed functional characterization. Applying a PS degradation assay, we found nine mostly secreted, PS-active hydrolases with varying hydrolytic activity. All active hydrolases showed a serine-histidine-aspartate/glutamate catalytical triad. Further, we subjected the active hydrolases to pH-screenings and revealed a diverse range of activity optima, which can facilitate the identification of residual hydrolases during bioprocess development. Ultimately, we compiled our dataset in a risk matrix identifying PAF-AH, LIPA, PPT1, and LPLA2 as highly critical hydrolases based on their cellular expression, detection in purified antibodies, active secretion, and PS degradation activity. With this work, we pave the way toward a comprehensive functional characterization of PS-degrading hydrolases and provide a basis for a future reduction of PS degradation in biopharmaceutical drug products.

Small molecule substrates for the rapid quantification of acyl transfer activity of nylon hydrolase NylCA.

The widespread use of polyamides such as nylons has led to the accumulation of nylon waste, which is particularly resistant to decomposition due to the intrinsic stability of the amide bond. New methods are required for the true recycling of these waste materials by depolymerization. Enzymes that are capable of hydrolyzing polyamides have been proposed as biocatalysts that may be suitable for this application. NylC is an enzyme that can mediate the hydrolysis of aminohexanoic acid oligomers, and to some extent, bulk polymers. However, current assays to characterize the activity of this enzyme require long reaction times and/or rely on secondary reactions to quantify hydrolysis. Herein, we have designed structurally-optimized small molecule chromogenic esters that serve as substrate analogues for monitoring NylC acyltransferase activity in a continuous manner. This assay can be performed in minutes at room temperature, and the substrate N-acetyl-GABA-pNP ester (kcat = 0.37 s-1, KM = 256 µM) shows selectivity for NylC in complex biological media. We also demonstrate that activity towards this substrate analogue correlates with amide hydrolysis, which is the primary activity of this enzyme. Furthermore, our screening of substrate analogues provides insight into the substrate specificity of NylC, which is relevant to biocatalytic applications.

Biochemical Characterization and Application of Zearalenone Lactone Hydrolase Fused with a Multifunctional Short Peptide.

Zearalenone (ZEN) is an estrogenic mycotoxin causing reproductive toxicity in livestock. Currently, lactone hydrolases are used in the enzymatic degradation of ZEN. However, most lactone hydrolases suffer from low degradation efficiency and poor thermal stability. ZHD518, as a documented neutral enzyme for ZEN degradation, exhibits high enzymatic activity under neutral conditions. In this study, a multifunctional peptide S1v1-(AEAEAHAH)2 was fused to the N-terminus of ZHD518. Compared with the wild-type enzyme, the peptide fusion significantly enhanced protein expression by 1.28 times, enzyme activity by 9.27 times, thermal stability by 37.08 times after incubation at 45 °C for 10 min and enzyme stability during long-term storage. Moreover, ZEN concentrations in corn bran, corn germ meal, and corn gluten powder decreased from 5.29 ± 0.04, 5.31 ± 0.03, and 5.30 ± 0.01 µg/g to 0.48 ± 0.05, 0.48 ± 0.06, and 0.21 ± 0.04 µg/g, respectively, following a 60 min treatment with S1v1-GS-ZHD518, resulting in degradation rates of 90.98, 91.00, and 95.32%, respectively. In conclusion, the properties of S1v1-GS-ZHD518, such as its efficient degradability, high temperature resistance and storage resistance, offer the possibility of its application in food or feed.

An engineered enzyme embedded into PLA to make self-biodegradable plastic.

