葡萄糖氧化酶的有机相共价固定化

将葡萄糖氧化酶(GOD)在最适pH条件下冻干后,以戊二醛活化的壳聚糖为载体,分别在传统水相和1,4-二氧六环、乙醚、乙醇三种不同的有机相中进行共价固定化。通过比较水相固定化酶和有机相固定化酶的酶比活力、酶学性质及酶动力学参数,考察酶在有机相中的刚性特质对酶在共价固定化过程中保持酶活力的影响。结果表明,戊二醛浓度为0.1%、加酶量为80 mg/1 g载体、含水1.6%的1,4-二氧六环有机相固定化GOD与水相共价固定化GOD相比,酶比活力提高2.9倍,有效酶活回收率提高3倍;在连续使用7次后,1,4-二氧六环有机相固定化GOD的酶活力仍为相应水相固定化酶的3倍。在酶动力学参数方面,不论是表观米氏常数,最大反应速度还是转换数,1,4-二氧六环有机相固定化的GOD(Kmapp=5.63 mmol/L,Vmax=1.70μmol/(min.mgGOD),Kcat=0.304 s-1)都优于水相共价固定化GOD(Kmapp=7.33 mmol/L,Vmax=1.02μmol/(min.mg GOD),Kcat=0.221 s-1)。因此,相比于传统水相,GOD在合适的有机相中进行共价固定化可以获得具有更高酶活力和更优催化性质的固定化酶。该发现可能为酶蛋白在共价固定化时因构象改变而丢失生物活性的问题提供解决途径。     

Enzymatic activity of glucose oxidase covalently wired via viologen to electrically conductive polypyrrole films

The surface functionalization of an electrically conductive polypyrrole film (PPY) with a viologen, (N-(2-carboxyl-ethyl)-N'-(4-vinyl-benzyl)-4,4'-bipyridinium dichloride, or CVV) for the covalent immobilization of glucose oxidase (GOD) has been carried out. The viologen was first synthesized and graft polymerized on PPY film. It then served as an anchor via its carboxyl groups for the covalent immobilization of GOD. The surface composition of the as-functionalized substrates was characterized by X-ray photoelectron spectroscopy (XPS). The effects of the CVV monomer concentration on the CVV-graft polymer concentration and the amount of GOD immobilized on the surface were investigated. The activity of the immobilized GOD was compared with that of free GOD and the kinetic effects were also obtained. The cyclic voltammetric (CV) response of the GOD-functionalized PPY substrates was studied in a phosphate buffer solution under an argon atmosphere. The CV results support the mechanism in which CVV acts as a mediator to transfer electron between the electrode and enzyme, and hence regenerating the enzyme in the enzymatic reaction with glucose. High sensitivity and linear response of the enzyme electrode was observed with glucose concentration ranging from 0 to 20 mM.     

Activity and storage stability of immobilized glucose oxidase onto magnesium silicate

Glucose oxidase (GOD) was covalently immobilized onto florisil (magnesium silicate) carrier via glutaraldehyde. Immobilization conditions were optimized: the amount of initial GOD per grams of carrier as 5 mg, pH as 5.5, immobilization time as 120 min and temperature as 10 °C. Under the optimized reaction conditions activities of free and immobilized GOD were measured. Free and immobilized GOD samples were characterized with their kinetic parameters, and thermal and storage stabilities. K_M and V_max values were 68.2 mM and 435 U mg GOD⁻¹ for free and 259 mM and 217 U mg GOD⁻¹ for immobilized enzymes, respectively. Operational stability of the immobilized enzyme was also determined by using a stirred batch type column reactor. Immobilized GOD was retained 40% of its initial activity after 50 reuses. Storage stabilities of the immobilized GOD samples stored in the mediums with different relative humidity in the range of 0–100% were investigated during 2 months. The highest storage stability was determined for the samples stored in the medium of 60% relative humidity. Increased relative humidity from 0% to 60% caused increased storage stability of immobilized GODs, however, further increase in relative humidity from 80% to 100% caused a significant decrease in storage stability of samples.     

