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.

     

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.     

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.     

Membrane reactor immobilized with palladium‐loaded polymer nanogel for continuous‐flow Suzuki coupling reaction

A catalytic membrane reactor, which was immobilized with palladium‐loaded nanogel particles (NPs), was developed for continuous‐flow Suzuki coupling reaction. Palladium‐loaded membranes were prepared by immobilization of NPs, adsorption of palladium ions, and reduction into palladium(0). The presence of palladium in the membrane was confirmed by the scanning electron microscopy; palladium aggregation was not observed. The catalytic activity of the membrane reactor in continuous‐flow Suzuki coupling reaction was approximately double that of a comparable reactor in which palladium ions were directly adsorbed onto an aminated membrane. This was attributed to the formation of small palladium particles. The reusability in the continuous‐flow system was higher than that in a batch system, and the palladium‐loaded membrane reactor had high long‐term stability. © 2014 American Institute of Chemical Engineers AIChE J, 61: 582–589, 2015     

A new metal chelate sorbent for glucose oxidase: Cu(II)- and Co(II)-chelated poly(N-vinylimidazole) gels

Poly(N-vinylimidazole) (PVIm) gels were prepared by irradiating a binary mixture of N-vinylimidazole (VIm)–water in a ⁶⁰Co-γ source having 4.5 kGy/h dose rate. In the glucose oxidase (GOx) adsorption studies, affinity gels with a swelling ratio of 1100% for PVIm and 40 and 55% for Cu(II)- and Co(II)-chelated PVIm gels, respectively, at pH 6.5 in phosphate buffer were used. FTIR spectra were taken for PVIm and Cu(II)- and Co(II)-chelated PVIm, and glucose oxidase adsorption on these gels, to characterize the nature of the interactions in each species. The results show that PVIm–glucose oxidase interaction is mainly electrostatic and metal ion–chelated PVIm gel–glucose oxidase interaction is of coordinate covalent nature. Cu(II) and Co(II) ions were chelated within the gels via amine groups on the imidazole ring of the gel. Different amounts of Cu(II) and Co(II) ions [maximum 3.64 mmol/g dry gel for Cu(II) and 1.72 mmol/g dry gel for Co(II)] were loaded on the gels by changing the initial concentration of Cu(II) and Co(II) ions at pH 7.0. GOx adsorption on these gels from aqueous solutions containing different amounts of GOx at different pH was investigated in batch reactors. GOx adsorption capacity was further increased when Cu(II) and Co(II) ions were attached [up to 0.53 g GOx/g dry Co(II)-chelated PVIm gels]. More than 90% of the adsorbed GOx was desorbed in 5 h in desorption medium containing 1.0M KSCN at pH 7.0 for plain gel and 0.05M EDTA at pH 4.9 for metal-chelated gel. Nonspecific glucose oxidase adsorption on/in the metal ion–chelated PVIm gel was investigated using 0.02M of phosphate buffer solution. The nonspecific GOx adsorption was determined to be about 18% for PVIm and 8% for the metal ion–chelated PVIm gels. The ionic strength effect was investigated both on PVIm and on the metal ion–chelated PVIm gels for the glucose oxidase adsorption. It was found that ionic strength was more effective on the PVIm gel because of the electrostatic interaction between protonated gel and the deprotonated glucose oxidase side chain. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 82: 446–453, 2001     

硅藻土负载异养硝化-好氧反硝化菌的脱氮性能

将硅藻土经改性后作为异养硝化-好氧反硝化菌H1的载体,对负载条件以及固定化菌对环境的耐受性能进行了优化及研究.确定最佳吸附时间为24h,载体投加量为0.06g/mL(硅藻土/菌悬液).改性剂FeSO₄用量、pH值、温度不仅影响硅藻土载体吸附性,同时影响固定化H1活性.菌株经改性硅藻土负载后较游离菌对pH值及温度耐受性都有所增强,对溶解氧变化适应范围更广,当m(FeSO₄)/m(硅藻土)=3.5%、pH=7.5、温度为30℃、溶解氧为5.1mg/L左右时,固定化H1脱氮性能最佳.使用该固定化菌对生活污水进行连续式处理,8天后目标污染物的去除率趋于稳定,TN、NH₄⁺-N及COD去除率分别达到52.40%、55.64%与61.23%,表明改性硅藻土负载异养硝化-好氧反硝化菌在污水脱氮领域具有广阔的前景.     

