Hydrophobic salt-modified Nafion for enzyme immobilization and stabilization

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel cells. In most bioelectrochemical applications, enzymes or organelles are immobilized onto an electrode surface with the use of some type of polymer matrix. This polymer scaffold should keep the enzymes stable and allow for the facile diffusion of molecules and ions in and out of the matrix. Most polymers used for this type of immobilization are based on polyamines or polyalcohols - polymers that mimic the natural environment of the enzymes that they encapsulate and stabilize the enzyme through hydrogen or ionic bonding. Another method for stabilizing enzymes involves the use of micelles, which contain hydrophobic regions that can encapsulate and stabilize enzymes. In particular, the Minteer group has developed a micellar polymer based on commercially available Nafion. Nafion itself is a micellar polymer that allows for the channel-assisted diffusion of protons and other small cations, but the micelles and channels are extremely small and the polymer is very acidic due to sulfonic acid side chains, which is unfavorable for enzyme immobilization. However, when Nafion is mixed with an excess of hydrophobic alkyl ammonium salts such as tetrabutylammonium bromide (TBAB), the quaternary ammonium cations replace the protons and become the counter ions to the sulfonate groups on the polymer side chains (Figure 1). This results in larger micelles and channels within the polymer that allow for the diffusion of large substrates and ions that are necessary for enzymatic function such as nicotinamide adenine dinucleotide (NAD). This modified Nafion polymer has been used to immobilize many different types of enzymes as well as mitochondria for use in biosensors and biofuel cells. This paper describes a novel procedure for making this micellar polymer enzyme immobilization membrane that can stabilize enzymes. The synthesis of the micellar enzyme immobilization membrane, the procedure for immobilizing enzymes within the membrane, and the assays for studying enzymatic specific activity of the immobilized enzyme are detailed below.     

Purification of Penicillin G Amidase using Quaternary Ammonium Salts and effect on the activity of the immobilised enzymes

This paper describes the purification of Penicillin G Amidase (EC 3.5.1.11) using quaternary ammonium salts with the aim of increasing the activity of immobilised enzymes prepared from the purified solutions. Two different quaternary ammonium salts were tested with different solutions of the enzyme. It was concluded that the quaternary ammonium salts used selectively precipitated the non-enzymatic protein leaving in solution practically all the enzyme resulting in a high yield of purification. Optimal conditions for purification using the two types of quaternary ammonium salts were determined. Immobilisation studies were performed from various purified enzyme solutions, using different amounts of a quaternary ammonium salt. The immobilised enzymes so obtained showed a much higher activity than the immobilised enzyme obtained from non-purified enzyme solutions.     

Immobilization of a thermostable α-amylase, Termamyl, onto nitrocellulose membrane by Cibacron Blue F3GA dye binding

Highly porous nitrocellulose membranes were prepared by a solvent casting technique for the first time to immobilize α-amylase. An affinity dye, namely Cibacron Blue F3GA (CB), was incorporated covalently within the structure. The nitrocellulose–CB derivatized membranes were used for the immobilization of a starch degrading enzyme, α-amylase. Optimum conditions of immobilization for highest apparent activity were determined as pH 6.0, temperature 50°C and initial enzyme concentration 0.317 KNU/l. Under these optimum conditions, maximum enzyme immobilization yield was around 21% of the initial amount of the enzyme in the solution. Performance of free and immobilized enzymes at the same amount was compared for repeated runs. Up to the third use, immobilized enzyme showed higher activity than that of free enzyme mainly due to higher enzyme concentration in the membrane structure, then the apparent activity decreased gradually. However, when regenerated by switching pH to cause contraction/expansion of the structure, the membrane showed the highest activity, almost 2.5 times than that of the free enzyme. This unusual feature along with inexpensive cost may well make the nitrocellulose membrane an economical material for industrial application in glucose syrup production.     

Ceramic membrane microfilter as an immobilized enzyme reactor.

