Upregulation of Fatty Acid Synthase Increases Activity of ß-Catenin and Expression of NOTUM to Enhance Stem-like Properties of Colorectal Cancer Cells.

Dysregulated fatty acid metabolism is an attractive therapeutic target for colorectal cancer (CRC). We previously reported that fatty acid synthase (FASN), a key enzyme of de novo synthesis, promotes the initiation and progression of CRC. However, the mechanisms of how upregulation of FASN promotes the initiation and progression of CRC are not completely understood. Here, using Apc/VillinCre and ApcMin mouse models, we show that upregulation of FASN is associated with an increase in activity of ß-catenin and expression of multiple stem cell markers, including Notum. Genetic and pharmacological downregulation of FASN in mouse adenoma organoids decreases the activation of ß-catenin and expression of Notum and significantly inhibits organoid formation and growth. Consistently, we demonstrate that NOTUM is highly expressed in human CRC and its expression positively correlates with the expression of FASN in tumor tissues. Utilizing overexpression and shRNA-mediated knockdown of FASN, we demonstrate that upregulation of FASN increases ß-catenin transcriptional activity, NOTUM expression and secretion, and enhances stem-like properties of human CRC cells. Pharmacological inhibition of NOTUM decreases adenoma organoids growth and proliferation of cancer cells. In summary, upregulation of FASN enhances ß-catenin signaling, increases NOTUM expression and stem-like properties of CRC cells, thus suggesting that targeting FASN upstream of the ß-catenin/NOTUM axis may be an effective preventative therapeutic strategy for CRC.

Mammalian Esterase Activity: Implications for Peptide Prodrugs.

As a traceless, bioreversible modification, the esterification of carboxyl groups in peptides and proteins has the potential to increase their clinical utility. An impediment is the lack of strategies to quantify esterase-catalyzed hydrolysis rates for esters in esterified biologics. We have developed a continuous Förster resonance energy transfer (FRET) assay for esterase activity based on a peptidic substrate and a protease, Glu-C, that cleaves a glutamyl peptide bond only if the glutamyl side chain is a free acid. Using pig liver esterase (PLE) and human carboxylesterases, we validated the assay with substrates containing simple esters (e.g., ethyl) and esters designed to be released by self-immolation upon quinone methide elimination. We found that simple esters were not cleaved by esterases, likely for steric reasons. To account for the relatively low rate of quinone methide elimination, we extended the mathematics of the traditional Michaelis-Menten model to conclude with a first-order intermediate decay step. By exploring two regimes of our substrate → intermediate → product (SIP) model, we evaluated the rate constants for the PLE-catalyzed cleavage of an ester on a glutamyl side chain (kcat/KM = 1.63 × 103 M-1 s-1) and subsequent spontaneous quinone methide elimination to regenerate the unmodified peptide (kI = 0.00325 s-1; t1/2 = 3.55 min). The detection of esterase activity was also feasible in the human intestinal S9 fraction. Our assay and SIP model increase the understanding of the release kinetics of esterified biologics and facilitate the rational design of efficacious peptide prodrugs.

Isolation and purification of esterase enzyme from marine bacteria associated with biodegradation of polyvinyl chloride (PVC).

Polyvinyl chloride (PVC) is the third most produced synthetic plastic and releases the most harmful and lethal environmental component after incineration and landfilling. Few studies on microbial degradation of PVC have been reported but very little knowledge about the enzymes. In the present study, esterase enzyme was isolated and partially purified from marine bacterial isolates (T-1.3, BP-4.3 and S-237 identified as Vibrio sp., Alteromonas sp., and Cobetia sp., respectively) having the capability of PVC degradation. Initially, a plate assay was carried out for testing esterase production by studying bacteria using 1-naphthyl acetate as substrate. Enzyme assay showed higher production of esterase i.e. 0.57 U mL-1 (2nd day), 0.46 U mL-1 (2nd day) and 0.55 U mL-1 (5th day) by bacterial isolate Vibrio sp., Alteromonas sp. and Cobetia sp., respectively incubated with PVC. Other enzymes like lipase, laccase and manganese peroxidase were much less or negligible compared to esterase enzyme production. Sephadex G-50 column purification had shown 58.62, 42.35 and 223.70 units mg-1 of a specific activity by esterase for bacterial isolates Vibrio sp., Alteromonas sp. and Cobetia sp., respectively. Further, Sephadex G-50 column purification removed all the contamination and gave a clear appearance of the band at 38, 20 and 20 KD for bacterial isolates Vibrio sp., Alteromonas sp., and Cobetia sp., respectively. Esterase has shown maximum stability at a range of pH between 6.0 to 7.5, temperature between 30 to 35 °C and salinity concentration between 3 to 3.5 M for all bacterial isolates. In conclusion, esterase enzyme has promising potential to degrade PVC which can contribute to the decline the plastic pollution in an eco-friendly manner from the environment.

