Coupled Enzyme System Research Articles

Coupling of NAD(P)H:FMN-oxidoreductase and luciferase from luminous bacteria in a viscous medium: Finding the weakest link in the chain.

The study aims at revealing the mechanisms of the viscous medium effects on the kinetic features of NAD(P)H:FMN-oxidoreductase from luminous bacteria (Red), which are exhibited in a single enzyme assay and in coupling with bacterial luciferase (BLuc). Different concentrations of glycerol and sucrose were used to vary the medium viscosity. The activity of Red, alone and in the presence of BLuc, was analyzed, as well as BLuc activity in the presence of Red, whereas in the absence of BLuc, the Red activity was suppressed in viscous medium, and in the presence of BLuc, the increase in Red activity was observed at low glycerol concentrations (5-20 wt%). The interaction of glycerol and sucrose with Red substrates FMN and NADH was studied using absorption spectroscopy and molecular dynamics. Glycerol was found to form hydrogen bonds with the phosphate groups of the substrates, unlike sucrose. A mechanism for the activation of Red in the presence of BLuc in glycerol solutions through the acceleration of FMN reoxidation was proposed. Thus, it was concluded that, under the conditions used, the weakest link of the coupled enzyme system BLuc-Red in viscous medium is the FMN concentration, which depends on Red activity and the medium viscosity.

In situ H2 O2 Generation by Choline Oxidase and Its Application in Amino Polysaccharide Degradation by Coupling to Lytic Polysaccharide Monooxygenase.

Chitin, the most abundant amino polysaccharide in Nature, has many applications in different fields. However, processing of this recalcitrant biopolymer in an environmentally friendly manner remains a major challenge. In this context, lytic polysaccharide monooxygenases (LPMOs) are of interest, as they can act on the most recalcitrant parts of chitin and related insoluble biopolymers such as cellulose. Efficient LPMO catalysis can be achieved by feeding reactions with H2 O2 , but careful control of H2 O2 is required to avoid autocatalytic enzyme inactivation. Herein, we present a coupled enzyme system in which a choline oxidase from Arthrobacter globiformis is employed for controlled in situ generation of H2 O2 that fuels LPMO-catalyzed oxidative degradation of chitin. We show that the rate, stability and extent of the LPMO reaction can be manipulated by varying the amount of choline oxidase and/or its substrate, choline chloride, and that efficient peroxygenase reactions may be achieved using sub-μM concentrations of the H2 O2 -generating enzyme. This coupled system requires only sub-stoichiometric amounts of the reductant that is needed to keep the LPMO in its active, reduced state. It is conceivable that this enzyme system may be used for bioprocessing of chitin in choline-based natural deep eutectic solvents.

Co-Immobilization of RizA Variants with Acetate Kinase for the Production of Bioactive Arginyl Dipeptides.

The biocatalytic system comprised of RizA and acetate kinase (AckA) combines the specific synthesis of bioactive arginyl dipeptides with efficient ATP regeneration. Immobilization of this coupled enzyme system was performed and characterized in terms of activity, specificity and reusability of the immobilisates. Co-immobilization of RizA and AckA into a single immobilisate conferred no disadvantage in comparison to immobilization of only RizA, and a small addition of AckA (20:1) was sufficient for ATP regeneration. New variants of RizA were constructed by combining mutations to yield variants with increased biocatalytic activity and specificity. A selection of RizA variants were co-immobilized with AckA and used for the production of the salt-taste enhancers Arg-Ser and Arg-Ala and the antihypertensive Arg-Phe. The best variants yielded final dipeptide concentrations of 11.3 mM Arg-Ser (T81F_A158S) and 11.8 mM Arg-Phe (K83F_S156A), the latter of which represents a five-fold increase in comparison to the wild-type enzyme. T81F_A158S retained more than 50% activity for over 96 h and K83F_S156A for over 72 h. This study provides the first example of the successful co-immobilization of an l-amino acid ligase with an ATP-regenerating enzyme and paves the way towards a bioprocess for the production of bioactive dipeptides.

