Immobilization of tyrosinase in polysiloxane/polypyrrole copolymer matrices

Immobilization of tyrosinase in conducting copolymer matrices of pyrrole functionalized polydimethylsiloxane/polypyrrole (PDMS/PPy) was achieved by electrochemical polymerization. The polysiloxane/polypyrrole/tyrosinase electrode was constructed by the entrapment of enzyme in conducting matrices during electrochemical copolymerization. Maximum reaction rate (V(max)) and Michaelis-Menten constant (K(m)) were investigated for immobilized enzyme. Enzyme electrodes were prepared in two different electrolyte/solvent systems. The effect of supporting electrolytes, p-toluene sulfonic acid and sodium dodecyl sulfate on the enzyme activity and film morphology were determined. Temperature and pH optimization, operational stability and shelf-life of enzyme electrodes were also examined. Phenolic contents of green and black tea were determined by using enzyme electrodes.     

A new amperometric enzyme electrode for alcohol determination

A new enzyme electrode for the determination of alcohols was developed by immobilizing alcohol oxidase in polvinylferrocenium matrix coated on a Pt electrode surface. The amperometric response due to the electrooxidation of enzymatically generated H(2)O(2) was measured at a constant potential of +0.70 V versus SCE. The effects of substrate, buffer and enzyme concentrations, pH and temperature on the response of the electrode were investigated. The optimum pH was found to be pH 8.0 at 30 degrees C. The steady-state current of this enzyme electrode was reproducible within +/-5.0% of the relative error. The sensitivity of the enzyme electrode decreased in the following order: methanol>ethanol>n-butanol>benzyl alcohol. The linear response was observed up to 3.7 mM for methanol, 3.0 mM for ethanol, 6.2 mM for n-butanol, and 5.2 mM for benzyl alcohol. The apparent Michaelis-Menten constant (K(Mapp)) value and the activation energy, E(a), of this immobilized enzyme system were found to be 5.78 mM and 38.07 kJ/mol for methanol, respectively.     

Biosensing approach for alcohol determination using immobilized alcohol oxidase

Alcohol oxidase (AOD) was immobilized in polypyrrole (PPy) and a random copolymer containing 3-methylthienyl methacrylate and p-vinylbenzyloxy poly(ethyleneoxide) matrices. Immobilization of enzyme was performed via entrapment in conducting polymers during electrochemical polymerization of pyrrole through the thiophene moiety of the copolymer. Three different alcohols, namely methanol, ethanol and n-propanol, were used as substrates. Maximum reaction rates, Michaelis–Menten constants, optimum temperature and pH values, operational stabilities and shelf life of the enzyme electrodes were investigated.     

Electrodeposited nonconducting polytyramine for the development of glucose biosensors

Biosensors with the composition of carbon/Prussian blue/(glucose oxidase+glutaraldehyde+polytyramine) were constructed. Before tyramine monomers were electropolymerized, glucose oxidase and tyramine monomers were cross-linked with glutaraldehyde onto the surface of Prussian-blue-modified electrodes. The constructed biosensors produced highly reproducible and stable devices. The biosensors exhibited neglectable decrease in current response after 10 repeated uses or after 1 month of dry storage. The resultant biosensors had a linear range of 0.1-1 mM glucose and a detection limit of 0.05 mM. Since the following electrocatalytic process proceeds at a low electrode potential (ca. -0.3 V vs Ag/AgCl), ascorbate and uric acid do not produce observable interfering signal for the determination of glucose.     

Amperometric alcohol electrode with extended linearity and reduced interferences.

Conventional amperometric alcohol electrodes were constructed with oxygen- and hydrogen peroxide-base sensors and a much improved electrode was designed by placing a hydrophobic, gas-permeable membrane over the conventional hydrogen peroxide-based alcohol electrode. The immobilization of alcohol oxidase with glutaraldehyde was also studied and optimized. The upper linear ranges of the conventional and newly designed alcohol electrodes were 0.02 and 0.5% ethanol, respectively. The hydrophobic membrane of the new design eliminated the classical electrochemical interferences of hydrogen peroxide-based electrodes and the typical pH dependence of enzymatic systems.     

