Biodegradation of phenol by enzymes from Pseudomonas sp. immobilized onto ultrafiltration membranes

A method of immobilizing enzymes from Pseudomonas sp. that decompose phenol on polymeric ultrafiltration membranes is described. Transport-separation properties of neutral and enzymic membranes have been compared and the optimal ultrafiltration process parameters of a model phenol solution have been determined. The immobilized enzyme system was applied to the biodegradation of phenol in coke wastewaters.     

A new conception of enzymatic membrane reactor for the production of whey hydrolysates with low contents of phenylalanine

A novel concept of enzymatic membrane reactor (EMR) is presented, aiming at the production of cheese whey protein hydrolysates with low contents of phenylalanine (Phe) for the diet of phenylketonuria patients. Whey proteins were first hydrolyzed by chimotrypsin, followed by the action of carboxypeptidase A (CPA), immobilized on agarose gel particles, which were retained inside the reactor using a filter. The liquid medium passes through hollow fiber ultrafiltration unit (1 kDa cut-off), and the retentate is recycled to the reactor. The innovation here is that the membrane is not used to retain the enzyme neither physically nor as an immobilization support. The EMR provided a higher performance than the classical, sequential approach: batch reaction followed by ultrafiltration. It was confirmed that the removal of products promoted by the EMR enhances reaction rates, due to the reduction of inhibition effects. A mathematical model of the EMR is also presented and validated.     

Series-type enzyme deactivations: Influence of intermediate activity on deactivation kinetics

A series-type enzyme deactivation model involving an active enzyme precursor is proposed wherein the enzyme activity is a weighted function of the active enzyme states. The active enzyme precursor may be less active, as active or more active than the initial enzyme form. The proposed model is shown to fit the soluble and immobilized enzyme deactivation data presented reasonably well. Some enzymes exhibit a ‘compensation-like’ effect. In other enzymes, if the deactivation rate coefficient for the second step, k₂, is zero, then the activity may stabilize to a value that depends upon the relative activities of the two active enzyme states.     

Identification of l-Baikiain from Calcareous Red Algae Serraticardia maxima (Yendo) Silva and Its Distribution in Some Rhodophyta

The conjugated enzyme system of NAD kinase (NK) and acetate kinase (AK) was investigated to produce NADP continuously by the dynamic recycling of ATP. The enzymes were physically entrapped in an ultrafiltration hollow fiber tube and the cosubstrate, ATP, as well as the substrates were fed to the reactor continuously. The ATP turnover increased with a decrease in the concentration of ATP fed and with an increase in the concentration of immobilized enzymes. The maximal productivity of the reactor was 0.228 μmol/hr/ml-reactor with an ATP turnover of 8.35. The experimental results were approximately predicted using a theoretical model in which the equilibrium random mechanism was assumed for the AK reaction and neglecting the backward reaction for NK. When the concentration of ATP fed was much lower than the Michaelis constant of NK for ATP, the ATP turnover took a constant value irrespective of the ATP feed concentration, and this value was proportional to the concentration of NK. A rate limiting step was proved to exist in the reaction of NK. The half-life of the enzyme system was over 10 days in a continuous operation.     

Use of stable emulsion to improve stability, activity, and enantioselectivity of lipase immobilized in a membrane reactor

The enantiocatalytic performance of immobilized lipase in an emulsion membrane reactor using stable emulsion prepared by membrane emulsification technology was studied. The production of optical pure (S)-naproxen from racemic naproxen methyl ester was used as a model reaction system. The O/W emulsion, containing the substrate in the organic phase, was fed to the enzyme membrane reactor from shell-to-lumen. The enzyme was immobilized in the sponge layer (shell side) of capillary polyamide membrane with 50 kDa cut-off. The aqueous phase was able to permeate through the membrane while the microemulsion was retained by the thin selective layer. Therefore, the substrate was kept in the enzyme-loaded membrane while the water-soluble product was continuously removed from the reaction site. The results show that lipase maintained stable activity during the entire operation time (more than 250 h), showing an enantiomeric excess (96 +/- 2%) comparable to the free enzyme (98 +/- 1%) and much higher compared to similar lipase-loaded membrane reactors used in two-separate phase systems (90%). The results demonstrate that immobilized enzymes can achieve high stability as well as high catalytic activity and enantioselectivity.     