Bioluminescent-Inhibition-Based Biosensor for Full-Profile Soil Contamination Assessment.

A bioluminescent-enzyme-inhibition-based assay was applied to predict the potential toxicity of the full profile of the following soil samples: agricultural grassland, 10-year fallow land (treated with remediation processes for 10 years) and uncontaminated (virgin) land. This assay specifically detects the influence of aqueous soil extracts from soils on the activity of a coupled enzyme system of luminescent bacteria: NAD(P)H:FMN-oxidoreductase + luciferase (Red + Luc). It was shown that the inhibitory effect of the full-profile soil samples on the Red + Luc system decreased with depth for the 10-year fallow-land and virgin-land samples, which correlated with a decrease in the humic organic matter content in the soils. The inhibitory effect of the agricultural grassland on the Red + Luc enzyme system activity was more complex and involved the presence of the humic organic matter content, as well as the presence of pollutants in the whole-soil profile. However, if the interfering effect of humic organic substances on the Red + Luc system’s activity is taken into account during full-profile soil toxicity assessments, it might help to detect pollutant mobility and its leaching into the subsoil layer. Thus, this bioluminescent method, due to the technical simplicity, rapid response time and high sensitivity, has the potential to be developed as a biological part of the inhibition-based assay and/or biosensors for the preventive tracing of potential full-profile soil contamination.

Tenofovir‐Diphosphate Formation by Creatine Kinase Brain‐Type is Diminished by Naturally Occurring Mutations In Vitro

Tenofovir (TFV) is a nucleotide reverse transcriptase inhibitor that is administered as a prodrug for use in both HIV treatment and prevention. TFV requires two sequential phosphorylation steps to form the pharmacologically active metabolite, tenofovir‐diphosphate (TFV‐DP). Previous studies have shown that creatine kinase muscle‐type (CKM) can catalyze the final phosphorylation step in colon tissue. Thus, to discover other TFV activating enzymes, we used CKM as a starting point and identified creatine kinase brain‐type (CKB) as a candidate TFV activating enzyme, as it is 80% homologous to CKM. To test the hypothesis that CKB can phosphorylate TFV in a manner similar to CKM, we performed in vitro activity assays by incubating recombinantly expressed CKB or CKM with reaction substrates, phosphocreatine and tenofovir‐monophosphate. TFV metabolites were detected using ultra‐high performance liquid chromatography tandem mass spectrometry (uHPLC/MS). Analysis of TFV‐DP levels revealed no significant difference between CKB and CKM catalyzed reactions. These data indicate that CKB may contribute to TFV activation in tissues where it is expressed, thus genetic variation in CKB may play a role in inter‐individual variability observed in TFV efficacy. To investigate the potential impact of genetic variation, fifteen naturally occurring missense mutations were chosen and mutant CKB enzymes were recombinantly expressed in E. coli. The purified enzymes were used in our in vitro activity assay and TFV metabolites were subsequently detected by uHPLC/MS. Eight mutations (C74S, R96P, S128R, R132H, R172P, R236Q, R292Q, and H296R) demonstrated a statistically significant reduction in the formation of TFV‐DP compared to that of wild‐type CKB. To gain insight into how these mutations disrupt enzymatic function, we exploited the reverse canonical reaction (ATP dephosphorylation) in an enzyme coupled system, from which Michaelis‐Menten curves were constructed and resulting kinetic parameters were used to calculate catalytic efficiencies. Five mutations (R96P, R132H, R236Q, R292Q, and H296R) resulted in catalytic efficiencies less than 20% of wild‐type, which can be attributed to increases in Km. Lastly, thermal stability was examined using differential scanning fluorimetry and showed four mutations (C74S, R96P, R172P, and H296P) had significantly lower melting temperatures than wild‐type. Additionally, seven mutations (C74S, R96P, R132H, R172G, R172P, R236Q, and H296P) displayed melting curves with double peaks or shouldering, suggesting local domain melting or dimer instability induced by the mutation. Together these data support our hypothesis that CKB contributes to TFV activation and indicates that naturally occurring mutations can diminish TFV‐DP formation, catalytic efficiency, and thermal stability in vitro.

