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。     

Investigation on the pH-dependent binding of benzocaine and lysozyme by fluorescence and absorbance

The interaction mechanism between benzocaine (BZC) and lysozyme (Lys) has been investigated by fluorescence, synchronous fluorescence, ultraviolet–vis (UV) absorption spectra, and three-dimensional fluorescence (3-D) in various pH medium. The observations of fluorescence spectra were mainly rationalized in terms of a static quenching process at lower concentration of BZC (C_BZC/C_Lys < 9) and a combined quenching process at higher concentration of BZC (C_BZC/C_Lys > 9) at pH 7.4 and 8.4. However, the fluorescence quenching was mainly arisen from static quenching by complex formation in all studied drug concentrations at pH 3.5. The structural characteristics of BZC and Lys were probed, and their binding affinities were determined under different pH conditions (pH 3.5, 7.4, and 8.4). The results indicated that the binding abilities of BZC to Lys decreased at the pH below and above the simulative physiological condition (pH 7.4) due to the alterations of the protein secondary and tertiary structures or the structural change of BZC. The effect of BZC on the conformation of Lys was analyzed using UV, synchronous fluorescence and three-dimensional fluorescence under different pH conditions. These results indicate that the binding of BZC to Lys causes apparent change in the secondary and tertiary structures of Lys. The effect of Zn²⁺ on the binding constant of BZC with Lys under various pH conditions (pH 3.5, 7.4, and 8.4) was also studied.     

Conformational changes and sequence analysis in cellulase from <Emphasis Type="Italic">Aspergillus niger</Emphasis> with cationic surfactant

The present study evaluates the binding of cetylpyridinium chloride (CPC) with cellulase in various experimental conditions using potentiometric, fluorescence spectroscopy and turbidimetric techniques. The analysis of binding curves revealed the existence of two sets of binding sets for CPC. The binding parameters were estimated and interpreted in terms of structural viewpoints of cellulase. The observation of turbidity suggests that CPC molecules individually nucleate around cellulase/CMC complex to form micelle-like structures. Fluorescence spectroscopy analysis of cellulase/CMC-surfactant system showed that these complexes could be compact to elucidate the mechanism of binding cellulase/CMC complex to CPC. The differential response of the enzyme/CMC to surfactant, indicates that the interaction on the complex surface is strongly ionic and hydrophobic(cooperative) in nature. A sequencing analysis was also conducted on β-1, 4-endoglucanase from A. niger (EglA) and others from family 12 in order to examine the nature of interaction involved in binding process and structure of carbohydrate-protein complexes. The results suggest that the conserved residues are located in a more hydrophobic microenvironment and apolar area energy is more than polar within enzyme structure.     

Thermal inactivation and conformational lock studies on glucose oxidase

In this study, the dissociative thermal inactivation and conformational lock theories are applied for the homodimeric enzyme glucose oxidase (GOD) in order to analyze its structure. For this purpose, the rate of activity reduction of glucose oxidase is studied at various temperatures using β-d-glucose as the substrate by incubation of enzyme at various temperatures in the wide range between 40 and 70 °C using UV–Vis spectrophotometry. It was observed that in the two ranges of temperatures, the enzyme has two different forms. In relatively low temperatures, the enzyme is in its dimeric state and has normal activity. In high temperatures, the activity almost disappears and it aggregates. The above achievements are confirmed by dynamic light scattering. The experimental parameter “n” as the obvious number of conformational locks at the dimer interface of glucose oxidase is obtained by kinetic data, and the value is near to two. To confirm the above results, the X-ray crystallography structure of the enzyme, GOD (pdb, 1gal), was also studied. The secondary and tertiary structures of the enzyme to track the thermal inactivation were studied by circular dichroism and fluorescence spectroscopy, respectively. We proposed a mechanism model for thermal inactivation of GOD based on the absence of the monomeric form of the enzyme by circular dichroism and fluorescence spectroscopy.     

