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.     

Cutoff performance of Escherichia coli by charged and noncharged polyacrylonitrile ultrafiltration membranes

Cutoff performance of Escherichia coli (E. coli) was studied by three types of polyacrylonitrile (PAN) ultrafiltration (UF) membranes without and with charge groups of sulfonate sodium salt (SSS) and trimethylammonium chloride (TMA). These UF membranes were prepared by phase inversion method in water coagulation bath with various concentrations of dimethylsulfoxide (DMSO) and used for the E. coli cutoff experiments under 2.5 kPa applied pressure. With the increase of the polymer concentration in the DMSO cast solution, the pore size of the molecular size exclusion effect of the resultant UF membrane decreased. For UF experiments of E. coli suspension solution with 10⁷ colony forming unit/unit volume (cfu/ml), the permeability of the bacteria through the membrane was in the range of about 10⁻³% in PAN homopolymer membranes. It was found that E. coli permeation through copolymer UF membranes with SSS and TMA groups was completely restricted. Difference of the E. coli cutoff performance in these UF membranes was discussed in comparison with membrane filtration properties such as molecular sieve effect, permeation rate of solute and membrane morphology.     

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.     

Polyethersulfone/polyacrylonitrile blend ultrafiltration membranes with different molecular weight of polyethylene glycol: preparation,morphology and antifouling properties

Asymmetric ultrafiltration (UF) membranes were prepared from blends of polyethersulfone (PES)/polyacrylonitrile (PAN) via phase inversion method induced by immersion precipitation. Polyethylene glycol (PEG) with four different molecular weights was used as pore former and hydrophilic polymeric additive. N,N‐dimethylformamide (DMF) and water were used as solvent and coagulant (nonsolvent), respectively. The effects of different proportion of PES/PAN and molecular weight of PEG on morphology and performance of the prepared membranes were investigated. Performance of the membranes was evaluated using UF experiments of pure water and buffered bovine serum albumin (BSA) solution as feed. The contact angle measurements indicated that the hydrophilicities of PES/PAN membrane increase by increasing the PAN concentration in the casting solution. However, performance of the membranes improves by increasing the PAN concentration in the casting solution up to 20% and then decreases with further addition of PAN. It was found out that the rejection of BSA decreases with increasing the PAN concentration in the casting solution. Furthermore, it was found that the performance of the membranes increases by increasing the molecular weight of PEG up to 1500 Da and then decreases with the higher molecular weights. The morphology of the prepared membranes was studied by scanning electron microscopy. Copyright © 2011 John Wiley & Sons, Ltd.     

Diffusion and calibration properties of microdialysis sampling membranes in biological media

The diffusion and calibration properties for three commercially available microdialysis membranes (polycarbonate-polyether (PC), polyacrylonitrile (PAN), and cuprophan (CUP)) were evaluated. The analytes studied had molecular weights between 94 (phenol) and 1355 (vitamin B12). For each analyte-membrane pair, an effective membrane diffusion coefficient was calculated. Effective membrane diffusion coefficients varied considerably between the microdialysis membranes. For Vitamin B12, CUP and PAN membranes gave relative recovery values of greater than 20% at 0.5 microl min(-1), while the PC membrane had a 1% recovery. When backpressure was applied. PC and PAN membranes exhibited more ultrafiltration than CUP membranes. Ultrafiltration did not affect analyte relative recovery through either PC or PAN membranes. Effective membrane diffusion coefficients were not significantly altered for some membrane-analyte combinations when exposed to 4% bovine serum albumin or 0.3% fibrinogen. These data suggest that reductions in relative recovery during long-term microdialysis sampling experiments may be due to other physiologically relevant proteins or to tissue reactions near the dialysis membrane.     