Synthesis of Linoleic Acid 13-Hydroperoxides from Safflower Oil Utilizing Lipoxygenase in a Coupled Enzyme System with In-Situ Oxygen Generation

Linoleic acid hydroperoxides are versatile intermediates for the production of green note aroma compounds and bifunctional ω-oxo-acids. An enzyme cascade consisting of lipoxygenase, lipase and catalase was developed for one-pot synthesis of 13-hydroperoxyoctadecadienoic acid starting from safflower oil. Reaction conditions were optimized for hydroperoxidation using lipoxygenase 1 from Glycine max (LOX-1) in a solvent-free system. The addition of green surfactant Triton CG-110 improved the reaction more than two-fold and yields of >50% were obtained at linoleic acid concentrations up to 100 mM. To combine hydroperoxidation and oil hydrolysis, 12 lipases were screened for safflower oil hydrolysis under the reaction conditions optimized for LOX-1. Lipases from Candida rugosa and Pseudomonas fluorescens were able to hydrolyze safflower oil to >75% within 5 h at a pH of 8.0. In contrast to C. rugosa lipase, the enzyme from P. fluorescens did not exhibit a lag phase. Combination of P. fluorescens lipase and LOX-1 worked well upon LOX-1 dosage and a synergistic effect was observed leading to >80% of hydroperoxides. Catalase from Micrococcus lysodeikticus was used for in-situ oxygen production with continuous H2O2 dosage in the LOX-1/lipase reaction system. Foam generation was significantly reduced in the 3-enzyme cascade in comparison to the aerated reaction system. Safflower oil concentration was increased up to 300 mM linoleic acid equivalent and 13-hydroperoxides could be produced in a yield of 70 g/L and a regioselectivity of 90% within 7 h.

Design of bioluminescent biosensors for assessing contamination of complex matrices

The presence of potentially toxic xenobiotics in complex matrices has become rather the rule than the exception. Therefore, there is a need for highly sensitive inexpensive techniques for analyzing environmental and food matrices for toxicants. Enzymes are selectively sensitive to various toxic compounds, and, thus, they can be used as the basis for detection of contaminants in complex matrices. There are, however, a number of difficulties associated with the analysis of complex matrices using enzyme assays, including the necessity to take into account properties and effects of the natural components of the test media for accurate interpretation of results. The present study describes the six-stage procedure for designing new enzyme sensors intended for assessing the quality of complex matrices. This procedure should be followed both to achieve the highest possible sensitivity of the biosensor to potentially toxic substances and to minimize the effect of the uncontaminated components of complex mixtures on the activity of the biosensor. The proposed strategy has been tested in designing a bioluminescent biosensor for integrated rapid assessment of the safety of fruits and vegetables. The biosensor is based on the coupled enzyme system NAD(P)H:FMN-oxidoreductase and luciferase as the biorecognition element. The study describes methods and techniques for attaining the desired result in each stage. The proposed six-stage procedure for designing bioluminescent enzyme biosensors can be used to design the enzymatic biosensors based on other enzymes.

Conversion of Racemic Unnatural Amino Acids to Optically Pure Forms by a Coupled Enzymatic Reaction.

Genetic code expansion (GCE) technology is a useful tool for the site-specific modification of proteins. An unnatural amino acid (UAA) is one of the essential components of this technique, typically required at high concentration (1 mM or higher) in growth medium. The supply of UAAs is an important limitation to the application of GCE technology, as many UAAs are either expansive or commercially unavailable. In this study, two UAAs in a racemic mixture were converted into optically pure forms using two enzymes, the d-amino acid oxidase (RgDAAO) from Rhodotorula gracilis and the aminotransferase (TtAT) from Thermus thermophilus. In the coupled enzyme system, RgDAAO oxidizes the d-form of UAAs in a stereospecific manner and produces the corresponding α-keto acids, which are then converted into the l-form of UAAs by TtAT, resulting in the quantitative and stereospecific conversion of racemic UAAs to optically pure forms. The genetic incorporation of the optically pure UAAs into a target protein produced a better protein yield than the same experiments using the racemic mixtures of the UAAs. This method could not only be used for the preparation of optically pure UAAs from racemic mixtures, but also the broad substrate specificity of both enzymes would allow for its expansion to structurally diverse UAAs.