Protein analysis using enzymes immobilized to paramagnetic beads.

A new method combining protein chemistry with enzymes immobilized to paramagnetic beads is presented. The immobilized enzymes can substitute for regular enzymes in a number of protein chemistry protocols, resulting in faster reaction times, less sample contamination, and improved interfacing to modern procedures, like mass spectrometry. Trypsin was used as a model enzyme to test the amount of protein coupled to glass beads and the degree of autodigestion when analyzed by MALDI-MS and HPLC. Immobilization of trypsin resulted in digestions comparable with standard solution digestions using fetuin as a model substrate. Furthermore, fetuin was used to test the stability of the enzyme-coated beads. No apparent loss of enzyme activity was observed after 10 times reuse of trypsin-coated beads. Immobilization of exo- and endoglycosidases to paramagnetic beads resulted in high sensitivity, faster sequential glycosidase digestion of glycopeptides, and reduced sample contamination. All digestions could be performed in less than 24 h, when a tryptic glycopeptide from human lung proteinosis surfactant protein A was used as model compound.     

Modeling of a Catalyzed Reaction Using Lipase Immobilized in a Poly(vinyl alcohol) Membrane

A mathematical model for the hydrolysis reaction of p‐nitro phenol laurate catalyzed by a lipase immobilized in a membrane was developed. In an earlier study this model reaction was found to show very different reaction rates when it was performed in aqueous micellar solution with free enzyme and with membrane immobilized enzyme. It was assumed that a local accumulation of substrate in the membrane is responsible for the observed rate enhancement. The conversion of p‐nitro phenol ester within the membrane was modeled by considering a combination of the convective flow through poly(vinyl alcohol) membrane pores, concentration polarization of substrate containing micelles at the membrane surface and the kinetics of the reaction with free enzymes. It was demonstrated that the model offered a comprehensive understanding of the interaction of the involved phenomena. The modeling results are in good agreement with the experimental data from 10 runs with different enzyme and substrate concentrations. The substrate concentration at the membrane surface increased by up to a factor of 3 compared to the feed concentration. This effect explains the observed rate enhancement. Moreover, the model was used to determine the unknown parameters, i.e., the intrinsic retention and the mass transfer coefficient, by fitting the model to the experimental data. The model may also be used to calculate the optimum operating conditions and design parameters of such a reactor.     

Lysozyme activity in the presence of nonionic detergent micelles

The effect of a nonionic surfactant, polyoxyethylenesorbitan monolaurate (Tween 20), on the hen egg-white lysozyme catalyzed lysis of a dried cell suspension of Micrococcus lysodeikticus is analysed. A rate enhancement of up to 70% is observed in the presence of surfactant at concentrations above the critical micelle concentration. This activity increase may be explained by postulating the existence of a micelle-enzyme complex in which enzyme molecules are bound to micelles with preferential orientation of their active sites. The reaction is found to be second order with respect to substrate. A mechanism is postulated in which a substrate particle is assumed to be an energy-furnishing collision partner to the enzyme-substrate complex. This mechanism correlated data over a wide range of enzyme and substrate concentrations. Data from kinetic, ultrafiltration, ultraviolet, and fluorescence studies provide convincing evidence for the existence of a micelle-lysozyme complex. The results suggest that it is possible that immobilized enzymes mat in general be more reactive than corresponding free enzymes.     

