Characterization of a novel immobilized biocatalyst obtained by matrix-assisted refolding of recombinant polyhydroxyoctanoate depolymerase from <Emphasis Type="Italic">Pseudomonas putida</Emphasis> KT2442 isolated from inclusion bodies

Purification and matrix-assisted refolding of recombinant His-tagged polyhydroxyalkanoate (PhaZ) depolymerase from Pseudomonas putida KT2442 was carried out. His-tagged enzyme was overproduced as inclusion bodies in recombinant E. coli M15 (pREP4, pPAZ3), which were denatured by 8 M urea, immobilized on Ni²⁺-nitrilotriacetate-agarose matrix, and refolded by gradual removal of the chaotropic agent. The refolded enzyme could not be eluted with 1 M imidazole buffer, leading to an immobilized biocatalyst where PhaZ depolymerase was homogeneously distributed in the agarose support as shown by confocal scanning microscopy. Polyhydroxyoctanoate could not be hydrolyzed by this novel immobilized biocatalyst, whereas the attached enzyme was active in the hydrolysis of p-nitrophenyl alkanoate esters, which differed in their alkyl chain length. Taking advantage of the observed esterase activity on p-nitrophenylacetate, functional characterization of immobilized PhaZ depolymerase was carried out. The immobilized enzyme was more stable than its soluble counterpart and showed optimal hydrolytic activity at 37°C and 50 mM phosphate buffer pH 8.0. Kinetic parameters were obtained with both p-nitrophenylacetate and p-nitrophenyloctanoate, which had not been described so far for the soluble enzyme, representing an attractive and alternative chromogenic assay for the study of this paradigmatic enzyme.     

Increase of electrical properties using a novel mixed buffer system in an enzyme fuel cell

Environment-friendly biocatalytic energy is considered to represent an attractive alternative to chemical catalystbased cells due to its renewability and better operation at low temperature. However, electrical biocatalysts have a low activity and electrical power. For increasing electrical properties of biocatalyst, a novel mixed buffer (phosphate and 3-morpholinopropanesulfonic acid (MOPS)) system was applied to an enzyme-based biofuel cell with microperoxidase (MP-11)-modified Au electrode. The cathodic electrical properties were increased by the phosphate and MOPS-mixed buffer solution. It was identified that the novel mixed buffer system obtained stronger ionic strength from phosphate buffer and better enzyme activity from MOPS buffer. The highest results of cyclic voltammetry were obtained when the proportion of phosphate to MOPS was nearly 1:1 and the pH was 7.0∼7.3. In addition, the novel mixed buffer led to the maximum power density (ca. 62.7 μW/cm²) in a basic enzymatic fuel cell (EFC).     

An electrochemical method for the measurements of substrate-oxidizing activity of acetic acid bacteria using a carbon-paste electrode modified with immobilized bacteria

In order to measure the substrate-oxidizing activity of intact cells of Acetobacter pasteurianus no. 2, a given amount of the bacterial cells was immobilized on a carbon-paste electrode, and the current at the electrode was measured in a buffer solution. When Fe(CN)³⁻ ₆ was added to the buffer solution, an anodic current was observed at 0.5 V (against Ag/AgCl). Further, when ethanol was added to the solution, the current started to increase to reach a steady-state within 3 min. The electrode had a good response to acetaldehyde and lactic acid as well as ethanol. Culture conditions affected the current response to various substances; the response of the electrode modified with the cells grown in static culture was much higher than that of the electrode with the cells grown in shaking culture, and the electrode with ethanol-grown cells had a high response to ethanol and acetaldehyde compared with that of the electrode with glucose-grown cells. The increase in the amount of the current after the addition of ethanol (ΔI _EtOH) was linearly proportional to the total number of immobilized cells per electrode in the range 1.0 × 10⁴–1.0 × 10⁸ cells. The ΔI _EtOH values were measured with the electrode prepared with a fixed volume of the cell suspensions taken from the culture at 6-h intervals; the dependence of the ΔI _EtOH value on time agreed well with the cell growth measured by colony counting and turbidity in the lag and logarithmic phase. After the logarithmic phase, the value of ΔI _EtOH sharply decreased, resembling to the growth measured by colony counting, rather than by turbidity. Received: 30 October 1998 / Received revision: 2 February 1999 / Accepted: 5 February 1999     

