Time Course Of Reaction Research Articles

Oxidative cross-linking of potato proteins by fungal laccases: Reaction kinetics and effects on the structural and functional properties

Aiming at developing biocatalytic approach to modulate potato proteins functionalities, laccase-catalyzed oxidative cross-linking of potato proteins was investigated in terms of enzyme kinetics, reaction time course and product structural-functional properties. The catalytic efficiency (kcat/Km, mM−1S−1) of fungal laccases in oxidizing patatin-enriched potato protein (PAT, 0.010–0.748) was higher than that of potato protease inhibitors (PIs, 0.008–0.184). While PIs formed more efficiently oxidative cross-linked products. Cross-linking of potato proteins were achieved by laccase alone or by laccase-ferulic acid system; ferulic acid contributed to higher cross-linking extent and to the antioxidant activity of modified proteins. Potato proteins exhibited similar or slightly enhanced solubility upon cross-linking, except that highly cross-linked PIs (cross-linking extent >30%) obtained from Coriolus hirsutus laccase (LacCh)-treatments showed reduced solubility. General improvement in the emulsification property of potato proteins was observed upon cross-linking. Cross-linked potato proteins from LacCh-6 h treatment showed good foaming property; these proteins exhibited enhanced molecular flexibility and the characteristics of mild cross-linking extent (9.6–17.1%) with the accumulation of moderate molecular weight fraction (PAT:60–80 kDa, PIs:30–40 kDa). This study will lay foundations for the exploitation of potato proteins as functional ingredients. Industrial relevanceThe exploitation of novel proteins as functional ingredients is needed in order to meet the high demands for natural, healthy-oriented and sustainable food products. Laccase-catalyzed protein cross-linking is a promising green technology for tailoring the functionality of protein ingredients and achieving the desired textural/rheological quality of final products. The current study demonstrated the effectiveness of this enzymatic approach to enhance foaming and emulsifying properties of potato proteins. This approach can be adopted to modulate the functionality of other plant proteins and to develop plant proteins-based functional ingredients for various applications including stable delivery system, fat-replacing biopolymer matrix, meat analog/vegan products and gluten-replacement formulations.

Liquid fuel synthesis via CO2 hydrogenation by coupling homogeneous and heterogeneous catalysis

Summary Synthesis of liquid fuel (C5+ hydrocarbons) via CO2 hydrogenation generally involves cascade catalysis of reverse water gas shift (RWGS) and Fischer-Tropsch synthesis (FTS) reactions over heterogeneous catalysts. Because of thermodynamic limitations of the cascade reactions, the reported heterogeneous catalysts usually suffer from high temperature (above 300°C) and low selectivity. Here, we report the liquid fuel production from CO2 hydrogenation by coupling homogeneous RuCl3 and heterogeneous Ru0 catalysts, which proceeded at the lowest temperature reported so far (180°C) and reached the highest C5+ selectivity to date (71.1%). The TOF of the CO2 hydrogenation reached 9.5 h−1, which is comparable with that of the reported Ru0 catalyzed FTS reaction using syngas. Detailed study indicates that synergy of homogeneous and heterogeneous catalysis is the key for the outstanding performance.

Magnetic cellulose beads as support for enzyme immobilization using polyelectrolytes through electrostatic adsorption

Pectinase was immobilized on magnetic regenerated cellulose microspheres using cationic polyacrylamide (CPAM) or polyethyleneimine (PEI) via electrostatic adsorption. PEI-immobilized enzyme (PEI-EMCB) had higher activity (2711 U/g) than CPAM-immobilized enzyme (CPAM-EMCB). The time course of PEI-immobilized enzyme catalytic reaction was similar to the free form. PEI-EMCB reached its maximal activity at pH 4.8, while the optimal pH for CPAM-EMCB was the same with the free form (4.4). CPAM3-EMCB was most tolerant to pH variation, which can be possibly ascribed to the high molecular weight of CPAM3. After immobilization, the optimum temperature for the enzyme declined from 45 °C to different degrees. PEI-EMCB exhibited good storage stability with 67% of the initial activity maintained after 7 days and with moderate reusability. The magnetic properties of the regenerated cellulose beads provided convenience for the immobilized enzyme to be used and recycled. The results indicate a potential route for utilization of cellulose as enzyme support via a simple method.

