Apparent Michaelis Constant Research Articles

Identification and characterization of an Endo-glucanase secreted from cellulolytic Escherichia coli ZH-4

BackgroundIn the previous study, the cellulolytic Escherichia coli ZH-4 isolated from bovine rumen was found to show extracellular cellulase activity and could degrade cellulose in the culture. The goal of this work was to identify and characterize the secreted cellulase of E. coli ZH-4. It will be helpful to re-understand E. coli and extend its application in industry.ResultsA secreted cellulase was confirmed to be endo-glucanase BcsZ which was encoded by bcsZ gene and located in the cellulose synthase operon bcsABZC in cellulolytic E. coli ZH-4 by western blotting. Characterization of BcsZ indicated that a broad range of pH and temperature tolerance with optima at pH 6.0 and 50 °C, respectively. The apparent Michaelis–Menten constant (Km) and maximal reaction rate (Vmax) for BcsZ were 8.86 mg/mL and 0.3 μM/min·mg, respectively. Enzyme activity of BcsZ was enhanced by Mg2+ and inhibited by Zn2+, Cu2+ and Fe3+. BcsZ could hydrolyze carboxymethylcellulose (CMC) to produce cello-oligosaccharides, cellotriose, cellobiose and glucose.ConclusionsIt is confirmed that extracellular cellulolytic capability of E. coli ZH-4 was attributed to BcsZ, which explained why E. coli ZH-4 can grow on cellulose. The endo-glucanase BcsZ from E. coli-ZH4 has some new characteristics which will extend the understanding of endo-glucanase. Analysis of the secretion characteristics of BcsZ provided a great reference for applying E. coli in multiple industrial fields.

Meso-Cellular Silicate Foam-Modified Reduced Graphene Oxide with a Sandwich Structure for Enzymatic Immobilization and Bioelectrocatalysis.

An integrated composite of meso-cellular silicate foam (MCF)-modified reduced graphene oxide (MCF@rGO) was designed and synthesized based on polyethylene oxide-polypropylene oxide-polyethylene oxide (P123)-modified rGO (P123-rGO). As the polymeric template for the fabrication of mesoporous silicates, modified P123 greatly improved the affinity between the nanosheet and the in situ formed MCFs, resulting in the formation of thin layers of MCFs on both sides of rGO. Therefore, the MCFs@rGO formed exhibited a unique sandwich structure with an inner skeleton of rGO and two outer layers of MCFs. The outer modification by MCFs, with the presence of large mesopores, not only shifted the surface property of rGO from hydrophobic to hydrophilic but also offered immobilized enzymes a favorable microenvironment to maintain their bioactivity. Meanwhile, the inner skeleton of rGO compensated for the weak conductivity of MCFs, providing a pathway for the direct electron transfer (DET) of various redox enzymes or proteins, such as hemoglobin (Hb), horseradish peroxidase, glucose oxidase (GOD), and cholesterol oxidase. It was found that the DET signal obtained from Hb-MCFs@rGO/glassy carbon electrode (GCE) was much larger than the sum of the signals from two components-based modified electrodes of Hb-P123-rGO/GCE and Hb-MCFs/GCE. A similar improvement in DET signal was also observed using GOD-MCFs@rGO/GCE. The significant enhancement of DET signals for both protein electrodes can be ascribed to the synergistic effects generated from the integration of the two components, one of which enhances biocompatibility and the other enhances conductivity. The bioelectrocatalytic performance of Hb and GOD electrodes was further investigated. As for Hb-MCFs@rGO/GCE, the GOD electrode displayed excellent analytical performance for the detection of hydrogen peroxide (H2O2), including a good sensitivity of 0.25 μA μmol-1 L cm-2, a low detection limit of 63.6 nmol L-1 based on S/N = 3, and a low apparent Michaelis-Menten constant (KMapp) of 49.05 μmol L-1. GOD-MCFs@rGO/GCE also exhibited good analytical performance for the detection of glucose, with a wide linear range of 0.25-8.0 mmol L-1. In addition, blood glucose detection in samples of human serum was successfully achieved using GOD-MCFs@rGO/GCE with a low quantification limit.