Simultaneous directed evolution of coupled enzymes for efficient asymmetric synthesis of l-phosphinothricin.

The traditional strategy to improve the efficiency of an entire coupled enzyme system relies on separate direction of the evolution of enzymes involved in their respective enzymatic reactions. This strategy can lead to enhanced single-enzyme catalytic efficiency but may also lead to loss of coordination among enzymes. This study aimed to overcome such shortcomings by executing a directed evolution strategy on multiple enzymes in one combined group that catalyzes the asymmetric biosynthesis of l-phosphinothricin. The genes of a glutamate dehydrogenase from Pseudomonas moorei (PmGluDH) and a glucose dehydrogenase from Exiguobacterium sibiricum (EsGDH), along with other gene parts (promoters, ribosomal binding sites (RBSs), and terminators) were simultaneously evolved. The catalytic efficiency of PmGluDH was boosted by introducing the beneficial mutation A164G (from 1.29 s-1mM-1 to 183.52 s-1mM-1), and the EsGDH expression level was improved by optimizing the linker length between the RBS and the start codon of gdh. The total turnover numbers of the bioreaction increased from 115 (GluDH WTNADPH) to 5846 (A164GNADPH coupled with low expression of EsGDH), and to 33950 (A164GNADPH coupled with high expression of EsGDH). The coupling efficiency was increased from ∼30% (GluDH_WT with low expression of GDH) to 83.3% (GluDH_A164G with high expression of GDH). In the batch production of l-phosphinothricin utilizing whole-cell catalysis, the strongest biocatalytic reaction exhibited a high space-time yield (6410 g·L-1·d-1) with strict stereoselectivity (>99% enantiomeric excess).Importance: The traditional strategy to improve multienzyme-catalyzed reaction efficiency may lead to enhanced single-enzyme catalytic efficiency but may also result in loss of coordination among enzymes. We describe a directed evolution strategy of an entire coupled enzyme system to simultaneously enhance enzyme coordination and catalytic efficiency. The simultaneous evolution strategy was applied to a multienzyme-catalyzed reaction for the asymmetric synthesis of l-phosphinothricin, which not only enhanced the catalytic efficiency of GluDH but also improved the coordination between GluDH and GDH. Since this strategy is enzyme-independent, it may be applicable to other coupled enzyme systems for chiral chemical synthesis.

Enzymatic Responses to Low-Intensity Radiation of Tritium.

The present study considers a possible role of enzymatic reactions in the adaptive response of cells to the beta-emitting radionuclide tritium under conditions of low-dose exposures. Effects of tritiated water (HTO) on the reactions of bacterial luciferase and NAD(P)H:FMN-oxidoreductase, as well as a coupled system of these two reactions, were studied at radioactivity concentrations ≤ 200 MBq/L. Additionally, one of the simplest enzymatic reactions, photobiochemical proton transfer in Coelenteramide-containing Fluorescent Protein (CLM-FP), was also investigated. We found that HTO increased the activity of NAD(P)H:FMN-oxidoreductase at the initial stage of its reaction (by up to 230%); however, a rise of luciferase activity was moderate (<20%). The CLM-FP samples did not show any increase in the rate of the photobiochemical proton transfer under the exposure to HTO. The responses of the enzyme systems were compared to the ‘hormetic’ response of luminous marine bacterial cells studied earlier. We conclude that (1) the oxidoreductase reaction contributes significantly to the activation of the coupled enzyme system and bacterial cells by tritium, and (2) an increase in the organization level of biological systems promotes the hormesis phenomenon.

A noninvasive and qualitative bioluminescent assay for express diagnostics of athletes' responses to physical exertion.

