Gluconic acid production in bioreactor with immobilized glucose oxidase plus catalase on polymer membrane adjacent to anion-exchange membrane

Gluconic acid was obtained in the permeate side of the bioreactor with glucose oxidase (GOD) immobilized onto anion-exchange membrane (AEM) of low-density polyethylene grafted with 4-vinylpiridine. The electric resistance of the anion-exchange membranes was increased after the enzyme immobilization on the membrane. The gluconic acid productions were relatively low with the GOD immobilized by any method on the AEM. To increase the enzyme reaction efficiency, GOD was immobilized on membrane of AN copolymer (PAN) adjacent to an anion-exchange membrane in bioreactor. Uses of anion-exchange membrane led to selective removal of the gluconic acid from the glucose solution and reduce the gluconic acid inhibition. The amount of gluconic acid obtained in the permeate side of the bioreactor with the GOD immobilized on the PAN membrane adjacent to the AEM under electrodialysis was about 30 times higher than that obtained with enzyme directly bound to the AEM. The optimal substrate concentration in the feed side was found to be about 1 g/l. Further experiments were carried out with the co-immobilized GOD plus Catalase (CAT) on the PAN membrane adjacent to the AEM to improve the efficiency of the immobilize system. The yield of this process was at least 95%. The storage stability of the co-immobilized GOD and CAT was studied (lost 20% of initial activity for 90 d). The results obtained clearly showed the higher potential of the dual membrane bioreactor with GOD plus CAT bound to ultrafiltration polymer membrane adjacent to the AEM. Storage stability of GOD activity in GOD plus CAT immobilized on PAN//AEM membranes and on AEM.     

氨基化树枝状介孔二氧化硅固定葡萄糖氧化酶用于检测葡萄糖

以三乙胺为碱源合成了树枝状介孔二氧化硅纳米粒子(DMSNs),并用3-氨基丙基三乙氧基硅烷(APTES)进行氨基修饰合成了氨基化树枝状介孔二氧化硅纳米粒子(DMSNs-NH₂),将其用于葡萄糖氧化酶(GOD)的固定化研究.采用扫描电子显微镜、透射电子显微镜、红外光谱仪、X射线衍射仪、氮气吸附仪及热重分析仪对固定化GOD(DMSNs-NH₂-GOD)进行了表征,测定了其活性及蛋白载量.结果表明,固定化GOD的直径约为200 nm,形状均一,呈分散的球形微粒;在最佳固定条件下,蛋白载量达225 mg/g,酶活性达215 U/mg;固定化GOD检测葡萄糖的最低检测限为0.0014 mg/mL.利用固定化GOD检测了血清和饮料中的葡萄糖,重复使用36次以上其相对酶活性仍剩余80%.该方法操作方便、准确度高,提高了酶的pH稳定性、热稳定性及重复使用性,降低了检测成本.     

Reversible denaturation behavior of immobilized glucose oxidase

Glucose oxidase (GOD) was immobilized by using glutaraldehyde crosslinking and various stabilizing agents such as BSA, gelatin, lysozyme, and polyethylenimine (PEI). Studies on the denaturation of the soluble as well as immobilized GOD were carried out for 1 h at various concentrations of guanidine hydrochloride (GdmCl) in 50 mM phosphate buffer, pH 6.0 at 25±1°C. The soluble enzyme required a GdmCl concentration of 5M for total activity loss, whereas for GOD immobilized with BSA, gelatin, lysozyme, and heat-inactivated lysozyme, the corresponding GdmCl concentration required was 8 M. GOD immobilized with PEI, however, was more stable and retained 25% activity when denatured for 1 h using 8 M GdmCl. However, after undergoing denaturation for 1 h, GOD immobilized with lysozyme regained 72% original activity within 20 min of renaturation, while GOD immobilized with BSA, PEI, gelatin, and heat-inactivated lysozyme regained only 39, 21, 20, and 25% of activity, respectively. After five cycles of repeated denaturation and renaturation with 8 M GdmCl, GOD immobilized with lysozyme retained 70% of the original activity. Refolding ability of lysozyme, glutaraldehyde crosslinkages between lysozyme and GOD, together with ionic interactions between them, appear to play an important role in the denaturation-renaturation behavior of the immobilized enzyme.     