Layer-by-layer self-assembly film of PEI-reduced graphene oxide composites and cholesterol oxidase for ultrasensitive cholesterol biosensing

A novel electrochemical platform composed of water soluble polyethylene imine-reduced graphene oxide (PEI-rGO) composites and cholesterol oxidase (ChOx) based on layer-by-layer (LBL) technique has been triumphantly developed. The PEI-rGO composites could effectively improve the electron transfer ability between the assembled enzyme and the surface of electrode. Furthermore, LBL technique could immobilize more enzyme under mild conditions, which was benefit to keep the bioactivity of redox enzyme. TEM, UV–vis and FT-IR showed that the PEI-rGO composites behaved as individual nanosheet structure and were successfully prepared. The self-assembly process of (ChOx/PEI-rGO)n film was monitored by UV–vis spectroscopy, demonstrating that the ChOx had been immobilized orderly without denaturation. Owing to the good conductivity of the PEI-rGO composites, the electron transfer ability of the immobilized ChOx was greatly improved on glassy carbon electrode (GCE). The PEI/(ChOx/PEI-rGO)3/GCE showed excellent electrochemical performances toward the detection of cholesterol. Under optimized conditions, the fabricated electrode exhibited a wide linear range in the cholesterol concentration from 0.10 × 10−6 to 9.331 × 10−3 mol L−1 with a detection limit of 0.021 μmol L−1 estimated at a signal-to-noise ratio of 3. The apparent Michaelis-Menten constant (Km) was 0.0431 mmol L−1. This result indicated that a good affinity between the immobilized ChOx and the cholesterol. Meanwhile, the biosensor presented good stability, reproducibility and anti-interference ability with good recoveries for real sample detection.

Effect of operative variables and kinetic study of butyl butyrate synthesis by Candida rugosa lipase activated by chitosan-reinforced nanocellulose derived from raw oil palm leaves

Currently, the chemically-assisted esterification to manufacture butyl butyrate employs corrosive homogeneous acid catalyst and liberates enormous quantities of hazardous by-products which complicate downstream treatment processes. This study aimed to identify the optimized esterification conditions, and the kinetic aspects of the enzyme-assisted synthesis of butyl butyrate using immobilized Candida rugosa lipase activated by chitosan-reinforced nanocellulose derived from raw oil palm leaves (CRL/CS-NC). The best process variables that gave the maximum conversion degree of butyl butyrate by CRL/CS-NC (90.2%) in just 3 h, as compared to free CRL (62.9%) are as follows: 50 °C, 1:2 M ratio of acid/alcohol, stirring rate of 200 rpm and a 3 mg/mL enzyme load. The enzymatic esterification followed the ping pong bi-bi mechanism with substrate inhibition, revealing a ˜1.1-fold higher Ki for CRL/CS-NC (55.55 mM) over free CRL (50.68 mM). This indicated that CRL/CS-NC was less inhibited by the substrates. Butanol was preferred over butyric acid as reflected by the higher apparent Michaelis-Menten constant of CRL/CS-NC for butanol (137 mM) than butyric acid (142.7 mM). Thus, the kinetics data conclusively showed that CRL/CS-NC (Vmax 0.48 mM min−1, Keff 0.07 min−1 mM−1) was catalytically more efficient than free CRL (Vmax 0.35 mM min-1, Keff 0.06 min-1 mM-1).

