Polystyrene Attached Pt(IV)�CAzomethine, Synthesis and Immobilization of Glucose Oxidase Enzyme

Modified polystyrene with Pt(IV)–azomethine (APS–Sch–Pt) was synthesized by means of condensation and demonstrated to be a promising enzyme support by studying the enzymatic properties of glucose oxidase enzyme (GOx) immobilized on it. The characteristics of the immobilized glucose oxidase (APS–Sch–Pt–GOx) enzyme showed two optimum pH values that were pH = 4.0 and pH = 7. The insertion of stable Pt(IV)–azomethine spacers between the polystyrene backbone and the immobilized GOx, (APS–Sch–Pt–GOx), increases the enzymes’ activity and improves their affinity towards the substrate even at pH = 4. The influence of temperature, reusability and storage capacity on the free and immobilized glucose oxidase enzyme was investigated. The storage stability of the immobilized glucose oxidase was shown to be eleven months in dry conditions at +4 °C.     

纤维素硫酸钠的批量制备及其NaCS-PDMDAAC微胶囊固定化黄色短杆菌培养过程研究

在非均相反应制备纤维素硫酸钠方法的基础上,通过增加液固比,引入搅拌,使体系的温度分布和浓度分布都得到很大改进,由此可以保证产品质量的均一性。用正交试验进行了500 mL反应器制备条件的摸索,并将其逐步放大到1 L、5 L反应器中进行,确立了5 L规模的生产工艺条件。通过对不同规模下制得的硫酸纤维素钠产品在理化性质、制备微胶囊及在微囊化培养等方面对比较,表明研制过程有利于合成出高质量的硫酸纤维素钠,用于制备微胶囊。用NaCS-PDMDAAC(聚二甲基二烯丙基氯化铵—硫酸纤维素钠)微胶囊固定化黄色短杆菌,考察了葡萄糖浓度、摇床转速对黄色短杆菌生长和代谢谷氨酸的影响,确定了一个较优培养条件。胶囊平均生产能力达15.29 g /(Lh)(对微胶囊)。微胶囊固定化黄色短杆菌连续培养12批后,仍具有良好的代谢谷氨酸性能。     

Porous Poly(vinyl alcohol) Composite Membranes for Immobilization of Glucose Oxidase

Particle loaded porous poly(vinyl alcohol) composite membranes were selected for immobilization of glucose oxidase (GOx) for their hydrophilicity and unique interactions with amino functional groups. GOx was immobilized on the membranes by adsorption at pH values between 3.5 and 7.1. The highest adsorption loading was observed at pH 7.1 and the highest catalytic activity was observed at pH 5.1. Infrared studies showed that the highest ratio of amide I to amide II at pH 5.1 is obtained for GOx immobilized on membranes loaded with amine-functionalized micro-particles, suggesting that the conformational changes of GOx on these membranes yield to higher catalytic activity than in other supports.

     

The effect of glucose oxidase enzyme on wool fibres

Glucose oxidase is a type of enzyme that converts glucose into hydrogen peroxide and gluconic acid by enzymatic reaction. Glucose oxidase is widely used in industry; however, in the textile industry, glucose oxidase has only received academic interest. Previously, wool was bleached by some reducing agents; however, currently in industry, hydrogen peroxide dominates the bleaching of wool fibres. In this study, the effect of glucose oxidase enzyme treatment on wool merino fibres and dyeability properties was investigated. Wool fibres were treated with glucose oxidase enzyme, after which the whiteness index (Stensby) and yellowness index (ASTM D 1925 and ASTM E 313) were investigated. Scanning electron microscopy and scanning electron microscopy with energy dispersive X-ray spectroscopy were used to identify the morphological structure of wool fibres and their atomic content. The chemical damage caused by enzyme was investigated using a fluorescence and a light microscope, and the alkali solubility (ASTM D 1283) was determined. After enzymatic treatment, the wool fibres were dyed at a 2.0% concentration with reactive dyes. Dyeability (K/S) and CIELab values were assessed with a Minolta CM 3600 D spectrophotometer (D65, 10°). The washing fastness of wool fibres was investigated according to TS EN ISO 105-C06 (A1S).     