Off to a slow start: Analyzing lag phases and accelerating rates in steady-state enzyme kinetics

Steady-state enzyme kinetics typically relies on the measurement of ‘initial rates’, obtained when the substrate is not significantly consumed and the amount of product formed is negligible. Although initial rates are usually faster than those measured later in the reaction time-course, sometimes the speed of the reaction appears instead to increase with time, reaching a steady level only after an initial delay or ‘lag phase’. This behavior needs to be interpreted by the experimentalists. To assist interpretation, this article analyzes the many reasons why, during an enzyme assay, the observed rate can be slow in the beginning and then progressively accelerate. The possible causes range from trivial artifacts to instances in which deeper mechanistic or biophysical factors are at play. We provide practical examples for most of these causes, based firstly on experiments conducted with ornithine δ-aminotransferase and with other pyridoxal-phosphate dependent enzymes that have been studied in our laboratory. On the side to this survey, we provide evidence that the product of the ornithine δ-aminotransferase reaction, glutamate 5-semialdehyde, cyclizes spontaneously to pyrroline 5-carboxylate with a rate constant greater than 3 s−1.

Time course and variability of tendinous vibration-induced postural reactions in forward and backward directions

Mechanical vibration of tendons induces large postural reactions (PR-VIB) but little is known about how these reactions vary within and between subjects. We investigated the intra- and inter-individual variability of PR-VIB and determined the reliability of center of pressure (COP) measures. Bipodal postural control (eyes closed) of 30 healthy adults were evaluated using a force platform under 02 conditions: bilateral VIB of the tibialis anterior (TA) and Achilles tendons (ACH-T) at 80 Hz. Each condition consisted of 03 trials of 30 s duration (Baseline: 10 s; VIB: 10 s; POST-VIB: 10 s). The Amplitude and Velocity of the COP in the antero-posterior/medio-lateral (AP/ML) directions were recorded and analyzed according to 5 time-windows incremented every 2 s of vibration (i.e. the first 2 s; 4 s; 6 s; 8 s & 10 s), whereas the COP position/AP was monitored every 0.5 s. All postural parameters increased significantly during TA and ACH-T vibration compared to the Baseline. The reliability of the COP measures showed good ICC scores (0.40-0.84) and measurement errors that varied depending on the duration of VIB time-windows. The COP position/AP reveals a lower intra- and inter-subject variability of PR-VIB in the first 2 s of VIB. The metrological characteristics of PR-VIB should be investigated further to guide their future use by clinicians and researchers.

Blood pressure reaction to negative stimuli: Insights from continuous recording and analysis.

Individuals with a tendency toward abnormally enhanced cardiovascular responses to stress are at greater risk of developing essential hypertension later in life. Accurate profiling of continuous blood pressure (BP) reactions in healthy populations is crucial for understanding normal and abnormal emotional reaction patterns. To this end, we examined the continuous time course of BP reactions to aversive pictures among healthy participants. In two experiments, we showed participants negative and neutral pictures while simultaneously measuring their continuous BP and heart rate (HR) reactions. In this study, BP reactions were analyzed continuously, in contrast to previous studies, in which BP responses were averaged across blocks. To compare time points along a temporal continuum, we applied a multi‐level B‐spline model, which is innovative in the context of BP analysis. Additionally, HR was similarly analyzed in order to examine its correlation with BP. Both experiments revealed a similar pattern of BP reactivity and association with HR. In line with previous studies, a decline in BP and HR levels was found in response to negative pictures compared to neutral pictures. In addition, in both conditions, we found an unexpected elevation of BP toward the end of the stimuli exposure period. These findings may be explained by the recruitment of attention resources in the presence of negative stimuli, which is alleviated toward the end of the stimulation. This study highlights the importance of continuous measurement and analysis for characterizing the time course of BP reactivity to emotional stimuli.