Plastic production reached 400 million tons in 2022 (ref. 1), with packaging and single-use plastics accounting for a substantial amount of this2. The resulting waste ends up in landfills, incineration or the environment, contributing to environmental pollution3. Shifting to biodegradable and compostable plastics is increasingly being considered as an efficient waste-management alternative4. Although polylactide (PLA) is the most widely used biosourced polymer5, its biodegradation rate under home-compost and soil conditions remains low6-8. Here we present a PLA-based plastic in which an optimized enzyme is embedded to ensure rapid biodegradation and compostability at room temperature, using a scalable industrial process. First, an 80-fold activity enhancement was achieved through structure-based rational engineering of a new hyperthermostable PLA hydrolase. Second, the enzyme was uniformly dispersed within the PLA matrix by means of a masterbatch-based melt extrusion process. The liquid enzyme formulation was incorporated in polycaprolactone, a low-melting-temperature polymer, through melt extrusion at 70 °C, forming an 'enzymated' polycaprolactone masterbatch. Masterbatch pellets were integrated into PLA by melt extrusion at 160 °C, producing an enzymated PLA film (0.02% w/w enzyme) that fully disintegrated under home-compost conditions within 20-24 weeks, meeting home-composting standards. The mechanical and degradation properties of the enzymated film were compatible with industrial packaging applications, and they remained intact during long-term storage. This innovative material not only opens new avenues for composters and biomethane production but also provides a feasible industrial solution for PLA degradation.

Engineered polyethylene terephthalate hydrolases: perspectives and limits.

Polyethylene terephthalate (PET) is a major component of plastic waste. Enzymatic PET hydrolysis is the most ecofriendly recycling technology. The biorecycling of PET waste requires the complete depolymerization of PET to terephthalate and ethylene glycol. The history of enzymatic PET depolymerization has revealed two critical issues for the industrial depolymerization of PET: industrially available PET hydrolases and pretreatment of PET waste to make it susceptible to full enzymatic hydrolysis. As none of the wild-type enzymes can satisfy the requirements for industrialization, various mutational improvements have been performed, through classical technology to state-of-the-art computational/machine-learning technology. Recent engineering studies on PET hydrolases have brought a new insight that flexibility of the substrate-binding groove may improve the efficiency of PET hydrolysis while maintaining sufficient thermostability, although the previous studies focused only on enzymatic thermostability above the glass transition temperature of PET. Industrial biorecycling of PET waste is scheduled to be implemented, using micronized amorphous PET. Next stage must be the development of PET hydrolases that can efficiently degrade crystalline parts of PET and expansion of target PET materials, not only bottles but also textiles, packages, and microplastics. This review discusses the current status of PET hydrolases, their potential applications, and their profespectal goals. KEY POINTS: • PET hydrolases must be thermophilic, but their operation must be below 70 °C • Classical and state-of-the-art engineering approaches are useful for PET hydrolases • Enzyme activity on crystalline PET is most expected for future PET biorecycling.

A Hydrophilic Metal Azolate Coordination Polymer for In Situ Encapsulation of Haloalkane Dehalogenase with Enhanced Enzymatic Performance.

Encapsulating enzymes within metal-organic frameworks such as zeolitic imidazolate framework-8 (ZIF-8) has been demonstrated to enhance enzymatic performance under harsh conditions. However, by computer-aided analysis, we revealed that highly hydrophobic organic ligands and unfavorable metal ions could greatly impair the activity of haloalkane dehalogenase DhaA by directly interacting with the catalytic sites, causing an extremely low activity of DhaA after encapsulating within ZIF-8. We also found that the presence of a protecting polymer could protect DhaA from the damage of organic ligands and metal ions and that a positively charged amino acid could increase the DhaA activity. Based on the simulations and experimental observations, we have designed to coencapsulate DhaA with poly(vinylpyrrolidone) (PVP) and lysine (Lys) within the amorphous Co-based metal azolate coordination polymer (CoCP). The as-prepared immobilized enzyme (DhaA/PVP/Lys@CoCP) exhibited significantly increased activity (91.5 times higher than that of DhaA@ZIF-8), dramatically enhanced thermostability at 50-70 °C, greatly improved catalytic performance in several organic solvent solutions, and good recyclability (over 75% of the initial activity after 10 cycles). The superiority of the immobilized enzyme was also demonstrated with a substrate frequently detected in the real world. In addition to the protective effect of PVP and positive effect of Lys, experimental and computational investigations unveiled other two favorable aspects that contributed to the enhanced enzymatic performance: (1) high hydrophilicity of the immobilization material and (2) the use of Co2+ with a minimal negative effect on DhaA. The research has thus provided a promising immobilized DhaA with favorable catalytic performance and great potential in industrial applications.