Application of polymaleimidostyrene as a convenient immobilization reagent of enzyme in biosensor

Sulfhydryl groups of glucose oxidase (GOD) were reacted with maleimide groups of polymaleimidostyrene (PMS) which was coated onto the porous carbon sheet, and the carbon sheet immobilized by GOD was combined with an oxygen electrode to fabricate a glucose sensor. The activity of thiolated GOD immobilized to PMS is much larger than that of native GOD immobilized to PMS. The good linear relationship of glucose and oxygen current response was obtained in a concentration range from 0.1 to 2 mM and upper limit of linear range was found to be 3.0 mM. The immobilized GOD activity is highly dependent on pH at immobilization and the maximum activity was obtained at pH 5.5, probably because the SH groups of GOD that are indispensable for generation of enzyme activity is not exposed at this pH. It was found that PMS is very effective reagent to immobilize enzyme strongly via covalent bond, because high density of maleimide groups of PMS can catch not only exposed SH groups but also buried SH groups.     

1-芘丁酸/石墨烯复合物的电化学性质及其在葡萄糖传感器上的应用

本文采用一步法制备了1-芘丁酸/石墨烯复合物(PBA/G),研究了其电化学性质. 采用铁氰化钾和亚铁氰化钾电化学探针测定了电化学阻抗滴定曲线,确定了PBA/G的表观pK_a为6.2. 此外,将葡萄糖氧化酶(GOD)共价键合在PBA/G表面构建了葡萄糖电化学传感器,其电化学响应与葡萄糖浓度（5 mmol L^-1浓度范围内）呈线性,检测限为0.085 mmol L^-1. 实验还测定了固定在PBA/G表面的GOD的表观米氏常数为5.40 mmol L^-1,表明固定化的GOD对葡萄糖有较高的催化活性。     

固定化葡萄糖氧化酶活性的X射线微区分析

利用X射线微区分析方法,对固定化活性葡萄糖氧化酶进行了定位分析;葡萄糖作为底物,FeSO4和KI作为捕捉剂,底物经固定化葡萄糖氧化酶催化产生H2O2,后者和捕捉剂反应生成沉淀,可以确定固定化葡萄糖氧化酶的催化活性部位。结果表明：颗粒越小,酶活越高,活性葡萄糖氧化酶在凝胶内分布均匀,且绝大多数葡萄糖氧化酶固定在凝胶的内部。作者还研究了固定化活性葡萄糖氧化酶定位的最佳条件。     

Direct electrochemistry of glucose oxidase immobilized on a hexagonal mesoporous silica-MCM-41 matrix

The direct electrochemistry of glucose oxidase (GOD) immobilized on a hexagonal mesoporous silica modified glassy carbon electrode was investigated. The adsorbed GOD displayed a pair of redox peaks with a formal potential of -417 mV in 0.1 M pH 6.1 phosphate buffer solution (PBS). The response showed a diffusion-controlled electrode process with a two-electron transfer coupled with a two-proton transfer reaction process. GOD immobilized on a hexagonal mesoporous silica retained its bioactivity and stability. In addition, the immobilized GOD could electrocatalyze the oxidation of glucose to gluconlactone by taking ferrocene monocarboxylic acid (FMCA) as a mediator in N(2) saturated solutions, indicating that the electrode may have the potential application in biosensors to analyze glucose. The sensor could exclude the interference of commonly coexisted uric acid, p-acetaminophenol and ascorbic acid and diagnose diabetes very fast and sensitively. This work demonstrated that the mesoporous silica provided a novel matrix for protein immobilization and the construction of biosensors.     

Immobilization and enzymatic activity of glucose oxidase on polystyrene surface modified with ozone aeration and UV irradiation in distilled water and/or aqueous ammonia solution

Adsorption condition and enzymatic activity of glucose oxidase (GOD) on polystyrene (PS) film surfaces modified with ozone aeration and UV irradiation (O3/UV) treatment were investigated. The total amount of GOD immobilized on the PS film modified with the O3/UV treatment in distilled water (PS-W film) was approximately twice as large as that on the film treated in an aqueous ammonia solution (PS-A film), whereas the specific activity of GOD on the PS-A film was four times higher than that on the PS-W film. In contrast, no enzymatic activity of GOD on the non-treated PS film was observed because of irreversible denaturation of the adsorbed GOD. We therefore conclude that the PS films modified by the O3/UV treatment in the aqueous media are effective in immobilizing GOD.     