This study investigated the use of a ceramic microfilter as an immobilized enzyme reactor. In this type of reactor, the substrate solution permeates the ceramic membrane and reacts with an enzyme that has been immobilized within its porous interior. The objective of this study was to examine the effect of permeation rate on the observed kinetic parameters for the immobilized enzyme in order to assess possible mass transfer influences or shear effects. Kinetic parameters were found to be independent of flow rate for immobilized penicillinase and lactate dehydrogenase. Therefore, neither mass transfer nor shear effects were observed for enzymes immobilized within the ceramic membrane. Both the residence time and the conversion in the microfilter reactor could be controlled simply by regulating the transmembrane pressure drop. This study suggests that a ceramic microfilter reactor can be a desirable alternative to a packed bed of porous particles, especially when an immobilized enzyme has high activity and a low Michaelis constant.     

New redox mediator-modified polysulfone composite films for the development of dehydrogenase-based biosensors

This work presents polysulfone membranes as new materials for the development of compact dehydrogenase-based biosensors. Composite films were prepared by mixing polysulfone with graphite and were deposited on epoxy-graphite composite electrodes. Redox mediators were successfully immobilized in the composite film leading to highly reproducible biosensors, without leakage of the immobilized species. This results in a more reliable analytical system as, at the same time, problems of electrode fouling related to the detection of the coenzyme nicotinamide adenine dinucleotide (NADH) on which is based the amperometric detection of dehydrogenase-based biosensors are avoided. Scanning electron microscopy was used to study the morphological characteristics of the surface and the cross-section of the polysulfone-graphite composite films. Several procedures to immobilize enzymes in these membranes were demonstrated. Glutamate dehydrogenase (GlDH) was immobilized as an example of dehydrogenase enzyme, in this case for the development of an ammonium biosensor. High sensitivity, good selectivity, wide linear ranges and short response times were obtained for the optimized sensors and biosensors. Their good performance combined with the simplicity of the construction method, make the polysulfone-graphite composite films attractive matrices for the development of new enzyme-based biosensors, especially those based on dehydrogenase enzymes.     

Interaction between model membranes and a new class of surfactants with antioxidant function.

The effect of two series of amphiphilic quaternary ammonium salts on some properties of phospholipid membranes was studied. The compounds of one series, N-benzyl-N,N-dimethyl-N-alkyl ammonium bromides, exert a destructive effect on membranes and are treated as reference compounds. The compounds of the other series, N-(3,5-di-t-butyl-4-hydroxy)benzyl-N,N-dimethyl-N-alkyl ammonium bromides, are derivatives of the former ones, exhibit antioxidant properties, and do only relatively slight damage to the membranes. The aim of the work was to explain the difference in molecular interaction with membranes between the two kinds of hydrophobic compounds. Thermodynamic methods, a new mixing technique, and monolayer and quantum calculation methods were used. It has been shown that the antioxidant molecules are less hydrophobic than those of the reference compounds and disturb the membrane organization to a lesser extent. On the basis of monolayer data, we suggest that the studied antioxidant behaves like a substitutional impurity, whereas the reference behaves like an interstitial one.     

Investigations on fungicides. XXI. The fungitoxicity and systemic antifungal activity of some aromatic quaternary ammonium salts

Root application of certain quaternary ammonium salts, most notably tri-n-butyl-and tri-n-pentyl-4-chlorobenzylammonium bromides, to wheat seedings grown in water culture enhanced their resistance to powdery mildew (Erysiphe graminis) but not to brown rust (Puccinia recondita). Anti-mildew activity was much less apparent if the compounds were applied to sand- or soil-grown seedlings. Systemic activity is considered in relation to the fungitoxicity, phytotoxicity and growth-retardant activity of these compounds.     