A gas–liquid chemical reaction treatment and phase inversion technique for formation of high permeability PAN UF membranes

Polyacrylonitrile (PAN) ultrafiltration (UF) membranes were prepared by the pretreatment (gas–liquid interfacial chemical reaction) and the phase inversion process from a casting solution containing dimethylacetamide (DMAC) as a solvent and CaCl₂/NH₃·H₂O/H₂O as a composite additive. Deionized (DI) water was used as a coagulant. The membranes were characterized in terms of the pure water fluxes, protein retention and direct field emission scanning electron microscopy (FESEM) observations. The effects of gas–liquid interfacial chemical reaction on the membrane performance were investigated by changing the pretreatment time, CO₂ contacting method (static or flowing) and Partial pressure of CO₂. The gas–liquid interfacial chemical reaction had great influence on reducing pore size and increasing porosity of PAN membrane.     

Surface-anchored poly(2-vinyl-4,4-dimethyl azlactone) brushes as templates for enzyme immobilization

We explored surface-anchored poly(2-vinyl-4,4-dimethyl azlactone) (PVDMA) brushes as potential templates for protein immobilization. The brushes were grown using atom transfer radical polymerization from surface-anchored initiators and characterized by a combination of ellipsometry, atomic force microscopy, and X-ray photoelectron spectroscopy. RNase A was immobilized as a model enzyme through the nucleophilic attack of azlactone by the amine groups in the lysines located in the protein. The surface density of RNase A increased linearly from 5 to 50 nm. For 50 nm thick poly(2-vinyl-4,4-dimethyl azlactone) brushes, 7.5 microg/cm2 of RNase A was bound. The kinetics and thermodynamics of RNase A immobilization, the activity relative to surface density, and the pH and temperature dependence were examined. A Langmuir-like model for binding kinetics indicates that the kinetics are controlled by the rate of adsorption of RNase A and has an adsorption rate constant, k(ads), of 2.8 x 10(-8) microg(-1) s(-1) cm3. A maximum relative activity of approximately 0.95, which is near the activity of free RNase A, was reached at 1.2 microg/cm2 (approximately 3.0 monolayers) of immobilized RNase A. The immobilized RNase A had a similar temperature and pH dependence as free RNase A, indicating no significant change in conformation. The PVDMA template was extended to other biotechnologically relevant enzymes, such as deoxyribonuclease I, glucose oxidase, glucoamylase, and trypsin, with relative activities higher than or comparable to those of enzymes immobilized by other means. PVDMA brushes offer an efficient route to immobilize proteins via the ring opening of azlactone without the need for activation or pretreatment while retaining high relative activities of the bound enzymes.     

Electron paramagnetic resonance spin label titration: a novel method to investigate random and site-specific immobilization of enzymes onto polymeric membranes with different properties

The immobilization of biological molecules onto polymeric membranes to produce biofunctional membranes is used for selective catalysis, separation, analysis, and artificial organs. Normally, random immobilization of enzymes onto polymeric membranes leads to dramatic reduction in activity due to chemical reactions involved in enzyme immobilization, multiple-point binding, etc., and the extent of activity reduction is a function of membrane hydrophilicity (e.g. activity in cellulosic membrane?polysulfone membrane). We have used molecular biology to effect site-specific immobilization of enzymes in a manner that orients the active site away from the polymeric membrane surface, thus resulting in higher enzyme activity that approaches that in solution and in increased stability of the enzyme relative to the enzyme in solution. A prediction of this site-specific method of enzyme immobilization, which in this study with subtilisin and organophosphorus hydrolase consists of a fusion tag genetically added to these enzymes and subsequent immobilization via the anti-tag antibody and membrane-bound protein A, is that the active site conformation will more closely resemble that of the enzyme in solution than is the case for random immobilization. This hypothesis was confirmed using a new electron paramagnetic resonance (EPR) spin label active site titration method that determines the amount of spin label bound to the active site of the immobilized enzyme. This value nearly perfectly matched the enzyme activity, and the results suggested: (a) a spectroscopic method for measuring activity and thus the extent of active enzyme immobilization in membrane, which may have advantages in cases where optical methods can not be used due to light scattering interference; (b) higher spin label incorporation (and hence activity) in enzymes that had been site-specifically immobilized versus random immobilization; (c) higher spin label incorporation in enzymes immobilized onto hydrophilic bacterial cellulose membranes versus hydrophobic modified poly(ether)sulfone membranes. These results are discussed with reference to analysis and utilization of biofunctional membranes.     

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.     

