Kinetic Characterization Research Articles

Thermal kinetic analysis and characterization of water hyacinth biomass for renewable energy application

Thermal kinetic analysis and characterization of water hyacinth biomass for renewable energy application

Unique biogenesis and kinetics of hornwort Rubiscos revealed by synthetic biology systems

Hornworts are the only land plants that employ a pyrenoid to optimize Rubisco’s CO2 fixation. Yet, hornwort Rubisco remains poorly characterized. Here we assemble the hornwort Anthoceros agrestis Rubisco (AaRubisco) using the Arabidopsis thaliana SynBio expression system and observed the formation of stalled intermediates, prompting us to develop a new SynBio system with A. agrestis cognate chaperones. We successfully assembled AaRubisco and Rubisco from three other hornwort species. Unlike A. thaliana Rubisco, AaRubisco assembly is not dependent on RbcX or Raf2. Kinetic characterization reveals that hornwort Rubiscos exhibit a range of catalytic rates (3-10 s-1), but with similar affinity (∼30 μM) and specificity (∼70) for CO2. In other words, hornwort Rubiscos do not comply with the long held canonical catalytic trade-off observed in other land plants, providing experimental support that Rubisco kinetics may be phylogenetically constrained. Unexpectedly, we observed a 50% increase in AaRubisco catalytic rates when RbcX was removed from our SynBio system, without any reduction in specificity. Structural biology, biochemistry and proteomic analysis suggest that subtle differences in Rubisco large subunit interactions, when RbcX is absent during biogenesis, increases the accessibility of active sites and catalytic turnover rate. This study uncovered a previously unknown Rubisco kinetic parameter space and provides a SynBio chassis to expand the survey of other Rubisco kinetics. Our discovery could thus reshape the approaches for engineering Rubisco with superior kinetics.

Impact of the key residues of 1-Deoxy-D-Xylulose-5-Phosphate synthase on its catalysis

1-Deoxy-D-xylulose-5-phosphate synthase (DXS) is the first rate-limiting enzyme in the 2-methyl-D-erythritol-4-phosphate (MEP) terpenoid biosynthetic pathway. To explore the active sites of the protein, we made a series of mutations to the key amino acid residues of Escherichia coli DXS including H49, R420, D427, and R478. The results show that several mutants including H49Q lose almost all their catalytic activity in DXP synthesis, but the double-point mutants H49QD427H and H49QD427 N recover 100 % activity of the wild type enzyme. The kinetic characterization displays that the affinity of the double-point mutants for pyruvate and D-glyceraldehyde-3-phosphate only slightly decreases. We also assessed impact of the mutation on the oxidative decarboxylation activity of all the mutants. Despite the fact that H49QD427H and H49QD427 N get back the full activity with regard to DXP synthesis, their oxidase activity remains unrestored. Furthermore, we assayed the acceptor substrate spectrum of the mutants and the results show that a large portion of them catalyzes ligation between pyruvate and nitrosobenzene to form N-phenyl-N-hydroxyacetamide, whereas R420D, R420F, and D427H exhibit activity in connection of pyruvate and benzaldehyde to afford phenylacetocarbinol (PAC), with the first two mutants yielding (S)-PAC (yields 21 %-25 %, ee values 71–75 %) while D427H furnishing (R)-PAC (yield 41 %, ee value 91 %), implying the key role of D427 for the acceptor substrate recognition and the stereoselectivity of the enzyme. It is worth noting that this is the first report of DXS mutants that can recognize benzaldehyde as an acceptor substrate and generate both enantiomers of PAC.

Discovery and Enzyme Kinetic Characterization of Novel CYP2D6 Variants.

Cytochrome P450 2D6 (CYP2D6) exhibits rich genetic polymorphism, and functional changes caused by variations are the key reasons for differences in substrate drug systemic exposure. Discovering novel variants and defining their enzymatic kinetic characteristics can contribute to the personalized application of drugs. In this study, a data chain of variant-function-structure was established through population-based sequencing, baculovirus insect cell expression, in vitro enzymatic incubation, and ultrahigh performance liquid chromatography tandem mass spectrometry. Results revealed nine novel missense mutations in the exonic regions. After the corresponding microsomes were obtained, the kinetics of the variants were investigated using dextromethorphan as a probe substrate. It was found that the activities of CYP2D6.2, 10, 17, 35, 65, R28G, T76M, and E215K were significantly reduced, while D301V almost led to loss of enzyme function. Additionally, the relative clearance rate of R25Q was significantly increased. From the molecular structure perspective, the mutation sites are distributed outside the dextromethorphan binding pocket, suggesting that they primarily influence CYP2D6 activity via allosteric modulation. These research findings provide fundamental data for the precise application of CYP2D6 substrate drugs.