A hydrophobin-based-biosensor layered by an immobilized lactate dehydrogenase enzyme for electrochemical determination of pyruvate

Appropriate enzyme immobilization on the electrode surface in order to access its active site has always been an important strategy for electrode modification. In this report, lactate dehydrogenase enzyme was appropriately immobilized on the glassy carbon electrode via hydrophobin (HFB1) and graphene oxide nanocomposite. The step-by-step modification was successfully confirmed by water contact analysis, cyclic voltammetry, and electrochemical impedance spectroscopy. Under optimum conditions, this biosensor demonstrated a detection limit of 8.69 nM and RSD of 4.3% and 3.6% (n = 5) for reproducibility and repeatability. The effect of scan rate on the oxidation behavior of NADH was investigated by cyclic voltammetry; and diffusion coefficient for NADH was estimated at 6.27 × 10−8 cm2.s−1. The apparent Michaelis–Menten constant (Kmapp) was amperometrically determined and it was lower than Kmapp for the free enzyme. Also, the modified electrode represented good stability after nine days with 6% decrease in current. The proposed assay was successfully used in real sample-serum-analysis and the obtained recoveries were between 93% and 104.0%.

Novel biomaterial based on monoamine oxidase-A and multi-walled carbon nanotubes for serotonin detection

Serotonin is a potential biomarker for diverse neuropsychiatric and neurodevelopmental disorders. In this study a novel biocomposite based on the enzyme monoamine oxidase-A (MAO-A) immobilized by covalent binding on multi-walled carbon nanotubes (MWCNT) has been developed for detection of serotonin. Immobilized MAO-A (MWCNT/MAO-A) retained 94.3% of its catalytic activity, showing good thermal and pH stability at temperatures from 35 to 40 °C and pH from 7–8.5. A decrease of the apparent Michaelis-Menten constant was observed compared to free MAO-A indicating an improvement of its affinity towards its substrate. Evaluation of MWCNT/MAO-A by scanning electrochemical microscopy (SECM) demonstrated that the immobilized enzyme maintains its activity. The biocomposite was employed for successfull detection of serotonin through quantification of the enzymatically produced hydrogen peroxide (H2O2). A MWCNT/MAOA modified glassy carbon electrode (GCE) was characterized by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Electrochemical measurements were performed in simulated body fluid (SBF), obtaining a linear response of 5.67 × 10-7 M - 2.26 × 10-6 M, with a low detection limit of 2 × 10-7 M (35.6 ng mL-1;S/N = 3) and a quantification limit of 6.73 × 10-7 M (118.7 ng mL-1;S/N = 3); values that are in range with serotonin levels found in different neurological disorders. The biocomposite have the potential to be employed in future development of biosensing devices for serotonin detection.

Mediatorless, Reversible Optical Nanosensor Enabled through Enzymatic Pocket Doping

Single-walled carbon nanotubes (SWCNTs) exhibit intrinsic near-infrared fluorescence that benefits from indefinite photostability and tissue transparency, offering a promising basis for in vivo biosensing. Existing SWCNT optical sensors that rely on charge transfer for signal transduction often require exogenous mediators that compromise the stability and biocompatibility of the sensors. This study presents a reversible, mediatorless, near-infrared glucose sensor based on glucose oxidase-wrapped SWCNTs (GOx-SWCNTs). GOx-SWCNTs undergo a selective fluorescence increase in the presence of aldohexoses, with the strongest response toward glucose. When incorporated into a custom-built membrane device, the sensor demonstrates a monotonic increase in initial response rates with increasing glucose concentrations between 3 × 10−3 and 30 × 10−3 M and an apparent Michaelis–Menten constant of KM(app) ≈ 13.9 × 10−3 M. A combination of fluorescence, absorption, and Raman spectroscopy measurements suggests a fluorescence enhancement mechanism based on localized enzymatic doping of SWCNT defect sites that does not rely on added mediators. Removal of glucose reverses the doping effects, resulting in full recovery of the fluorescence intensity. The cyclic addition and removal of glucose is shown to successively enhance and recover fluorescence, demonstrating reversibility that serves as a prerequisite for continuous glucose monitoring.