Biosensors with reversed micelle-enzyme sensitive membrane

The effect of reversed micelle on the conformation of enzyme was studied by sensor techniques. By means of measurement of the response current of GOD enzyme membrane electrode, the effects of enzyme embedded in AOT reversed micellar on GOD conformation and catalytic activity are discussed. The results show that the response current increased greatly with decreasing ratio of GOD/AOT, meaning that the catalytic activity and the conformation stability of enzyme were enhanced.     

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.     

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.     

Kinetic study of the thermal inactivation of glucose oxidase in the presence of denaturant and stabilizer by means of bioelectrocatalysis method

The thermal inactivation of glucose oxidase (GOD) in aqueous solution has been studied by the electrochemical method to follow the bioelectrocatalytic current due to the oxidation of glucose by GOD. Exponential time-dependent decrease in bioelectrocatalytic current, that is, the decrease in the enzymatic activity of GOD, was observed at given temperatures to determine the rate constant (k) of a simple inactivation process: GOD (active) → GOD (inactive). The ln[k] vs. T(-1) plots gave straight lines with all solution conditions tested, so that the resulting Arrhenius activation parameters including ΔH(?) and ΔS(?) can be compared with each other. In the 50 mmol/L phosphate buffer at 70°C, k was determined to be (6.6 ± 1.6)× 10(-4) s(-1), and ΔH(?) and ΔS(?) were calculated to be 202 ± 13 kJ mol(-1) and 282 ± 39 J K(-1) mol(-1), respectively. By addition of 3 mol/L guanidine hydrochloride, the k was increased to (4.7 ± 0.6)× 10(-3) s(-1), indicating that the denaturant accelerates the thermal inactivation. In this case, ΔH(?) was significantly reduced. By addition of 1 g/L ε-poly-L-lysine, which may adsorb onto the GOD surface to reduce the local disorder, k was decreased to (1.8 ± 0.6)× 10(-4) s(-1). In this case, ΔS(?) was reduced but ΔH(?) was not decreased much. This can be used as an important indication for selection of the enzyme stabilizer in solution.     

The Preparation and Enzyme Immobilization of Hydrophobic Polysiloxane Supports

Enzymes are versatile biocatalysts and find increasing applications in many areas. The major advantages of using enzymes in biocatalytic transformations are their chemo‐, regio‐, and stereospecificity, as well as the mild reaction conditions that can be used. However, even when an enzyme is identified as being useful for a given reaction, its application is often hampered by its lack of long‐term stability under process conditions, and also by difficulties in recovery and recycling. For ease of application and stabilization purposes, enzymes are often immobilized on solid supports. Among support matrices, hydrophobic biomaterials have been extensively used as supports for enzyme immobilization because the hydrophobic interactions not only can effectively increase the amount of enzyme immobilization, but also exhibit higher activity and retention of activity compared with hydrophilic supports. On the other hand, polysiloxane can evidently increase the amount of enzyme immobilization because of its hydrophobicity and strong affinity with enzyme. Therefore, this research details the first preparation and use of a hydrophobic polysiloxane support for enzyme immobilization in which the structural and functional characteristics of new supports have been investigated by using glucose oxidase (GOD) and a simple Fenton's assay method, and extremely interesting features were revealed. The results showed that the amount of GOD immobilization and the stability of GOD loaded, which are fundamental properties for enzyme separation and purification, can be significantly improved by adsorption. Moreover, the results indicated that hydrophobic polysiloxane supports can effectively increase the enzymatic affinity and durability of GOD, and decrease the rate of GOD desorbed.

     

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.     

