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1.
To realize coenzyme regeneration in the reduction of haloketones, a codon-optimized gene Sygdh encoding glucose 1-dehydrogenase (SyGDH) was synthesized based on the putative GDH gene sequence (Ta0897) in Thermoplasma acidophilum genomic DNA, and expressed in E. coli BL21(DE3). Recombinant SyGDH was purified to homogeneity by affinity chromatography with the specific activity of 86.3 U/mg protein towards D-glucose at the optimum pH and temperature of 7.5 and 40 °C. It was highly stable in a pH range of 4.5–8.0 and at 60 °C or below, and resistant to various organic solvents. The Km and catalytic efficiency (kcat/Km) of SyGDH towards NADP+ were 0.67 mM and 104.0 mM−1 s−1, respectively, while those towards NAD+ were 157.9 mM and 0.64 mM−1 s−1, suggesting that it preferred NADP+ as coenzyme to NAD+. Additionally, using whole cells of E. coli/Sygdh-Sys1, coexpressing SyGDH and carbonyl reductase (SyS1), as the biocatalyst, the asymmetric reduction of 60 mM m-chlorophenacyl chloride coupled with the regeneration of NADPH in situ was conducted in DMSO/phosphate buffer (2:8, v/v) system, producing (R)-2-chloro-1-(3-chlorophenyl)ethanol with over 99.9% eep and 99.2% yield. Similarly, the reduction of 40 mM α-bromoacetophenone in n-hexane/buffer (6:4, v/v) biphasic system produced (S)-2-bromo-1-phenylethanol with over 99.9% eep and 98.3% yield.  相似文献   

2.
GOX is the most widely used enzyme for the development of electrochemical glucose biosensors and biofuel cell in physiological conditions. The present work describes the production of a recombinant glucose oxidase from Penicillium amagasakiense (yGOXpenag) displaying a more efficient glucose catalysis (kcat/KM(glucose) = 93 μM−1 s−1) than the native GOX from Aspergillus niger (nGOXaspng), which is the most industrially used (kcat/KM(glucose) = 27 μM−1 s−1). Expression in Pichia pastoris allowed easy production and purification of the recombinant active enzyme, without overglycosylation. Its biotechnological interest was further evaluated by measuring kinetics of ferrocinium-methanol (FMox) reduction, which is commonly used for electron transfer to the electrode surface. Despite their homologies in sequence and structure, pH-dependant FMox reduction was different between the two enzymes. At physiological pH and temperature, we observed that electron transfer to the redox mediator is also more efficient for yGOXpenag than for nGOXaspng(kcat/KM(FMox) = 27 μM−1 s−1 and 17 μM−1 s−1 respectively). In our model system, the catalytic current observed in the presence of blood glucose concentration (5 mM) was two times higher with yGOXpenag than with nGOXaspng. All our results indicated that yGOXpenag is a better candidate for industrial development of efficient bioelectrochemical devices used in physiological conditions.  相似文献   

3.
In this study, a bioanode was developed by using layer-by-layer (LBL) assembly of sulfonated graphene (SG)/ferritin (Frt)/glucose oxidase (GOx). The SG/Frt biocomposite was used as an electron transfer elevator and mediator, respectively. Glucose oxidase (GOx) from Aspergillus niger was applied as a glucose oxidation biocatalyst. The electrocatalytic oxidation of glucose using GOx modified electrode increases with an increase in the concentration of glucose in the range of 10–50 mM. The electrochemical measurements of the electrode was carried out by using cyclic voltammetry (CV) at different scan rates (20–100 mV s−1) in 30 mM of glucose solution prepared in 0.3 M potassium ferrocyanide (K4Fe(CN)6) and linear sweep voltammetry (LSV). A saturation current density of 50 ± 2 mA cm−2 at a scan rate of 100 mV s−1 for the oxidation of 30 Mm glucose is achieved.  相似文献   