Purification and properties of cis-aconitic decarboxylase

Summary Lyophilized and stored in a deep-freeze, the mycelial material was found to retain cis-aconitic decarboxylase activity unimpaired at the end of 2 months. Mycelia could be stored also in the frozen condition but after squeezing hard to remove as much of adherent water as possible. Extracts with maximum cis-aconitic decarboxylase activity were obtained when the frozen or better the lyophilized mycelia of Aspergillus terreus were ground in a mortar with phosphate buffer using pyrex glass powder as abrasive. Cis-aconitic decarboxylase was purified 25-fold by fractionation with ammonium sulfate, starting from extracts of the mycelia in phosphate buffer. The purified enzyme was considerably more stable than the crude extracts to storage and dialysis. The optimum pH was 5.8 using 0.2 m phosphate buffer; Km value was 5×10^-3 m at pH 5.8 and 37°C. EDTA and 8-hydroxyquinoline activated the enzyme; all metals tested inhibited the enzyme, Zn⁺⁺ and Cu⁺⁺ leading to complete inactivation. Fluoride, arsenite and azide also inhibited the enzyme activity.     

Amperometric determination of sodium nitrite by a microbial sensor

Summary A microbial sensor was prepared to determine sodium nitrite. This microbial sensor consisted of immobilized Nitrobacter sp. and an oxygen electrode. When a sample solution containing sodium nitrite was tested, nitrite was changed to NO₂ gas in the buffer (pH 2.0) and the current of the electrode decreased with time until a steady state was reached. The steady state current was attained within 10 min and the maximum decrease in current was obtained at 30°C and pH 2.0. A linear relationship was observed between the current decrease and the sodium nitrite concentration below 0.59 mM, the minimum sodium nitrite concentration that could be determined was 0.01 mM. The current decrease was reproducible (5% relative error). The current output of the sensor was almost constant for more than 21 days and 400 assays.     

A Cu‐Based Nanoparticulate Film as Super‐Active and Robust Catalyst Surpasses Pt for Electrochemical H2 Production from Neutral and Weak Acidic Aqueous Solutions

Electrocatalysts that are stable and highly active at low overpotential (η) under mild conditions as well as cost‐effective and scalable are eagerly desired for potential use in photo‐ and electro‐driven hydrogen evolution devices. Here the fabrication and characterization of a super‐active and robust Cu‐Cu_xO‐Pt nanoparticulate electrocatalyst is reported, which displays a small Tafel slope (44 mV dec^?1) and a large exchange current density (1.601 mA cm^?2) in neutral buffer solution. The catalytic current density of this catalyst film reaches 500 mA cm^?2 at η = ?390 ± 12 mV and 20 mA cm^?2 at η = ?45 ± 3 mV, which are significantly higher than the values displayed by Pt foil and Pt/C electrodes in neutral buffer solution and even comparable with the activity of Pt electrode in 0.5 m H₂SO₄ solution.     

A Novel Catechol Oxidase Enzyme Electrode for the Specific Determination of Catechol

An enzyme electrode for the specific determination of catechol was developed by using catechol oxidase (EC 1.10.3.1) from eggplant (Solanum melangena L.) in combination with a dissolved oxygen probe. Optimization studies of the prepared catechol oxidase enzyme electrode established a phosphate buffer 50 mM at pH 7.0 and 35°C to provide the optimum conditions for affirmative electrode response. The enzyme electrode response depended linearly on a catechol concentration range of 5?10^-7-30?10^-5 M with a response time of 25 sec and substrate specificity of the catechol oxidase electrode of 100%. The biosensor retained its enzyme activity for at least 70 days.     