A novel matrix for the immobilization of acetylcholinesterase

In this study, a new matrix for immobilization of acetylcholinesterase was investigated by using alginate and kappa-carrageenan. The effects of pH, temperature, storage and thermal stability on the free and immobilized acetylcholinesterase activity were examined. Maximum reaction rate (V(max)) and Michaelis-Menten constant (K(m)) was also investigated for free and immobilized enzymes. For free and immobilized enzymes into Ca-alginate and alginate/kappa-carrageenan polymer blends, optimum pH and temperature was found to be 7 and 30 degrees C, respectively. For free enzyme, maximum reaction rate (V(max)) and Michaelis-Menten constant (K(m)) values were found to be 6.35 mM and 50 mM min(-1), respectively, the same values for immobilized enzymes were determined as 8.68, 12.7 mM and 39.7, 52.9 mM min(-1), respectively. Storage and thermal stability of acetylcholinesterase was increased by as a result of immobilization.     

Various applications of immobilized glucose oxidase and polyphenol oxidase in a conducting polymer matrix

In this study, glucose oxidase and polyphenol oxidase were immobilized in conducting polymer matrices; polypyrrole and poly(N-(4-(3-thienyl methylene)-oxycarbonyl phenyl) maleimide-co-pyrrole) via electrochemical method. Fourier transform infrared and scanning electron microscope were employed to characterize the copolymer of (N-(4-(3-thienyl methylene)-oxycarbonyl phenyl) maleimide) with pyrrole. Kinetic parameters, maximum reaction rate and Michealis-Menten constant, were determined. Effects of temperature and pH were examined for immobilized enzymes. Also, storage and operational stabilities of enzyme electrodes were investigated. Glucose and polyphenol oxidase enzyme electrodes were used for determination of the glucose amount in orange juices and human serum and phenolic amount in red wines, respectively.     

Immobilization of glucose oxidase in polypyrrole/polytetrahydrofuran graft copolymers

Glucose oxidase (GOD) was immobilized in four different conducting polymer matrices, namely: polypyrrole, (PPy), poly(pyrrole-graft-polytetrahydrofuran), (1) and (3); and poly(pyrrole-graft-polystyrene/polytetrahydrofuran), (2). The kinetic parameters V(max) and K(m), and the optimum temperature were determined for both immobilized and native enzymes. The effect of electrolysis time and several supporting electrolytes, p-toluenesulfonic acid, p-toluene sulfonic acid (PTSA), sodium p-toluene sulfonate, sodium p-toluene sulfonate (NaPTS), and sodium dodecyl sulfate, sodium dodecyl sulfate (SDS), on enzyme immobilization were investigated. The high K(m) value (59.9 mM) of enzyme immobilized in PPy was decreased via immobilization in graft copolymer matrices of pyrrole. V(max), which was 2.25 mM/min for pure PPy, was found as 4.71 mM/min for compound (3).     

Postnatal ontogeny of kinetics of porcine jejunal brush border membrane-bound alkaline phosphatase,aminopeptidase N and sucrase activities

Our objectives were to determine postnatal changes in the maximal enzyme activity (V(max)) and enzyme affinity (K(m)) of jejunal mucosal membrane-bound alkaline phosphatase, aminopeptidase N and sucrase using a porcine model which may more closely resemble the human intestine. Jejunal brush border membrane was prepared by Mg(2+)-precipitation and differential centrifugation from pigs of suckling (8 days), weaning (28 days), post-weaning (35 days) and adult (70 days) stages. p-Nitrophenyl phosphate (0-8 mM), L-alanine-p-nitroanilide hydrochloride (0-28 mM) and sucrose (0-100 mM) were used in alkaline phosphatase, aminopeptidase N and sucrase kinetic measurements. V(max) of alkaline phosphatase was the lowest in the adult (4.27 micromol.mg(-1) protein.min(-1)), intermediate in the suckling (9.75 micromol.mg(-l) protein.min(-l)) and the highest in the weaning and post-weaning stage (12.83 and 10.40 micromol.mg(-l) protein.min(-l)). K(m) of alkaline phosphatase was high in the suckling and weaning stages (5.14 and 9.93 mM) and low in the adult (0.66 mM). V(max) of aminopeptidase N was low in the suckling (7.04 micromol.mg protein(-1).min(-1)) and high in the post-weaning stage (13.36 micromol.mg(-l) protein.min(-l)). K(m) of aminopeptidase N was the highest in the two weaning stages (2.96 and 3.39 mM), intermediate in the adult (2.33 mM) and the lowest in the suckling stage (1.66 mM). V(max) of sucrase increased from the suckling to the adult (0.48-1.30 micromol.mg(-l) protein.min(-l)). K(m) of sucrase ranged from 11.19 to 16.57 mM. There are dramatic postnatal developmental changes in both the maximal enzyme activity and enzyme affinity of jejunal brush border membrane-bound alkaline phosphatase, aminopeptidase N and sucrase in the pig.     