耦合中空纤维膜超滤分离游离细胞催化合成ATP

对耦合中空纤维膜超滤分离进行游离细胞催化合成ATP过程进行了实验研究,考察了细胞的催化效率和膜组件的操作稳定性。结果显示,中空纤维超滤膜的耦合分离能有效地截留反应液中的游离酶,其中乙醇脱氢酶(ADH)和已糖激酶(HK)的稳态截留效率在95%以上。耦合膜分离的酵母细胞催化ATP合成反应可重复使用2.5～3.0次,酶的利用率比普通分离的细胞提高2.0～2.5倍。中空纤维超滤膜于0.1Mpa工作压力下连续11批耦合分离操作,膜的渗透性无明显下降,过滤速率保持在初速率的95%以上。在稀释速率0.25h-1下,反应体系保持了连续5h的ATP高转化率合成与分离耦合的拟稳态操作。     

Investigations of reaction kinetics for immobilized enzymes--identification of parameters in the presence of diffusion limitation

A method is proposed for identification of kinetic parameters when diffusion of substrates is limiting in reactions catalyzed by immobilized enzymes. This method overcomes conventional sequential procedures, which assume immobilization does not affect the conformation of the enzyme and, thus, consider intrinsic and inherent kinetics to be the same. The coupled equations describing intraparticle mass transport are solved simultaneously using numerical methods and are used for direct estimation of kinetic parameters by fitting modeling results to time-course measurements in a stirred tank reactor. While most traditional procedures were based on Michaelis-Menten kinetics, the method presented here is applicable to more complex kinetic mechanisms involving multiple state variables, such as ping-pong bi-bi. The method is applied to the kinetic resolution of (R/S)-1-methoxy-2-propanol with vinyl acetate catalyzed by Candida antarctica lipase B. A mathematical model is developed consisting of irreversible ping-pong bi-bi kinetics, including competitive inhibition of both enantiomers. The kinetic model, which fits to experimental data over a wide range of both substrates (5-95%) and temperatures (5-56 degrees C), is used for simulations to study typical behavior of immobilized enzyme systems.     

An Affinity Chromatographic Reactor for Highly Efficient Turnover of Dissociable Cofactors

A conjugated enzyme system of alcool dehydrogenase and lactate dehydrogenase was immobilized in an ultrafiltration hollow fiber tube, which was inserted in a fine nylon tube to form a hollow-fiber-capillary reactor. To this reactor, the substrates, pyruvate and ethanol, were supplied continuously. The necessary cofactor, NAD, was supplied as a pulse for a short time. The retention time of NAD in the reactor, estimated from the response curve of lactate produced, was much longer than those of the other substrates and products because of the strong adsorption of NAD to the immobilized enzymes through affinity. Therefore, the reactor could produce lactate from pyruvate for a long time without any more NAD. As a typical case, when the enzyme concentration is sufficiently high, the estimated retention time of NAD was 50 times as long as those of other materials so that the NAD turnover obtained was 412,000. The effects of NAD pulse concentration and the immobilized enzyme concentration on the retention time of NAD and NAD turnover were investigated experimentally and theoretically.     

A deactivation protection model for immobilized and soluble enzymes

A model is proposed in which a deactivated enzyme protects an active enzyme from deactivation. Enzyme modifiers, when present, may also appear to induce or enhance enzyme protection by providing intra-molecular covalent cross-links to ‘lock-in’ protein conformation and facilitate enzyme protection by the deactivated enzyme. In both cases a first-order deactivation protection model is shown to fit the immobilized and soluble enzyme deactivation data presented reasonably well. Steric hindrances and the immobile nature of the enzyme-support link of immobilized enzymes would appear to lessen the extent of this protection.     

Glutathione transferases immobilized on nanoporous alumina: Flow system kinetics,screening, and stability

The previously uncharacterized Drosophila melanogaster Epsilon-class glutathione transferases E6 and E7 were immobilized on nanoporous alumina. The nanoporous anodized alumina membranes were derivatized with 3-aminopropyl-triethoxysilane, and the amino groups were activated with carbonyldiimidazole to allow coupling of the enzymes via ε-amino groups. Kinetic analyses of the immobilized enzymes were carried out in a circulating flow system using CDNB (1-chloro-2,4-dinitrobenzene) as substrate, followed by specificity screening with alternative substrates. A good correlation was observed between the substrate screening data for immobilized enzyme and corresponding data for the enzyme in solution. A limited kinetic study was also carried out on immobilized human GST S1-1 (also known as hematopoietic prostaglandin D synthase). The stability of the immobilized enzymes was virtually identical to that of enzymes in solution, and no leakage of enzyme from the matrix could be observed.     