Novel enzyme-free immunomagnetic microfluidic device based on Co0.25Zn0.75Fe2O4 for cancer biomarker detection

Early diagnosis of cancer by biomarker detection has been widely studied since it can lead to an increase in patient survival rates. Magnetic nanoparticles (MNPs) play an important role in this field acting as a valuable tool in the biomarker immunocapture and detection. In this work, Co0.25Zn0.75Fe2O4 (CoZnFeONPs) nanoparticles were synthesized and applied as enzyme mimics of peroxidase-like catalysis in a disposable enzyme-free microfluidic immunoarray device (μID). The catalytic activity of CoZnFeONPs was evaluated by hydrogen peroxide detection using cyclic voltammetry and the apparent Michaelis-Menten constant was estimated by Lineweaver–Burk equation showing good Km values. In μID, the immunosensors were assembled with monoclonal antibody against CYFRA 21-1 covalently immobilized on graphene oxide previously deposited on the screen-printed carbon-based electrodes. Under optimized conditions, the method presented a good linear response for CYFRA 21-1 in the range of 3.9–1000 fg mL−1 achieving an ultralow limit of detection (LOD) of 0.19 fg mL−1. For comparison, Fe3O4 nanoparticles (FeONPs) was also synthetized and presented results slight inferior to that obtained with CoZnFeONPs. The methods developed using both MNPs exhibited countless advantages when compared with the immunosensors developed for CYFRA-21-1, previously reported in the literature. The methods were successful applied for the detection of CYFRA 21-1 in real serum samples of healthy and prostate cancer patients and showed good correlation with results obtained with the enzyme-linked immunosorbent assay (ELISA). The CoZnFeONPs associated with the disposable microfluidic immunoarray device provides a simple and effective method for biomarker detection that could satisfy the need for a low-cost and rapid test for early diagnosis of cancer.

Enzyme Reactor Based on Reversible pH-Controlled Catalytic Polymer Porous Membrane.

The challenge for polymeric enzyme reactors at present is to selectively control the enzymolysis rate in complex conditions. Additionally, the fabrication methodology is hindered by complex processes, especially for achieving diverse stimuli responsiveness and functions. Here, we reported a kind of pH-sensitive polymer, poly(styrene- co-maleic anhydride-acrylic acid) (PS-MAn-AA)-based hybrid enzyme reactor. It comprised magnetic nanoparticles and a pH-sensitive PS-MAn-AA porous polymer membrane made by breath figure method. The enzyme l-asparaginase (l-ASNase) could covalently bond on the surface of the pH-sensitive porous polymer membrane (pH-PPM), and the resultant enzyme reactor was characterized by Fourier transform infrared spectroscopy and vibrating sample magnetometer. The apparent Michaelis-Menten constants ( Km and Vmax) of the l-ASNase enzyme reactor at different pH values were determined by a chiral ligand-exchange capillary electrophoresis method with l-asparagine as the substrate. The Vmax value of the l-ASNase enzyme reactor (0.67 mM/min) was almost 3-fold of that of the free l-ASNase (0.23 mM/min) at pH 8.2. Its ability to precisely control the enzymolysis rate in complex conditions is triggered primarily by the pH of the buffer solution, allowing controlled enzymatic reactions and displaying excellent stability and reusability of the proposed pH-PPM. This strategy for porous polymer membrane enzyme reactor fabrication has established a platform for enzyme efficiency adjusting. These valve-like distinguished features highlight the outstanding potential of stimuli-responsive enzyme reactor applied for enzyme immobilization and enzyme-related disease treatment.

CoO-supported ordered mesoporous carbon nanocomposite based nanozyme with peroxidase-like activity for colorimetric detection of glucose

Abstract Nano-based mimetic enzymes have been widely used for catalysis due to their large specific surface area, high stability and activity. Herein a new nanocomposite cobalt oxide (CoO) assembled onto ordered-mesoporous carbon (CoO-OMC) is reported, which shows good peroxidase-like activity upon the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) by hydrogen peroxide (H2O2). The apparent maximum velocity (Vmax) and apparent Michaelis–Menten constant (Km) were 2.66 × 10−7 M s−1 and 3.29 mM, respectively. Since the glucose oxidation and TMB oxidation can be bridged by the quantitation of H2O2, therefore, coupled with glucose oxidase, the as-prepared CoO-OMC nanocomposite was applied for the colorimetric detection of glucose in the linear range of 0.1–5.0 mM and the limit of detection of 68 μM glucose. Further, the nanozyme catalyzing platform was used to detect glucose in human serums with good accuracy and reproducibility.