Determination of biocatalytic parameters of a copper radical oxidase using real-time reaction progress monitoring.

An Auxiliary Activity Family 5 (AA5) copper-radical alcohol oxidase (AlcOx) with promiscuous activity towards simple alkyl and aromatic alcohols was evaluated using real-time reaction progress monitoring. Reaction kinetics using variable time normalization analysis (VTNA) were determined from reaction progress curves. By this approach, a detailed view of the entire reaction time course under various conditions was obtained and used to identify parameters that will inform further process optimization development. Optimal activity was found impacted by several factors, including reaction pH, oxygen saturation, and the source of a co-oxidant, either HRP or a chemical alternative, potassium ferricyanide. Analysis of reaction progress curves demonstrated that reaction stalling occurred as a result of oxygen depletion and from a loss of enzyme activity over time. The cooperativity between AlcOx, horseradish peroxidase (HRP), and catalase that result in enhanced reactivity was explored, with reaction pH being identified as a key factor for optimal activity. The results show that a process with HRP is more robust than with potassium ferricyanide, but that both oxidants likely activate AlcOx by a similar mechanism. The phenomenon of product inhibition was investigated for representative reactants, revealing that reaction inhibition was more significant for butyraldehyde than for benzaldehyde. Our analysis suggests that this is linked to the greater proportion in which butyraldehyde exists in the hydrated form.

Microdroplet synthesis of azo compounds with simple microfluidics-based pH control.

Conventional solution-phase synthesis of azo compounds is complicated by the need for precise pH and temperature control, high concentrations of pH control reagents, and by-product removal. In this work, we exploited the advantages of microdroplet chemistry to realize the simple and highly efficient synthesis of an azo compound using microfluidics-based pH control. Owing to the small size of microdroplets, heat exchange between a microdroplet and its environment is extremely fast. Furthermore, chemical reactions in microdroplets occur rapidly due to the short diffusion distance and vortex flow. Formation of the azo compound reached completion in less than 3 s at room temperature, compared with 1 h at 0 °C under conventional conditions. pH control was simple, and the pH control reagent concentration could be reduced to less than one-tenth of that used in the conventional method. No by-products were generated, and thus this procedure did not require a recrystallization step. The time course of the chemical reaction was elucidated by observing the growth of azo compound microcrystals in droplets at various locations along the channel corresponding to different mixing times.

Cobalt-Catalyzed Borylation of Fluorinated Arenes: Thermodynamic Control of C(sp2)-H Oxidative Addition Results in ortho-to-Fluorine Selectivity.

The mechanism of C(sp2)-H borylation of fluorinated arenes with B2Pin2 (Pin = pinacolato) catalyzed by bis(phosphino)pyridine (iPrPNP) cobalt complexes was studied to understand the origins of the uniquely high ortho-to-fluorine regioselectivity observed in these reactions. Variable time normalization analysis (VTNA) of reaction time courses and deuterium kinetic isotope effect measurements established a kinetic regime wherein C(sp2)-H oxidative addition is fast and reversible. Monitoring the reaction by in situ NMR spectroscopy revealed the intermediacy of a cobalt(I)-aryl complex that was generated with the same high ortho-to-fluorine regioselectivity associated with the overall catalytic transformation. Deuterium labeling experiments and stoichiometric studies established C(sp2)-H oxidative addition of the fluorinated arene as the selectivity-determining step of the reaction. This step favors the formation of ortho-fluoroaryl cobalt intermediates due to the ortho fluorine effect, a phenomenon whereby ortho fluorine substituents stabilize transition metal-carbon bonds. Computational studies provided evidence that the cobalt-carbon bonds of the relevant intermediates in (iPrPNP)Co-catalyzed borylation are strengthened with increasing ortho fluorine substitution. The atypical kinetic regime involving fast and reversible C(sp2)-H oxidative addition in combination with the thermodynamic preference for forming cobalt-aryl bonds adjacent to fluorinated sites are the origin of the high regioselectivity in the catalytic borylation reaction.