Esterase responsive release of anti-cancer agents from conjugated lipid nanocarrier and the regulatory considerations.

The release of active agents in tumors rather than normal tissues, limits systemic exposure and toxicities. Targeting over-expressed esterase enzyme in the tumor microenvironment can selectively release immune-active agents like Programmed Death-1 (PD-1) and PD-1 ligand inhibitors from ester-sensitive lipid nanocarriers, offering a novel approach compared with conventional therapies. PD-1 and PD-L1 association cause T-cell inactivation, whereas blocking their association improves their cytotoxic mechanism. The patent application US2022/0080051-A1 discloses a novel immune-active agent conjugated with lipid to form a nanocarrier for esterase-sensitive release. These nanocarriers selectively enter leaky vasculature of tumors through enhanced permeability and retention effect, undergo ester cleavage to release agents, and are reported to increase bioavailability by 24 times. Further, with other agents or alone it achieves targeted synergistic cancer therapy. Also, the current patent spotlight delves into the crucial formulation considerations necessary for obtaining successful approval of lipidic nano products from relevant regulatory authorities.

[Box: see text].

Synthesis of a Zn(II)-2-aminoimidazole Framework as an Efficient Carbonic Anhydrase Mimic.

Carbonic anhydrase (CA) plays a crucial role in the CO2 capture processes by catalyzing the hydration of CO2. In this study, we synthesized a bioinspired carbonic anhydrase Zn-MOF (metal-organic framework) incorporating 2-aminoimidazole and Zn2+ as initial constituents. The synthesized Zn-MOF exhibited promising potential for efficiently catalyzing the CO2 hydration. Structural analyses such as SEM, XRD, and BET confirmed that the Zn-MOF crystal consisted of stacked grains with an average size of approximately 36 nm, forming a micron-sized spherical structure. Functionally, Zn-MOF exhibited effective catalytic activity toward both CO2 hydration and ester hydrolysis. The introduction of amino groups significantly enhanced the esterase activity of Zn-MOF to 0.28 U/mg at ambient temperature, which was twice that of ZIF-8. Furthermore, the introduction of amino groups resulted in remarkable hydrothermal stability, with the esterase activity reaching 0.72 U/mg after undergoing hydrothermal treatment at 80 °C for 12 h. Additionally, Zn-MOF exhibited enhanced capability in CO2 hydration at a pH value exceeding 8.5. After six repeated uses, ZIF-8 and Zn-MOF retained approximately 68 and 65% of their initial enzyme activity, respectively, underscoring the potential practical applicability of Zn-MOF in industrial CO2 capture processes. This work showcases the development of a novel Zn-MOF crystal as an efficient CA mimic, effectively emulating the active sites of natural CA using 2-aminoimidazole as a coordinating ligand for Zn2+ coordination. These findings not only advance the field of innovative enzyme mimics but also pave the way for further exploration of industrial CO2 capture catalysts.

Mechanistic Insight into the Enantioselective Degradation of Esterase QeH to (R)/(S)-Quizalofop-Ethyl with Molecular Dynamics Simulation Using a Residue-Specific Force Field.

The enantioselective mechanism of the esterase QeH against the two enantiomers of quizalofop-ethyl (QE) has been primitively studied using computational and experimental approaches. However, it is still unclear how the esterase QeH adjusts its conformation to adapt to substrate binding and promote enzyme-substrate interactions in the catalytic kinetics. The equilibrium processes of enzyme-substrate interactions and catalytic dynamics were reproduced by performing independent molecular dynamics (MD) runs on the QeH-(R)/(S)-QE complexes with a newly developed residue-specific force field (RSFF2C). Our results indicated that the benzene ring of the (R)-QE structure can simultaneously form anion-π and cation-π interactions with the side-chain group of Glu328 and Arg384 in the binding cavity of the QeH-(R)-QE complex, resulting in (R)-QE being closer to its catalytic triplet system (Ser78-Lys81-Tyr189) with the distances measured for the hydroxyl oxygen atom of the catalytic Ser78 of QeH and the carbonyl carbon atom of (R)-QE of 7.39 Å, compared to the 8.87 Å for (S)-QE, whereas the (S)-QE structure can only form an anion-π interaction with the side chain of Glu328 in the QeH-(S)-QE complex, being less close to its catalytic site. The computational alanine scanning mutation (CAS) calculations further demonstrated that the π-π stacking interaction between the indole ring of Trp351 and the benzene ring of (R)/(S)-QE contributed a lot to the binding stability of the enzyme-substrate (QeH-(R)/(S)-QE). These results facilitate the understanding of their catalytic processes and provide new theoretical guidance for the directional design of other key enzymes for the initial degradation of aryloxyphenoxypropionate (AOPP) herbicides with higher catalytic efficiencies.