Preparation of glucose oxidase membrane for the application of glucose sensor by plasma activation

Plasma activation is a method that takes the advantage of low temperature plasma to immobilize the bioactive materials. An active immobilized glucose oxidase membrane was obtained via plasma-initiated polymerization of acrylic acid. The obtained immobilized enzyme is very active and stable. After 100 tests, the response error of the glucose sensor is less than 5% and its linear detecting range is 0–300 ppm. A hydrophilic PP film treated by Ar or NH₃ plasma was used for the preparation of the immobilized glucose oxidase membrane. The obtained immobilized enzyme is also very active and stable. After 180 tests, the response error of the glucose sensor is less than 4% and its linear detecting range is 0–300 ppm. Furthermore, SEM was used to study the mophology of the glucose oxidase membranes obtained via both methods. And ESCA was used to analyze the plasm-initiated polymerization products and the plasma-treated PP films so as to obtain the optimum conditions for the immobilization of bioactive materials.     

Preparation of glucose oxidase immobilized in different carriers using radiation polymerization

Glucose oxidase (EC 1.1.3.4) was immobilized on different polymeric materials using different immobilization techniques (entrapping by γ‐irradiation, and covalent binding using epichlorohydrin). Studies were carried out to increase the thermal stability of glucose oxidase (GOD) for different applications. The activity and stability of the resulting biopolymers have been compared with those of free GOD. The effect of different polyvinyl alcohol/polyacrylamide (PVA/PAAm) compositions of the copolymer carrier on the enzymatic activity of the immobilized GOD was studied. The maximum enzymatic activity was obtained with the composition ratio of PVA/PAAm of 60:40. The behaviour of the free and immobilized enzyme was analysed as a function of pH. A broadening in the pH profile (5.5–8) was observed for immobilized preparations. The activity and stability of the resulting biopolymers produced by immobilization of GOD onto different carriers have been compared, in both aqueous and organic media, with those of the free GOD. The enzyme's tolerance toward both heat and organic solvent was enhanced by immobilization onto polymers. The addition of different concentrations of organic solvents (10–50%, v/v) to the enzyme at higher temperature (60 °C) was found to stabilize the enzyme molecule. The strongest stabilizing effect on the enzymatic activity was achieved at a concentration of 10%. Copyright © 2005 Society of Chemical Industry     

The effect of oxygen upon the kinetics of glucose oxidase inactivation

The effect of oxygen upon the inactivation rate of glucose oxidase was studied. Tests on enzyme stability during catalytic turnover were conducted under a range of oxygen partial pressures. A standard activity assay was used to monitor changes in glucose oxidase activity. Studies revealed that oxygen influenced the inactivation of glucose oxidase. Inactivation rates during catalytic turnover ranged between 0.01143 +/- 0.0016 h^-1 under 10 kPa oxygen to 0.04879 +/- 0.0023 h^-1under 101 kPa oxygen. Statistically different inactivation rates were obtained at oxygen partial pressures of 10,15, 21, 51, and 76 kPa. The enzymatic turnover number decreased from 11.0 X 10⁴ to 5.7 X 10⁴when the oxygen partial pressure increased from 10 to 101 kPa, suggesting that enzyme utilization was most efficient at low oxygen partial pressures (<15 kPa).     

固定化酵母细胞发酵玉米芯酶解液生产木糖醇

用木聚糖酶对玉米芯的自水解液进行酶解以获得可发酵的木糖溶液。该法与直接用酶对玉米芯水解相比,水解速度快,木糖得率较高。所得酶解液发酵木糖醇的性能虽不如纯木糖,但明显优于玉米芯酸水解液。海藻酸钙/壳聚糖(ACA)微胶囊的最佳成膜时间和液化时间分别是18 min和20 min,微胶囊使用的环境pH范围是3～6。用微胶囊固定化细胞发酵玉米芯酶解液,重复培养了8批,平均木糖醇得率为61.4%。     

Porous Poly(vinyl alcohol) Composite Membranes for Immobilization of Glucose Oxidase

Particle loaded porous poly(vinyl alcohol) composite membranes were selected for immobilization of glucose oxidase (GOx) for their hydrophilicity and unique interactions with amino functional groups. GOx was immobilized on the membranes by adsorption at pH values between 3.5 and 7.1. The highest adsorption loading was observed at pH 7.1 and the highest catalytic activity was observed at pH 5.1. Infrared studies showed that the highest ratio of amide I to amide II at pH 5.1 is obtained for GOx immobilized on membranes loaded with amine-functionalized micro-particles, suggesting that the conformational changes of GOx on these membranes yield to higher catalytic activity than in other supports.     