Development of Novel Glucose oxidase Immobilization on Graphene/Gold nanoparticles/Poly Neutral red modified electrode

Glucose oxidase (GOx) was immobilized onto glassy carbon electrode (GCE) that modified by reduced graphene oxide-gold nanoparticles- poly neutral red (RGO/AuNPs/PNR) nanocomposite. The composite was analyzed by scanning electron microscope (SEM), energy dispersive x-ray (EDX) spectroscopy, atomic force microscopy (AFM), attenuated total reflectance (ATR), cyclic voltammetry (CV), chronoamperometry and electrochemical impedance spectroscopy (EIS). SEM/EDX analysis showed the morphological of the nanocomposite. AFM results showed the morphology and structure of the RGO/AuNPs and RGO surfaces. The covalent bonding between glucose oxidase and composite was confirmed by ATR technique. The electrochemical experiments were done in 100 mM phosphate buffer at pH 7 and temperature of 25 °C with three electrodes including Ag/AgCl, platinum wire and the modified GCE as the reference electrode, the auxiliary electrode and working electrode respectively. The electrochemical results confirmed the activity and direct electron transfer of immobilized enzyme. The immobilized electroactive GOx concentration was estimated 3.06 × 10⁻¹¹ mol cm⁻². The results showed the immobilized enzyme had a good stability and maintained 90% of its performance after two weeks. The nanocomposite bioanode in an air-birthing biofuel cell and 100 mM glucose concentration showed 176 μWcm⁻² Power density. This strategy could be used for GOx-based biofuel cells.     

Immobilization of -glucose oxidase onto a hydrogen peroxide permselective membrane and application for an enzyme electrode

A hydrogen peroxide permselective membrane with asymmetric structure was prepared and -glucose oxidase (EC 1.1.3.4) was immobilized onto the porous layer. The activity of the immobilized -glucose oxidase membrane was 0.34 units cm⁻² and the activity yield was 6.8% of that of the native enzyme. Optimum pH, optimum temperature, pH stability and temperature stability were found to be pH 5.0, 30–40°C, pH 4.0–7.0 and below 55°C, respectively. The apparent Michaelis constant of the immobilized -glucose oxidase membrane was 1.6 × 10⁻³ mol l⁻¹ and that of free enzyme was 4.8 × 10⁻² mol l⁻¹. An enzyme electrode was constructed by combination of a hydrogen peroxide electrode with the immobilized -glucose oxidase membrane. The enzyme electrode responded linearly to -glucose over the concentration 0–1000 mg dl⁻¹ within 10 s. When the enzyme electrode was applied to the determination of -glucose in human serum, within day precision (CV) was 1.29% for -glucose concentration with a mean value of 106.8 mg dl⁻¹. The correlation coefficient between the enzyme electrode method and the conventional colorimetric method using a free enzyme was 0.984. The immobilized -glucose oxidase membrane was sufficiently stable to perform 1000 assays (2 to 4 weeks operation) for the determination of -glucose in human whole blood. The dried membrane retained 77% of its initial activity after storage at 4°C for 16 months.     

Characterization of microbial membranes used for the estimation of biochemical oxygen demand with a biosensor

Summary A bacterial mixed culture was immobilized in Millipore filters to construct microbial-membranes for BOD determination using an oxygen electrode. The biosensor response was best when 0.5 mg cells were immobilized per cm² of membrane, at 30°C, pH 7 and 0.05 M phosphate buffer. Reproducible microbial-membranes can be constructed and they can be stored for up to 20 days without appreciable loss of their response characteristics.     

Photochemical energy conversion system using immobilized chloroplasts

Immobilized chloroplasts and Clostridium butyricum were employed for a photochemical energy conversion system. Spinach chloroplasts were immobilized in 2% agar gel. The optimum temperature of immobilized chloroplasts was 30°C. The maximum activity was obtained in the phosphate buffer solution (pH 8.0) containing 8μM of ferredoxin under an N₂ bubbling condition. Hydrogen was evolved under illumination by immobilized chloroplasts and C. butyricum. Hydrogen produced by this system was applied to a hydrogen-oxygen fuel cell. Photoinduced current was obtained from this photochemical energy conversion system. A photocurrent of 0.4?1.5 mA was continuously obtained for 4 h. The conversion ratio from hydrogen to current was 80?100%.     