Synthesis of a natural core substrate with lignin-xylan cross-linkage for unveiling the productive kinetic parameters of glucuronoyl esterase.

Lignin-carbohydrate complexes (LCCs) present a considerable hurdle to the economic utilization of lignocellulosic biomass. Glucuronoyl esterase (GE) is an LCC-degrading enzyme that catalyzes the cleavage of the cross-linkages between lignin and xylan in LCCs. Benzyl-d-glucuronate (Bn-GlcA), a commercially available substrate, is widely used to evaluate GE activity assays. However, since Bn-GlcA lacks the structural backbone of naturally occurring LCCs, the mechanisms underlying the activity of GEs and their diversity in the structure-activity relationship are not fully understood. Herein, we provided a synthesis scheme for designing 1,23-α-d-(6-benzyl-4-O-methyl-glucuronyl)-1,4-ß-d-xylotriose (Bn-MeGlcA3Xyl3) as a natural core substrate bearing cross-linkage between lignin and glucuronoxylan. A well-defined and yet more realistic synthetic substrate was successfully synthesized via a key step of the benzyl esterification of 4-O-methyl-glucuronyl-1,4-ß-d-xylotriose (MeGlcA3Xyl3), a minimized fragment of glucuronoxylan enzymatically digested by ß-1,4-xylanase. To the best of our knowledge, this is the first report of the productive GE kinetic analysis using this substrate. Kinetic parameters of the GE from the fungal Pestalotiopsis sp. AN-7 (PesGE), i.e., the Km, Vmax, and kcat of Bn-MeGlcA3Xyl3, were 0.43 mM, 55.5 µmol min-1·mg-1, and 35.8 s-1, respectively. On the other hand, as reported to date, the productive kinetic parameters for Bn-GlcA were not obtained because of its excessively high Km value (>16 mM). The substantial variance in the enzymatic activity of PesGE regarding substrate-binding affinity between Bn-MeGlcA3Xyl3 and Bn-GlcA was also demonstrated using in silico docking simulation. These results suggested that the extended xylan fragment is a key structural determinant affecting PesGE's substrate recognition. Furthermore, the presence of a natural xylan backbone allows for evaluating the enzyme activity of xylan-degrading enzymes. Accordingly, the synthesized substrate with the natural core structure of LCC allowed us to unveil the productive kinetic parameters of GEs, serving as a versatile substrate for further elucidating the cascade reaction of GE and xylan-degrading enzymes.

A radical containing micellar probe for assessing esterase enzymatic activity with ultra-low field Overhauser-enhanced magnetic resonance imaging.

The ability to track altered enzyme activity using a non-invasive imaging protocol is crucial for the early diagnosis of many diseases but is often challenging. Herein, we show that Overhauser magnetic resonance imaging (OMRI) can be used to monitor enzymatic conversion at an ultra-low field (206 µT) using a highly sensitive "off/on" probe with a nitroxide stable radical containing ester, named T2C12-T80. This TEMPO derivative containing probe forms stable electron paramagnetic resonance (EPR) silent micelles in water that are hydrolysed by esterases, thus yielding narrow EPR signals whose intensities correlate directly with specific enzymatic activity. The responsiveness of the probe to tumours, facilitated by increased esterase activity, was initially determined by comparing EPR signals measured upon incubation with 3T3 (healthy fibroblasts used as control), HepG2 (human hepatoma) and Hs766T (human pancreatic cancer cells) cell lysates and then with Hs766T and 3T3 living cells. Next, Overhauser MR images were detected on a phantom containing the probe and the esterases to show that the approach is well suited for being translated to the in vivo detection at the earth's magnetic field. Regarding detection sensitivity, ultra-low field OMRI (ULF-OMRI) is beneficial over OMRI at higher fields (e.g. 0.2 T) since Overhauser enhancements are significantly higher and the technique is safe in terms of the specific absorption rate.