Development of glucose oxidase-based bioanodes for enzyme fuel cell applications

We fabricated an enzyme fuel cell (EFC) device based on glucose as fuel and glucose oxidase (GOx) as biocatalyst. As a strategy to improve GOx stability, preserving at the same time the enzyme catalytic activity, we propose an immobilization procedure to entrap GOx in a polymer matrix based on Nafion and multiwalled carbon nanotubes. Circular dichroism (CD) spectra were recorded to study changes in the 3D structure of GOx that might be generated by the immobilization procedure. The comparison between the CD features of GOx immobilized and free in solution indicates that the shape of the spectra and position of peaks do not significantly change. The bioelectrocatalytic activity toward glucose oxidation of immobilized GOx was studied by cyclic voltammetry and chronoamperometry experiments. Such electrochemical experiments allow monitoring the rate of GOx-catalyzed glucose oxidation and extrapolating GOx kinetic parameters. Results demonstrate that immobilized GOx has high catalytic efficiency, due the maintaining of regular and well-ordered structure of the immobilized enzyme, as indicated by spectroscopic findings. Once investigated the electrode structure–property relationship, an EFC device was assembled using the GOx-based bioanode, and sulfonated poly ether ether ketone as electrolyte membrane. Polarization and power density curves of the complete EFC device were acquired, demonstrating the suitability of the immobilization strategy and materials to be used in EFCs.     

Research on the Repeated Use of Novel Ferrocene-Tagged Nanomaterial for Determination of Glucose

A good novel double ferrocene-tagged nanomaterial is designed for glucose determination and its recycling stability. Double Ferrocene-tagged nanomaterial displays higher catalytic activity toward the glucose oxidation than non–ferrocene-tagged nanomaterial. In this study novel nanoparticles including double ferrocene, N-{2-[Bis(2-aminoethyl)amino]ethyl}aminomethyl-polystyrene (2AEPS) and ferrocene aldehyde (Fc) have been synthesized by means of condensation and investigated the enzymatic properties of glucose oxidase enzyme (GOD) immobilized on there. Double ferrocene-tagged nanomaterial-GOD shows high reusability and storage capacity and fast incubation time determination of glucose. 2AEPSFc-GOD retains more than 15% of the initial activity after forty five successive cycles, which is a perfect performance.     

Polyaniline synthesis catalysed by glucose oxidase

A novel method for synthesis of polyaniline (PANI) in aqueous media based on application of oxidizing-enzyme glucose oxidase (GOx) is reported. Hydrogen peroxide was produced during catalytic reaction of oxidizing-enzyme glucose oxidase from Penicillium vitale and initiated the polymerization of aniline. The increase in optical absorbance in the range of 340-700 nm was exploited for the monitoring of PANI polymerization process. The role of GOx in the formation of PANI, influence of the initial concentrations of GOx, and glucose and aniline monomer on the aniline polymerization rate was studied. The study of pH influence on polymerization rate showed that PANI polymerization was occurring in a broad pH range from the pH 2.0 to 9.0. Optimal polymerization/oligomerization temperature was found to be at 37 °C, which is also optimal for GOx-catalysed enzymatic reaction. After 10 days of continuous GOx-catalysed polymerization PANI appeared as colloid-microparticles visible by an optical microscope.     

Immobilization strategy of accessible transmission for trypsin to catalyze synthesis of dipeptide in mesoporous support

Immobilized trypsin in mesoporous silica foams was used to catalyze dipeptide synthesis in hydrophilic organic solvent instead of soluble form. The area surface of nano support was measured. The catalytic activity, coupled yield and kinetic characterization of immobilized trypsin were examined. Bz-Arg-OEt was chosen as the acyl donor with Lys-OH as the nucleophile. The trypsin-catalyzed synthesis condition was optimized, such as catalytic temperature, pH, reaction time, physical properties and content of organic solvents, together with the added enzyme amount. The immobilized trypsin showed 112.8% of residual activity with 91.9% of coupled yield, and the kinetic parameters exhibited accessibility for transmission. The product yield of 5.8% was reached at the optimum conditions for enzymatic synthesis of dipeptide: 800 mg of wet immobilized trypsin (200 mg/g support) was used in Tris-HCl buffer (0.1 mol/L, pH 8.0) containing 80% (v/v) ethanol solvents for 6 h of reaction time at 35 °C. This attempt of immobilized strategy for trypsin in nanopores renders the possibility of wide application of inorganic nano-sized support in catalytic synthesis process, which can avoid usage of large amounts of organic solvents in washing steps by chemical methods and reduce the tedious purification process of its soluble form.     