Purification and characterization of carbonic anhydrase from bovine stomach and effects of some known inhibitors on enzyme activity

Carbonic anhydrase (CA) was purified from four different cell localisation (outer peripheral, cytosolic, inner peripheral and integral) in bovine stomach using affinity chromatography with Sepharose-4B-L-tyrosine sulphanilamide. During the purification steps, the activity of the enzyme was measured using p-nitrophenyl acetate at pH 7.4. Optimum pH and optimum temperature values for all CA samples were determined, and their K(m) and V(max) values for the same substrate by Lineweaver-Burk graphics. The extent of purification for all CA localizations was controlled by SDS-PAGE. The K(m) values at optimum pH and 20 degrees C were 0.625 mM, 0.541 mM, 0.785 mM and 0.862 mM with p-nitro phenyl acetate, for all CA localizations. The respective V(max) values at optimum pH and 20 degrees C were 0.875 micromol/L min, 0.186 micromol/L min, 0.214 micromol/L min and 0.253 micromol/L min with the same substrate. The K(i) and I50 values for the inhibitors sulphanilamide, KSCN, NaN3 and acetazolamide were determined for all the CA localizations.     

Characterization of kinetics and thermostability of Acremonium strictum glucooligosaccharide oxidase

The kinetic and thermostability properties of a glucooligosaccharide oxidase from Acremonium strictum were determined. This enzyme produces only maltobionic acid from maltose. It is most active at pH 9 to 10.5, and is most stable at pH 6.5. Values of both K(M) and V(max) on maltose are highest at pH 10. The highest values of K(M) and V(max) occur with glucose, maltopentaose, and maltoheptaose, whereas the lowest values of K(M) are with maltotriose and of V(max) are with maltohexaose. Values of K(M) with any substrate and at any pH are always substantially above 1 mM. Activation energies for catalysis and thermoinactivation are 23 kJ/mol and 421 kJ/mol, respectively. The N-terminal sequence is not homologous with any other oxidase, but has some homology with other proteins having different functions. These unusual properties suggest that glucooligosaccharides may not be the primary substrates of this enzyme.     

Immobilization of glucose oxidase in conducting graft copolymers and determination of glucose amount in orange juices with enzyme electrodes

Glucose oxidase was immobilized in conducting copolymers of three different types of poly(methyl methacrylate-co-thienyl methacrylate). Immobilization of enzyme was carried out by the entrapment in conducting polymers during electrochemical polymerization of pyrrole on the copolymer electrodes. Maximum reaction rate, Michaelis-Menten constants, temperature, pH and operational stabilities were determined for immobilized enzyme. The amount of glucose in orange juices of Turkey was investigated by using enzyme electrodes.     

Fabrication of bienzyme nanobiocomposite electrode using functionalized carbon nanotubes for biosensing applications

Mediated biosensors consisting of an oxidase and peroxidase (POx) have attracted increasing attention because of their wider applicability. This work presents a novel approach to fabricate nanobiocomposite bienzymatic biosensor based on functionalized multiwalled carbon nanotubes (MWNTs) with the aim of evaluating their ability as sensing elements in amperometric transducers. Electrochemical behavior of the bienzymatic nanobiocomposite biosensor is investigated by Faradaic impedance spectroscopy and cyclic voltammetry. The results indicate that glucose oxidase (GOD) and horseradish peroxidase (HRP) are strongly adsorbed on the surface of the thionin (TH) functionalized MWNTs and demonstrate a facile electron transfer between immobilized GOD/HRP and the electrode via the functionalized MWNTs in a Nafion film. The functionalized carbon nanotubes act as molecular wires to allow efficient electron transfer between the underlying electrode and the redox centres of enzymes through TH. Linear ranges for these electrodes are from 10 nM to 10 mM for glucose and 17 nM to 56 mM for hydrogen peroxide with the detection limit of 3 and 6 nM, respectively. A remarkable feature of the bienzyme electrode is the possibility to determine glucose and hydrogen peroxide at a very low applied potential where the noise level and interferences from other electroactive compounds are minimal. Performance of the biosensor is evaluated with respect to response time, detection limit, selectivity, temperature and pH as well as operating and storage stability.     