Esterase-Responsive Floxuridine-Tethered Multifunctional Nanoparticles for Targeted Cancer Therapy.

Floxuridine is a potential clinical anticancer drug for the treatment of various cancers. However, floxuridine typically causes unfavorable side effects due to its very poor tumor selectivity, and, hence, there is a high demand for the development of novel approaches that permit the targeted delivery of floxuridine into cancerous cells. Herein, the design and synthesis of an esterase-responsive multifunctional nanoformulation for the targeted delivery of floxuridine in esterase-overexpressed cancer cells is reported. Photopolymerization of floxuridine-tethered lipoic acid results in the formation of amphiphilic floxuridine-tethered poly(disulfide). Self-assembly of the amphiphilic polymer results in the formation of nanoparticles with floxuridine decorated on the surfaces of the particles. Integration of aptamer DNA for nucleolin onto the surface of the nanoparticle is demonstrated by exploring the base-pairing interaction of floxuridine with adenine. Targeted internalization of the aptamer-decorated nanoparticle into nucleolin-expressed cancer cells is demonstrated. Esterase triggered cleavage of the ester bond connecting floxuridine with the polymer backbone, and the subsequent targeted delivery of floxuridine into cancer cells is also shown. Excellent therapeutic efficacy is observed both in vitro and also in the 3D tumor spheroid model. This noncovalent strategy provides a simple yet effective strategy for the targeted delivery of floxuridine into cancer cells in a less laborious fashion.

Thermoascus aurantiacus harbors an esterase/lipase that is highly activated by anionic surfactant.

Fungal lipolytic enzymes play crucial roles in various lipid bio-industry processes. Here, we elucidated the biochemical and structural characteristics of an unexplored fungal lipolytic enzyme (TaLip) from Thermoascus aurantiacus var. levisporus, a strain renowned for its significant industrial relevance in carbohydrate-active enzyme production. TaLip belongs to a poorly understood phylogenetic branch within the class 3 lipase family and prefers to hydrolyze mainly short-chain esters. Nonetheless, it also displays activity against natural long-chain triacylglycerols. Furthermore, our analyses revealed that the surfactant sodium dodecyl sulfate (SDS) enhances the hydrolytic activity of TaLip on pNP butyrate by up to 5.0-fold. Biophysical studies suggest that interactions with SDS may prevent TaLip aggregation, thereby preserving the integrity and stability of its monomeric form and improving its performance. These findings highlight the resilience of TaLip as a lipolytic enzyme capable of functioning in tandem with surfactants, offering an intriguing enzymatic model for further exploration of surfactant tolerance and activation in biotechnological applications.

Esterase-Responsive Fluorogenic Prodrugs of Aldose Reductase Inhibitor Epalrestat: An Innovative Strategy toward Enhanced Anticancer Activity.

In addition to the conventional chemotherapeutic drugs, potent inhibitors of key enzymes that are differentially overexpressed in cancer cells and associated with its progression are often considered as the drugs of choice for treating cancer. Aldose reductase (AR), which is primarily associated with complications of diabetes, is known to be closely related to the development of cancer and drug resistance. Epalrestat (EPA), an FDA-approved drug, is a potent inhibitor of AR and exhibits anticancer activity. However, its poor pharmacokinetic properties limit its bioavailability and therapeutic benefits. We report herein the first examples of esterase-responsive turn-on fluorogenic prodrugs for the sustained release of EPA to cancer cells with a turn-on fluorescence readout. Carboxylesterases are known to be overexpressed in several organ-specific cancer cells and help in selective uncaging of drug from the prodrugs. The prodrugs were synthesized using a multistep organic synthesis and successfully characterized. Absorption and emission spectroscopic studies indicated successful activation of the prodrugs in the presence of porcine liver esterase (PLE) under physiological condition. HPLC studies revealed a simultaneous release of both the drug and the fluorophore from the prodrugs over time with mechanistic insights. While the inhibitory potential of EPA released from the prodrugs toward the enzyme AR was validated in the aqueous medium, the anticancer activity of the prodrugs was studied in a representative cervical cancer cell line. Interestingly, our results revealed that the development of the prodrugs can significantly enhance the anticancer potential of EPA. Finally, the drug uncaging process from the prodrugs by the intracellular esterases was studied in the cellular medium by measuring the turn-on fluorescence using fluorescence microscopy. Therefore, the present study highlights the rational development of the fluorogenic prodrugs of EPA, which will help enhance its anticancer potential with better therapeutic potential.