Cu-Promoted ipso-Hydroxylation of sp2 Bonds with Concomitant Aromatic 1,2-Rearrangement Involving a Cu-oxyl-hydroxo Species.

Herein, we report the first example of Cu-promoted β ipso-hydroxylation of substituted benzophenones using a bidentate directing group (DG) and H2O2 as an oxidant. In addition to the new C-O bond formed, the ipso-oxidation induces a very unusual 1,2-rearrangement of the iminyl group to the vicinal γ position. This transformation is highly dependent on the substrate utilized (favored for 4-methoxy-substituted benzophenones) and on the DG used (2-picolylamine leads to selective γ-C-H functionalization, while β ipso-oxidation requires 2-(2-aminoethyl)pyridine). An analysis of the oxidation of substrate-ligands derived from 2-(2-aminoethyl)pyridine and unsymmetrical 4-MeO-substituted benzophenones indicates high regioselectivity (up to 89:11 for the MeO-substituted arene ring and up to 92:8 for β ipso- vs γ-C-H hydroxylation). Mechanistic studies (which include spectroscopic characterization of reaction intermediates, kinetics, and calculations) suggest the formation of a mononuclear CuIIOOH species before the rate-determining step (rds) of the reaction. DFT calculations suggest that the γ-C-H hydroxylation pathway involves a one-step concerted O-O cleavage and electrophilic aromatic attack. Conversely, β ipso-hydroxylation occurs in a stepwise fashion, in which O-O bond cleavage produces a CuIII(O·)(OH) before electrophilic aromatic attack. Calculations also shed light on the mechanism of the 1,2-rearrangement step, which involves strain release from a spiro 5-membered to a 6-membered Cu chelate.

Vitis vinifera Lipoxygenase LoxA is an Allosteric Dimer Activated by Lipidic Surfaces

Lipoxygenases catalyze the peroxidation of poly-unsaturated fatty acid chains either free or esterified in membrane lipids. Vitis vinifera LoxA is transcriptionally induced at ripening onset and localizes at the inner chloroplastic membrane where it is responsible for galactolipid regiospecific mono- and di-peroxidation. Here we present a kinetic and structural characterization of LoxA. Our X-ray structures reveal a constitutive dimer with detergent induced conformational changes affecting substrate binding and catalysis. In a closed conformation, a LID domain prevents substrate access to the catalytic site by steric hindrance. Detergent addition above the CMC destabilizes the LID and opens the dimer with both catalytic sites accessible from the same surface framed by the PLAT domains. As a consequence, detergent molecules occupy allosteric sites in the PLAT/catalytic domain interfaces. These structural changes are mirrored by increased enzymatic activity and positive cooperativity when the substrate is provided in micelles. The ability to interact with micelles is lost upon dimer destabilization by site-directed mutagenesis as assessed by tryptophan fluorescence. Our data allow to propose a model for protein activation at the membrane, classifying LoxA as an interfacial enzyme acting on fatty acid chains directly from the membrane similar to mammalian 15-LOX and 5-LOX.

Catalytic oxidation of Mn(II) in the co-presence of Fe(II) by free chlorine: significance of in situ formed Mn(II)-doped Fe(III) oxides

Fe(II) and Mn(II) are abundant in groundwater and require operationally simple and efficient method to remove in drinking water treatment. The rapid oxidation of Mn(II) is essential in water treatment. This study investigates the efficiency of Mn(II) oxidation by free chlorine in the presence of Fe(II). The results demonstrate that the presence of Fe(II) significantly accelerates the oxidation rate of Mn(II) by free chlorine under neutral and alkaline conditions. The rapid oxidation of Fe(II) by free chlorine and the presence of Mn(II) promote the formation of in situ Mn(II)-doped ferrihydrite. Kinetic modeling and characterization of Fe(III) oxides confirm that the heterogeneous catalytic effect of the Mn(II)-doped ferrihydrite, rather than manganese oxides or their coupled catalytic effect, is responsible for the enhanced oxidation rates. The doped Mn(II) substitutes the tetrahedral Fe(III) ions in the ferrihydrite, introducing additional negative charges at the doped sites. The increased charge enhances Mn(II) adsorption and lowers its redox potential, thereby accelerating Mn(II) oxidation rate through direct electron transfer with adjacent free chlorine. Additionally, the lepidocrocite formed by the reaction between Fe(II) and dissolved oxygen significantly impedes the catalytic performance. These findings provide new insights into the catalytic co-oxidation mechanism of Fe(II) and Mn(II), and help the optimization of water treatment engineering practices.