Upcoming professional sports authorities seek rapid noninvasive biosensing tools for regular monitoring of athletes' physiological states. The analysis of saliva through luminescence-based biosensors has been perceived as a suitable candidate for such purposes. The present study reports a qualitative bioluminescence assay based on a coupled enzyme system that consists of bacterial luciferase (BLuc) and nicotinamide adenine dinucleotide (NADH):flavin mononucleotide (FMN) oxidoreductase (Red), BLuc-Red, for the express diagnostics of athletes' stress levels before and after physical exertion. The volunteers who participated in the study were grouped as freestyle wrestlers and students who adapted to different levels of physical activities. Under physical exertion modelling conditions, the influence of participant saliva on BLuc-Red catalyzed light emission was investigated. Results showed a significant increase in residual luminescence (Iexp , mean maximum bioluminescence intensity of the experimental measurement (Iexp ); Ic , luminescence intensity in control; Iexp /Ic , %) values for participants in the wrestler group while a decrease in the student group (P < 0.05). Such contrasting residual luminescence values in both groups were found to be dependent on the catalase activity of saliva. The proposed bioluminescence assay can be utilized as a potential nonspecific biosensing tool for determining the physical state of athletes under high loads.

Thermostable arginase from Sulfobacillus acidophilus with neutral pH optimum applied for high-efficiency L-ornithine production.

This study aims to use neutral pH optimum arginase as the catalyst for high-efficiency L-ornithine production. Sulfobacillus acidophilus arginase was firstly cloned and overexpressed in Escherichia coli. The purified enzyme was obtained, and the molecular mass determination showed that this arginase was a hexamer. S. acidophilus arginase possessed similarities with the other arginases such as the conserved sequences, purification behavior, and the necessity for Mn2+ as a cofactor. The maximum enzyme activity was obtained at pH7.5 and 70°C. Thermostability and pH stability analysis showed that the arginase was stable at 30-60°C and pH7.0-8.5, respectively. The kinetic parameters suggested that S. acidophilus arginase could efficiently hydrolyze L-arginine. Bioconversion with this neutral pH optimum arginase had the advantages of avoiding producing by-product, high molar yield, and high-level production of L-ornithine. When the bioconversion was performed with a fed-batch strategy and a coupled-enzyme system involving S. acidophilus arginase and Jack bean urease, the final production of 2.87mol/L was obtained with only 1.72mmol/L L-arginine residue, and the molar yield was 99.9%. The highest production record suggests that S. acidophilus arginase has a great prospect in industrial L-ornithine production.

PH-Neutralization, Redox-Balanced Process with Coupled Formate Dehydrogenase and Glucose Dehydrogenase Supports Efficient Xylitol Production in Pure Water.

Enzymatic production of xylitol is a promising alternative to the chemical hydrogenation process. However, it encounters problems that are largely due to protein susceptibility to environmental factors. In this study, to develop a robust, practical enzymatic process for xylitol production, a coupled enzyme system consisting of formate dehydrogenase (FDH), glucose dehydrogenase (GDH), and xylose reductase (XR) was constructed, wherein the alkaline product produced by FDH and the acidic product produced by GDH could neutralize each other during cofactor regeneration. After optimization of conditions, a pH-neutralization, redox-balanced process was developed that could be carried out in pure water requiring no pH regulation. As a result, a xylitol production of 273.6 g/L that is much higher than those yet reported was obtained from 2 M xylose in 24 h, with a relatively high productivity of 11.4 g/(L h). The strategy demonstrated here can be adapted for the production of other NADH-consuming products.

One-Pot Multienzymatic Transformation of NH3, CO2, and Ornithine into the Organic Nitrogen Plant Fertilizer Citrulline Using a Single Recombinant Lysate of E. coli

Biocatalytic transformation of carbamate formed readily from CO2 and NH3 provides attractive green routes for mitigation of these important environmental pollutants. Accordingly, a coupled-enzyme system was developed for the one-pot production of citrulline through carbamoylation of ornithine in aqueous solutions of CO2 and NH3. Hyperthermophilic ornithine carbamoyltransferases are produced recombinantly in E. coli with carbamate kinases known to have a propensity for carbamoyl phosphate synthesis. Importantly, in vitro biocatalysis is carried out by E. coli cell lysate prepared through coexpression of the required recombinant enzymes in a single bacterial culture, greatly reducing limitations normally associated with protein production and purification. Acetate kinase that is endogenous in the lysate also recycles the required ATP cofactor, which would otherwise have been required in costly stoichiometric amounts. Recombinant lysates catalyze the production of carbamoyl phosphate with substoichiometric A...