4.
The graphene nanosheets and carbon nanospheres mixture (GNS–CNS) was prepared by electrolyzing graphite rob in KNO3 solution under constant current, which was characterized by TEM, AFM, SEM, FT-IR, XRD, XPS, TGA and UV–vis. The nano-mixture can keep stable in water for more than one month. Based on this kind of mixture material, a novel electrochemical biosensing platform for glucose determination was developed. Cyclic voltammetry of glucose oxidase (GOD) immobilized on GNS–CNS/GCE exhibited a pair of well-defined quasi-reversible redox peaks at −0.488 V (Epa) and −0.509 V (Epc) by direct electron transfer between the protein and the electrode. The charge-transfer coefficient (α) was 0.51, the electron transfer rate constant was 2.64 s−1 and the surface coverage of HRP was 3.18 × 10−10 mol cm−2. The immobilized GOD could retain its bioactivity and catalyze the reduction of dissolved oxygen. The glucose biosensor has a linear range from 0.4 to 20 mM with detection limit of 0.1 mM. Moreover, the biosensor exhibits acceptable reproducibility and storage stability. The fabricated biosensor was further used to determine glucose in human plasma sample with the recoveries from 96.83% to 105.52%. Therefore, GOD/GNS–CNS/GCE could be promisingly applied to determine blood sugar concentration in the practical clinical analysis.  相似文献   

5.
Polymers and copolymers of horseradish root peroxidase (HRP) and Penicillium funiculosum 46.1 glucose oxidase (GO) have been synthesized and their catalytic properties have been characterized (free and immobilized forms of each enzyme were studied). The cooxidation reaction of phenol and 4-aminoantipyrin (4-AAP), performed in an aqueous medium in the presence of equimolar amounts of GO and HRP, was characterized by effective K M and k cat of 0.58 mM and 20.9 s?1 (for phenol), and 14.6 mM and 18.4 s?1 (glucose), respectively. The catalytic efficiency of polymerization products (PPs) of GO (GO-PPs) depended on the extent of their aggregation. The combinations GO + HRP-PP and HRP + GO-PP, as well as the copolymer HRP*-GO-PP, proved promising as reagents for enzyme-based analytical systems. When adsorbed on aluminum hydroxide gels, GO-PPs exhibited higher catalytic activity than the non-polymeric enzyme. Maximum retention of GO-PP activity on the inorganic carrier was observed in the case of GO-PP copolymers with an activated HRP. Polymerization of HRP in the presence of a zinc hydroxide gel, paralleled by HRP-PP immobilization onto the gel, increased both the activity of the enzyme and its operational stability.  相似文献   

6.
In this article we report a selective urea electrochemical biosensor based on electro-co-deposited zirconia-polypropylene imine dendrimer (ZrO2-PPI) nanocomposite modified screen printed carbon electrode (SPCE). ZrO2 nanoparticles, prepared by modified sol–gel method were dispersed in PPI solution, and electro-co-deposited by cyclic voltammetry onto a SPCE surface. The material and the modified electrodes were characterised using FTIR, electron microscopy and electrochemistry. The synergistic effect of the high active surface area of both materials, i.e. PPI and ZrO2 nanoparticles, gave rise to a remarkable improvement in the electrocatalytic properties of the biosensor and aided the immobilisation of the urease enzyme. The biosensor has an ampereometric response time of ∼4 s in urea concentration ranging from 0.01 mM to 2.99 mM with a correlation coefficient of 0.9985 and sensitivity of 3.89 μA mM−1 cm−2. The biosensor was selective in the presence of interferences. Photochemical study of the immobilised enzyme revealed high stability and reactivity.  相似文献   