An in vitro platform for the enzymatic characterization of the rhomboid protease RHBDL4.

Rhomboid proteases are ubiquitous intramembrane serine proteases that can cleave transmembrane substrates within lipid bilayers. They exhibit many and diverse functions, such as but not limited to, growth factor signaling, immune and inflammatory response, protein quality control, and parasitic invasion. Human rhomboid protease RHBDL4 has been demonstrated to play a critical role in removing misfolded proteins from the Endoplasmic Reticulum and is implicated in severe diseases such as various cancers and Alzheimer's disease. Therefore, RHBDL4 is expected to constitute an important therapeutic target for such devastating diseases. Despite its critical role in many biological processes, the enzymatic properties of RHBDL4 remain largely unknown. To enable a comprehensive characterization of RHBDL4's kinetics, catalytic parameters, substrate specificity, and binding modality we expressed and purified recombinant RHBDL4, and employed it in a Förster Resonance Energy Transfer-based cleavage assay. Until now, kinetic studies have been limited mostly to bacterial rhomboid proteases. Our in vitro platform offers a new method for studying RHBDL4's enzymatic function and substrate preferences. Furthermore, we developed and tested potential inhibitors using our assay and successfully identifying peptidyl α-ketoamide inhibitors of RHBDL4 that are highly effective against recombinant RHBDL4. We utilize ensemble docking and molecular dynamics (MD) simulations to explore the binding modality of substrate-derived peptides bound to RHBDL4. Our analysis focused on key interactions and dynamic movements within RHBDL4's active site that contributed to binding stability, offering valuable insights for optimizing the non-prime side of RHBDL4 ketoamide inhibitors. In summary, our study offers fundamental insights into RHBDL4's catalytic activities and substrate preferences, laying the foundation for downstream applications such as drug inhibitor screenings and structure-function studies, which will enable the identification of lead drug compounds for RHBDL4.

Slow Conformational Exchange between Partially Folded and Near-Native States of Ubiquitin: Evidence for a Multistate Folding Model.

The mechanism by which small proteins fold, i.e., via intermediates or via a two-state mechanism, is a subject of intense investigation. Intermediate states in the folding pathways of these proteins are sparsely populated due to transient lifetimes under normal conditions rendering them transparent to a majority of the biophysical methods employed for structural, thermodynamic, and kinetic characterization, which attributes are essential for understanding the cooperative folding/unfolding of such proteins. Dynamic NMR spectroscopy has enabled the characterization of folding intermediates of ubiquitin that exist in equilibrium under conditions of low pH and denaturants. At low pH, an unlocked state defined as N' is in fast exchange with an invisible state, U″, as observed by CEST NMR. Addition of urea to ubiquitin at pH 2 creates two new states F' and U', which are in slow exchange (kF'→U' = 0.14 and kU'→F' = 0.28 s-1) as indicated by longitudinal ZZ-magnetization exchange spectroscopy. High-resolution solution NMR structures of F' show it to be in an "unlocked" conformation with measurable changes in rotational diffusion, translational diffusion, and rotational correlational times. U' is characterized by the presence of just the highly conserved N-terminal β1-β2 hairpin. The folding of ubiquitin is cooperative and is nucleated by the formation of an N-terminal β-hairpin followed by significant hydrophobic collapse of the protein core resulting in the formation of bulk of the secondary structural elements stabilized by extensive tertiary contacts. U' and F' may thus be described as early and late folding intermediates in the ubiquitin folding pathway.