Immobilization of glucose oxidase and lactate dehydrogenase onto magnetic nanoparticles for bioprocess monitoring system

Glucose oxidase (GOD) and lactate dehydrogenase (LDH) were immobilized onto magnetic nanoparticles, viz. Fe₃O₄, via carbodiimide and glutaraldehyde. The immobilization efficiency was largely dependent upon the immobilization time and concentration of glutaraldehyde. The magnetic nanoparticles had a mean diameter of 9.3 nm and were superparamagnetic. The immobilization of GOD and LDH on the nanoparticles slightly decreased their saturation magnetization. However, the FT-IR spectra showed that GOD and LDH were immobilized onto the nanoparticles by different binding mechanisms, the reason for which was not well explained. The optimum pH values of the immobilized GOD and LDH were changed to 8 and 10, respectively. The free and immobilized enzyme kinetic parameters (K_m and V_max) were determined by Michaelis-Menten enzyme kinetics. The Km values for free and immobilized GOD were 0.168 and 0.324 mM, respectively, while those for free and immobilized LDH were 0.19 and 0.163 mM for NAD, and 2.976 and 4.785 mM for lactate, respectively. High operational stability was observed, with more than 80% of the initial enzyme activity being retained for the immobilized GOD up to 12 h and for the immobilized LDH up to 24 h. The immobilized GOD was applied to a sequential injection analysis system for the application of bioprocess monitoring.     

Simultaneous co-immobilization of glucose oxidase and catalase in their substrates

Glucose oxidase (GOD) and catalase (CAT) were simultaneously co-immobilized onto magnesium silicate (Florisil®) by covalent coupling. Glucose was added in immobilization mixture and hydrogen peroxide, which is the substrate of CAT, was produced in coupling mixture during immobilization time. Therefore, co-immobilization of GOD and CAT was carried out in the presence of both their substrates: glucose and hydrogen peroxide, respectively. The effect of glucose concentration in immobilization mixture on activities of GOD and CAT of co-immobilized samples were investigated. Maximum GOD and CAT activities were determined for samples co-immobilized in the presence of 15 and 20 mM glucose, respectively. Co-immobilization of GOD and CAT in the presence of their substrates highly improved the activity and reusability of both enzymes.     

Simultaneous co-immobilization of glucose oxidase and catalase in their substrates

Glucose oxidase (GOD) and catalase (CAT) were simultaneously co-immobilized onto magnesium silicate (florisil) by covalent coupling. Glucose was added in immobilization mixture and hydrogen peroxide which is the substrate of CAT was produced in coupling mixture during immobilization time. Therefore, co-immobilization of GOD and CAT was carried out in presence of both their substrate: glucose and hydrogen peroxide, respectively. The effect of glucose concentration in immobilization mixture on activities of GOD and CAT of co-immobilized samples were investigated. Maximum GOD and CAT activities were determined for samples co-immobilized in presence of 15 and 20 mM glucose, respectively. Co-immobilization of GOD and CAT in presence of their substrates highly improved the activity and reusability of both enzymes.     

Surface functionalization of polypyrrole film with glucose oxidase and viologen

A surface modification technique was developed for the functionalization of polypyrrole (PPY) film with glucose oxidase (GOD) and viologen moieties. The PPY film was first graft copolymerized with acrylic acid (AAc) and GOD was then covalently immobilized through the amide linkage formation between the amino groups of the GOD and the carboxyl groups of the grafted AAc polymer chains in the presence of a water-soluble carbodiimide. Viologen moieties could also be attached to the PPY film via graft-copolymerization of vinyl benzyl chloride with the PPY film surface followed by reaction with 4,4'-bipyridine and alpha,alpha'-dichloro-p-xylene. X-ray photoelectron spectroscopy (XPS) was used to characterize the PPY films after each surface modification step. Increasing the AAc graft concentration would allow a greater amount of GOD to be immobilized but this would decrease the electrical conductivity of the PPY film. The activity of the immobilized GOD was compared with that of free GOD and the kinetic effects were also studied. The immobilized GOD was found to be less sensitive to temperature deactivation as compared to the free GOD. The results showed that the covalent immobilization technique offers advantages over the technique involving the entrapment of GOD in PPY films during electropolymerization. The presence of viologen in the vicinity of the immobilized GOD also enabled the GOD-catalyzed oxidation of glucose to proceed under UV irradiation in the absence of O(2).     

Glucose oxidase immobilization on a novel cellulose acetate-polymethylmethacrylate membrane

Glucose oxidase (GOD) was immobilized on cellulose acetate-polymethylmethacrylate (CA-PMMA) membrane. The immobilized GOD showed better performance as compared to the free enzyme in terms of thermal stability retaining 46% of the original activity at 70 degrees C where the original activity corresponded to that obtained at 20 degrees C. FT-IR and SEM were employed to study the membrane morphology and structure after treatment at 70 degrees C. The pH profile of the immobilized and the free enzyme was found to be similar. A 2.4-fold increase in Km value was observed after immobilization whereas Vmax value was lower for the immobilized GOD. Immobilized glucose oxidase showed improved operational stability by maintaining 33% of the initial activity after 35 cycles of repeated use and was found to retain 94% of activity after 1 month storage period. Improved resistance against urea denaturation was achieved and the immobilized glucose oxidase retained 50% of the activity without urea in the presence of 5M urea whereas free enzyme retained only 8% activity.     