7.
Biopolymer pectin stabilized gold nanoparticles were prepared at graphene and multiwalled carbon nanotubes (GR-MWNTs/AuNPs) and employed for the determination of glucose. The formation of GR-MWNTs/AuNPs was confirmed by scanning electron microscopy, X-ray diffraction, UV–vis and FTIR spectroscopy methods. Glucose oxidase (GOx) was successfully immobilized on GR-MWNTs/AuNPs film and direct electron transfer of GOx was investigated. GOx exhibits highly enhanced redox peaks with formal potential of −0.40 V (vs. Ag/AgCl). The amount of electroactive GOx and electron transfer rate constant were found to be 10.5 × 10−10 mol cm−2 and 3.36 s−1, respectively, which were significantly larger than the previous reports. The fabricated amperometric glucose biosensor sensitively detects glucose and showed two linear ranges: (1) 10 μM  2 mM with LOD of 4.1 μM, (2) 2 mM  5.2 mM with LOD of 0.95 mM. The comparison of the biosensor performance with reported sensors reveals the significant improvement in overall sensor performance. Moreover, the biosensor exhibited appreciable stability, repeatability, reproducibility and practicality. The other advantages of the fabricated biosensor are simple and green fabrication approach, roughed and stable electrode surface, fast in sensing and highly reproducible.  相似文献   

8.
Abstract

The direct electron transfer of immobilized haemoglobin (Hb) on nano-TiO2 and dodecyltrimethylammonium bromide (DTAB) film modified carbon paste electrode (CPE) and its application as a hydrogen peroxide (H2O2) biosensor were investigated. On nano-TiO2/DTAB/Hb/CPE, Hb displayed a rapid electron transfer process with participation of one proton and with an electron transfer rate constant which estimated as 0.29 s??1. Thus, the proposed biosensor exhibited a high sensitivity and excellent electrocatalytic activity for the reduction of H2O2. The catalytic reduction current of H2O2 was proportional to H2O2 concentration in the range of 0.2–4.0 mM with a detection limit of 0.07 mM. The apparent Michaelis–Menten constant (Kmapp) of the biosensor was calculated to be 0.127 mM, exhibiting a high enzymatic activity and affinity. This sensor for H2O2 can potentially be applied in determination of other reactive oxygen species as well.  相似文献   

9.
Novel Pt nanoclusters embedded polypyrrole nanowires (PPy-Pt) composite was electrosynthesized on a glassy carbon electrode, denoted as PPy-Pt/GCE. A glucose biosensor was further fabricated based on immobilization of glucose oxidase (GOD) in an electropolymerized non-conducting poly(o-aminophenol) (POAP) film that was deposited on the PPy-Pt/GCE. The morphologies of the PPy nanowires and PPy-Pt nanocomposite were characterized by field emission scanning electron microscope (FE-SEM). Effect of experimental conditions involving the cycle numbers for POAP deposition and Pt nanoclusters deposition, applied potential used in glucose determination, temperature and pH value of the detection solution were investigated for optimization. The biosensor exhibited an excellent current response to glucose over a wide linear range from 1.5 × 10−6 to 1.3 × 10−2 M (r = 0.9982) with a detection limit of 4.5 × 10−7 M (s/n = 3). Based on the combination of permselectivity of the POAP and the PPy films, the sensor had good anti-interference ability to ascorbic acid (AA), uric acid (UA) and acetaminophen. The apparent Michaelis–Menten constant (Km) and the maximum current density (Im) were estimated to be 23.9 mM and 378 μA/cm2, respectively. In addition, the biosensor had also good sensitivity, stability and reproducibility.  相似文献   

10.
Herein, a novel third-generation glucose biosensor based on unique hollow nanostructured Pt decorated multiwall carbon nanotubes (HPt-CNTs) composites was successfully constructed. The HPt-CNTs composites were successfully prepared and cast on the glassy carbon electrode (GCE) surface directly. With the help of electrostatic adsorption and covalent attachment, the negative l-cysteine (l-cys) and the positive poly(diallydimethylammonium) chloride (PDDA) protected gold nanoparticles (PDDA-Au) were modified on the resulting electrode surface subsequently, which provided further immobilization of glucose oxidase (GOD). Exploitation of the unique properties of HPt-CNTs composites led to the achievement of direct electron transfer between the electrode and the redox active centers of GOD, and the electrode exhibited a pair of well-defined reversible redox peaks with a fast heterogeneous electron transfer rate. In particular, the detection limit (4 × 10−7 M) of this biosensor was significantly lower and the linear range (1.2 μM–8.4 mM) was much wider than similar carbon nanotubes (CNTs) and Pt-based glucose biosensors. The resulted biosensor also showed high sensitivity and freedom of interference from other co-existing electroactive species, indicating that our facile procedure of immobilizing GOD exhibited better response and had potential application for glucose analysis.  相似文献   