Integrated Computational Model of Lung Tissue Bioenergetics.

Altered lung tissue bioenergetics plays a key role in the pathogenesis of lung diseases. A wealth of information exists regarding the bioenergetic processes in mitochondria isolated from rat lungs, cultured pulmonary endothelial cells, and intact rat lungs under physiological and pathophysiological conditions. However, the interdependence of those processes makes it difficult to quantify the impact of a change in a single or multiple process(es) on overall lung tissue bioenergetics. Integrated computational modeling provides a mechanistic and quantitative framework for the bioenergetic data at different levels of biological organization. The objective of this study was to develop and validate an integrated computational model of lung bioenergetics using existing experimental data from isolated perfused rat lungs. The model expands our recently developed computational model of the bioenergetics of mitochondria isolated from rat lungs by accounting for glucose uptake and phosphorylation, glycolysis, and the pentose phosphate pathway. For the mitochondrial region of the model, values of kinetic parameters were fixed at those estimated in our recent model of the bioenergetics of mitochondria isolated from rat lungs. For the cytosolic region of the model, intrinsic parameters such as apparent Michaelis constants were determined based on previously published enzyme kinetics data, whereas extrinsic parameters such as maximal reaction and transport velocities were estimated by fitting the model solution to published data from isolated rat lungs. The model was then validated by assessing its ability to predict existing experimental data not used for parameter estimation, including relationships between lung nucleotides content, lung lactate production rate, and lung energy charge under different experimental conditions. In addition, the model was used to gain novel insights on how lung tissue glycolytic rate is regulated by exogenous substrates such as glucose and lactate, and assess differences in the bioenergetics of mitochondria isolated from lung tissue and those of mitochondria in intact lungs. To the best of our knowledge, this is the first model of lung tissue bioenergetics. The model provides a mechanistic and quantitative framework for integrating available lung tissue bioenergetics data, and for testing novel hypotheses regarding the role of different cytosolic and mitochondrial processes in lung tissue bioenergetics.

Characterization of conjugated and unconjugated bile acid transport via human organic solute transporter α/β

Bile acids are biosynthesized from cholesterol in hepatocytes and usually localize in the enterohepatic circulation system. This system is regulated by several transporters that are expressed in the liver and intestine. Organic solute transporter (OST) α/β, which is known as a bidirectional transporter for some organic anions, contributes to the transport of bile acids; however, the transport properties of individual bile acids are not well understood. In this study, we investigated the transport properties of five bile acids (cholic acid [CA], chenodeoxycholic acid [CDCA], deoxycholic acid [DCA], ursodeoxycholic acid [UDCA], and lithocholic acid [LCA]) together with their glycine and taurine conjugates mediated by OSTα/β. Of the unconjugated bile acids, CA, CDCA, DCA, and LCA were taken up by OSTαβ/MDCKII cells more rapidly than mock cells, but no significant increase in the uptake of UDCA was observed. On the contrary, all glycine- and taurine-conjugated bile acids showed a significant increase in the uptake by OSTαβ/MDCKII cells. Saturable OSTα/β-mediated transports of CDCA, DCA, glycochenodeoxycholic acid (GCDCA), glycodeoxycholic acid (GDCA), glycolithocholic acid (GLCA), taurochenodeoxycholic acid (TCDCA), and taurolithocholic acid (TLCA) were observed. The apparent Michaelis constants of CDCA, DCA, GCDCA, GDCA, GLCA, TCDCA, and TLCA for OSTα/β were 23.0 ± 4.0, 14.9 ± 1.9, 864.2 ± 80.7, 586.4 ± 43.2, 12.8 ± 0.5, 723.7 ± 4.8, and 23.9 ± 0.3 μM, respectively. However, the transport of other bile acids was not saturable. Our results indicate that OSTα/β has a low affinity but a high capacity for transporting bile acids.