In Vitro Assays to Monitor the Enzymatic Activities of zDHHC Protein Acyltransferases.

A family of zDHHC protein acyltransferase (PAT) enzymes catalyze the S-palmitoylation of target proteins via a two-step mechanism. The first step involves transfer of palmitate from the palmitoyl-CoA donor to the active site cysteine of the zDHHC PAT enzyme, releasing reduced CoA (CoASH). In the second step, the palmitoyl-PAT intermediate thioester reacts with a cysteine side chain within the target substrate to produce the palmitoylated substrate product or, in the absence of a protein substrate, the palmitoyl-PAT intermediate thioester is hydrolyzed and releases palmitate. Formation and resolution of the palmitoyl-PAT intermediate complex (autopalmitoylation) is measured using a coupled enzyme system that monitors the production of CoASH via reduction of NAD+ by the α-ketoglutarate dehydrogenase complex. This assay can be used to isolate and characterize modulators of autopalmitoylation and is scalable to high-throughput screening (HTS). A second fluorescence-based assay is described that monitors the hydrolysis of the palmitoyl-PAT thioester linked intermediate by thin-layer chromatography using a palmitoyl-CoA analog, BODIPY®-C12:0-CoA, as a substrate. These two assays provide a methodology to quantify the first enzymatic step of the two-step zDHHC PAT reaction.

Systematically redesigning and optimizing the expression of D-lactate dehydrogenase efficiently produces high-optical-purity D-lactic acid in Saccharomyces cerevisiae

D-lactic acid (D-LA) is gaining increased attention as it can improve the thermostability of poly lactic acid. Acid-tolerant Saccharomyces cerevisiae is a good host for D-LA fermentation. High catalytic efficiency of D-lactate dehydrogenase (D-LDH, EC 1.1.1.28) is crucial for the production of D-LA in yeast. Here, a synthetic biology approach was used to construct high-producing D-LA strains by redesigning and optimization of D-LDH expression by a combination of different promoters, terminators and D-LDHs. The pyruvate decarboxylase-deficient mutant strain TAMH was used as host strain for optimizing the 40 D-LDH expression cassettes. The TCSt strain harboring the pTCSt plasmid with the TEF1 promoter, E. coliD-LDH and Synth25 synthetic short terminator produced 5.8 g/L D-LA with an optical purity of 99.9%. The production of D-LA was further improved by integrating this high expression cassette into the Ty1 transposable element of the YIP-01 strain with deleted Pdc1and Pdc6. The resulting strain YIP-pTCSt-301(CGMCC2.5726) was screened by a double enzyme-coupled system. Genomes sequencing of the strain revealed three copies of the D-LDH expression cassette. This strain was further improved by deleting the Jen1, Cyb2, Dld1, and Adh1 genes and the resulting strain YIP-J-C-D-A1 (CGMCC2.5783) produced 80.0 g/L D-LA with a yield of 0.6 g/g glucose and a volumetric productivity of 1.1 g/L/h in fed-batch fermentation under non-neutralization conditions.

Expression of genes encoding the luciferase from Photobacterium leiognathi in Escherichia coli Rosetta (DE3) and its application in NADH detection.