Review of thermal neutron scintillators: Evaluation metrics and future prospects for demanding applications

Neutron applications are progressing rapidly, requiring detectors to meet increasingly rigorous criteria. Specifications such as high neutron counting rates, fast timing resolution and low background have a significant impact on choice of scintillator and how scintillators are evaluated. This work considers detector requirements for neutron scattering instruments that are becoming more stringent as the sources and scattering technologies continue to improve. Meanwhile, both prominent scintillators used for neutron scattering detectors, ZnS:Ag/6LiF and GS-glass, remain stagnant. While these scintillators have been suitable for many years, they are becoming a limiting factor for neutron scattering instruments. ZnS:Ag is inhibiting count rate capability and GS-glass is too gamma sensitive so alternatives need to be found. A brief explanation of neutron scattering, its detector requirements and how those relate to scintillator properties will be discussed. Furthermore, this work explains how standard characterization methods and reporting of scintillation properties are not entirely suitable for high count rate, low background applications like neutron scattering. As a result, modifications to characterization of gamma discrimination, light output, and decay kinetics are suggested. Additional steps are suggested including neutron activation and a more appropriate assessment of count rate capability. A non-exhaustive set of 6Li containing neutron sensitive scintillators including ZnS:Ag/6LiF, GS20 glass, ZnO:Zn/6LiF, Cs6LiYCl:Ce, 6LiI:Eu, 6LiI:Ce, 6LiCaAlF:Eu, LiCaAlF:Ce, transparent rubber sheet (TRUST) 6LiCaAlF:Eu, TRUST 6LiCaAlF:Eu with added fluorophore and EJ-270 (6Li containing plastic) are reviewed and studied from this different perspective. Finally, new scintillator developments relevant to high count rate, low background applications are discussed.

High Temperature Oxidation Behavior of ODS Ferritic Stainless Steel Fe-16Cr-4Al-1Ni-0.4Y<sub>2</sub>O<sub>3</sub>

This study investigates the isothermic oxidation behavior of the new ODS alloy Fe-16Cr-4Al-1Ni-0.4Y2O3 (% by weight) at 700, 800 and 900 °C, with exposure times of 5, 20, 50, and 100 h at each temperature. The purpose is to obtain new data on its high-temperature parabolic oxidation constant for assessing oxidation resistance. The methods used include isothermal oxidation testing, XRD, SEM-EDS characterization, and analysis of oxidation kinetics by monitoring changes in oxide thickness using microscopy and SEM-EDS. The oxide products formed on the sample surface are Fe2O3, Fe3O4, AlFe2O4, and (Fe,Cr)2O3. Al and Cr oxides are located under the dominant Fe oxide layer on the surface of the sample. The oxidation test results showed that the most protective sample was obtained at a temperature of 700 °C for 100 h with an oxide thickness of 263.99 μm. The kinetics analysis correlates strongly with the parabolic equation (R2 ≈ 1). The oxidation rate constants at temperatures of 700, 800, and 900 °C were 681.76, 2957.5, and 12300 μm2 h−1, respectively. The activation energy required by the oxidation reaction in this alloy is 136.5 kJ mol−1. This research enhances understanding and potential applications of the Fe-16Cr-4Al-1Ni-0.4Y2O3 alloy in high-temperature environments.

Kinetic characterization of amino acid activation by aminoacyl-tRNA synthetases using radiolabelled γ-[32P]ATP.

Aminoacyl-tRNA synthetases (AARSs) are fundamental enzymes that pair amino acids and tRNAs for protein synthesis. Aminoacylation occurs in two discrete steps. The amino acid is first activated by ATP, leading to an aminoacyl-adenylate intermediate and pyrophosphate (PPi) formation. In a subsequent step, the aminoacyl moiety is transferred to the tRNA. Kinetic assays were developed to follow each of these steps independently, as well as cumulative two-step aminoacylation. The main advantage of following the activation step over two-step aminoacylation is that most AARSs can activate amino acids in the absence of the tRNA, the production of which is laborious. Hence, the activation step is often tested first in the kinetic analysis, including large screens exploring AARS-targeting inhibitors. Since the 1960s, the activation reaction has been routinely followed by the standard ATP/[32P]PPi exchange assay, which relies on the equilibrium exchange of radiolabel between PPi and ATP using [32P]PPi as a labelled compound. However, this method became much less convenient when [32P]PPi was discontinued in 2022. As a solution, we developed a modified assay that uses easily attainable γ-[32P]ATP as a labelled compound in the equilibrium-based assay. Using this assay, herein named the [32P]ATP/PPi assay, we followed the activation step of several AARSs. The obtained data are in good agreement with the previously published kinetic constants obtained with the standard ATP/[32P]PPi exchange assay.