Effect of individual plasma components on the performance of a glucose enzyme electrode based on redox polymer mediation of a flavin adenine dinucleotide-dependent glucose dehydrogenase

The performance of glucose enzyme electrodes, consisting of crosslinked flavin adenine dinucleotide glucose dehydrogenase (FADGDH), an osmium redox polymer and multi-walled carbon nanotubes on graphite electrodes, was tested in phosphate buffered saline, artificial plasma and the individual components of artificial plasma to assess the effect of each component on current response and operational stability of the response to better understand the decrease in electrode performance observed in blood. Electrodes tested in artificial plasma show a significant decrease in current response in 5 mM glucose, and operational stability of the response in 100 mM glucose, compared to electrodes tested in buffer. The lowest current response for the enzyme electrodes was observed in the presence of physiological level of uric acid although the largest alteration to enzyme affinity, as estimated from the apparent Michaelis-Menten constant, occurred upon addition of physiological level of sodium bicarbonate. The operational stability observed in the presence of uric acid was the lowest of all components tested, with only 46% of initial current response after 12 h, and was comparable to the 27% of current remaining after 12 h for electrodes operating in artificial plasma. The effect of uric acid on glucose oxidation by enzyme electrodes prepared using both glucose oxidase (GOx) and a recombinant cellobiose dehydrogenase (CDH) was assessed. The maximum current decreased for both FADGDH and GOx enzyme electrodes in the presence of uric acid, with no significant change to the enzyme affinity, suggesting non-competitive inhibition. The CDH based electrodes provided highest stability of current signal in buffer, with 86% of the initial signal present after 12 h, but display significant change in enzyme affinity, maximum current and operational stability, dropping to only 33%, in the presence of uric acid. In contrast the operational stability of the GOx-based enzyme electrodes was unaffected by the presence of physiological level of uric acid. As uric acid and sodium bicarbonate are present in blood, these results highlight the importance of enzyme selection for in vivo biosensing and biofuel cell applications. Further work is required to understand the mechanism of uric acid inhibition on each of the enzymes.

The synergistic effect of lignin peroxidase and cellulase in Aspergillus oryzae solid‐state fermentation substrate on enzyme‐catalyzed oxidative degradation of lignin

AbstractBACKGROUNDWe have reported the oxidative degradation of lignin catalyzed by lignin peroxidase (LiP) from Aspergillus oxyzae broth in previous studies. The aim of the present paper is to obtain the maximal yield of total reducing sugar (Ytrs) by synergistic effect analysis between LiP and three components of cellulase (endo‐β‐1,4‐glucanase, exo‐β‐1,4‐glucanase and β‐glucosidase).RESULTSThe Michaelis–Menten analogous model was applied to fit experimental data of lignin oxidative degradation. The apparent Michaelis constant of purified LiP product (3.02 mg mL–1) was lower than that of the crude LiP solution (4.74 mg mL−1), and the apparent maximum reaction rate of the purified LiP product (1.15 mg mL−1 h−1) was higher than that of the crude LiP solution (0.86 mg mL−1 h−1). The maximum Ytrs (49.794%) was obtained when the β‐glucosidase activity was 0; the activities of LiP, endo‐β‐1,4‐glucanase and exo‐β‐1,4‐glucanase were 10.77, 3.360 and 3.427 U mL−1 (3:1:1), respectively. The components and structure of the substrate was extremely different before and after synergistic action under optimal conditions.CONCLUSIONA synergistic effect exists between LiP and cellulase. β‐glucosidase is the key enzyme that inhibits the oxidative degradation of lignin. The results lay the foundation for screening and optimizing fermentation process conditions of the LiP strain, and improving the efficiency of enzyme‐catalyzed oxidative degradation of lignin. © 2018 Society of Chemical Industry