Understanding the pH-dependent immobilization efficacy of feruloyl esterase-C on mesoporous silica and its structure–activity changes

The purpose of the present investigation was to study the pH dependence of both the immobilization process and the enzyme activity of a feruloyl esterase (FoFaeC from Fusarium oxysporum) immobilized in mesoporous silica. This was done by interpreting experimental results with theoretical molecular modeling of the enzyme structure. Modeling of the 3D structure of the enzyme together with calculations of the electrostatic surface potential showed that changes in the electrostatic potential of the protein surface were correlated with the pH dependence of the immobilization process. High immobilization yields were associated with an increase in pH. The transesterification activity of both immobilized and free enzyme was studied at different values of pH and the optimal pH of the immobilized enzyme was found to be one unit lower than that for the free enzyme. The surface charge distribution around the binding pocket was identified as being a crucial factor for the accessibility of the active site of the immobilized enzyme, indicating that the orientation of the enzyme inside the pores is pH dependent. Interestingly, it was observed that the immobilization pH affects the specific activity, irrespective of the changes in reaction pH. This was identified as a pH memory effect for the immobilized enzyme. On the other hand, a change in product selectivity of the immobilized enzyme was also observed when the transesterification reaction was run in MOPS buffer instead of citrate phosphate buffer. Molecular docking studies revealed that the MOPS buffer molecule can bind to the enzyme binding pocket, and can therefore be assumed to modulate the product selectivity of the immobilized enzyme toward transesterification.     

Purification and some properties of glyceraldehyde 3-phosphate dehydrogenase fromSynechococcus sp.

Glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.13) was purified 386 fold to apparent homogeneity from the thermophilic cyanobacteriumSynechococcus sp. grown at optimum light intensities in batch cultures. The molecular mass of the tetrameric form of the enzyme was 160 kDa as determined by gel filtration and sucrose gradient centrifugation in a phosphate buffer containing DTT. The pH optimum for the oxidation of NADPH was broad (6–8) and the enzyme had a pI of 4.5. The turnover number was 36,000 min^–1 at 40° C. The activation energy was 12.4 Kcal for t>29° C and 20.6 Kcal for t<29° C. The specific absorption coefficient, A _{280 mm} ^{1% 1cm} of the pure enzyme in phosphate buffer at pH 6.8 was 15.2.By SDS gel electrophoresis molecular masses of 78 kDa and 39 kDa were found, indicating that the purified enzyme is a tetramer, probably a homotetramer.When Tris was used as buffer in the homogenization and phosphate and DTT were omitted, a high molecular form with a molecular mass above 500 kDa was found. This form was less active than the purified tetrameric form. Acetone and other organic solvents stimulated the native enzyme several fold.     

Electrochemically induced oxidative damage to double-stranded calf thymus DNA adsorbed on gold electrodes

Electrochemically induced oxidative damage to DNA was studied with double-stranded calf thymus DNA immobilized directly on a gold electrode surface. Pre-polarization of the DNA-modified electrodes at +0.5 V versus Ag/AgCl reference electrode, in a free from DNA blank buffer solution, pH 7.4, allowed for subsequent detection of direct electrochemical oxidation of adsorbed on gold DNA, in the potential range from +0.7 to +0.8 V. The redox potential of the process corresponded to the potentials of the oxidation of guanine bases in DNA. It is shown that with increasing potential scan rate, v, the mechanism of electrochemical oxidation of DNA changes from the irreversible 4e^– oxidative damage of DNA at low v to reversible 1e^– oxidation at high v, keeping the electrochemical activity of the adsorbed DNA layer virtually the same.     