Direct Electrochemistry of Glucose Oxidase at a Gold Electrode Modified with Graphene Nanosheets

In this work, we report the direct electrochemistry of glucose oxidase (GOD) observed at a gold electrode modified with graphene nanosheets. Initially, graphene nanosheets were synthesized and conjugated to the enzyme GOD and immobilized on to a gold electrode surface. Cyclic voltammetry was then performed using Gold-Graphene-GOD modified electrodes in a pH 7.2 phosphate buffered saline (PBS). A pair of well-defined redox peaks was obtained for GOD with the reduction peak centered at +180 mV and a peak separation of 70 mV in PBS under physiological conditions. Moreover, the electron transfer rate of GOD redox reaction was greatly enhanced and the peak potential was found to be pH dependent at the graphene-GOD surface. Further, the performance of the Gold-Graphene-GOD was found to be stable and excellent under physiological conditions indicating the possibility of employing this platform for real time analysis. The observed results indicated that the 2D-graphene holds great promise for conjugation ability with a variety of enzymes. Further, our results also confirmed that graphene is capable of holding the enzyme GOD in a favorable position and retains its original structure and functionality that are essential for biosensing.     

Inhibition and enhancement of glucose oxidase activity in a chitosan-based electrode filled with silver nanoparticles

A chitosan-based electrode filled with silver nanoparticles (AgNPs) and glucose oxidase (GOD) was used as an enzyme electrode to investigate the effect of aging process of AgNPs on the GOD activity. Freshly prepared AgNPs inhibit the GOD activity, however, the inhibitory effect decreased with the increase of aging time. After aged for a period of time, AgNPs showed enhancement effect on the GOD activity. The effect of aging was studied by the measurements of Ag⁺ ions concentration, zeta (ζ) potential and X-ray photoelectron spectroscopy (XPS). And the results indicated that the concentration of Ag⁺ ions in the silver sol decreased during the aging period (i.e. Ag⁺ ions converted to more inert silver metal Ag⁰). The effect of AgNPs on the GOD activity can be changed by controlling the aging time of AgNPs. This research provides a new and simple approach to mediate AgNPs property, which is of great value in potential application of AgNPs in biosensors and nanoscale devices.     

生物功能电极 Ⅲ.葡萄糖氧化酶的电化学固定化研究

利用磷酸盐缓冲溶液中吡咯的电聚合,将葡萄糖氧化酶(GOD)包埋在聚吡咯(PPy)基质中以构成生物功能电极。讨论了溶液pH和聚合电位对酶固定化的影响,并用IR和交流阻抗谱对酶膜进行表征。GOD的固定化只有当pH>5.5时才能实现,由此推测酶是以带负电的粒子嵌入PPy的。交流阻抗谱表明这一电极具有有界多孔电极的特征。探索了酶与电子传递体Fe(CN)_6~(3-)同时固定化的可行性。电化学固定化的GOD保持其生物催化活性,酶反应表观上遵循Michealis-Menten动力学。     

生物功能电极 III. 葡萄糖氧化酶的电化学固定化研究

利用磷酸盐缓冲溶液中吡咯的电聚合, 将葡萄糖氧化酶(GOD)包埋在聚吡咯(PPy)基质中以构成生物功能电极。讨论了溶液pH和聚合电位对酶固定化的影响, 并用IR和交流阻抗谱对酶膜进行表征。GOD的固定化只有当pH>5.5时才能实现, 由此推测酶是以带负电的粒子嵌入PPy的。交流阻抗谱表明这一电极具有有界多孔电极的特征。探索了酶与电子传递体Fe(CN)_6~(3-)同时固定化的可行性。电化学固定化的GOD保持其生物催化活性, 酶反应表观上遵循Michealis-Menten动力学。     

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.     

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.     

Entrapment of glucose oxidase in non-porous poly(vinyl chloride)