Use of white grape pomace for removal of cationic dyes from aqueous solution: kinetic, isotherm and thermodynamic characterization

Use of white grape pomace for removal of cationic dyes from aqueous solution: kinetic, isotherm and thermodynamic characterization

Isoflavone-rich extracts from okara using supercritical fluid extraction: Kinetic modeling and characterization

The lipid fraction and bioactive compounds from okara, a by-product of soybean processing, have been extracted by supercritical fluid extraction. The effect of operating parameters, pressure (20–40 MPa), temperature (40–80 ºC), and cosolvent usage (0–10 %wt. ethanol), on the extraction kinetics has been investigated, satisfactorily correlating the experimental data to the Sovová’s model. Extraction yield by SFE ranged from 0.1014 g extract/g insoluble solid (IS) at 20 MPa and 80 ºC to 0.1081 g extract/g IS at 30 MPa and 60 ºC (from 9.2 to 9.8 g extract/100 g okara, respectively), similar to Soxhlet extraction with n-hexane (9.2 ± 0.2 g extract/100 g). Pressure positively affected the initial extraction rate, which also increased with temperature except for the lowest pressure of 20 MPa, exhibiting a typical cross-over behavior. The use of ethanol as cosolvent promoted the extraction of phenolic compounds and isoflavones, and was less important in the extraction of tocopherols. Extraction yield using ethanol increased up to 0.1100 g extract/g IS at 40 MPa and 40 ºC. As expected, the higher content of bioactive compounds in the sc-CO2 + ethanol extracts positively affected the antioxidant capacity, with the maximum found at 20 MPa, 40 ºC and 10 %wt. ethanol.

Optical coherence tomography characterization of degradation kinetics between second- and third-generation resorbable magnesium scaffold.

This preclinical study aimed to establish optical coherence tomography (OCT)-derived parameters that could be used in the clinical setting for assessing strut degradation in the third-generation drug-eluting resorbable magnesium scaffold (DREAMS-3G), and characterize the comparative degradation profile against its precursor device (MagmarisTM scaffold). Twelve DREAMS-3G and 10 MagmarisTM scaffolds were implanted in juvenile pigs, and OCT images obtained at baseline and follow-up (6 or 12 months). Strut degradation was assessed by planimetric analysis and compared with OCT-derived indices to validate their diagnostic accuracy. A total of 3327 struts of DREAMS-3G and 2995 struts of the MagmarisTM scaffold were delineated by OCT. DREAMS-3G exhibited a significantly higher number of visible struts per analyzed frame at 6 months than the MagmarisTM scaffold, in the absence of significant differences at 12 months. Attenuation index (AtI) analysis indicated DREAMS-3G degradation was less advanced at 6 months but more advanced at 12 months compared to the MagmarisTM scaffold. These OCT-derived indices significantly correlated with the results of the planimetric analysis. The current preclinical study validated OCT indices that may serve as clinical surrogate markers for scaffold degradation. AtI analysis indicated that DREAMS-3G showed less degradation at 6 months but more advanced degradation at 12 months compared to the MagmarisTM scaffold, which corroborates the findings from planimetric analysis.

Computer-aided pattern scoring (C@PS): a novel cheminformatic workflow to predict ligands with rare modes-of-action

The identification, establishment, and exploration of potential pharmacological drug targets are major steps of the drug development pipeline. Target validation requires diverse chemical tools that come with a spectrum of functionality, e.g., inhibitors, activators, and other modulators. Particularly tools with rare modes-of-action allow for a proper kinetic and functional characterization of the targets-of-interest (e.g., channels, enzymes, receptors, or transporters). Despite, functional innovation is a prime criterion for patentability and commercial exploitation, which may lead to therapeutic benefit. Unfortunately, data on new, and thus, undruggable or barely druggable targets are scarce and mostly available for mainstream modes-of-action only (e.g., inhibition). Here we present a novel cheminformatic workflow—computer-aided pattern scoring (C@PS)—which was specifically designed to project its prediction capabilities into an uncharted domain of applicability.

Biosynthesis of Piceatannol from Resveratrol in Grapevine Can Be Mediated by Cresolase-Dependent Ortho-Hydroxylation Activity of Polyphenol Oxidase.