Direct electrochemistry and electrocatalysis of glucose oxidase immobilized on glassy carbon electrode modified by Nafion and ordered mesoporous silica-SBA-15

In this paper, it was found that glucose oxidase (GOD) has been stably immobilized on glassy carbon electrode modified by ordered mesoporous silica-SBA-15 and Nafion. The sorption behavior of GOD immobilized on SBA-15 matrix was characterized by transmission electron microscopy (TEM), ultraviolet–visible (UV–vis), FTIR, respectively, which demonstrated that SBA-15 can facilitate the electron exchange between the electroactive center of GOD and electrode. The direct electrochemistry and electrocatalysis behavior of GOD on modified electrode were characterized by cyclic voltammogram (CV) which indicated that GOD immobilized on Nafion and SBA-15 matrices displays direct, nearly reversible and surface-controlled redox reaction with an enhanced electron transfer rate constant of 3.89 s⁻¹ in 0.1 M phosphate buffer solution (PBS) (pH 7.12). Furthermore, it was also discovered that, in the absence of O₂, GOD immobilized on Nafion and SBA-15 matrices can produce a wide linear response to glucose in the positive potential range. Thus, Nafion/GOD-SBA-15/GC electrode is hopeful to be used in the third non-mediator's glucose biosensor. In addition, GOD immobilized on SBA-15 and Nafion matrices possesses an excellent bioelectrocatalytic activity for the reduction of O₂. The Nafion/GOD-SBA-15/GC electrode can be utilized as the cathode in biofuel cell.     

Polyphenol biosensor based on laccase immobilized onto silver nanoparticles/multiwalled carbon nanotube/polyaniline gold electrode

Laccase purified from Ganoderma sp. was immobilized covalently onto electrochemically deposited silver nanoparticles (AgNPs)/carboxylated multiwalled carbon nanotubes (cMWCNT)/polyaniline (PANI) layer on the surface of gold (Au) electrode. A polyphenol biosensor was fabricated using this enzyme electrode (laccase/AgNPs/cMWCNT/PANI/Au electrode) as the working electrode, Ag/AgCl as the reference electrode, and platinum (Pt) wire as the auxiliary electrode connected through a potentiostat. The biosensor showed optimal response at pH 5.5 (0.1 M acetate buffer) and 35 °C when operated at a scan rate of 50 mV s⁻¹. Linear range, response time, and detection limit were 0.1–500 μM, 6 s, and 0.1 μM, respectively. The sensor was employed for the determination of total phenolic content in tea, alcoholic beverages, and pharmaceutical formulations. The enzyme electrode was used 200 times over a period of 4 months when stored at 4 °C. The biosensor has an advantage over earlier enzyme sensors in that it has no leakage of enzyme during reuse and is unaffected by the external environment due to the protective PANI microenvironment.     

Electroreduction of O(2) to water at 0.6 V (SHE) at pH 7 on the "wired" Pleurotus ostreatus laccase cathode

O(2) was electroreduced to water at 0.6 V (SHE) near neutral pH on the "wired" Pleurotus ostreatus laccase cathode. We previously reported high-current density (5 mA cm(-2)), four-electron electroreduction of O(2) to water on a "wired" Coriolus hirsutus laccase electrode at +0.7 V (SHE) in pH 5 in citrate buffer. Since the enzyme was inhibited by chloride and because its activity declined steeply when the pH was raised to neutral, the rate of O(2) electroreduction in a physiological buffer solution was only approximately 1% of that at pH 5 in absence of chloride. Here we show that substitution of the C. hirsutus laccase by laccase from P. ostreatus allows the upward extension of the pH range of O(2) electroreduction. The current density of the electrode made with laccase from P. ostreatus in pH 7 citrate buffer was approximately 100 microA cm(-2) and at pH 7 and in phosphate buffered NaCl (PBS, 20 mM phosphate, 0.1 M NaCl) it still retained 6% of its maximal (1 mA cm(-2)) current density at pH 5 in citrate buffer. The electrocatalyst consisted of the crosslinked P. ostreatus laccase and the electron conducting redox polymer PVI-Os(dmebpy)(tpy)(2+/3+) [PVI=poly(N-vinyl imidazole) with about 1/5th of the rings complexed with (Os-dmebpy-tpy)(2+/3+); dmebpy=4,4'-dimethyl-2,2'-bipyridine; tpy=2,2',6',2"-terpyridine].     