Porous polydimethylsiloxane membranes for enzyme immobilization

Porous polydimethylsiloxane (PDMS) membranes with α-amylase or glucose oxidase activity were prepared by catalytic hydrosilylation cure of PDMS in the presence of the enzymewater solution. The pores in the membrane are the result of hydrogen foams, which are generated during the curing reaction. The enzyme reactions were examined in batch and permeation experiments by using glucose and starch solutions as substrates. For the permeation set-up, the reaction yields of the immobilized α -amylase increased as the permeation rate of the starch solution decreased. The Michaelis-Menten type of reaction kinetics for the immobilized enzyme indicated that the permeation system is effective for the diffusion through the solute of the matrix, as compared with the batch system. © 1996 John Wiley & Sons, Inc.     

Simultaneous covalent immobilization of glucose oxidase and catalase onto chemically modified acrylonitrile copolymer membranes

Ultrafiltration membranes from acrylonitrile copolymer were chemically modified with different concentrations of hydrogen peroxide (from 5 to 30% H₂O₂). The amount of the amide groups in the modified membranes was determined. The water flow and permeability coefficients of the initial and modified membranes were also researched. The modified membranes were used as carriers for covalent immobilization of the dual enzyme system of glucose oxidase and catalase (GOD+CAT). It was found that the best matrices for immobilization of the dual system were membranes modified with 20% H₂O₂ and the optimal activity ratio was GOD : CAT = 1 : 5. The glucose conversion efficiency with the dual enzyme system was twice as high as that of bound GOD alone. Some of the basic characteristics (optimum pH, optimum temperature, pH, temperature stability, and storage stability) of the dual enzyme system were determined and compared with characteristics of free and bound enzymes. The catalytic parameters of the enzyme reaction (K_m and V_max) were determined with GOD immobilized alone and with the dual system GOD+CAT. The higher rate observed with the dual enzyme system clearly showed the advantage and the efficiency of the immobilized system. Glucose oxidase without catalase was deactivated by H₂O₂ more rapidly than the immobilized dual GOD+CAT system. These experimental evidences can be explained by the protecting effect of catalase on glucose oxidase from inhibition by H₂O₂. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 91: 4057–4063, 2004     

Multicommuted flow system for the determination of glucose in animal blood serum exploiting enzymatic reaction and chemiluminescence detection

An automatic flow procedure based on multicommutation dedicated for the determination of glucose in animal blood serum using glucose oxidase with chemiluminescence detection is described. The flow manifold consisted of a set of three-way solenoid valves assembled to implement multicommutation. A microcomputer furnished with an electronic interface and software written in Quick BASIC 4.5 controlled the manifold and performed data acquisition. Glucose oxidase was immobilized on porous silica beads (glass aminopropyl) and packed in a minicolumn (15 x 5 mm). The procedure was based on the enzymatic degradation of glucose, producing hydrogen peroxide, which oxidized luminol in the presence of hexacyanoferrate(III), causing the chemiluminescence. The system was tested by analysing a set of serum animal samples without previous treatment. Results were in agreement with those obtained with the conventional method (LABTEST Kit) at the 95% confidence level. The detection limit and variation coefficient were estimated as 12.0 mg l(-1) (99.7% confidence level) and 3.5% (n = 20), respectively. The sampling rate was about 60 determinations h(-1) with sample concentrations ranging from 50 to 600 mg l(-1) glucose. The consumptions of serum sample, hexacyanoferrate(III) and luminol were 46 mul, 10.0 mg and 0.2 mg/determination, respectively.     