11.
Glycine oxidase (GO) has great potential for use in biosensors, industrial catalysis and agricultural biotechnology. In this study, a novel GO (BliGO) from a marine bacteria Bacillus licheniformis was cloned and characterized. BliGO showed 62% similarity to the well-studied GO from Bacillus subtilis. The optimal activity of BliGO was observed at pH 8.5 and 40 °C. Interestingly, BliGO retained 60% of the maximum activity at 0 °C, suggesting it is a cold-adapted enzyme. The kinetic parameters on glyphosate (Km, kcat and kcat/Km) of BliGO were 11.22 mM, 0.08 s−1, and 0.01 mM−1 s−1, respectively. To improve the catalytic activity to glyphosate, the BliGO was engineered by directed evolution. With error-prone PCR and two rounds of DNA shuffling, the most evolved mutant SCF-4 was obtained from 45,000 colonies, which showed 7.1- and 8-fold increase of affinity (1.58 mM) and catalytic efficiency (0.08 mM−1 s−1) to glyphosate, respectively. In contrast, its activity to glycine (the natural substrate of GO) decreased by 113-fold. Structure modeling and site-directed mutation study indicated that Ser51 in SCF-4 involved in the binding of enzyme with glyphosate and played a crucial role in the improvement of catalytic efficiency.  相似文献   

12.
Glucose potentiometric biosensor was prepared by immobilizing glucose oxidase on iodide-selective electrode. The hydrogen peroxide formed after the oxidation of glucose catalysed by glucose oxidase (GOD) was oxidized by sodium molybdate (SMo) at iodide electrode in the presence of dichlorometane. The glucose concentration was calculated from the decrease of iodide concentration determined by iodide-selective sensor. The sensitivity of glucose biosensor towards iodide ions and glucose was in the concentration ranges of 1.0 × 10?1–1.0 × 10?6 M and 1.0 × 10?2?1.0 × 10?4 M, respectively. The characterization of proposed glucose biosensor and glucose assay in human serum were also investigated.  相似文献   

13.
A modified electrode, nickel(II)-baicalein complex modified multiwall carbon nanotube paste electrode (Ni(II)-BA-MWCNT-PE), has been fabricated by electrodepositing Ni(II)-BA complex on the surface of MWCNT-PE in alkaline solution. The Ni(II)-BA-MWCNT-PE exhibits the characteristic of improved reversibility and enhanced current responses of the Ni(III)/Ni(II) couple compared with Ni(II)-BA-carbon paste electrode (CPE). It also shows better electrocatalytic activity toward the oxidation of glycine than Ni(II)-MWCNT-PE. Kinetic parameters such as the electron transfer coefficient α, rate constant ks of the electrode reaction, the diffusion coefficient D of glycine, and the catalytic rate constant kcat of the catalytic reaction are determined. Moreover, the catalytic currents present linear dependence on the concentration of glycine from 20 μM to 1.0 mM by amperometry. The detection limit and sensitivity are 9.2 μM and 3.92 μA mM−1, respectively. The modified electrode for glycine determination is of the property of simple preparation, fast response, and good stability.  相似文献   