Integrated Computational Model of Lung Tissue Bioenergetics

Altered lung tissue bioenergetics plays a key role in the pathogenesis of lung diseases. A wealth of information exists regarding the bioenergetic processes in mitochondria isolated from rat lungs, cultured pulmonary endothelial cells, and intact rat lungs under physiological and pathophysiological conditions. However, the interdependence of those processes makes it difficult to quantify the impact of a change in a single or multiple process(es) on overall lung tissue bioenergetics. Integrated computational modeling provides a mechanistic and quantitative framework for the bioenergetic data at different levels of biological organization. The objective of this study was to develop and validate an integrated computational model of lung bioenergetics using existing experimental data from isolated perfused rat lungs. The model expands our recently developed computational model of the bioenergetics of mitochondria isolated from rat lungs by accounting for glucose uptake and phosphorylation, glycolysis, and the pentose phosphate pathway. For the mitochondrial region of the model, values of kinetic parameters were fixed at those estimated in our recent model of the bioenergetics of mitochondria isolated from rat lungs. For the cytosolic region of the model, intrinsic parameters such as apparent Michaelis constants were determined based on previously published enzyme kinetics data, whereas extrinsic parameters such as maximal reaction and transport velocities were estimated by fitting the model solution to published data from isolated rat lungs. The model was then validated by assessing its ability to predict existing experimental data not used for parameter estimation, including relationships between lung nucleotides content, lung lactate production rate, and lung energy charge under different experimental conditions. In addition, the model was used to gain novel insights on how lung tissue glycolytic rate is regulated by exogenous substrates such as glucose and lactate, and assess differences in the bioenergetics of mitochondria isolated from lung tissue and those of mitochondria in intact lungs. To the best of our knowledge, this is the first model of lung tissue bioenergetics. The model provides a mechanistic and quantitative framework for integrating available lung tissue bioenergetics data, and for testing novel hypotheses regarding the role of different cytosolic and mitochondrial processes in lung tissue bioenergetics.

Thiolated DAB Dendrimer-Gold Nanoparticles Self-Assembled Monolayer as Covalent Support for Direct Electrochemistry of HRP and Sensing Applications

A thiolated DAB dendrimer has been employed to bond gold nanoparticles of several sizes in order to obtain an electrocatalytic framework for the covalent immobilization and direct electrochemistry of horseradish peroxidase (HRP). This biosensor must represent the basis for developing a lot of oxidase-peroxidase bienzymatic biosensors and heavy metals biosensors based on the HRP inhibition. The kinetic study of the modified electrodes showed that the 5 nm and 16 nm gold nanoparticles are the most efficient to contact with the HRP active centre and the optimized biosensor allow to measure hydrogen peroxide at -0.3 V applied potential in linear ranges of 1-5000 and 1-140 or 140-5000 respectively with high sensitivities of 418.6 and 266.3 u A mM-1 cm-2 respectively too with low detection limits of 5 and 9 nM and fast response. The obtained apparent Michaelis-Menten constants, were 0.16 and 0.84 mM respectively. Both are significantly lower than the intrinsic K´M revealing the very high enzymatic efficiency of the developed devices.

Glucose Oxidase Immobilized on a Functional Polymer Modified Glassy Carbon Electrode and Its Molecule Recognition of Glucose.