Influence of remote plasma on PEDOT:PSS-coated carbon felt for improved activity of glucose oxidase

Increasing wettability of carbon felts is an important strategy to improve their efficiency in bio-electrochemical applications. Herein, influence of cold remote plasma (N₂ + O₂) treatment on surface properties of carbon felts with poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) coating was tested, aiming to improve immobilizing of glucose oxidase enzyme (GOx). Spectra of N 1s and O 1s confirmed the integration of carbonyl and ether as well as amide and amine groups on bare carbon fiber surface, while on coated fibers, carbonyl groups were pre-dominant. S 2p spectra confirmed oxidation of PEDOT:PSS coating with reduction of (S⁻) compared to (SO³⁻) group. GOx immobilized on different samples showed highest activity for PEDOT:PSS coating subjected to plasma with 2% O₂, maintaining up to 60% after immobilization, and 37% of its activity after six cycles for some samples. Enzymes immobilized on samples without plasma treatment lost their activity after four cycles. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2020 , 137, 48521.     

TiO2纳米管阵列孔径调控葡萄糖氧化酶生物传感器性能

采用电化学阳极氧化法制备出不同孔径（21、62、83、102 nm）的TiO₂纳米管阵列（TNA）,研究了孔径对固定化葡萄糖氧化酶（GOx）的传感器性能的影响。循环伏安测试结果表明固定在不同孔径大小的TNA上的GOx在葡萄糖溶液中均具有良好的酶活性。计时电流法和交流阻抗法测试发现,当孔径是83 nm时,灵敏度达到最大值27.2 μA·（mmol·L^-1）^-1·cm^-2。调控TNA的孔径可改变固定化GOx的活性及溶液扩散阻抗,从而显著提高生物传感器性能。     

树脂吸附法固定Candida rugosa脂肪酶

Candida rugosa脂肪酶具有优良的催化性能,对其进行固定化可以很方便地实现酶的回收和再利用。采用南开大学化工厂生产的4种阴离子交换树脂和4种大孔吸附树脂为载体,对来源于Candida rugosa的脂肪酶进行了吸附固定化,结果表明,以大孔吸附树脂AB-8为载体的固定酶比活性最高。固定化酶制备过程中缓冲液的最适宜pH值为7.2,最佳固定化时间为1 h,载体和酶的最佳质量配比为10∶1。与游离酶相比,固定化后酶活损失大约30%,但稳定性平均约提高60%。     

Dye derived and metal incorporated affinity poly(2-hydroxyethyl methacrylate) membranes for use in enzyme immobilization

Microporous poly(2-hydroxyethyl methacrylate) (PHEMA) membranes were prepared by UV-initiated photopolymerization of HEMA in the presence of an initiator (α,α′-azobisisobutyronitrile, AIBN). An affinity dye Cibacron Blue F3GA (CB) was attached covalently and then Fe³⁺ ions incorporated. The PHEMA-CB and PHEMA-CB-Fe³⁺ membranes derived were used for adsorption of glucose oxidase (GOD). The adsorption capacities of these membranes were determined under conditions of different pH and with different concentrations of the adsorbate in the medium. The adsorption phenomena appeared to follow a typical Langmuir isotherm. The glucose oxidase adsorption capacity of the Fe³⁺ incorporated membrane (87μgcm^-2) was greater than that of the dye-derived membrane (66μgcm^-2). Non-specific adsorption of the glucose oxidase on the PHEMA membranes was negligible. The K_m values for both immobilized glucose oxidase PHEMA-CB-GOD (8·3) and PHEMA-CB-Fe³⁺-GOD (7·6) were higher than that of the free enzyme (6·2mM). Optimum reaction pH was 5·5 for the free and 6·0 for both immobilized preparations. The optimum reaction temperature of the adsorbed enzymes was 5°C higher than that of the free enzyme and was significantly broader. After 15 successive uses the retained activity of the adsorbed enzyme was 87%. It was observed that enzymes could be repeatedly adsorbed and desorbed on the derived PHEMA membranes without significant loss in adsorption capacity or enzymic activity. © 1998 SCI.     

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     

Covalent immobilization of penicillin G acylase onto amine-functionalized PVC membranes for 6-APA production from penicillin hydrolysis process. II. Enzyme immobilization and characterization