Miniature Direct Electron Transfer Based Enzymatic Fuel Cell Operating in Human Sweat and Saliva

We present data on operation of a miniature membrane‐less, direct electron transfer based enzymatic fuel cell in human sweat and saliva. The enzymatic fuel cell was fabricated following our previous reports on miniature biofuel cells, utilizing gold nanoparticle modified gold microwires with immobilized cellobiose dehydrogenase and bilirubin oxidase. The following average characteristics of miniature glucose/oxygen biodevices operating in human sweat and saliva, respectively, were registered: 580 and 560 mV open‐circuit voltage, 0.26 and 0.1 μW cm^–2 power density at a cell voltage of 0.5 V, with up to ten times higher power output at 0.2 V. When saliva collected after meal ingestion was used, roughly a two‐fold increase in power output was obtained, with a further two‐fold increase by addition of 500 μM glucose. Likewise, the power generated in sweat at 0.5 V increased two‐fold by addition of 500 μM glucose.     

An Synthesepulp immobilisierte Enzyme,I.

Synthetic pulp—a polyethylene fibrid which contains poly(vinyl alcohol)—was reacted with 2-(3-aminophenyl)-1,3-dioxolane. The amino derivative was transferred into the reactive diazonium compound. The content of reactive diazonium groups was determined by reaction with tyrosine. The diazonium derivatives of synthetic pulp were used for the immobilization of the hydrolases trypsin, chymotrypsin, papain, aminoacrylase, esterase and urease of the oxidoreductases glucose oxidase, catalase, peroxidase, and glucose-6-phosphate-dehydrogenase, and of the transferase hexokinase. Furthermore soybean trypsin inhibitor was immobilized on synthetic pulp via azo coupling. The protein binding ability of the reactive carriers and the enzymatic properties of the immobilization products were investigated. The thermostability of immobilized trypsin and immobilized urease, as well as the dependence of the immobilized tryptic activity on the temperature were studied. The activities of immobilized trypsin were assayed with low molecular weight substrates as well as with high molecular weight substates. The pH-optima of immobilized chymotrypsin, papain and urease were studied and the influence of buffers on the pH-activity profiles was investigated. Hexokinase and glucose-6-phosphatedehydrogenase were co-immobilized on synthetic pulp as an example for a two enzyme system and the properties of the immobilization products were investigate.     

Characterization of regenerated silk fibroin membranes for immobilizing glucose oxidase and construction of a tetrathiafulvalene-mediating glucose sensor

The IR spectra, the electronic absorption bands, and scanning electron microscopy (SEM) of the blend membranes of regenerated silk fibroin and glucose oxidase were reported for the first time and the second generation of glucose sensor based on glucose oxidase immobilized in the regenerated silk fibroin membrane was first constructed. The IR absorption spectra of the pure regenerated silk fibroin membrane were ascribed to its structural characteristics by which the part transition from silk I to silk II was recognized in ethanol immersion. Those spectra of the blend membrane of regenerated silk fibroin and glucose oxidase were identified as a composite of the absorption bands characteristic of both macromolecules. The electronic absorption bands showed that the glucose oxidase in the membrane exists in aggregates. A sea islands' structure was observed by SEM. These findings suggest that the regenerated silk fibroin and glucose oxidase are incompatible and their molecular interactions are very weak. A tetrathiafulvalene-mediating glucose sensor, employing immobilization of glucose oxidase by regenerated silk fibroin, was fabricated. The influences of temperature, applied potential, and pH on steady-state electrocatalytic oxidation of glucose at the sensor were evaluated. The response of the sensor to glucose under N₂ saturation reached 95% steady-state current within 40 s. The sensor could be used repeatedly for 1.5 months without deterioration of the response. © 1995 John Wiley & Sons, Inc.     

A hybrid photocatalytic and enzymatic process using glucose oxidase immobilized on TiO2/polyurethane for removal of a dye

A rectangular recycling photo-bioreactor using glucose oxidase (GOx) immobilized on TiO₂/polyurethane (PU) was developed as a novel coupling of photodegradation and enzymatic process. This method was tested for removal of Acid Orange 7 (AO7), as a model pollutant. High efficiency of decolorization (>99%) was achieved after 22 min using the GOx/TiO₂/PU photo-biocatalyst. Roles of various processes including photodegradation (TiO₂/PU), enzymatic process (GOx/PU) and a coupling of photocatalytic–enzymatic (GOx/TiO₂/PU) process were investigated in the presence and absence of UV light. All the experiments were performed in a circulation photoreactor equipped with a 6 W UV lamp with rate of 5 mL/min.