Immobilization of d-glucose oxidase onto a hydrogen peroxide permselective membrane and application for an enzyme electrode

A hydrogen peroxide permselective membrane with asymmetric structure was prepared and d-glucose oxidase (EC 1.1.3.4) was immobilized onto the porous layer. The activity of the immobilized d-glucose oxidase membrane was 0.34 units cm^?2 and the activity yield was 6.8% of that of the native enzyme. Optimum pH, optimum temperature, pH stability and temperature stability were found to be pH 5.0, 30–40°C, pH 4.0–7.0 and below 55°C, respectively. The apparent Michaelis constant of the immobilized d-glucose oxidase membrane was 1.6 × 10^?3 mol l^?1 and that of free enzyme was 4.8 × 10^?2 mol l^?1. An enzyme electrode was constructed by combination of a hydrogen peroxide electrode with the immobilized d-glucose oxidase membrane. The enzyme electrode responded linearly to d-glucose over the concentration 0–1000 mg dl^?1 within 10 s. When the enzyme electrode was applied to the determination of d-glucose in human serum, within day precision (CV) was 1.29% for d-glucose concentration with a mean value of 106.8 mg dl^?1. The correlation coefficient between the enzyme electrode method and the conventional colorimetric method using a free enzyme was 0.984. The immobilized d-glucose oxidase membrane was sufficiently stable to perform 1000 assays (2 to 4 weeks operation) for the determination of d-glucose in human whole blood. The dried membrane retained 77% of its initial activity after storage at 4°C for 16 months.     

Yeast alcohol dehydrogenase immobilized in a glutaraldehyde–albumin matrix: Kinetics and cofactor diffusional effects*

This study was carried out to define how the overall rate of reaction would be influenced by different degrees of diffusional resistance to cofactor transport within an oxidoreductase membrane matrix. To accomplish this, 0.7–6.6μM yeast alcohol dehydrogenase was immobilized in an albumin matrix crosslinked with 2.5 or 5.0% glutaraldehyde to give 102–1685 μM thick membranes. The enzyme half-life was at least doubled at pH 7.5 or 8.8 on immobilization. Values of the kinetic constants for the soluble and immobilized enzyme were determined at 25°C and pH 8.8 over the range of 0.01–1.0M bulk solution concentration of ethanol as substrate and 140–1000μM bulk solution concentration of nicotinamide adenine dinucleotide (NAD⁺) as cofactor, to give essentially single substrate kinetics in NAD⁺. Equilibrium partitioning of ethanol and NAD⁺ between the solution and membrane was measured and used in the data analysis. The four kinetic constants for the soluble enzyme agreed with literature values; and all increased with immobilization of the enzyme. The Michaelis constants for NAD⁺ and for ethanol were greater for the immobilized enzyme. The diffusional resistance to NAD⁺ transport, presented in terms of the Thiele modulus, showed that the overall rate of reaction was decreased by about 50% even at values of the modulus as low as 2.0.     

Third-generation superoxide anion sensor based on superoxide dismutase directly immobilized by sol-gel thin film on gold electrode

A third-generation biosensor for superoxide anion (O(2)*-) was developed based on superoxide dismutase (SOD) immobilized by thin silica-PVA sol-gel film on gold electrode surface. A rapid and direct electron transfer of SOD in the thin sol-gel film at the gold electrode was realized without any mediators or promoters. The characterization of the SOD electrodes showed a quasi-reversible electrochemical redox behavior with a formal potential of 80 + 5 mV (versus SCE) in 50 mmol l(-1) phosphate buffer solution (PBS), pH 7.0. The heterogeneous electron transfer rate constant was evaluated to be about 2.1s(-1). The anodic and cathodic transfer coefficients are 0.6 and 0.4, respectively. Based on biomolecular recognition for specific reactivity of SOD toward O(2)*- the SOD electrode was applied to a sensitive and selective measurement of O(2)*- with the low operation potential (-0.15 V versus SCE) in phosphate buffer solution, pH 7.0. The amperometric response was proportional to O(2)*- concentration in the range of 0.2-1.6 micromol l(-1) and the detection limit was 0.1 micromol l(-1) at a signal-to-noise ration of 3. The preparation of SOD electrode is easy and simple. The uniform porous structure of the silica-PVA sol-gel matrix results in a fast response rate of immobilized SOD and is very efficient for stabilizing the enzyme activity.