Enzymatic treatment for removal of hazardous aqueous arylamines, 4,4′-methylenedianiline and 4,4′-thiodianiline

The search for an effective and sustainable treatment method to remove the recalcitrant atom-bridged bis-anilino compounds, 4,4′-methylenedianiline (MDA) and 4,4′-thiodianiline (TDA) from water is a major challenge and focus of this study. The escalating discharge of these two toxic and carcinogenic pollutants from industrial sources may pose a serious threat to the environment. Crude soybean peroxidase (SBP), isolated from soybean seed hulls (coats), catalyzes the oxidative polymerization of these aqueous pollutants in the presence of hydrogen peroxide. The effects of several process parameters, i.e., pH, hydrogen peroxide-to-substrate concentration ratio and SBP concentration, were investigated to optimize the performance of enzymatic treatment. The minimum effective SBP concentration required for removal of MDA was 0.70 U/mL, which was higher than that of TDA (0.15 U/mL). The reaction time course to achieve ≥95% removal of these compounds from water was determined under those optimum conditions. Identification of the transformed products was performed by means of high-resolution electrospray ionization mass spectrometry. The products generally observed were protonated oxidized oxidative dimers and higher oligomers (most commonly azo-coupled products). Michaelis constant, KM, and maximum reaction velocity, Vmax, obtained from the Michaelis-Menten (M-M) model revealed that TDA had a 65-fold lower KM than MDA (indicating TDA's higher affinity for SBP), and almost 5-fold higher Vmax than MDA. A pro-forma cost analysis is presented to assess the possibility of commercialization of enzymatic treatment as an alternative to conventional/traditional treatment methods.

Facile synthesis of D-xylulose-5-phosphate and L-xylulose-5-phosphate by xylulokinase-catalyzed phosphorylation

The enantiomeric metabolites D-xylulose-5-phosphate and L-xylulose-5-phosphate are highly relevant in numerous important pathways from microbial to human metabolism. As these phosphorylated chiral metabolites are required for the analysis of enzyme activities, pathways and biological functions in health and disease, efficient and straightforward methodologies for their synthesis are needed. Biocatalytic phosphorylations of their corresponding monosaccharides offer single step synthetic routes. Therefore, D- and L-xylulokinases have been selected, the XKS1 gene from Saccharomyces cerevisiae and the lyx gene from Escherichia coli have been synthesized and cloned into an appropriate expression vector to allow for the production of the enzymes in E. coli. The expression has led to highly active xylulokinases in the soluble fractions, which could be directly used for the biocatalytic phosphorylation of D- and L-xylulose, using the phosphoenolpyruvate(PEP)/pyruvatekinase(PK)-system for the regeneration of the ATP cofactor. The time course of the phosphorylation reactions has been monitored by 31P-NMR and 1H-NMR spectroscopy and the monosaccharide substrates could be phosphorylated with nearly 100% conversion. After standard workup, both the D-xylulose-5-phosphate and the L-xylulose-5-phosphate have been obtained in high yield and purity.

Slow-Binding Inhibition of Tyrosinase by Ecklonia cava Phlorotannins.

Tyrosinase inhibitors improve skin whitening by inhibiting the formation of melanin precursors in the skin. The inhibitory activity of seven phlorotannins (1–7), triphlorethol A (1), eckol (2), 2-phloroeckol (3), phlorofucofuroeckol A (4), 2-O-(2,4,6-trihydroxyphenyl)-6,6′-bieckol (5), 6,8′-bieckol (6), and 8,8′-bieckol (7), from Ecklonia cava was tested against tyrosinase, which converts tyrosine into dihydroxyphenylalanine. Compounds 3 and 5 had IC50 values of 7.0 ± 0.2 and 8.8 ± 0.1 μM, respectively, in competitive mode, with Ki values of 8.2 ± 1.1 and 5.8 ± 0.8 μM. Both compounds showed the characteristics of slow-binding inhibitors over the time course of the enzyme reaction. Compound 3 had a single-step binding mechanism and compound 5 a two-step-binding mechanism. With stable AutoDock scores of −6.59 and −6.68 kcal/mol, respectively, compounds 3 and 5 both interacted with His85 and Asn260 at the active site.