Piceatannol is a naturally occurring hydroxylated analogue of the stilbene phytoalexin resveratrol that can be found in grape fruit and derived products. Piceatannol has aroused great interest as it has been shown to surpass some human health-beneficial properties of resveratrol including antioxidant activity, several pharmacological activities and also bioavailability. The plant biosynthetic pathway of piceatannol is still poorly understood, which is a bottleneck for the development of both plant defence and bioproduction strategies. Cell cultures of Vitis vinifera cv. Gamay, when elicited with dimethyl-β-cyclodextrin (MBCD) and methyl jasmonate (MeJA), lead to large increases in the accumulation of resveratrol, and after 120 h of elicitation, piceatannol is also detected due to the regiospecific hydroxylation of resveratrol. Therefore, an ortho-hydroxylase must participate in the biosynthesis of piceatannol. Herein, three possible types of resveratrol hydroxylation enzymatic reactions have been tested, specifically, a reaction catalyzed by an NADPH-dependent cytochrome, P450 hydroxylase, a 2-oxoglutarate-dependent dioxygenase and ortho-hydroxylation, similar to polyphenol oxidase (PPO) cresolase activity. Compared with P450 hydoxylase and the dioxygenase activities, PPO displayed the highest specific activity detected either in the crude extract, the particulate or the soluble fraction obtained from cell cultures elicited with MBCD and MeJA for 120 h. The overall yield of PPO activity present in the crude extract (107.42 EU) was distributed mostly in the soluble fraction (66.15 EU) rather than in the particulate fraction (3.71 EU). Thus, partial purification of the soluble fraction by precipitation with ammonium sulphate, dialysis and ion exchange chromatography was carried out. The soluble fraction precipitated with 80% ammonium sulphate and the chromatographic fractions also showed high levels of PPO activity, and the presence of the PPO protein was confirmed by Western blot and LC-MS/MS. In addition, a kinetic characterization of the cresolase activity of partially purified PPO was carried out for the resveratrol substrate, including Vmax and Km parameters. The Km value was 118.35 ± 49.84 µM, and the Vmax value was 2.18 ± 0.46 µmol min-1 mg-1.

Assessment of Blast Furnace Slags as a Potential Catalyst in Ozonation to Degrade Bezafibrate: Degradation Study and Kinetic Study via Non-Parametric Modeling

This study investigates the effectiveness of blast furnace slags (BFSs) as catalysts in the ozonation process to degrade complex contaminants such as bezafibrate (BFZ) at different pH levels. The findings reveal that the presence of BFS enhances degradation efficiency, achieving a 10% improvement at pH 10 and a 30% improvement at pH 5.5 compared to simple ozonation. The highest degradation efficiency was observed in the Ozonation–BFS system at pH 10, with 90% decomposition of BFZ. These results were corroborated through ozone consumption analysis, BOD5 measurements, and the identification of oxalic acid as the final decomposition product. Due to the complexity of the reaction system, kinetic characterization was performed using non-parametric modeling based on differential neural networks. The model indicated that the observed reaction rate for BFZ degradation in the presence of ozone and BFS was 4.12 times higher at pH 5.0 and 1.08 times higher at pH 10.0 compared to simple ozonation. These results underscore the potential of using BFS in catalytic ozonation processes for the effective treatment of recalcitrant contaminants in wastewater.

Identification of Plant Peroxidases Catalyzing the Degradation of Fluorinated Aromatics Using a Peroxidase Library Approach.

In this work, the degradation of mono- and polyfluorinated phenolic compounds was demonstrated by a series of crude plant peroxidases, including horseradish root (HRP) and six members of the Cucurbita genus. Highly active samples were identified using a library screening approach in which more than 50 crude plant samples were initially evaluated for defluorination activity toward 4-fluorophenol. The highest concentrations were observed in the HRP, pumpkin skin (PKS), and butternut squash skin (BNS), which consistently gave the highest intrinsic rates of decomposition for all the substrates tested. Although HRP exhibited a significant decrease in activity with increased fluorination of the phenolic substrate, PKS showed only minor reductions. Furthermore, in silico studies indicated that the active site of HRP poorly accommodates the steric bulk of additional fluorines, causing the substrate to dock farther from the catalytic heme and thus slowing the catalysis rate. We propose that the PKS active site might be larger, allowing closer access to the perfluorinated substrate, and therefore maintaining higher activity compared to the HRP enzyme. However, detailed kinetic characterization studies of the peroxidases are recommended. Conclusively, the high catalytic activity of PKS and its high yield per gram of tissue make it an excellent candidate for developing environmentally friendly biocatalytic methods for degrading fluorinated aromatics. Finally, the success of the library approach in identifying highly active samples for polyfluorinated aromatic compound (PFAC) degradation suggests the method may find utility in the quest for other advanced catalysts for PFAS degradation.

Kinetic characterization of sera antibodies – A surface plasmon resonance study

Kinetic characterization of sera antibodies – A surface plasmon resonance study