Cloning of genes encoding the luciferase from Photobacterium leiognathi YL in Escherichia coli Rosetta (DE3) was performed successfully and the expressed forms of lux AB were purified to homogeneity. Experimental measurements revealed that luciferase from Photobacterium leiognathi YL has good thermal stability and a high residual activity at extreme pH values, which are extremely important for its various ecological, industrial and medical applications. Furthermore, we made a first attempt for quantitative detection of NADH by recombinant E. coli Rosetta (DE3) coupled enzyme system. A good linear relationship between luminescence intensity and NADH with low (1-12nmol/L) and high (10-500nmol/L) concentration was observed, whose standard curve was y=772.97×+4041.1, R2 =0.9884 and y=1710×+4.99×105 , R2 =0.9727, respectively. Our results demonstrate a high sensitivity of recombinant E. coli coupled enzyme system to NADH on the basis of high soluble expression of recombinant luciferase and continuous and stable expression of some NAD(P)H-dependent flavin mononucleotide (FMN) reductases.

Graphene Quantum Dots and Enzyme-Coupled Biosensor for Highly Sensitive Determination of Hydrogen Peroxide and Glucose.

In this paper, a simple and specific graphene quantum dots (GQDs)-based fluorescent biosensor adopted for the determination of glucose based on the combination of the enzyme-coupled method and fluorescence quenching mechanism is demonstrated. Glucose was oxidized by the enzyme glucose oxidase (GOx), forming hydrogen peroxide (HO) via the catalysis by horseradish peroxidase (HRP). HO was then employed to oxidize phenol to quinone, which led to effective quenching effect in the GQDs–GOx–HRP–phenol system. By optimizing the reaction conditions of the GQDs-enzyme system, a linear relationship between the concentration of glucose and the fluorescence intensity over a range of 0.2–10 mol/L was obtained. The limit of detection for glucose is 0.08 mol/L. The present biosensor for the determination of glucose showed satisfactory reproducibility and accuracy in human serum samples. Since the enzymes have high specificity and unique affinity to the certain substance, the enzyme-coupled system promises a sensitive way for further detection of those chemicals which could be oxidized by enzymes and generated HO or glucose. GQDs and other fluorescent materials coupled with several enzymes can be applied to extensive sensing field.

Identifying the Oscillatory Mechanism of the Glucose Oxidase-Catalase Coupled Enzyme System.

We provide experimental evidence of periodic and aperiodic oscillations in an enzymatic system of glucose oxidase-catalase in a continuous-flow stirred reactor coupled by a membrane with a continuous-flow reservoir supplied with hydrogen peroxide. To describe such dynamics, we formulate a detailed mechanism based on partial results in the literature. Finally, we introduce a novel method for estimation of unknown kinetic parameters. The method is based on matching experimental data at an oscillatory instability with stoichiometric constraints of the mechanism formulated by applying the stability theory of reaction networks. This approach has been used to estimate rate coefficients in the catalase part of the mechanism. Remarkably, model simulations show good agreement with the observed oscillatory dynamics, including apparently chaotic intermittent behavior. Our method can be applied to any reaction system with an experimentally observable dynamical instability.

Bioluminescent enzyme inhibition-based assay to predict the potential toxicity of carbon nanomaterials

A bioluminescent enzyme inhibition-based assay was applied to predict the potential toxicity of carbon nanomaterials (CNM) presented by single- and multi-walled nanotubes (SWCNT and MWCNT) and aqueous solutions of hydrated fullerene С60 (C60HyFn). This assay specifically detects the influence of substances on parameters of the soluble or immobilised coupled enzyme system of luminescent bacteria: NAD(P)Н:FMN-oxidoreductase+luciferase (Red+Luc). A protocol based on the optical properties of CNM for correcting the results of the bioluminescent assay was also developed. It was shown that the inhibitory activity of CNM on Red+Luc decreased in the following order: MWCNT>SWCNT>C60HyFn. The soluble enzyme system Red+Luc had high sensitivity to MWCNT and SWCNT, with values of the inhibition parameter IC50 equal to 0.012 and 0.16mg/L, respectively. The immobilised enzyme system was more vulnerable to C60HyFn than its soluble form, with an IC50 equal to 1.4mg/L. Due to its technical simplicity, rapid response time and high sensitivity, this bioluminescent method has the potential to be developed as a general enzyme inhibition-based assay for a wide variety of nanomaterials.