Purification and properties of the trimethylamine dehydrogenase of Bacterium 4B6

1. The trimethylamine dehydrogenase of bacterium 4B6 was purified to homogeneity as judged by analytical polyacrylamide-gel electrophoresis. The specific activity of the purified enzyme is 30-fold higher than that of crude sonic extracts. 2. The molecular weight of the enzyme is 161000. 3. The kinetic properties of the purified enzyme were studied by using an anaerobic spectrophotometric assay method allowing the determination of trimethylamine dehydrogenase activity at pH8.5, the optimum pH. The apparent K(m) for trimethylamine is 2.0+/-0.3mum and the apparent K(m) for the primary hydrogen acceptor, phenazine methosulphate, is 1.25mm. 4. Of 13 hydrogen acceptors tested, only Brilliant Cresyl Blue and Methylene Blue replace phenazine methosulphate. 5. A number of secondary and tertiary amines with N-methyl and/or N-ethyl groups are oxidized by the purified enzyme; primary amines and quaternary ammonium salts are not oxidized. Of the compounds that are oxidized by the purified enzyme, only trimethylamine and ethyldimethylamine support the growth of bacterium 4B6. 6. Trimethylamine dehydrogenase catalyses the anaerobic oxidative N-demethylation of trimethylamine with the formation of stoicheiometric amounts of dimethylamine and formaldehyde. Ethyldimethylamine is also oxidatively N-demethylated yielding ethylmethylamine and formaldehyde; diethylamine is oxidatively N-de-ethylated. 7. The activity of the purified enzyme is unaffected by chelating agents and carbonyl reagents, but is inhibited by some thiol-binding reagents and by Cu(2+), Co(2+), Ni(2+), Ag(+) and Hg(2+). Trimethylamine dehydrogenase activity is potently inhibited by trimethylsulphonium chloride, by tetramethylammonium chloride and other quaternary ammonium salts, and by monoamine oxidase inhibitors of the substituted hydrazine and the non-hydrazine types. 8. Inhibition by the substituted hydrazines is time-dependent, is prevented by the presence of trimethylamine or trimethylamine analogues and in some cases requires the presence of the hydrogen acceptor phenazine methosulphate. The inhibition was irreversible with the four substituted hydrazines that were tested.     

Enzyme-based high-performance liquid chromatography supports as probes of enzyme activity and inhibition: the immobilization of trypsin and alpha-chymotrypsin on an immobilized artificial membrane high-performance liquid chromatography support.

Immobilized artificial membrane (IAM) HPLC supports have been used to immobilize the enzymes alpha-chymotrypsin and trypsin. The enzymes were trapped in hydrophobic cavities on the support and were not covalently attached to the IAM surface. The resulting IAM-enzyme supports retained the hydrolytic activity of the immobilized enzymes: the IAM-trypsin support catalyzed the hydrolysis of N alpha-benzoyl-DL-arginine-p-nitroanilide (BAPNA), and the IAM-alpha-chymotrypsin support (IAM-ACHT) catalyzed the hydrolysis of a number of substrates, including tryptophan methyl ester. The activities of both supports were decreased by known enzyme inhibitors and the activity of the IAM-ACHT was affected by changes in pH and temperature. When a substrate was chromatographed on an IAM-ACHT HPLC, the hydrolytic activity of the immobilized enzyme could be determined from the resulting substrate/product ratios. These data were obtained either directly from the IAM-ACHT chromatogram or from the chromatogram produced by a coupled column system. The results of this study indicate that IAM-immobilized alpha-chymotrypsin and trypsin can be used as chromatographic probes for the qualitative determination of enzyme/substrate and enzyme/inhibitor interactions.     

NAD recycling in the collagen membrane.

NAD recycling in the collagen membrane was investigated as follows: (1) Alcohol dehydrogenase and lactate dehydrogenase were co-immobilized in the collagen membrane and the rate of lactate production by immobilized enzymes was compared with that of free enzymes by using free NAD. An increased rate was observed in the case of immobilized enzyme. (2) The soluble high molecular weight derivatives of NAD (dextran-NAD) were immobilized in the collagen membrane with the two dehydrogenases and recycling of dextran-NAD in the membrane was examined. Lactate was produced by the membrane without adding free NAD. The interaction between the high molecular weight NAD derivatives and enzymes are also discussed.     