We have used solvent casting techniques to immobilise glucose oxidase (GOD) within unplasticised and plasticised poly(vinyl chloride) (PVC) matrices. The plasticisers studied were the cationic surfactant, tricaprylmethylammonium chloride (Aliquat 336s), the anionic surfactant bis(2-ethylhexyl) hydrogenphosphate (BEP) and the lipid, isopropylmyristate (IPM). The activity of the enzyme-membrane was tested by amperometric electrode. Changes in enzyme-membrane electrode response are rationalised on the basis of membrane permselective properties. The Aliquat and IPM modified PVC membranes gave amplified signals due to better retention and subsequent concentration of the H₂O₂ signal species. Effectively, less was being lost to the bulk solution. In the case of the BEP-modified membrane, while there was a linear step change in response up to 50 mM, at higher concentrations, responses did not reach steady-state; they were characterised by an upward drift in response of 0.050 nA/min. This characteristic is thought to be due to a build up of gluconic acid resulting in a pH reduction in the membrane microenvironment and hydrogen bonding between neighbouring BEP molecules. Under these conditions, we have previously shown that the membrane permeability to hydrophilic species is attenuated and it is tentatively suggested that the upward drift due to the build up of H₂O₂ on the electrode side with less permeating through the acidified membrane into bulk solution.The results were compared against using variously plasticised PVC (but no enzyme entrapped) as an outer membrane of a classical dual-membrane glucose enzyme electrode construct. In the latter case, the enzyme was chemically crosslinked between the membranes using glutaraldehyde.     

Binding and fluorescence study on interaction of human serum albumin (HSA) with cetylpyridinium chloride (CPC)

Human serum albumin (HSA) is frequently used in biophysical and biochemical studies since it has a well-known primary structure and it has been associated with the binding of many different categories of small molecules. In the present study, results are presented for the binding of cetylpyridinium chloride (CPC) with HSA at various pH and 25 degrees C, as monitored using ion selective membrane electrodes and fluorescence spectroscopy of intrinsic tryptophan. The obtained binding isotherms were analyzed on basis of binding capacity concept and Hill plot in order to determine the Hill parameters of binding sets. The system behaved as a system with two sets of binding sites in all studied situations. The results represent a positive cooperative behavior and the essential role of hydrophobic interactions in both binding sets. The intrinsic binding affinity of second binding set have a similar values and trends at acidic and neutral pHs, that represents the similar unfolded structure at these pHs. CPC quenched the fluorescence arising from Trp group incorporated to HSA. A biphasic behavior was observed in quenching process that confirmed the results of binding study correspond to the existence of two binding sets. The similarity of unfolded structure in acidic and neutral pH was also confirmed by fluorescence study. The quenching of HSA fluorescence takes place with a Stern-Volmer constant of 0.643 x 10(4), 1.23 x 10(4) and 7.40 x 10(4) at pH 3.5, 6.8 and 9.5, respectively. The Stern-Volmer behavior observed at low molar ratio of [CPC]/[HSA] (about 6), that represents the occurrence of conformational changes after this molar ratio. Comparing, the K(SV) values and binding parameters indicate that the binding is dominated by hydrophobic effects and, in minor degree, by electrostatic interactions.     

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.     

人类细胞色素P450 2E1酶在不同乙醇浓度下构象变化的分子动力学模拟

细胞色素P450(CYP) 2E1家族酶是一种具有双重功能的单加氧酶, 能够参与市场上6%药物的代谢而具有重要的作用. 这类酶与酒精的消耗、 糖尿病、 肥胖症以及厌食症等密切相关, 引起了广泛的研究兴趣. 目前尚未见从原子水平上对这种酶在不同乙醇浓度下构象行为的研究. 基于此, 本文研究了花生四烯酸(AA)与CYP2E1复合物结构在不同乙醇浓度下构象与能量变化的特点. 对于在不同乙醇浓度下AA与CYP2E1的复合物结构, 采用分子动力学模拟结合自由能计算的方法进行研究. 分子动力学模拟结果表明, His109和Lys243氨基酸残基对AA与CYP2E1的结合起到了至关重要的作用. 当体系的乙醇浓度较高时, AA的结合能力有所下降, 这种结合能力的下降是由于AA与CYP2E1之间氢键相互作用力的减弱所致. 本研究对于AA与CYP2E1复合物结构在不同乙醇浓度下, AA分子与CYP2E1分子结合能力下降以及CYP2E1的构象变化给出了详细的解释. 本研究工作得到的结论对于实验和理论研究均有重要意义, 可为后续细胞色素P450酶类催化活性的研究提供理论支持.