Immobilized Fullerene C60‐Enzyme‐Based Electrochemical Glucose Sensor

A mixed‐valence cluster of cobalt(II) hexacyanoferrate and fullerene C60‐enzyme‐based electrochemical glucose sensor was developed. A water insoluble fullerene C60‐glucose oxidase (C60‐GOD) was prepared and applied as an immobilized enzyme on a glassy carbon electrode with cobalt(II) hexacyanoferrate for analysis of glucose. The glucose in 0.1 M KCl/phosphate buffer solution at pH = 6 was measured with an applied electrode potential at 0.0 mV (vs Ag/AgCl reference electrode). The C60‐GOD‐based electrochemical glucose sensor exhibited efficient electro‐catalytic activity toward the liberated hydrogen peroxide and allowed cathodic detection of glucose. The C60‐GOD electrochemical glucose sensor also showed quite good selectivity to glucose with no interference from easily oxidizable biospecies, e.g. uric acid, ascorbic acid, cysteine, tyrosine, acetaminophen and galactose. The current of H₂O₂ reduced by cobalt(II) hexacyanoferrate was found to be proportional to the concentration of glucose in aqueous solutions. The immobilized C60‐GOD enzyme‐based glucose sensor exhibited a good linear response up to 8 mM glucose with a sensitivity of 5.60 × 10² nA/mM and a quite short response time of 5 sec. The C60‐GOD‐based glucose sensor also showed a good sensitivity with a detection limit of 1.6 × 10^‐6 M and a high reproducibility with a relative standard deviation (RSD) of 4.26%. Effects of pH and temperature on the responses of the immobilized C60‐GOD/cobalt(II) hexacyanoferrate‐based electrochemical glucose sensor were also studied and discussed.     

Covalent immobilization of glucose oxidase onto new modified acrylonitrile copolymer/silica gel hybrid supports

New polymer/silica gel hybrid supports were prepared by coating high surface area of silica gel with modified acrylonitrile copolymer. The concentrations of the modifying agent (NaOH) and the modified polymer were varied. GOD was covalently immobilized on these hybrid supports and the relative activity and the amount of bound protein were determined. The highest relative activity and sufficient amount of bound protein of the immobilized GOD were achieved in 10% NaOH and 2% solution of modified acrylonitrile copolymer. The influence of glutaraldehyde concentration and the storage time on enzyme efficiency were examined. Glutaraldehyde concentration of 0.5% is optimal for the immobilized GOD. It was shown that the covalently bound enzyme (using 0.5% glutaraldehyde) had higher relative activity than the activity of the adsorbed enzyme. Covalently immobilized GOD with 0.5% glutaraldehyde was more stable for four months in comparison with the one immobilized on pure silica gel, hybrid support with 10% glutaraldehyde and the free enzyme. The effect of the pore size on the enzyme efficiency was studied on four types of silica gel with different pore size. Silica with large pores (CPC-Silica carrier, 375 A) presented higher relative activity than those with smaller pore size (Silica gel with 4, 40 and 100 A). The amount of bound protein was also reduced with decreasing the pore size. The effect of particle size was studied and it was found out that the smaller the particle size was, the greater the activity and the amount of immobilized enzyme were. The obtained results proved that these new polymer/silica gel hybrid supports were suitable for GOD immobilization.     

Alginate beads containing pH-sensitive liposomes and glucose oxidase: glucose-sensitive release

Egg PC (EPC) liposomes bearing a copolymer of N-isopropylacrylamide, methacrylic acid, and octadecylacrylate (P(NIPAM-co-MAA-co-ODA)) were prepared as pH-sensitive liposomes. They were embedded in glucose oxidase (GOD)-immobilized alginate beads. The ratio of EPC/GOD/alginate in the beads was 7.8:1.0:140.4, and the beads were added to glucose solutions so that the concentration of GOD was 0.0068 mg/ml. The enzymatic activity of the immobilized GOD was one fifth to half of that of native enzyme. As the glucose concentration increased from 0 to 400 mg/dl, the degree of calcein release increased from 17% to 75%. The acidification induced by the enzymatic reaction would be responsible for the glucose-triggered release.     

New approach to the immobilization of glucose oxidase on non-porous silica microspheres functionalized by (3-aminopropyl)trimethoxysilane (APTMS)

The immobilization and encapsulation of glucose oxidase (GOD) onto the mesoporous and the non-porous silica spheres prepared by co-condensation of tetraethylorthosilicate (TEOS) and (3-aminopropyl)trimethoxysilane (APTMS) in the water-in-oil (W/O) emulsion system were studied. The terminal amine group was used as the important functionality for GOD immobilization on the silica substrate. When only TEOS is used as a silica source, the disordered mesoporous silica microspheres are obtained. As the molar ratio of APTMS to TEOS (R_AT) increases, the surface area and pore volume of the silica particles measured by nitrogen adsorption and desorption method and SEM decrease rapidly. Particularly, the largest change of the surface morphology is observed between R_AT = 0.20 and R_AT = 0.25. The amount and the adsorption time of immobilized enzyme were measured by UV spectroscopy. About 20 wt% of GOD was immobilized into the silica substrates above R_AT = 0.60 and was completely adsorbed into the substrate of R_AT = 0.80 with lapse of 4 h after addition. In the measurement of the thermal stability, GOD dissolved in buffer solution loses nearly all of its activity after 30 min at 65 °C. In contrast, GOD immobilized on the surface-modified silica particles still retains about 90% of its activity after the same treatment. At this temperature, the immobilized glucose oxidase retained half of its initial activity after 4 h. It is shown that the suitable usage of functionalizing agent like APTMS as well as the control of surface morphology is very important on the immobilization of enzyme.     