Enzymic and permeability activity ofPseudomonas aeruginosa after treatment with sub-MICs of organic ammonium salts

The effects of subinhibitory concentrations (1/4, 1/8, 1/16 of the MIC) of 12 organic ammonium salts of A (hard— alkyltrimethylammonium bromides) and B (soft—2-(dodecanoylamino)ethylalkyldimethylammonium bromides) homologous series on phospholipase C, proteinase, elastase and permeability activity were studied. The substances with longer substituents were more effective in reducing phospholipase C activity (hard and soft series) as well as proteinase (hard series). Phospholipase C was the most frequently and the most markedly inhibited enzyme. The organic ammonium salts were less effective in inhibiting elastase and permeability activity. Only one of the substances under study reduced all the tested activities.     

Stabilization of a multimeric beta-galactosidase from Thermus sp. strain T2 by immobilization on novel heterofunctional epoxy supports plus aldehyde-dextran cross-linking

This work exemplifies the advantages of using a battery of new heterofunctional epoxy supports to immobilize enzymes. We have compared the performance of a standard Sepabeads-epoxy support with other Sepabeads-epoxy supports partially modified with boronate, iminodiacetic, metal chelates, and ethylenediamine in the immobilization of the thermostable beta-galactosidase from Thermus sp. strain T2 as a model system. Immobilization yields depended on the support, ranging from 95% using Sepabeads-epoxy-chelate, Sepabeads-epoxy-amino, or Sepabeads-epoxy-boronic to 5% using Sepabeads-epoxy-IDA. Moreover, immobilization rates were also very different when using different supports. Remarkably, the immobilized beta-galactosidase derivatives showed very improved but different stabilities after favoring multipoint covalent attachment by long-term alkaline incubation, the enzyme immobilized on Sepabeads-epoxy-boronic being the most stable. This derivative had some subunits of the enzyme not covalently attached to the support (detected by SDS-PAGE). This is a problem if the biocatalysts were to be used in food technology. The optimization of the cross-linking with aldehyde-dextran permitted the full stabilization of the quaternary structure of the enzyme. The optimal derivative was very active in lactose hydrolysis even at 70 degrees C (over 1000 IU/g), maintaining its activity after long incubation times under these conditions and with no risk of product contamination with enzyme subunits.     

Electrogenerated chemiluminescence biosensor with alcohol dehydrogenase and tris(2,2'-bipyridyl)ruthenium (II) immobilized in sol-gel hybrid material

An ethanol biosensor based on electrogenerated chemiluminescence detection was developed. Electrogenerated chemiluminescence reagent tris(2,2'-bipyridyl)ruthenium (II) and alcohol dehydrogenase were immobilized in the same sol-gel hybrid film. The copolymer poly(vinyl alcohol) with 4-vinylpyridine and cation exchanger Nafion were incorporated into sol-gel film to provide the microenvironment for retaining the activity of enzyme and immobilize tris(2,2'-bipyridyl)ruthenium (II). The design was simpler than the previous two-layer format. The experimental conditions, such as scan rate, pH and concentration of the cofactor were investigated. The intensity of electrogenerated chemiluminescence increased linearly with ethanol concentration from 2.5x10(-5) to 5.0x10(-2) M and detection limit was 1.0x10(-5) M. The prepared biosensor exhibited high sensitivity, wide linear range and good stability.     

Enzymatic activity and stability of D-fructose dehydrogenase and sarcosine dehydrogenase immobilized onto giant vesicles

Stable vesicles with diameters between about 1 and 10 mum were prepared by a particular emulsification technology that involved the use of the surfactants Span 80 and Tween 80 and the phospholipid lecithin (phosphatidylcholine from soybeans). Two membrane enzymes, d-fructose dehydrogenase from Gluconobacter sp. (FDH) and sarcosine dehydrogenase from Pseudomonas putida (SDH), were for the first time immobilized onto the bilayer membranes of these type of vesicles; and the catalytic activity and enzymatic stability were measured and compared with the enzymes in a vesicle-free solution. The enzyme activity as well as stability considerably increased upon immobilization. In particular, immobilized FDH at 25 degrees C was stable for at least 20 days, while the activity of the free enzyme dropped to about 20% of its initial value during the same period of time.In contrast to FDH and SDH, immobilization of sorbitol dehydrogenase from Gluconobacter suboxydans (SODH) was not successful, as no improved activity or stability could be obtained.     