14.
In this paper, a mediatorless amperometric glucose biosensor based on direct covalent immobilisation of monomolecular layer of glucose oxidase (GOx) on a semiconducting indium-tin oxide (ITO) is demonstrated. The abundance of surface hydroxyl functional group of ITO allows it to be used as a suitable platform for direct covalent immobilisation of the enzyme for sensor architecture. The anodic current corresponding to electrochemical oxidation of the enzymatic product, hydrogen peroxide, at a sputtered Pt electrode at 0.500 V (vs. SCE) was obtained as the sensor signal. It was found that the biosensor based on the direct immobilisation scheme shows a fast biosensor response, minimum interference from other common metabolic species and ease of biosensor miniaturisation. A linear range of 0-10 mM of glucose was demonstrated, which exhibits a high sensitivity as far as performance per immobilised GOx molecule is concerned. A detection limit as low as 0.05 mM and long-term stability were observed. Even more important, the biosensor design allows fabrication through a dry process. These characteristics make it possible to achieve mass production of biosensor compatible with the current electronic integrated circuit manufacturing technologies.  相似文献   

15.
We constructed a fusion protein (GOx-R5) consisting of R5 (a polypeptide component of silaffin) and glucose oxidase (GOx) that was expressed in Pichia pastoris. Silaffin proteins are responsible for the formation of a silica-based cell matrix of diatoms, and synthetic variants of the R5 protein can perform silicification in vitro[1]. GOx secreted by P. pastoris was self-immobilized (biosilicification) in a pH 5 citric buffer using 0.1 M tetramethoxysilane as a silica source. This self-entrapment property of GOx-R5 was used to immobilize GOx on a graphite rod electrode. An electric cell designed as a biosensor was prepared to monitor the glucose concentrations. The electric cell consisted of an Ag/AgCl reference electrode, a platinum counter electrode, and a working electrode modified with poly(neutral red) (PNR)/GOx/Nafion. Glucose oxidase was immobilized by fused protein on poly(neutral red) and covered by Nafion to protect diffusion to the solution. The morphology of the resulting composite PNR/GOx/Nafion material was analyzed by scanning electron microscopy (SEM). This amperometric transducer was characterized electrochemically using cyclic voltammetry and amperometry in the presence of glucose. An image produced by scanning electron microscopy supported the formation of a PNR/GOx complex and the current was increased to 1.58 μA cm−1 by adding 1 mM glucose at an applied potential of −0.5 V. The current was detected by way of PNR-reduced hydrogen peroxide, a product of the glucose oxidation by GOx. The detection limit was 0.67 mM (S/N = 3). The biosensor containing the graphite rod/PNR/GOx/Nafion detected glucose at various concentrations in mixed samples, which contained interfering molecules. In this study, we report the first expression of R5 fused to glucose oxidase in eukaryotic cells and demonstrate an application of self-entrapped GOx to a glucose biosensor.  相似文献   

16.
The α-carbonic anhydrase gene from Helicobacter pylori strain 26695 has been cloned and sequenced. The full-length protein appears to be toxic to Escherichia coli, so we prepared a modified form of the gene lacking a part that presumably encodes a cleavable signal peptide. This truncated gene could be expressed in E. coli yielding an active enzyme comprising 229 amino acid residues. The amino acid sequence shows 36% identity with that of the enzyme from Neisseria gonorrhoeae and 28% with that of human carbonic anhydrase II. The H. pylori enzyme was purified by sulfonamide affinity chromatography and its circular dichroism spectrum and denaturation profile in guanidine hydrochloride have been measured. Kinetic parameters for CO2 hydration catalyzed by the H. pylori enzyme at pH 8.9 and 25°C are kcat=2.4×105 s−1, KM=17 mM and kcat/KM=1.4×107 M−1 s−1. The pH dependence of kcat/KM fits with a simple titration curve with pKa=7.5. Thiocyanate yields an uncompetitive inhibition pattern at pH 9 indicating that the maximal rate of CO2 hydration is limited by proton transfer between a zinc-bound water molecule and the reaction medium in analogy to other forms of the enzyme. The 4-nitrophenyl acetate hydrolase activity of the H. pylori enzyme is quite low with an apparent catalytic second-order rate constant, kenz, of 24 M−1 s−1 at pH 8.8 and 25°C. However, with 2-nitrophenyl acetate as substrate a kenz value of 665 M−1 s−1 was obtained under similar conditions.  相似文献   