Covalent immobilization of glucose oxidase on well-defined poly(glycidyl methacrylate)-Si(111) hybrids from surface-initiated atom-transfer radical polymerization

A simple one-step procedure was employed for the covalent immobilization of an atom-transfer radical polymerization (ATRP) initiator, via the robust Si-C bond, on the hydrogen-terminated Si(111) surface (Si-H surface). Well-defined poly(glycidyl methacrylate) [P(GMA)] brushes, tethered directly on the (111)-oriented single-crystal silicon surface, were prepared via surface-initiated ATRP. Kinetics study on the surface-initiated ATRP of glycidyl methacrylate revealed that the chain growth from the silicon surface was consistent with a "controlled" process. A relatively high concentration of glucose oxidase (GOD; above 0.2 mg/cm2) could be coupled directly to the well-defined P(GMA) brushes via the ring-opening reaction of the epoxide groups with the amine moieties of the enzyme. The resultant GOD-functionalized P(GMA) brushes, with the accompanying hydroxyl groups from the ring-opening reaction of the epoxide groups, serves as an effective spacer to provide the GOD with a higher degree of conformational freedom and a more hydrophilic environment. An equivalent enzyme activity above 1.6 units/cm2 [micromoles of beta-D-(+)-glucose oxidized to d-gluconolactone per minute per square centimeter] and a corresponding relative activity of about 60% could be readily achieved. The immobilized GOD also exhibited an improved stability during storage over that of the free enzyme. The GOD-functionalized silicon substrates are potentially useful to the development of silicon-based glucose biosensors.     

Immobilization of invertase onto poly(3-methylthienyl methacrylate)/poly(3-thiopheneacetic acid) matrix

A novel immobilization matrix, poly(3-methylthienyl methacrylate)–poly(3-thiopheneacetic acid) (PMTM–PTAA), was synthesized and used for the covalent immobilization of Saccharomyces cerevisiae invertase to produce invert sugar. The immobilization resulted in 87% immobilization efficiency. Optimum conditions for activity were not affected by immobilization and the optimum pH and temperature for both free and immobilized enzyme were found to be 4.5 and 55 °C, respectively. However, immobilized invertase was more stable at high pH and temperatures. The kinetic parameters for free and immobilized invertase were also determined using the Lineweaver–Burk plot. The K_m values were 35 and 38 mM for free and immobilized enzyme, respectively. The V_max values were 29 and 24 mg glucose/mg enzyme min for free and immobilized enzyme, respectively. Immobilized enzyme could be used for the production of glucose and fructose from sucrose since it retained almost all the initial activity for a month in storage and retained the whole activity in repeated 50 batch reactions.     

Stabilization of activity of oxidoreductases by their immobilization onto special functionalized glass and novel aminocellulose film using different coupling reagents

Glucose oxidase (GOD), horseradish peroxidase (HRP), and lactate oxidase (LOD) were covalently immobilized on special NH(2)-functionalized glass and on a novel NH(2)-cellulose film via 13 different coupling reagents. The properties of these immobilized enzymes, such as activity, storage stability, and thermostability, are strongly dependent on the coupling reagent. For example, GOD immobilized by cyanuric chloride on the NH(2)-cellulose film loses approximately half of its immobilized activity after 30 days of storage at 4 degrees C or after treatment at 65 degrees C for 30 min. In contrast, GOD immobilized by L-ascorbic acid onto the same NH(2)-cellulose film retains 90% of its initial activity after 1 year of storage at 4 degrees C and 92% after heat treatment at 65 degrees C for 30 min. Unlike GOD, in the case of LOD only immobilization on special NH(2)-functionalized glass, e.g., via cyanuric chloride, led to a stabilization of the enzyme activity in comparison to the native enzyme. The operational stability of immobilized HRP was up to 40 times higher than that of the native enzyme if coupling to the new NH(2)-cellulose film led to an amide or sulfonamide bond. Regarding the kinetics of the immobilized enzymes, the coupling reagent plays a minor role for the enzyme substrate affinity, which is characterized by the apparent Michaelis constant (K(M,app)). The NH(2)-functionalized support material as well as the immobilized density of the protein and/or immobilized activity has a strong influence on the K(M,app) value. In all cases, K(M,app) decreases with increasing immobilized enzyme protein density and particularly drastically for GOD.     