Biosensing nitrite using the system nitrite redutase/Nafion/methyl viologen--a voltammetric study

This work describes the construction and voltammetric characterization of a nitrite biosensor based on a cytochrome c-type nitrite reductase (ccNiR) and the Nafion ionomeric matrix loaded with methyl viologen as redox mediator. Despite the potential electrostatic repulsions between the anionic substrate and the Nafion sulfonate groups, the resulting bioelectrode exhibited electrocatalytic activity toward nitrite. This phenomenon must be due to the nonuniformity of the enzyme/Nafion membrane, which allows the direct interaction between the substrate and numerous enzyme molecules. Nevertheless, the anionic nature of Nafion exerted a certain diffusion barrier to nitrite, as revealed by the unusually elevated limits of the linear dynamic range and k(m)(app). The irregularity of the composite membrane also contributed to slow down the rate of charge transfer throughout the Nafion polymer. The level of viologens incorporated within the Nafion membrane had a strong influence in the analytical parameters: as much mediator was present, lower was the sensitivity and wider was the linear range. For an optimized ratio enzyme/mediator the sensitivity was 445+/-8 mA M(-1)cm(-2), within the linear range 75-800 microM; the lowest detected nitrite concentration was 60 microM. The operational stability of the biosensor and the influence of some possible interferences were evaluated.     

Purification and stabilization of a glutamate dehygrogenase from Thermus thermophilus via oriented multisubunit plus multipoint covalent immobilization

The immobilization of a glutamate dehydrogenase from Thermus thermophilus (GDH) on glyoxyl agarose beads at pH 7 has permitted to perform the immobilization, purification and stabilization of this interesting enzyme. It was cloned in Escherichia coli and a first thermal shock of the crude preparation destroyed most mesophilic multimeric proteins. Glyoxyl agarose can only immobilize enzymes via a multipoint and simultaneous attachment. Therefore, only proteins having several terminal amino groups in a position that permits their interaction with a flat surface can be immobilized. GDH became rapidly immobilized at pH 7 and its multimeric structure became stabilized as evidenced by SDS-PAGE. This derivative was stable at acidic pH value while the non-stabilized enzyme was very unstable under these conditions due to subunit dissociation. After immobilization, a further incubation at pH 10 improved enzyme stability under any inactivating conditions by increasing the enzyme–support bonds. In fact, GDH immobilized at pH 7 and incubated at pH 10 preserved more activity than GDH directly immobilized at pH 10 (50% versus 15% after 24 h of incubation) and was also more stable (1.5- to 3-fold, depending on the conditions).This method could be extended to any other multimeric enzyme expressed in mesophilic hosts.     

Stability improvement of immobilized lactoperoxidase using polyaniline polymer

Enzyme engineering via immobilization techniques is perfectly compatible against the other chemical or biological approximate to improve enzyme functions and stability. In this study lactoperoxidase was immobilized onto polyaniline polymer activated with glutaraldehyde as a bifunctional agent, to improve enzyme properties. Polyaniline polymer was used due its unique physical and chemical properties to immobilize lactoperoxidase (LPO). The optimum activity of immobilized LPO was observed at pH 6 and 55?°C, which has been increased about 10?°C for the immobilized enzyme. The immobilized enzyme maintained absolutely active for 60?days whereas the native enzyme lost 80?% of its initial activity within this period of time. Moreover, the immobilized enzyme can be reused for several times without loss of activity. The kinetic parameter studies showed slight differences between free and immobilized enzymes. The K_m and K_m.app were calculated to be 0.6 and 0.4; also V_max and V_max.app were 1.3 and 0.9 respectively.     