Physiological and enzymatic properties of the ram epididymal soluble form of germinal angiotensin I-converting enzyme.

The 94-kDa ram epididymal fluid form of the sperm membrane-derived germinal angiotensin I-converting enzyme (ACE) was purified by chromatography, and some of its enzymatic properties were studied. For the artificial substrate furanacryloyl-L-phenylalanylglycylglycine (FAPGG), the enzyme exhibited a Michaelis constant (K(m)) of 0.18 mM and a V(max) of 34 micromoles/(min x mg) and for hippuryl-L-histidyl-L-leucine a K(m) of 2.65 mM and a V(max) of 163 micromoles/(min x mg) under the defined standard conditions (300 mM NaCl and 50 mM Tris; pH 7.5 and 8.3, respectively). The FAPGG hydrolysis was decreased by 82.5% and 67.5% by EDTA and dithioerythritol, respectively, and was totally inhibited by specific ACE inhibitors such as captopril, P-Glu-Trp-Pro-Arg-Pro-Glu-Ile-Pro-Pro, and lisinopril. Optimum activity for FAPGG was with pH 6.0, 50 mM chloride, and 500 microM zinc. Under the various conditions tested, bradykinin, angiotensin (Ang) I, Ang II, and LHRH were competitors for FAPGG. Bradykinin and angiotensin I were the best competitors. The enzyme cleaved Ang I into Ang II, and the optimal conditions were with pH 7.5 and 300 mM chloride. The relationship between the carboxypeptidase activity in seminal plasma and the prediction of fertility of young rams was also studied. These results indicated a correlation between sperm concentration and ACE activity in semen but showed no statistically significant correlation between such activity and fertility of the animal. Finally, we tested the role of ACE in fertilization; no difference in the in vitro fertilization rate was observed in the presence of 10(-4) M captopril.     

Alcohol biosensing by polyamidoamine (PAMAM)/cysteamine/alcohol oxidase‐modified gold electrode

A highly stable and sensitive amperometric alcohol biosensor was developed by immobilizing alcohol oxidase (AOX) through Polyamidoamine (PAMAM) dendrimers on a cysteamine‐modified gold electrode surface. Ethanol determination is based on the consumption of dissolved oxygen content due to the enzymatic reaction. The decrease in oxygen level was monitored at ?0.7 V vs. Ag/AgCl and correlated with ethanol concentration. Optimization of variables affecting the system was performed. The optimized ethanol biosensor showed a wide linearity from 0.025 to 1.0 mM with 100 s response time and detection limit of (LOD) 0.016 mM. In the characterization studies, besides linearity some parameters such as operational and storage stability, reproducibility, repeatability, and substrate specificity were studied in detail. Stability studies showed a good preservation of the bioanalytical properties of the sensor, 67% of its initial sensitivity was kept after 1 month storage at 4°C. The analytical characteristics of the system were also evaluated for alcohol determination in flow injection analysis (FIA) mode. Finally, proposed biosensor was applied for ethanol analysis in various alcoholic beverage as well as offline monitoring of alcohol production through the yeast cultivation. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Glucose oxidase-polypyrrole electrodes synthesized in p-toluenesulfonic acid and sodium p-toluenesulfonate