Enzyme microgels in packed-bed bioreactors with downstream amperometric detection using microfabricated interdigitated microsensor electrode arrays.

In this article, we describe the use of pH- responsive hydrogels as matrices for the immobilization of two enzymes, glucose oxidase (GOx) and glutamate oxidase (GlutOx). Spherical hydrogel beads were prepared by inverse suspension polymerization and the enzymes were immobilized by either physical entrapment or covalent immobilization within or on the hydrogel surface. Packed-bed bioreactors were prepared containing the bioactive hydrogels and these incorporated into flow injection (FI) systems for the quantitation of glucose and monosodium glutamate (MSG) respectively. The FI amperometric detector comprised a microfabricated interdigitated array within a thin-layer flow cell. For the FI manifold incorporating immobilized GOx, glucose response curves were found to be linear over the concentration range 1.8-280 mg dL(-1) (0.1-15.5 mM) with a detection limit of 1.4 mg dL(-1) (0.08 mM). Up to 20 samples can be manually analyzed per hour, with the hydrogel-GOx bioreactor exhibiting good within-day (0.19%) precision. The optimized FI manifold for MSG quantitation yielded a linear response range of up to 135 mg dL(-1) (8 mM) with a detection limit of 3.38 mg dL(-1) (0.2 mM) and a throughput of 30 samples h(-1). Analysis of commercially produced soup samples gave a within-day precision of 3.6%. Bioreactors containing these two physically entrapped enzymes retained > 60% of their initial activities after a storage period of up to 1 year.     

Co-immobilization of glucose oxidase and hexokinase on silicate hybrid sol-gel membrane for glucose and ATP detections

The co-immobilization of glucose oxidase (GOD) and hexokinase/glucose-6-phosphate dehydrogenase (HEX) in the silica hybrid sol-gel film for development of amperometric biosensors was investigated. The silica hybrid film fabricated by hydrolysis of the mixture of tetraethyl orthosilicate and 3-(trimethoxysiyl)propyl methacrylate possessed a three-dimension vesicle structure and good uniformity and conformability, and was ready for enzyme immobilization. The electrochemical and spectroscopic measurements showed that the silica hybrid sol-gel provided excellent matrice for the enzyme immobilization and that the immobilized enzyme retained its bioactivity effectively. The immobilized GOD could catalyze the oxidation of glucose, which could be used to determine glucose at +1.0 V without help of any mediator. The competition between GOD and HEX for the substrate glucose involving ATP as a co-substrate led to a decrease of the glucose response, which allowed us to develop an ATP sensor with a good stability. The fabricated silica hybrid sol-gel matrice offered a stage for further study of immobilization and electrochemistry of proteins.     

Immobilization of biocatalysts with bombyx mori silk fibroin by several kinds of physical treatment and its application to glucose sensors

Biocatalyst-immobilized Bombyx mori silk fibroin membrane was prepared. The insolubilization of the water-soluble membranes was performed by physical treatments only, i.e. stretching, compressing and standing under high humidity and methanol-immersion treatment, without any use ofcovalently binding reagent. All physical treatments performed were effective for the purpose of the immobilization of the enzymes in the membranes. The structural characterization of the glucose oxidase (GOD) immobilized membrane was performed in detail. The permeability of the substrate depends on the crystalline structure, i.e. the fraction of Silk I and Silk II of the membrane. The activity yield of the immobilized GOD was more than 80% of the value of free enzyme when 0–002% of the enzyme was entrapped in the membrane, but it decreased with increasing the concentration of the GOD in the membrane. This seems to result from diffusion limitation of the substrate. The pH and thermal stabilities of the immobilized enzyme were much improved, and were essentially independent of the methods of the immobilization. Development of the GOD or microorganism, Pseudomonas fluorescens immobilized silk fibroin membranes as glucose sensors are described.     

Properties of immobilized pepsin on Modified PMMA microspheres

In this work we use micro-size poly(methyl methacrylate)/acrylaldehyde microspheres as a support for pepsin immobilization. The aldehyde groups on the microspheres offer a very simple, mild and firm combination for enzyme immobilization. The amount of enzyme we can bind to this support reaches 82 mg/g, which is much higher than for other supports (mostly less than 10 mg/g). Compared to free enzyme, the Km of immobilized enzyme is increased, whereas the Vmax is decreased. Further, the Vmax/Km value for immobilized pepsin is about 50% of the value for free enzyme. This is better than values reported previously, generally lower than 35%. The optimum temperature shifts from 43 degrees C for free pepsin to 47 degrees C. However, the optimum pH does not change between free and immobilized enzyme. This improved resistance of the immobilized enzyme towards changes in temperature and pH also shows that the aldehyde modified poly(methyl methacrylate)/acrylaldehyde microspheres can be a valuable support for pepsin immobilization.     