Development of sensors for direct detection of organophosphates. Part I: Immobilization, characterization and stabilization of acetylcholinesterase and organophosphate hydrolase on silica supports

Biosensors for organophosphates in solution may be constructed by monitoring the activity of acetylcholinesterase (AChE) or organophosphate hydrolase (OPH) immobilized to a variety of microsensor platforms. The area available for enzyme immobilization is small (< 1 mm2) for microsensors. In order to construct microsensors with increased surface area for enzyme immobilization, we used a sol-gel process to create highly porous and stable silica matrices. Surface porosity of sol-gel coated surfaces was characterized using scanning electron microscopy; pore structure was found to be very similar to that of commercially available porous silica supports. Based upon this analysis, porous and non-porous silica beads were used as model substrates of sol-gel coated and uncoated sensor surfaces. Two different covalent chemistries were used to immobilize AChE and OPH to these porous and non-porous silica beads. The first chemistry used amine-silanization of silica followed by enzyme attachment using the homobifunctional linker glutaraldehyde. The second chemistry used sulfhydryl-silanization followed by enzyme attachment using the heterobifunctional linker N-gamma-maleimidobutyryloxy succinimide ester (GMBS). Surfaces were characterized in terms of total enzyme immobilized, total and specific enzyme activity, and long term stability of enzyme activity. Amine derivitization followed by glutaraldehyde linking yielded supports with greater amounts of immobilized enzyme and activity. Use of porous supports not only yielded greater amounts of immobilized enzyme and activity, but also significantly improved long term stability of enzyme activity. Enzyme was also immobilized to sol-gel coated glass slides. The mass of immobilized enzyme increased linearly with thickness of coating. However, immobilized enzyme activity saturated at a porous silica thickness of approximately 800 nm.     

The antioxidant activity of BHT and new phenolic compounds PYA and PPA measured by chemiluminescence.

The antioxidative properties of two series of new phenolic, amphiphilic compounds were evaluated using the chemiluminescence (CL) method. 2,2'-Azobis (2-amidinopropane) dihydrochloride (AAPH) was used as a source of free radicals, to obtain high and prolonged CL. Three different kinds of buffers (organic and inorganic) were tested. The CL level varied only slightly depending on the buffer but increased significantly with the pH. Twelve newly synthesised compounds were compared with butylated hydroxytoluene (BHT), a commercially used antioxidant. The new antioxidants included two classes of quaternary ammonium salts with a phenol substituent functioning as an antioxidant. The salts were synthesised by quaternarization of pyrrolidine ethyl esters of dihydrocinnamic acid by n-alkoxymethyl bromides (PYA-n) or quaternarization of 2-dimethylaminoethyl esters by n-alkyl bromides (PPA-n). All the tested compounds quenched CL proportionally to their concentrations. In our experimental conditions 8.5 microM BHT quenched 50% of the CL. The PYA and PPA compounds had IC50 two to six times lower than BHT. CL inhibition was proportional to the pH for all antioxidants. The relationships between the structure and activity of the tested compounds are discussed.     

Effect of Polyelectrolytes on Catalytic Activity of Alcohol Dehydrogenase

Fluorescent and optical spectroscopy were used to study the interaction of alcohol dehydrogenase (ADH) with negatively charged polystyrene sulfonate (PSS) and dextran sulfate (DS), as well as positively charged poly(diallyldimethylammonium) (PDADMA). As found, DS and PDADMA did not affect the structural and catalytic enzyme properties. In contrast, PSS slightly decreased the protein self-fluorescence over 1 h of incubation, which is associated with partial destruction of its quaternary (globular) structure. Investigation of the ADH activity with and without PSS showed its dependency on the incubation time and the PSS presence. Sodium chloride (2.0 and 0.2 M) or ammonium sulfate (0.1 M) added to the reaction mixture did not completely protect the enzyme quaternary structure from the PSS action. However ammonium sulfate or 0.2 M sodium chloride stabilized the enzyme and partially inhibited the negative PSS effect.