17.
In order to eliminate the interference of impurities, such as ascorbic acid, a noninterference polypyrrole glucose biosensor was constructed with a four-electrode cell consisting of a polypyrrole film electrode, a polypyrrole-glucose oxidase electrode, a counter electrode and a reference electrode. The pure catalytic current of glucose oxidase (GOD) can be obtained from the difference between response currents of two working electrodes with and without GOD. The effects of potential, pH and temperature on analytical performance of the glucose biosensor were discussed. The optimum pH and apparent activation energy of enzyme-catalyzed reaction are 5.5 and 25 kJ mol(-1), respectively. The response current of the biosensor increases linearly with the increasing glucose concentration from 0.005 to 20.0 mmol dm(-3). The results show the glucose biosensor with under 2% of relative deviation has good ability of anti-interference. The glucose biosensor was also characterized with FT-IR and UV-vis spectra.  相似文献   

18.
We have cloned, purified and investigated the catalytic activity and anion inhibition profiles of a full catalytic domain (358 amino acid residues) carbonic anhydrase (CA, EC 4.2.1.1) from Plasmodium falciparum, PfCAdom, an enzyme belonging to the η-CA class and identified in the genome of the malaria-producing protozoa. A truncated such enzyme, PfCA1, containing 235 residues was investigated earlier for its catalytic and inhibition profiles. The two enzymes were efficient catalysts for CO2 hydration: PfCAdom showed a kcat of 3.8 × 105 s−1 and kcat/Km of 7.2 × 107 M−1 × s−1, whereas PfCA showed a lower activity compared to PfCAdom, with a kcat of 1.4 × 105 s−1 and kcat/Km of 5.4 × 106 M−1 × s−1. PfCAdom was generally less inhibited by most anions and small molecules compared to PfCA1. The best PfCAdom inhibitors were sulfamide, sulfamic acid, phenylboronic acid and phenylarsonic acid, which showed KIs in the range of 9–68 μM, followed by bicarbonate, hydrogensulfide, stannate and N,N-diethyldithiocarbamate, which were submillimolar inhibitors, with KIs in the range of 0.53–0.97 mM. Malaria parasites CA inhibition was proposed as a new strategy to develop antimalarial drugs, with a novel mechanism of action.  相似文献   

19.
A glucose biosensor using a glucose oxidase (GOx)-immobilized nylon net with glutaraldehyde as cross-linking reagent and an oxygen (O2) electrode for the determination of glucose has been fabricated. The detection scheme was based on the utilization of dissolved O2 in oxidation of glucose by the membrane bound GOx. Crucial factors including O-alkylation temperature, reaction times of nylon net with dimethyl sulfate, l-lysine, and glutaraldehyde, and enzyme loading were examined to determine the optimal enzyme immobilization conditions for the best sensitivity of the developed glucose biosensor. In addition, the effects of pH and concentration of phosphate buffer on the response of the biosensor were studied. The glucose biosensor had a linear range of 18 μM to 1.10 mM with the detection limit of 9.0 μM (S/N = 3) and response time of 80 s. The biosensor exhibited both good operational stability with over 200 measurements and long-term storage stability. The results from this biosensor compared well with those of a commercial glucose assay kit in analyzing human serum glucose samples.  相似文献   

20.
In this study, a novel glucose biosensor was fabricated by reconstitutional immobilization of glucose oxidase (GOx) onto a poly(glycidyl methacrylate-co-vinylferrocene) (poly(GMA-co-VFc)) film coated pencil graphite electrode (PGE). The amperometric current response of poly(GMA-co-VFc)-GOx to glucose is linear in the concentration range between 1 and 16 mM (correlation coefficient of 0.9998) with a detection limit of 2.7 μM (S/N = 3). Experimental parameters were studied in detail and optimized, including the pH and temperature governing the analytical performance of the biosensor. The stability and reusability of the biosensor as well as its kinetic parameters have also been studied.  相似文献   

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