Glucose oxidase/colloidal gold nanoparticles immobilized in Nafion film on glassy carbon electrode: Direct electron transfer and electrocatalysis

The direct electron transfer of glucose oxidase (GOD) was achieved based on the immobilization of GOD/colloidal gold nanoparticles on a glassy carbon electrode by a Nafion film. The immobilized GOD displayed a pair of well-defined and nearly reversible redox peaks with a formal potential (Eo ') of -0.434 V in 0.1 M pH 7.0 phosphate buffer solution and the response showed a surface-controlled electrode process. The dependence of Eo ' on solution pH indicated that the direct electron transfer reaction of GOD was a two-electron-transfer coupled with a two-proton-transfer reaction process. The experimental results also demonstrated that the immobilized GOD retained its electrocatalytic activity for the oxidation of glucose. So the resulting modified electrode can be used as a biosensor for detecting glucose.     

IMMOBILIZATION OF GLUCOSE OXIDASE AND CELLULASE BY CHITOSAN— POLYACRYLIC ACID COMPLEX

This study is concerned with chitosan-polyacrylic acid complex as a carrier to immobilize glucose oxidase (GOD)and cellulase. The optimum emperature of the immobilized GOD (IG) was determined to be 60℃which is higher than that of the native GOD about 40℃. The optimum temperature of the immobilized cellulase (IC) was determined to be about 30℃higher than that of native cellulase. Both of the optimum pH of IG and IC shifted one pH unit to acid. Immobilized enzyme may be used in more wide pH range. Their storage life are much longer compared with their native states. Both of them can be reused at least 12 times.     

Flow-through chemiluminescence sensor using immobilized oxidases for the selective determination of L-glutamate in a flow-injection system.

A selective and sensitive chemiluminometric flow sensor for the determination of L-glutamate in serum, based on immobilized oxidases such as glutamate oxidase (GOD), uricase (UC) and peroxidase (POD), is described herein. The principle for the selective chemiluminometric detection for L-glutamate is based on coupled reactions of four sequentially aligned immobilized oxidases, UC/POD/GOD/POD in a flow cell. The immobilized UC was employed to decompose urate, which is one of the major interfering components in serum for a luminol-H2O2 chemiluminescence reaction. The H2O2 produced from the UC reaction readily reacted with reducing components, such as ascorbate and glutathione, and then the excess H2O2 was decomposed by the immobilized POD. L-Glutamate in the sample plug was enzymatically converted to H2O2 with immobilized GOD. Subsequently, the peroxide reacts with luminol on the immobilized POD to produce chemiluminescence, proportional to glutamate concentration. The enzymes were immobilized on tresylated poly(vinyl alcohol beads). The immobilized enzymes were packed into TPFE tube (1.0 mm i.d. x 60 cm), in turn, and used as a flow cell. The sampling rate was 30 h-1. The calibration graph for L-glutamate is linear for 20 nM-5 microM; the detection limit (signal-to-noise = 3) is 10 nM.     

Poly(acrylic acid)-silicon Hybrids Prepared via a RAFT-mediated Process and Covalent Immobilization of Glucose Oxidase

A surface modification technique was proposed for the modification of silicon surface with glucose oxidase (GOD). The silicon surface was first graft copolymerized with acrylic acid (AAc) via surface-initiated reversible addition-fragmentation chain-transfer (RAFT)-mediated process. With the aid of a water-soluble carbodiimide, GOD was then covalently immobilized on the silicon surface through the amide linkage between the amino group of GOD and the carboxyl group of the grafted AAc polymer. The changes in the surface composition after polymer grafting and enzyme immobilization on the silicon surface were investigated using X-ray photoelectron spectroscopy (XPS). The amount of GOD immobilized could be varied by changing the thickness of the polymer layer and the immobilization time. The GOD-functionalized silicon hybrids are potential useful in the application of the silicon-based biosensors.     