Esterase-Activated Hydrogen Sulfide Donors with Self-Reporting Fluorescence Properties and Highly Tunable Rates of Delivery.

Hydrogen sulfide (H2S) has emerged as a significant biomolecule with diverse activities, akin to other gaseous signaling molecules such as nitric oxide (NO) and carbon monoxide (CO). In the present study, we report on the development of esterase-activated donors that track their direct cellular donation of H2S by enlisting a cyclization reaction onto a thioamide that forms a fluorogenic byproduct. This simple donor design provides a noninvasive method for monitoring the biological delivery and activity of H2S, along with access to a library of compounds with highly variable rates of H2S delivery. These studies culminated with the identification of a slow-release, yet highly efficient, donor (ZL-DMA-Ph) that was shown to self-report its gradual and continuous cellular donation of H2S for up to 24 h which, in addition to better mimicking the natural biosynthesis of H2S, provided impressive cytoprotection in a cellular cardiotoxicity model, even at submicromolar concentrations. In total, these findings indicate that the esterase-triggered fluorogenic donors identified in this study will offer new opportunities for exploring the chemical biology and therapeutic potential of exogenous H2S supplementation.

Biomolecular Condensation of Trypsin Prevents Autolysis and Promotes Ca2+-Mediated Activation of Esterase Activity.

The presence of Ca2+ ions is known to facilitate the activity of trypsin-like serine proteases via structural stabilization against thermal denaturation and autolysis. Herein, we report a new and hidden regulatory role of Ca2+ in the catalytic pathways of trypsin and α-chymotrypsin under physiological conditions. We discovered that macromolecular crowding promotes spontaneous homotypic condensation of trypsin via liquid-liquid phase separation to yield membraneless condensates over a broad range of concentrations, pH, and temperature, which are stabilized by multivalent hydrophobic interactions. Interestingly, we found that Ca2+ binding in the calcium binding loop reversibly regulates the condensation of trypsin and α-chymotrypsin. Spontaneous condensation effectively prevents autolysis of trypsin and preserves its native-like esterase activity for a prolonged period of time. It has also been found that phase-separated trypsin responds to Ca2+-dependent activation of its esterase activity even after 14 days of storage while free trypsin failed to do so. The present study highlights an important physiological aspect by which cells can spatiotemporally regulate the biocatalytic efficacy of trypsin-like serine proteases via Ca2+-signaling.

Structural, Biochemical, and Phylogenetic Analysis of Bacterial and Fungal Carbohydrate Esterase Family 15 Glucuronoyl Esterases in the Rumen.

Glucuronoyl esterases (GEs) are carbohydrate active enzymes in carbohydrate esterase family 15 which are involved in the hydrolysis of lignin-carbohydrate complexes. They are encoded by a wide range of aerobic and anaerobic fungi and bacteria inhabiting diverse environments. The rumen microbiome is a complex microbial community with a wide array of enzymes that specialize in deconstructing plant cell wall carbohydrates. Enzymes from the rumen tend to show low similarity to homologues found in other environments, making the rumen microbiome a promising source for the discovery of novel enzymes. Using a combination of phylogenetic and structural analysis, we investigated the structure-function relationship of GEs from the rumen bacteria Fibrobacter succinogenes and Ruminococcus flavefaciens, and from the rumen fungus, Piromyces rhizinflata. All adopt a canonical α/ß hydrolase fold and possess a structurally conserved Ser-His-Glu/Asp catalytic triad. Structural variations in the enzymes are localized to loops surrounding the active site. Analysis of the active site structures in these enzymes emphasized the importance of structural plasticity in GEs with non-canonical active site conformations. We hypothesize that interkingdom HGT events may have contributed to the diversity of GEs in the rumen, and this is demonstrated by the phylogenetic and structural similarity observed between rumen bacterial and fungal GEs. This study advances our understanding of the structure-function relationship in glucuronoyl esterases and illuminates the evolutionary dynamics that contribute to enzyme diversity in the rumen microbiome.

Crofton weed derived isomers of ageraphorone as potent antifeedant against Plutella xylostella (L.).