This article describes the covalent immobilization of penicillin G acylase (PGA) onto glutaraldehyde-activated NH₂-PVC membranes. The immobilized enzyme was used for 6-aminopenicillanic acid production from penicillin hydrolysis. Parameters affecting the immobilization process, which affecting the catalytic activity of the immobilized enzyme, such as enzyme concentration, immobilization's time and temperature were investigated. Enzyme concentration and immobilization's time were found of determine effect. Higher activity was obtained through performing enzyme immobilization at room temperature. Both optimum temperature (35°C) and pH (8.0) of immobilized enzyme have not been altered upon immobilization. However, immobilized enzyme acquires stability against changes in the substrate's pH and temperature values especially in the higher temperature region and lower pH region. The residual relative activities after incubation at 60°C were more than 75% compared to 45% for free enzyme and above 50% compared to 20% for free enzyme after incubation at pH 4.5. The apparent kinetic parameters K_M and V_M were determined. K_M of the immobilized PGA (125.8 mM) was higher than that of the free enzyme (5.4 mM), indicating a lower substrate affinity of the immobilized PGA. Operational stability for immobilized PGA was monitored over 21 repeated cycles. The catalytic membranes were retained up to 40% of its initial activity after 10.5 working h. © 2012 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

Immobilization of Phospholipase C for the Production of Ceramide from Sphingomyelin Hydrolysis

The immobilization of Clostridium perfringens phospholipase C was studied for the first time and the catalytic properties of the immobilized enzyme were investigated for the hydrolysis of sphingomyelin to produce ceramide. Ceramide is of great commercial value in the cosmetic and pharmaceutical industries for use in, for example, hair and skin care products, owing to its major role in maintaining the water-retaining properties of the epidermis. The feasibility of enzymatic production of ceramide through hydrolysis of sphingomyelin has previously been proven. In order to improve the reusability of the enzyme, the present study focused on the immobilization of phospholipase C in the production of ceramide from sphingomyelin. By screening nine different carriers, we found that the enzyme immobilized on Lewatit had the highest catalytic activity towards sphingomyelin hydrolysis. Prewetting Lewatit with ethanol led to higher enzyme fixation on the carrier, but the activity of the enzyme was decreased. Increasing the initial enzyme concentration resulted in more enzyme adsorption on the carrier, where the specific activity was increased. Through optimization of the reaction using the immobilized enzyme, the optimal temperature was around 46 °C and the optimal water volume was 3.5%. The reaction had little dependence on pH. After seven recycles, immobilized enzyme retained around 70% of the initial activity. Immobilized enzyme was deactivated irregularly when stored at room temperature, but followed first-order deactivation when stored at 40 °C.     

UV-curable methacrylated/fumaric acid modified epoxy as a potential support for enzyme immobilization

The immobilization procedure of UV-curing coating is simple and causes less loss of enzymatic activity. UV-curable methacrylated/fumaric acid modified cycloaliphatic epoxide is here proposed as a rigid support material for covalent immobilization of α-amylase. The immobilized enzyme is analyzed in terms of bioactivity retention as a function of repeated use ability, pH, storage, as well as stability under various experimental conditions, taking starch as a substrate. The properties of immobilized enzyme were also compared with those of the free enzyme. The highest activity of free enzyme was obtained at pH 7.0 while this value was shifted to pH 7.5 for immobilized system. Optimum catalytic activity was observed at 30 °C, for both free and immobilized enzyme; however, the immobilized enzyme had a higher activity than the free one. The immobilized enzyme that was used 35 times in 8 h in repeated batch experiments demonstrated that about 73% of the enzyme activity was retained. The free enzyme lost all its activity with in 15 days. The retained activity of immobilized enzyme was found to be around 80%. The amount of bound α-amylase was found 94 mg per gram polymeric support material.     

Papain Adsorption on Chitosan-Coated Nylon-Based Immobilized Metal Ion (Cu2+, Ni2+, Zn2+, Co2+) Affinity Membranes

A novel immobilized metal affinity membrane was prepared for papain adsorption in this article. Higher papain adsorption capacity between 43-67 mg/g was observed and the adsorption isotherm fitted the Freundlich equation. Experimental data were analyzed using two adsorption kinetic models. The pseudo-second-order kinetic model provided better correlation to the experimental results. A significant amount of the adsorbed papain was eluted by 1.0 M NaSCN at pH 5.0 for all affinity membranes. It was concluded that the novel chitosan-coated nylon-based immobilized metal ion affinity membrane could be applied for the large-scale isolation of papain without resulting in enzyme denaturation.     