Active transport of glycerol-3-phosphate with artificial enzyme membranes: A new kinetic model for active transport processes

An approach to the mechanism which may govern translocation and active transport system is presented. Two artificial enzyme membranes with immobilized kinase and immobilized phosphatase, respectively, were used close together to separate two unequal compartments of a specially designed diffusion cell in order to mimic solute active transport. Experiments were conducted and both translocation and active transport of glycerol-3-phosphate were obtained. The theoretical analysis of this active transport-like phenomenon, which underlines the key role played by the charge distribution on the membrane and the diffusion layers existing close to the membrane-bound enzymes is presented and is in good agreement with the experimental data. Our results mainly demonstrate that under specific conditions, the association of kinase and phosphatase activities on both parts of a porous membrane functions as an enzymic pump which performs active transport. Such results may be of general significance and lead us to suggest that a carrier could be substituted by two catalytic activities bound on both parts of a structure of channel type and catalysing two opposite reactions in diffusion layers.     

Immobilization of urease onto membranes of modified acrylonitrile copolymer

Urease was immobilized onto membranes prepared from acrylonitrile (AN) copolymer (powder) modified preliminarily with 2-dimethylaminoethyl methacrylate (DMAEM) and diacrylamido-2-methylpropanesulfonic acid (AMPSA). The results obtained were compared with those from commercial acrylonitrile copolymer membranes surface modified with DMAEM and AMPSA under the same conditions. The preliminary treatment was found to give higher amount of active groups and higher modification degree compared to surface modified acrylonitrile copolymer membranes. The modified membranes were used as carriers for immobilization of urease. The basic characteristics of the immobilized urease (amount of bound protein and relative activity) were studied. Temperature and pH optima and thermal stability of the immobilized urease were also studied. The membranes prepared from AN copolymer (powder) modified with AMPSA and DMAEM were used to manufacture diagnostic test-strips for analysis of urea in blood.     

Enzyme-entrapping membranes for enzyme sensors

A reliable method of physically immobilizing enzymes in cellulose triacetate (TAC) membranes was developed. The method has several advantages compared with analogous ones currently employed; it was possible to prepare enzyme sensors based on immobilized glucose oxidase (GOD) for determination of glucose in standard solutions and control sera, and based on GOD and invertase for determination of sucrose.     

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.     

The relationship between membrane surface pore characteristics and flux for ultrafiltration membranes

Surface porosities of Amicon XM100A and XM300 membranes have been measured by electron microscopy and found to be less than 1 per cent. From the measured pore size distributions it is deduced that 50 per cent of the solvent flow is through 20 to 25 per cent of the pores.The conventional model for concentration polarisation in ultrafiltration (UF), which assumes a homogeneously permeable membrane surface, has been modified to account for regions of differing permeability. An effective free area correction factor (≤ 1.0) has been introduced to allow for the effect of membrane surface properties on gel-polarised UF flux.Ultrafiltration experiments with protein solutions and membranes with a range of water fluxes confirm that gel-polarised UF flux is dependent on membrane permeability and surface properties. Effective free area correction factors vary from about 0.4 to 1.0 with values < 1.0 obtained for membranes with water fluxes typically < 150 1/m² hr at 100 kPaSupport for the effective free area concept in UF is provided by an analogy between a gel-polarised UF membrane and a composite reverse osmosis membrane. In both cases the magnitude of the upper ‘controlling’ resistance may be influenced by the pore size and spacing of the lower supporting structure.     

环糊精聚合物与苯醌的分子包合作用及其在酶电极中的应用

研究了水溶性环糊精预聚合物的存在对苯醌/氢醌体系在铂电极上氧化还原行为的影响, 根据伏安曲线讨论了该预聚合物与苯醌的分子包合作用。环糊精预聚合物与戊二醛缩聚反应而形成的不溶性聚合物膜用于葡萄糖氧化酶的固定化, 以制得新型的第二代葡萄糖电极。由于分子包合作用, 作为电子受体的苯醌在含酶的环糊精聚合物膜中具有较高的浓度, 从而加速了固定化酶的电子传递。测定了酶电极上BQ反应的动力学参数。     

Structure and dynamics of crystalline protein layers bound to supported lipid bilayers