Amperometric glucose biosensors have been developed based on entrapment on platinum (Pt) electrode using cyclic voltammetry technique in glucose oxidase (GOD) and pyrrole containing p-toluenesulfonic acid (pTSA) or sodium p-toluenesulfonate (NapTS) as supporting electrolyte solutions. Both of electrolyte solutions were suitable media for the formation and deposition of polypyrrole-GOD (PPy-GOD) layers on Pt substrate. Pt/PPy-GOD electrodes brought about in different morphological properties as well as different electrochemical and biochemical response. The highest responses obtained in pTSA and NapTS electrolytes were observed at pH of 4.5 and 7.0 for Pt/PPy-GOD electrodes, respectively. While linearity was observed between 0.0-1.0 mM glucose substrate for both electrodes, I(max) value of Pt/PPy-GOD(NapTS) electrode was approximately twice as high as that of Pt/PPy-GOD(pTSA) electrode as 25.4 and 14.2 microA, respectively. Five commercial drinks were tested with enzyme electrodes and compared with results obtained spectrophotometrically using glucose kit. Results revealed that Pt/PPy-GOD(NapTS) electrode exhibited better biosensor response.     

Extended shelf life of enzyme-based biosensors using a novel stabilization system

The storage stability of amperometric enzyme electrodes has been enhanced by a combination of a soluble, positively charged polymer, diethylaminoethyl (DEAE)-dextran, and a sugar alcohol, lactitol. Two different types of alcohol biosensor have been produced using the enzyme alcohol oxidase, isolated from the methylotrophic yeast Hansenula polymorpha. The first employs enzyme entrapment between two membranes with direct hydrogen peroxide amperometry at +0·65 V. The second was based on the mediated, coupled reaction with horseradish peroxidase and N-methyl phenazimiumtetracyanoquinonedimethane (NMP-TCNQ) on a graphite electrode. In both cases, addition of the stabilizers promoted a considerable increase in the storage stability of the enzyme component, as indicated by an increase in the shelf life of desiccated biosensors under conditions of thermal stress at 37°C. In addition, an L-glutamate biosensor constructed from NMP-TCNQ-modified graphite electrodes and L-glutamate oxidase also exhibited an increase in shelf life when stored, desiccated in the presence of stabilizers.     

Development of acetylcholine sensor using carbon fiber (amperometric determination)

An enzyme sensor is developed using carbon fiber to measure acetylcholine concentration. The mechanism is based on the detection of H₂O₂ which is a product of the sequential enzyme reactions of acetylcholinesterase and choline oxidase. The fabrication of the electrode is described. The sensor is polarized at 1.2 V. Enzymes are co-immobilized in polyvinyl alcohol containing styryl pyrydinium (photo-crosslinkable polymer). A fast response time of 0.8 min is obtained. A linear correlation is observed between 0.2 and 1.0 mM. Other optimal operational conditions with respect to pH, temperature and stability are discussed. The use of carbon fiber containing co-immobilized enzymes could offer several model advantages especially in neuroscience research. In conclusion, the aims of the present work are centered on carbon fiber electrode fabrication, immobilization electrochemical measurements.     

Immobilization of Enzyme into Poly(vinyl alcohol) Membrane

Glucoamylase, invertase, and cellulase were entrapped within poly(vinyl alcohol) (PVA) membrane cross-linked by means of irradiation of ultraviolet light. The conditions for immobilization of glucoamylase were examined with respect to enzyme concentration in PVA, sensitizer (sodium benzoate) concentration in PVA, irradiation time, and membrane thickness. Various characteristics of immobilized glucoamylase were evaluated. Among them, the pH activity curve for the immobilized enzyme was superior to that for the native one, and thermal stability was improved by immobilization with bovine albumin. The apparent K(m) was larger for immobilized glucoamylase than for the native one, while V(max) was smaller for the immobilized enzyme. Also, the apparent K(m) appeared to be affected by the molecular size of the substrate. Further, immobilized invertase and cellulase showed good stabilities in repeating usage.     

Purification and characterization of a dimethoate-degrading enzyme of Aspergillus niger ZHY256, isolated from sewage

A dimethoate-degrading enzyme from Aspergillus niger ZHY256 was purified to homogeneity with a specific activity of 227.6 U/mg of protein. The molecular mass of the purified enzyme was estimated to be 66 kDa by gel filtration and 67 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was found to be 5.4, and the enzyme activity was optimal at 50 degrees C and pH 7.0. The activity was inhibited by most of the metal ions and reagents, while it was induced by Cu(2+). The Michaelis constant (K(m)) and V(max) for dimethoate were 1.25 mM and 292 micromol min(-1) mg of protein(-1), respectively.