The nature of β-cyclodextrin inhibition of potato polyphenol oxidase-catalyzed reactions

There is general interest in strategies to control polyphenol oxidase (PPO)-initiated enzymatic browning because it is often associated with declining food quality. Cyclodextrins are cyclic glucan oligosaccharides that form inclusion complexes with a number of PPO substrates. This study focuses on the effect of β-cyclodextrins (βCyD) on PPO-catalyzed reactions. Potato enzyme extracts and semi-purified potato PPO served as enzyme sources. Substrates included phenolics endogenous to potatoes. Reaction time-courses were followed spectrophotometrically; rates were compared by analysis of variance. Extents of βCyD inhibition of PPO-catalyzed reactions are shown to be substrate specific and can be quantitatively accounted for based on degrees of βCyD substrate sequestration. There was no evidence for direct irreversible βCyD inactivation of potato PPO. An apparent “direct PPO inactivation” by βCyD is shown to result from a sequence of sequestration-dependent reactions that occur in commonly employed assay systems for the quantification of PPO in fruits and vegetables.

Mouse Strain and Sex-Dependent Differences in Long-term Behavioral Abnormalities and Neuropathologies after Developmental Zika Infection.

Exposure of the developing fetus to Zika virus (ZIKV) results in a set of brain abnormalities described as the congenital Zika syndrome. Although microcephaly is the most obvious outcome, neuropathologies, such as intracranial calcifications and polymicrogyria, can occur in the absence of microcephaly. Moreover, the full impact of exposure on motor, social, and cognitive skills during development remains uncharacterized. We examined the long-term neurobehavioral consequences of neonatal ZIKV exposure in four genetically divergent inbred mouse strains (C57BL/6J, 129S1/SvImJ, FVB/NJ, and DBA/2J). Male and female mice were infected on postnatal day 1, considered comparable with exposure late in the second trimester of humans. We demonstrate strain differences in early susceptibility to the virus and the time course of glial reaction in the brain. These changes were associated with strain- and sex-dependent differences in long-term behavioral abnormalities that include hyperactivity, impulsiveness, and motor incoordination. In addition, the adult brains of susceptible mice exhibited widespread calcifications that may underlie the behavioral deficits observed. Characterization of the neuropathological sequelae of developmental exposure to the Zika virus in different immunocompetent mouse strains provides a foundation for identifying genetic and immune factors that contribute to long-term neurobehavioral consequences in susceptible individuals.SIGNIFICANCE STATEMENT Developmental Zika virus (ZIKV) infection is now known to cause brain abnormalities in infants that do not display microcephaly at birth, and the full impact of these more subtle neuropathologies has yet to be determined. We demonstrate in a mouse model that long-lasting behavioral aberrations occur after developmental ZIKV exposure. We compare four divergent mouse strains and find that the effects of Zika infection differ greatly between strains, in terms of behavioral changes, sex differences, and the intracranial calcifications that develop in the brains of susceptible mice. These findings provide a foundation for identifying susceptibility factors that lead to the development of abnormal behaviors secondary to ZIKV infection early in life.

Laccase-Catalyzed Oxidation of Mixed Aqueous Phenolic Substrates at Low Concentrations

It has been proposed that Trametes versicolor laccase can be used to detoxify wastewaters that are contaminated with phenolic pollutants. However, the oxidation of phenols at low concentrations may be impacted if other substrates tend to interfere with or enhance the oxidation of the target substrate. To test this, experiments were conducted to evaluate effects arising from the simultaneous presence of mixed substrates including phenol (P), estradiol (E2), cumylphenol (CP), and triclosan (TCL), each of which are characterized by different rates of oxidation and tendencies to inactivate laccase. Slower and faster substrates were found to have only minor negative impacts upon the rate of conversion of targeted substrates, except where they tended to cause inactivation. No enhancements in substrate oxidation were observed. A multi-substrate kinetic model was shown to be able to accurately predict the time course of reactions of mixed substrates over extended periods at micromolar and sub-micromolar concentrations, except when estradiol and triclosan were simultaneously present. In this case, more enzyme inactivation was observed than would be expected from the oxidation of individual substrates alone. The utility of the model for providing insights into the reaction phenomenon and for evaluating the feasibility of oxidizing targeted substrates in the presence of other substrates is demonstrated.