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).     

Inner epidermis of onion bulb scale: As natural support for immobilization of glucose oxidase and its application in dissolved oxygen based biosensor

Inner epidermal membrane of the onion bulb scales was studied as a natural polymer support for immobilization of the glucose oxidase (GOD) enzyme for biosensor application. Onion epidermal membrane was used for immobilization of glucose oxidase and was associated with dissolved oxygen (DO) probe for biosensor reading. Glucose was detected on the basis of depletion of oxygen, when immobilized GOD oxidizes glucose into gluconolactone. A wide detection range between 22.5 and 450 mg/dl was estimated from calibration plot. A single membrane was reused for 127 reactions with retention of approximately 90% of its initial enzyme activity. Membrane was stable for 45 days ( approximately 90% activity) when stored in buffer at 4 degrees C. Surface structure studies of the immobilized membranes were carried under SEM. To our knowledge, this is the first report on employing inner epidermal membrane of onion bulb scales as the solid support for immobilization of enzyme.     

Covalent immobilization of enzymes within micro-aqueous organic media

Traditional covalent immobilization of enzymes was mostly operated within water phase. However, most of enzymes are flexible when they are in water environment, and the covalent reactions generally lead to complete or partial activity losing due to the protein conformational changes.This paper examined enzyme covalent immobilization operated in micro-aqueous organic media, to display the differences between two environments of immobilization within water and micro-aqueous organic solvent by activity and stability determination of the resulting immobilized enzymes. Catalase, trypsin, horseradish peroxidase, laccase and glucose oxidase have been employed as model enzymes. Results showed the thermal, pH and reusable stabilities of the micro-aqueous organic covalently immobilized enzymes were improved when compared with the immobilized enzymes within water. Micro-aqueous covalent immobilization showed a remarkable advantage in remaining the enzymes catalytic activity for all the five enzymes compared with the traditional water phase immobilization. And the optimum pH values for both immobilization within water and micro-aqueous organic media shifted slightly.     

Immobilization and direct electrochemistry of glucose oxidase on a tetragonal pyramid-shaped porous ZnO nanostructure for a glucose biosensor

A tetragonal pyramid-shaped porous ZnO (TPSP-ZnO) nanostructure is used for the immobilization, direct electrochemistry and biosensing of proteins. The prepared ZnO has a large surface area and good biocompatibility. Using glucose oxidase (GOD) as a model, this shaped ZnO is tested for immobilization of proteins and the construction of electrochemical biosensors with good electrochemical performances. The interaction between GOD and TPSP-ZnO is examined by using AFM, N(2) adsorption isotherms and electrochemical methods. The immobilized GOD at a TPSP-ZnO-modified glassy carbon electrode shows a good direct electrochemical behavior, which depends on the properties of the TPSP-ZnO. Based on a decrease of the electrocatalytic response of the reduced form of GOD to dissolved oxygen, the proposed biosensor exhibits a linear response to glucose concentrations ranging from 0.05 to 8.2mM with a detection limit of 0.01mM at an applied potential of -0.50V which has better biosensing properties than those from other morphological ZnO nanoparticles. The biosensor shows good stability, reproducibility, low interferences and can diagnose diabetes very fast and sensitively. Such the TPSP-ZnO nanostructure provides a good matrix for protein immobilization and biosensor preparation.     

Immobilization of glucose oxidase on Fe<Subscript>3</Subscript>O<Subscript>4</Subscript>/SiO<Subscript>2</Subscript> magnetic nanoparticles

Glucose oxidase (GOD) was covalently immobilized onto Fe₃O₄/SiO₂ magnetic nanoparticles (FSMNs) using glutaraldehyde (GA). Optimal immobilization was at pH 6 with 3-aminopropyltriethoxysilane at 2% (v/v), GA at 3% (v/v) and 0.143 g GOD per g carrier. The activity of immobilized GOD was 4,570 U/g at pH 7 and 50°C. The immobilized GOD retained 80% of its initial activity after 6 h at 45°C while free enzyme retained only 20% activity. The immobilized GOD maintained 60% of its initial activity after 6 cycles of repeated use and retained 75% of its initial activity after 1 month at 4°C whereas free enzymes retained 62% of its activity.