We study proteins at the surface of bilayer membranes using streptavidin and avidin bound to biotinylated lipids in a supported lipid bilayer (SLB) at the solid-liquid interface. Using X-ray reflectivity and simultaneous fluorescence microscopy, we characterize the structure and fluidity of protein layers with varied relative surface coverages of crystalline and noncrystalline protein. With continuous bleaching, we measure a 10-15% decrease in the fluidity of the SLB after the full protein layer is formed. We propose that this reduction in lipid mobility is due to a small fraction (0.04) of immobilized lipids bound to the protein layer that create obstacles to membrane diffusion. Our X-ray reflectivity data show a 40 A thick layer of protein, and we resolve an 8 A layer separating the protein layer from the bilayer. We suggest that the separation provided by this water layer allows the underlying lipid bilayer to retain its fluidity and stability.     

Porous latex composite membranes: fabrication and properties

A new class of microfiltration (MF) and ultrafiltration (UF) membranes has been developed. By placing latex particles onto the surface of a microporous substrate and stabilizing the porous array, voids are formed between the particles which provide narrowly distributed pores that serve as separation channels. The size of the interstitial voids in the array is governed by the diameter of the latex particle. This aqueous based technology has advantages relative to other membrane fabrication processes in terms of the high asymmetry of the membranes, the facile adjustment of pore sizes, and the ability to easily modify pore surfaces during the synthesis of particles.A number of approaches were examined for placement of particles and stabilization of latex composite membranes (LCMs). Filtration of particles with reactive surface groups that provide covalent linkages at the contact points in the particle array proved most effective in obtaining stable membranes. These membranes had narrow size distributions in both the UF and MF range and were capable of being cleaned and backflushed. The membranes were characterized in terms of gas permeabilities, pure water permeabilities and electron microscopy. The rejection properties of LCMs were also examined during filtration of monodispersed latex particles and a broadly dispersed dextran mixture.     

Catalytic biofunctional membranes containing site-specifically immobilized enzyme arrays: a review

Biofunctional membranes normally involve the random immobilization of biomolecules to porous, polymeric membranes, often through the numerous lysine residues on the protein. In this process, bioactivity is significantly decreased largely due to different orientations of the biomolecule with respect to the membrane or to multiple point attachment. To circumvent this difficulty, while still taking advantage of the immobilization of biomolecules, site-specific immobilization of the biomolecule with the active (or binding) site directed away from the membrane is essential. In this review, we summarize our efforts involving biophysical and bioanalytical chemistry and chemical engineering, together with molecular biology, to develop and characterize such site-specifically membrane immobilized catalytic enzyme bioreactors. Site-directed mutagenesis, gene fusion technology, and post-translational modification methods are employed to effectuate the site-specific membrane immobilization. Electron paramagnetic resonance, in conjunction with active-site specific spin labels, kinetic analyses, and membrane properties are used to characterize these systems. Biofunctional membranes incorporating site-specifically immobilized biomolecules provide greater efficiency of biocatalysis, bioseparations, and bioanalysis.     

Evaluation of asymmetrical structure dialysis membrane by tortuous capillary pore diffusion model

The tortuous capillary pore diffusion model (TCPDM) has been used for estimating diffusive and pure water permeability from simple structure parameters such as pore diameter, surface porosity, wall thickness and tortuosity. The validity of this model for evaluation of homogeneous membrane has been already confirmed. Recently, there is a trend toward the use of asymmetrical dialysis membranes made of synthetic polymer such as poly(acrylonitrile) (PAN), polysulfone (PS) and a polyethersulfone polyarylate (PEPA) blend polymer. The purpose of the present study is to apply the TCPDM to evaluation of commercially available hollow-fiber dialysis membranes with asymmetrical structures by simplifying them to a double-layer membrane. The TCPDM is capable of estimating pore tortuosity of asymmetrical dialysis membranes having skin and supporting layers from data on membrane thickness, pore diameter, pure water permeability and water content. Values for diffusive permeability obtained by the TCPDM are in a good agreement with experimental data. This TCPDM model is useful for evaluation of not only homogeneous membrane but also asymmetrical membrane.