New assay method based on Raman spectroscopy for enzymes reacting with gaseous substrates.

Enzyme activity is typically assayed by quantitatively measuring the initial and final concentrations of the substrates and/or products over a defined time period. For enzymatic reactions involving gaseous substrates, the substrate concentrations can be estimated either directly by gas chromatography or mass spectrometry, or indirectly by absorption spectroscopy, if the catalytic reactions involve electron transfer with electron mediators that exhibit redox‐dependent spectral changes. We have developed a new assay system for measuring the time course of enzymatic reactions involving gaseous substrates based on Raman spectroscopy. This system permits continuous monitoring of the gas composition in the reaction cuvette in a non‐invasive manner over a prolonged time period. We have applied this system to the kinetic study of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F. This enzyme physiologically catalyzes the reversible oxidation of H2 and also possesses the nonphysiological functions of H/D exchange and nuclear spin isomer conversion reactions. The proposed system has the additional advantage of enabling us to measure all of the hydrogenase‐mediated reactions simultaneously. Using the proposed system, we confirmed that H2 (the fully exchanged product) is concomitantly produced alongside HD by the H/D exchange reaction in the D2/H2O system. Based on a kinetic model, the ratio of the rate constants of the H/D exchange reaction (k) at the active site and product release rate (k out) was estimated to be 1.9 ± 0.2. The proposed assay method based on Raman spectroscopy can be applied to the investigation of other enzymes involving gaseous substrates.

Two step-production of acylglycerols containing a high proportion of docosapentaenoic acid from marine omega-3 oil and their oxidative stability

The objective of this study was to concentrate polyunsaturated fatty acid (PUFA) from seal blubber oil in an innovative manner to produce a high content of docosapentaenoic acid (DPA) in the resultant product. It also aimed at investigating the use of lipases as catalysts for synthesizing acylglycerols from glycerol and polyunsaturated fatty acid concentrates. Additionally, study of the oxidative stability of acylglycerols synthesized by lipases was intended. A two-stage urea complexation process was used to concentrate PUFA from seal blubber oil, giving rise to a DPA content of up to 24.0% in the product. Enzymatic synthesis of acyglycerols directly from glycerol and fatty acid concentrate was studied. Three lipases were used as biocatalysts for esterification. Lipase SP435 from Candida antarctica showed the highest activity for esterification. Effects of reaction parameters, namely temperature, time course and mole ratio of glycerol to fatty acid were followed with all three lipases. The optimal reaction time was 24 hr at 30 °C at a mole ratio of glycerol to fatty acid of 14:1. The maximum degree of acylglycerol synthesis was > 90%. The effect of time course and mole ratio of glycerol to fatty acid on acylglycerols distribution was also determined. The oxidative stability of different samples under Schaal-oven conditions at 60 °C showed that the oxidative stability of acylglycerols was better than that of the corresponding fatty acid esters.

Allergic reactions to lower concentrations of nickel sulfate and formaldehyde often appear later than reactions to higher concentrations.

A late-appearing patch test reaction may be a sign of active sensitization or represent a delayed elicitation reaction. To retrospectively study the effect of concentration on the time course of allergic reactions to routine concentration dilution series of formaldehyde and nickel sulfate. We tested concentration dilution series of 2%, 1%, 0.32% and 0.1% formaldehyde and 5%, 1.6%, 0.5% and 0.16% nickel sulfate, respectively. The last readings were performed on day 4 to day 6. We included patients with allergic reactions to either of the two lowest concentrations in each dilution series and whose tests had been read three times. Forty-two nickel-allergic and 23 formaldehyde-allergic patients fulfilled the inclusion criteria. In 26 (62%) of the nickel-sensitive patients, reactions to lower concentrations appeared later than reactions to the highest concentration. Of the formaldehyde-sensitive patients, 17 (74%) developed one or two allergic reactions to lower concentrations later than reactions to the highest concentration, and one (4%) patient developed allergic reactions to lower concentrations sooner than a reaction to the highest concentration. The remaining patients showed all allergic reactions at the same reading. In these selected, relatively strongly sensitized patients, allergic reactions to lower concentrations quite regularly appeared later than reactions to higher concentrations.