In the present study, a glucose oxidase (GluOx) direct electron transfer was realized on an aminated polyethylene glycol (mPEG), carboxylic acid functionalized multi-walled carbon nanotubes (fMWCNTs), and ionic liquid (IL) composite functional polymer modified glassy carbon electrode (GCE). The amino groups in PEG, carboxyl groups in multi-walled carbon nanotubes, and IL may have a better synergistic effect, thus more effectively adjust the hydrophobicity, stability, conductivity, and biocompatibility of the composite functional polymer film. The composite polymer membranes were characterized by cyclic voltammetry (CV), ultraviolet-visible (UV-Vis) spectrophotometer, fluorescence spectroscopy, electrochemical impedance spectroscopy (EIS), and transmission electron microscopy (TEM), respectively. In 50 mM, pH 7.0 phosphate buffer solution, the formal potential and heterogeneous electron transfer constant (ks) of GluOx on the composite functional polymer modified GCE were −0.27 V and 6.5 s−1, respectively. The modified electrode could recognize and detect glucose linearly in the range of 20 to 950 μM with a detection limit of 0.2 μM. The apparent Michaelis-Menten constant (Kmapp) of the modified electrode was 143 μM. The IL/mPEG-fMWCNTs functional polymer could preserve the conformational structure and catalytic activity of GluOx and lead to high sensitivity, stability, and selectivity of the biosensors for glucose recognition and detection.

Characterization and in vitro expression of arginine kinase gene in the invasive western flower thrips, Frankliniella occidentalis.

Arginine kinase (AK) plays a critical role in insect energy metabolism and has been proposed to be a potential insecticide target for commercial exploitation. In this study, the full length cDNA encoding a typical group 1 insect AK (FoAK) was isolated from the western flower thrips (WFT), Frankliniella occidentalis (Pergande). Sequence analysis showed that FoAK contains an open reading frame of 1068 nucleotides, which encods a protein of 355 amino acid residues including the signature sequence pattern of ATP-guanidino kinases. Genomic structure analysis showed that the coding region of FoAK contains five exons connected by four introns. RT-qPCR analysis revealed that the mRNA expression of FoAK was developmentally regulated with the lowest level in prepupal stage. Enzymatic activity analysis of the recombinant enzymes expressed in Escherichia coli showed that FoAK is highly stereo specific for L-arginine versus D-arginine and the apparent Michaelis constant for L-arginine (KmArg) is comparable to that of AKs from a variety of species. This research should enable further investigation of the function as well as in vitro screening for inhibitors of FoAK.

Direct Electron Transfer of Catalase and its Biosensing for H2O2

By immobilizing catalase on a functional nanocomposites containing ion-liquid, TiO2 nano particles and carboxylic acid functionalized multi-walled carbon nanotubes, direct electron transfer of CAT was realized on the nanocomposites modified glassy carbon electrode. The electrochemical study showed that the formal potential and heterogeneous electron transfer constant of the prepared modified glassy carbon electrode were −0.163 V and 5.24 s−1, respectively. The electrocatalysis study showed that the linear response range toward H2O2 and the apparent Michaelis–Menten constant were from 5 μM to 1000 μM and 54.3 μM, respectively.

A broad spectrum amperometric pesticide biosensor based on glutathione S-transferase immobilized on graphene oxide-gelatin matrix

In this work we have fabricated a glutathione-S-transferase based amperometric biosensor for pesticides by immobilizing the enzyme on platinum electrode using graphene oxide-gelatin matrix, evaluated various biosensor parameters, applied the same for detection and quantification of four different classes of pesticides and validated the biosensor results with GC–MS analysis. The enzyme immobilization was confirmed through scanning electron microscopy, electrochemical impedance spectroscopy, cyclic voltammetry and chronoamperometry. The apparent Michaelis-Menten constant for the immobilized glutathione-S-transferase in the said matrix was found to be 0.083 mmol L−1 and 0.15 mmol L−1 respectively for glutathione and 1-Chloro-2,4-dinitrobenzene. Substrate specificity found to be 2.56 × 107 s−1 M−1 for glutathione and 2.15 × 107 s−1 M−1 for 1-Chloro-2,4-dinitrobenzene. Pesticide analysis was done in 25% methanol solution. The biosensor is a promising new tool for pesticide analysis as it can be applied for analysis of a broad spectrum of pesticides covering at least six different classes namely - benzamidazole, organochlorine, organothiophosphate, organocarbamate, polyphenol and pyrethroid.