Global agricultural production is significantly hampered by insect pests, and the demand for natural pragmatic pesticides with environmental concern remains unfulfilled. Ageratina adenophora (Spreng.) also known as Crofton weed, is an invasive perennial herbaceous plant that is known to possess multiple bioactive compounds. In our study, two isomers of ageraphorone metabolites i.e, 10 Hα-9-oxo-ageraphorone (10HA) and 10 Hß-9-oxo-ageraphorone (10HB), were identified from Crofton weed, exhibiting potent antifeedant and larvicidal activities against Plutella xylostella. For antifeedant activity, the median effective concentration (EC50) values for 10HA and 10HB in the choice method were 2279 mg/L and 3233 mg/L, respectively, and for the no choice method, EC50 values were 1721 mg/L and 2394 mg/L, respectively. For larvicidal activity, lethal concentration (LC50) values for 10HA and 10HB were 2421 mg/L and 4109 mg/L at 48 h and 2101 mg/L and 3550 mg/L at 72 h. Furthermore, both in- vivo and in-vitro studies revealed that the isomers 10HA and 10HB exhibited potent detoxifying enzymes inhibition activity such as carboxylesterase and glutathione S-transferases. Molecular docking and MD simulation analysis provide insight into the possible interaction between isomers of ageraphorone metabolites and Carboxylic Ester Hydrolase protein (Gene: pxCCE016b) of P. xylostella, which led to a finding that CarEH protein plays a significant role in the detoxification of the two compounds in P. xylostella. Finally, our findings show that the primary enzymes undergoing inhibition by isomers of ageraphorone metabolites, causing toxicity in insects, are Carboxylesterase and glutathione S-transferase.

Parallel Evolution in Mosquito Vectors-A Duplicated Esterase Locus is Associated With Resistance to Pirimiphos-methyl in Anopheles gambiae.

The primary control methods for the African malaria mosquito, Anopheles gambiae, are based on insecticidal interventions. Emerging resistance to these compounds is therefore of major concern to malaria control programs. The organophosphate (OP), pirimiphos-methyl, is a relatively new chemical in the vector control armory but is now widely used in indoor-residual spray campaigns. While generally effective, phenotypic resistance has developed in some areas in malaria vectors. Here, we used a population genomic approach to identify novel mechanisms of resistance to pirimiphos-methyl in A. gambiae s.l mosquitoes. In multiple populations, we found large and repeated signals of selection at a locus containing a cluster of detoxification enzymes, some of whose orthologs are known to confer resistance to OPs in Culex pipiens. Close examination revealed a pair of alpha-esterases, Coeae1f and Coeae2f, and a complex and diverse pattern of haplotypes under selection in A. gambiae, A. coluzzii and A. arabiensis. As in C. pipiens, copy number variants have arisen at this locus. We used diplotype clustering to examine whether these signals arise from parallel evolution or adaptive introgression. Using whole-genome sequenced phenotyped samples, we found that in West Africa, a copy number variant in A. gambiae is associated with resistance to pirimiphos-methyl. Overall, we demonstrate a striking example of contemporary parallel evolution which has important implications for malaria control programs.

Exploring the Esterase Catalytic Activity of Minimalist Heptapeptide Amyloid Fibers.

This paper investigates the esterase activity of minimalist amyloid fibers composed of short seven-residue peptides, IHIHIHI (IH7) and IHIHIQI (IH7Q), with a particular focus on the role of the sixth residue position within the peptide sequence. Through computational simulations and analyses, we explore the molecular mechanisms underlying catalysis in these amyloid-based enzymes. Contrary to initial hypotheses, our study reveals that the twist angle of the fiber, and thus the catalytic site's environment, is not notably affected by the sixth residue. Instead, the sixth residue interacts with the p-nitrophenylacetate (pNPA) substrate, particularly through its -NO2 group, potentially enhancing catalysis. Quantum mechanics/molecular mechanics (QM/MM) simulations of the reaction mechanism suggest that the polarizing effect of glutamine enhances catalytic activity by forming a stabilizing network of hydrogen bonds with pNPA, leading to lower energy barriers and a more exergonic reaction. Our findings provide valuable insights into the intricate interplay between peptide sequence, structural arrangement, and catalytic function in amyloid-based enzymes, offering potentially valuable information for the design and optimization of biomimetic catalysts.