Optimization of polyethylene terephthalate biodegradation using a self-assembled multi-enzyme cascade strategy.

Although many efforts have been devoted to the modification of polyethylene terephthalate (PET) hydrolases for improving the efficiency of PET degradation, the catalytic performance of these enzymes at near-ambient temperatures remains a challenge. Herein, a multi-enzyme cascade system (PT-EC) was developed and validated by assembling three well-developed PETases, PETaseEHA, Fast-PETase, and Z1-PETase, respectively, together with carboxylesterase TfCa, and hydrophobic binding module CBM3a using scaffold proteins. The resulting PT-ECEHA, PT-ECFPE, PT-ECZPE all demonstrated outstanding PET degradation efficacy. Notably, PT-ECEHA exhibited a 16.5-fold increase in product release compared to PETaseEHA, and PT-ECZPE yielded the highest amount of product. Subsequently, PT-ECs were displayed on the surface of Escherichia coli, respectively, and their degradation efficiency toward three PET types was investigated. The displayed PT-ECEHA exhibited a 20-fold increase in degradation efficiency with PET film compared to the surface-displayed PETaseEHA. Remarkably, an almost linear increase in product release was observed for the displayed PT-ECZPE over a one-week degradation period, reaching 11.56 ± 0.64 mM after 7 days. TfCaI69W/L281Y evolved using a docking-based virtual screening strategy showed a further 2.5-fold increase in the product release of PET degradation. Collectively, these advantages of PT-EC demonstrated the potential of a multi-enzyme cascade system for PET bio-cycling.

Enabling high-throughput enzyme discovery and engineering with a low-cost, robot-assisted pipeline.

As genomic databases expand and artificial intelligence tools advance, there is a growing demand for efficient characterization of large numbers of proteins. To this end, here we describe a generalizable pipeline for high-throughput protein purification using small-scale expression in E. coli and an affordable liquid-handling robot. This low-cost platform enables the purification of 96 proteins in parallel with minimal waste and is scalable for processing hundreds of proteins weekly per user. We demonstrate the performance of this method with the expression and purification of the leading poly(ethylene terephthalate) hydrolases reported in the literature. Replicate experiments demonstrated reproducibility and enzyme purity and yields (up to 400 µg) sufficient for comprehensive analyses of both thermostability and activity, generating a standardized benchmark dataset for comparing these plastic-degrading enzymes. The cost-effectiveness and ease of implementation of this platform render it broadly applicable to diverse protein characterization challenges in the biological sciences.

Efficient thermophilic polyethylene terephthalate hydrolase enhanced by cross correlation-based accumulated mutagenesis strategy.

Polyethylene terephthalate (PET) has caused significant pollution issues. Compared to chemical degradation with high energy consumption and cost, enzymatic degradation offers a sustainable solution for PET waste recycling. However, the hydrolytic activity of current PET hydrolases still requires improvement. In this study, a cross-correlation-based accumulated mutagenesis (CAM) strategy was developed to enhance the hydrolysis activity. By mitigating epistatic effect and combinational mutations, we achieved a highly active variant LCC-YGA (H183Y/L124G/S29A) with 2.1-fold hydrolytic activity on amorphous PET films of LCC-ICCG. Conformational analysis elucidated how the introduction of distal mutations enhanced activity. The dynamic correlation among different regions facilitated a synergistic effect, enhancing binding pocket flexibility through remote interactions. Totally, this work offers novel insights and methods for PET hydrolases engineering and provides an efficient enzyme for PET degradation and recycling.