Rapid characterization of the activities of lignin-modifying enzymes based on nanostructure-initiator mass spectrometry (NIMS)

BackgroundProducing valuable fuels and chemicals from lignin is a key factor for making lignocellulosic biomass economically feasible; however, significant roadblocks exist due to our lack of detailed understanding of how lignin is enzymatically depolymerized and of the range of possible lignin fragments that can be produced. Development of suitable enzymatic assays for characterization of putative lignin active enzymes is an important step towards improving our understanding of the catalytic activities of relevant enzymes. Previously, we have successfully built an assay platform based on glycan substrates containing a charged perfluorinated tag and nanostructure-initiator mass spectrometry to study carbohydrate active enzymes, especially various glycosyl hydrolyses. Here, we extend this approach to develop a reliable and rapid assay to study lignin-modifying enzymes.ResultsTwo β-aryl ether bond containing model lignin dimer substrates, designed to be suitable for studying the activities of lignin-modifying enzymes (LMEs) by nanostructure-initiator mass spectrometry (NIMS), were successful synthesized. Small-angle neutron scattering experiments showed that these substrates form micelles in solution. Two LMEs, laccase from the polypore mushroom Trametes versicolor, and manganese peroxidase (MnP) from white rot fungus Nematoloma frowardii, were tested for catalytic activity against the two model substrates. We show that the reaction of laccase and MnP with phenolic substrate yields products that arise from the cleavage of the carbon–carbon single bond between the α-carbon and the adjacent aryl carbon, consistent with the mechanism for producing phenoxy radical as reaction intermediates. Reactions of the nonphenolic substrate with laccase, on the other hand, adopt a different pathway by producing an α-oxidation product; as well as the cleavage of the β-aryl ether bond. No cleavage of the carbon–carbon bond between the α-carbon and the aryl carbon was observed. To facilitate understanding of reaction kinetics, the reaction time course for laccase activity on the phenolic substrate (I) was generated by the simultaneous measurement of all products at different time points of the reaction. Withdrawal of only a small sample aliquot (0.2 μL at each time point) ensured minimum perturbation of the reaction. The time course can help us to understand the enzyme kinetics.ConclusionsA new assay procedure has been developed for studying lignin-modifying enzymes by nanostructure-initiator mass spectrometry. Enzyme assays of a laccase and a MnP on phenolic and nonphenolic β-aryl ether substrates revealed different primary reaction pathways due to the availability of the phenoxy radical intermediates. Our assay provides a wealth of information on bond cleavage events not available using conventional colorimetric assays and can easily be carried out in microliter volumes and the quantitative analysis of product formation and kinetics is rapidly achieved by NIMS. This is the first time that NIMS technology was applied to study the activities of lignin-modifying enzymes. Unlike other previous works, our use of amphiphilic guaiacylglycerol β-O-4 substrate (I) enables the formation of micelles. This approach helps avoid the re-polymerization of the resulting monomeric product. As a result, our assay can clearly demonstrate the degradation pathways of phenolic guaiacylglycerol β-O-4 type of molecules with laccase and MnP.

In Vivo Use of 1D and 2D 1H NMR to Examine the Glycosylation of Scopoletin in Duboisia myoporoides Cell Suspensions

Cell suspensions initiated from Duboisia myoporoides-a shrub belonging to the Solanaceae family and being a rich source of tropane alkaloids-previously showed their ability to glycosylate scopoletin into scopolin, which represent coumarins showing health benefits. To investigate the time course of this glycosylation reaction, an in vivo NMR approach was developed using a perfusion system in an 8-mm NMR tube and 1H NMR with 1D and 2D (TOCSY and NOESY) experiments. The time course of metabolic changes could therefore be followed without any labeling.