孔蛋白-磷脂仿生膜的葡萄糖氧化酶电化学

采用孔蛋白（MspA）和双肉豆蔻磷脂酰胆碱（DMPC）在玻碳（GC）基底表面成功构建有仿生特性的纳米通道膜,同时将葡萄糖氧化酶（GOD）修饰于膜上. 使用循环伏安法研究GOD/MspA-DMPC/GC电极的GOD直接电化学过程以及其对氧气和葡萄糖的响应. 研究发现,MspA与DMPC形成的仿生纳米通道膜内,GOD在接近生物体系FAD/FADH标准电位处实现了自身两质子、两电子表面控制的电化学反应. MspA与DMPC的仿生纳米通道膜体系为GOD提供了理想活性环境.     

Novel Composite of Multiwalled Carbon Nanotubes and Gold Nanoparticles Stabilized by Chitosan and Hydrophilic Ionic Liquid for Direct Electron Transfer of Glucose Oxidase

A novel composite was fabricated through dispersing multiwalled carbon nanotubes (MWNTs) in gold nanoparticle (GPs) colloid stabilized by chitosan and ionic liquid (i.e., 1‐butyl‐3‐methylimidazolium tetrafluoroborate, BMIMBF₄). Transmission electron microscopy (TEM) experiment showed that the GPs highly dispersed on the MWNTs probably due to the electrostatic interaction among GPs, MWNTs and the imidazolium cation of BMIMBF₄. X‐ray photoelectron spectroscopy (XPS) indicated that thus‐formed gold nanostructure was mediated by BMIMBF₄. When glucose oxidase (GOD) was immobilized on the composite (MWNTs‐GPs) its ultraviolet‐visible absorption spectrum kept almost unchanged. The immobilized GOD coated glassy carbon electrode (GOD/MWNTs‐GPs/GC) exhibited a pair of well‐defined peaks in 0.10 M pH 7.0 phosphate buffer solution (PBS), with a formal potential of ?0.463 V (vs. SCE). The electrochemical process involved two‐electron transfer. The electron transfer coefficient was ca.0.56 and the electron transfer rate constant was 9.36 s^?1. Furthermore, the immobilized GOD presented good catalytic activity to the oxidation of glucose in air‐saturated PBS. The K_m and I_m values were estimated to be 13.7 μM and 0.619 μA. The GOD/MWNTs‐GPs/GC electrode displayed good stability and reproducibility.     

Reagentless Glucose Biosensor Based on the Direct Electrochemistry of Glucose Oxidase on Carbon Nanotube‐Modified Electrodes

The direct electrochemistry of glucose oxidase (GOD) was revealed at a carbon nanotube (CNT)‐modified glassy carbon electrode, where the enzyme was immobilized with a chitosan film containing gold nanoparticles. The immobilized GOD displays a pair of redox peaks in pH 7.4 phosphate buffer solutions (PBS) with the formal potential of about ?455 mV (vs. Ag/AgCl) and shows a surface‐controlled electrode process. Bioactivity remains good, along with effective catalysis of the reduction of oxygen. In the presence of dissolved oxygen, the reduction peak current decreased gradually with the addition of glucose, which could be used for reagentless detection of glucose with a linear range from 0.04 to 1.0 mM. The proposed glucose biosensor exhibited high sensitivity, good stability and reproducibility, and was also insensitive to common interferences such as ascorbic and uric acid. The excellent performance of the reagentless biosensor is attributed to the effective enhancement of electron transfer between enzyme and electrode surface by CNTs, and the biocompatible environment that the chitosan film containing gold nanoparticles provides for immobilized GOD.     

Towards the development of an integrated capillary electrophoresis optical biosensor

Extending the previous preliminary study on the construction of a capillary electrophoresis (CE)/sensor for the detection of reducing analytes, we focus the interest on the simultaneous detection of redox active species, which are important indicators of the oxidative damage in tissues, of food preservation, and of pollution. The CE/sensor was built by modifying the detector-portion of the capillary with the redox-sensitive polymer polyaniline (PANI). The analyte is detected by monitoring the changes in optical absorption of the PANI film. The CE/sensor was tested, with good results, with ascorbic acid, glutathione (GSH), as well as with compounds with very close similarity (ascorbic and isoascorbic acid). The kinetics of oxidation and reduction of PANI were evaluated. Further a PANI/CE-biological sensor was developed by coupling an enzyme, glucose oxidase (GOD), to the PANI-modified portion of the capillary. The stability of the immobilized GOD and the sensitivity of the CE/biosensor were studied, by using glucose as test analyte in concentrations within the physiological range. The results indicate that the CE/biosensor had good stability (more than 75% of original activity retained after 30 operational days), manufacturing reproducibility and a sensing range convenient for monitoring physiological glucose (1-24 mM).