Immobilization of catalases on amidoxime polyacrylonitrile nanofibrous membranes

Amidoxime polyacrylonitrile (AOPAN) nanofibrous membranes were generated by the reaction between electrospun polyacrylonitrile nanofibrous membranes and hydroxylamine hydrochloride. AOPAN nanofibrous membranes were further modified by Fe(III) chelation for immobilizing catalases with coordination bonds. The surface morphologies of the nanofibrous membranes and immobilized catalases were observed by field emission scanning electron microscopy. Chelation of Fe(III) onto AOPAN nanofibrous membranes was studied by the Langmuir isothermal adsorption model. It was found that the maximum amount of coordinated Fe(III) (q_m) was 4.5045 mmol g^?1 (dry nanofibrous membranes) and the binding constant (K_l) was 0.0698 L mmol^?1. The amounts of immobilized enzymes were determined by the method of Bradford. Kinetic parameters were analyzed for both immobilized and free catalases. The value of V_max (7122.6 µmol mg^?1 min^?1) for the immobilized catalases was smaller than that for the free catalases (9203.2 µmol mg^?1 min^?1), and the K_m for the immobilized catalases was larger. The immobilized catalases showed better resistance to pH and temperature change than the free catalases, and the storage stability of immobilized catalases was higher than that of free catalases. As for reusability, the immobilized catalases retained 71% of their activity after eight repeated uses. © 2012 Society of Chemical Industry     

Covalent immobilization of naringinase for the transformation of a flavonoid

Naringinase (EC 3.2.1.40) from Penicillium sp was immobilized by covalent binding to woodchips to improve its catalytic activity. The immobilization of naringinase on glutaraldehyde‐coated woodchips (600 mg woodchips, 10 U naringinase, 45 °C, pH 4.0 and 12h) through 1% glutaraldehyde cross‐linking was optimized. The pH–activity curve of the immobilized enzyme shifted toward a lower pH compared with that of the soluble enzyme. The immobilization caused a marked increase in thermal stability of the enzyme. The immobilized naringinase was stable during storage at 4 °C. No loss of activity was observed when the immobilized enzyme was used for seven consecutive cycles of operations. The efficiency of immobilization was 120%, while soluble naringinase afforded 82% efficacy for the hydrolysis of standard naringin under optimal conditions. Its applicability for debittering kinnow mandarin juice afforded 76% debittering efficiency. Copyright © 2005 Society of Chemical Industry     

Fabrication of hydrophilic nanoporous PMMA/O-MMT composite microfibrous membrane and its use in enzyme immobilization

The electrospun PMMA/O-MMT microfibrous membranes were pretreated by oxygen plasma to create substrates with better adsorption capability. The amount of O-MMT and conditions of plasma treatment were optimized for maximum laccase immobilization on these pretreated surfaces of the microfibrous membranes. The surface morphology and chemistry of the composite microfibrous membranes after plasma treatment and laccase immobilization were investigated by SEM and FTIR. The immobilized laccase showed better resistance to pH and temperature changes than that of the free form laccase, and after 10 successive runs of repeated use, the immobilized laccase still retained 30 % of its initial activity. Reactive X-3B was successfully degraded by both free and immobilized laccase.     

Fabrication of bioanode by using electrically conducting polythiophene via entrapment technique

A glassy carbon electrode (GCE) was tailored with conducting polymer polythiophene and further immobilized by an enzyme glucose oxidase (GOx). A thin film of polymer was developed by electrochemical polymerization of thiophene monomer. During electrochemical polymerization of the monomer the enzyme GOx and the redox active mediator ferritin (Frt) were entrapped within this polymer matrix. In this novel approach, the entrapment of enzyme and mediators within a polymer matrix occurs without chemical reaction that could affect their activity. The entrapment of enzyme and mediator within the conducting polymer matrices increases the surface area of the electrode. The tailored GCE/Ptp/Frt/GOx electrode showed a high catalytic activity. The increased surface area causes a high rate of electron transfer between the electrode and Frt engaged as an electron transfer mediator. The electrochemical properties of the electrode were determined by cyclic voltammetry (CV) and linear sweep voltammetry (LSV). The fabricated bioanode showed a current density of 3.9mA cm^?2 at 1.0 V vs. Ag/AgCl in a 45 mM glucose solution and suggests proficient chances in biofuel cells (BFCs) applications.     

Immobilization of hydrophobic lipase derivatives on to organic polymer beads

A simple and effective method of lipase immobilization is described. Lipase from Candida rugosa was first modified with several hydrophobic modifiers before being adsorbed on to organic polymer beads. The soluble hydrophobic lipase derivatives adsorbed more strongly on to the various polymers as compared with the native lipase. The optimal adsorption temperature of the native and modified lipases on all the polymers was 40°C. The optimal pH of adsorption was between 6 and 7. Lipase immobilized in this manner produced high catalytic recoveries which were affected by the type of modifiers, degree of modification and type of supports used. Monomethoxypolyethylene glycol (1900) activated with p-nitrophenyl chloroformate was found to be the best modifier of the enzyme at 95% modification, for adsorption to the polymers. Increasing the degree of modification of the enzyme increased the activity which was immobilized. Generally, both native and hydrophobic lipase derivatives showed higher specific activities when immobilized on polar polymers compared with non-polar polymers.