Selective recovery of esterase from Trichoderma harzianum through adsorption: Insights on enzymatic catalysis, adsorption isotherms and kinetics.

In recent years, numerous attempts have been made to develop a low-cost adsorbent for selectively recovering industrially important products from fermentation broth or complex mixtures. The current study is a novel attempt to selectively adsorb esterase from Trichoderma harzianum using cheap adsorbents like bentonite (BT), activated charcoal (AC), silicon dioxide (SiO2), and titanium dioxide (TiO2). AC had the highest esterase adsorption of 97.58% due to its larger surface area of 594.45 m3/g. SiO2 was found to have the highest selectivity over esterase, with an estimated purification fold of 7.2. Interestingly, the purification fold of 5.5 was found in the BT-extracted fermentation broth. The functional (FT-IR) and morphological analysis (SEM-EDX) were used to characterize the adsorption of esterase. Esterase adsorption on AC, SiO2, and TiO2 was well fitted by Freundlich isotherm, demonstrating multilayer adsorption of esterase. A pseudo-second-order kinetic model was developed for esterase adsorption in various adsorbents. Thermodynamic analysis revealed that adsorption is an endothermic process. AC has the lowest Gibbs free energy of -10.96 kJ/mol, which supports the spontaneous maximum adsorption of both esterase and protein. In the desorption study, the maximum recovery of esterase from TiO2 using sodium chloride was 41.34 %. Unlike other adsorbents, the AC-adsorbed esterase maintained its catalytic activity and stability, implying that it could be used as an immobilization system for commercial applications. According to the kinetic analysis, the overall rate of the reaction was controlled by reaction kinetics rather than external mass transfer resistance, as indicated by the Damkohler number.

Enzymatic degradation of polylactic acid (PLA).

Environmental concerns arising from the increasing use of polluting plastics highlight polylactic acid (PLA) as a promising eco-friendly alternative. PLA is a biodegradable polyester that can be produced through the fermentation of renewable resources. Together with its excellent properties, suitable for a wide range of applications, the use of PLA has increased significantly over the years and is expected to further grow. However, insufficient degradability under natural conditions emphasizes the need for the exploration of biodegradation mechanisms, intending to develop more efficient techniques for waste disposal and recycling or upcycling. Biodegradation occurs through the secretion of depolymerizing enzymes, mainly proteases, lipases, cutinases, and esterases, by various microorganisms. This review focuses on the enzymatic degradation of PLA and presents different enzymes that were isolated and purified from natural PLA-degrading microorganisms, or recombinantly expressed. The review depicts the main characteristics of the enzymes, including recent advances and analytical methods used to evaluate enantiopurity and depolymerizing activity. While complete degradation of solid PLA particles is still difficult to achieve, future research and improvement of enzyme properties may provide an avenue for the development of advanced procedures for PLA degradation and upcycling, utilizing its building blocks for further applications as envisaged by circular economy principles. KEY POINTS: • Enzymes can be promisingly utilized for PLA upcycling. • Natural and recombinant PLA depolymerases and methods for activity evaluation are summarized. • Approaches to improve enzymatic degradation of PLA are discussed.

Efficient Biodegradation of Multiple Aryloxyphenoxypropionate Herbicides by Corynebacterium sp. Z-1 and the Proposed Degradation Mechanism.

Esterases are crucial for aryloxyphenoxypropionate herbicide (AOPP) biodegradation. However, the underlying molecular mechanisms of AOPP biodegradation by esterases are poorly understood. In the current work, Corynebacterium sp. Z-1 was isolated and found to degrade multiple AOPPs, including quizalofop-p-ethyl (QPE), haloxyfop-p-methyl (HPM), fenoxaprop-p-ethyl (FPE), cyhalofop-butyl (CYB), and clodinafop-propargyl (CFP). A novel esterase, QfeH, which catalyzes the cleavage of ester bonds in AOPPs to form AOPP acids, was identified from strain Z-1. The catalytic activities of QfeH toward AOPPs decreased in the following order: CFP > FPE > CYB > QPE > HPM. Molecular docking, computational analyses, and site-directed mutagenesis indicated the catalytic mechanisms of QfeH-mediated degradation of different AOPPs. Notably, the key residue S159 is essential for the activity of QfeH. Moreover, V222Y, T227M, T227A, A271R, and M275K mutants, exhibiting 2.9-5.0 times greater activity than QfeH, were constructed. This study facilitates the mechanistic understanding of AOPPs bioremediation by esterases.