Immobilization of lipase by ultrafiltration and cross-linking onto the polysulfone membrane surface

In this study, a biphasic enzymatic membrane reactor was made by immobilizing Candida Rugosa lipase onto the dense surface of polysulfone ultrafiltration membrane by filtration and then cross-linking with glutaraldehyde solution. The reactor was further applied for the hydrolysis of olive oil, the performance of which was evaluated in respect of apparent reaction rate based on the amount of fatty acids extracted into the aqueous phase per minute and per membrane surface. It was found that the ultrafiltration and cross-linking process greatly improved the reaction rate per unit membrane area and the enzyme lifetime. The highest reaction rate reached 0.089 micromol FFA/min cm2 when the enzyme loading density was 0.098 mg/cm2. The results also indicated that the performance of lipase immobilized on the membrane surface was superior to that immobilized in the pores, and the apparent reaction rate and stability of immobilized lipases were improved greatly after cross-linking. It suggested that immobilization of enzymes by filtration and then cross-linking the enzymes onto the membrane surface is a simple and convenient way to prepare a high-activity immobilized enzyme membrane.     

Continuous monitoring of adenosine 5'-triphosphate in the microenvironment of immobilized enzymes by firefly luciferase

The study of enzymes sequestered in artificial or biological systems is generally conducted by indirect methodology with macroscopic measurements of reactants in the bulk medium. This paper describes a new approach with firefly luciferase to monitor ATP concentration directly in the microenvironment of enzymes producing or consuming ATP. Upon addition of ATP to immobilized firefly luciferase, the onset of light production is slower than that observed with the soluble enzyme, due to a slower diffusion of ATP to the immobilized enzyme. With immobilized pyruvate kinase, a relative accumulation of ATP inside the beads is demonstrated, as measured with coimmobilized firefly luciferase. The accumulation of product (ATP) is enhanced when the bead suspension is not stirred. This ATP in the beads is relatively inaccessible to soluble hexokinase added to the bulk medium. Similarly, a rapid ATP depletion in the microenvironment of immobilized hexokinase is demonstrated. This microscopic event is kinetically distinguishable from the slower macroscopic depletion of substrate in the bulk medium. The rate of depletion in the microenvironment depends on the local activity of the immobilized enzyme but not on the total amount of enzyme in suspension, as does the macroscopic phenomenon. The theoretical principles for the interaction of diffusion and catalysis in these systems are briefly summarized and discussed. These results are relevant to various molecular mechanisms proposed for membrane-bound enzyme action and regulation, derived from macroscopic kinetic measurements assuming a negligible diffusion control.     

Immobilization of urease within a thin channel ultrafiltration cell

Urease [urea amidohydrolase, EC 3.5.1.5] has been immobilized within a thin channel ultrafiltration cell. Loss of enzymic activity as a result of concentration polarization and other causes was minimized. The flow characteristics of the reactor were fully characterized by analysis of the distribution of residence times (using F diagrams) and kinetic data were also obtained for the immobilized enzyme. These data show that under certain conditions the thin channel ultrafiltration reactor can be considered to be an ideally mixed vessel. After almost 8 days of continuous operation it was found that 15% of the original enzyme activity remained.     

Effects of temperature and shear on the activity of acid phosphatase in a tubular membrane reactor

The effect of temperature on the activity of acid phosphatase [orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2] immobilized as a gel layer on the inner wall of ultrafiltration tubular membranes by both copolymerization/gelation and cogelation has been investigated. Both forms of gel-immobilized enzyme showed fairly good stability, the activation energy of their inactivation being significantly lower than that of the free enzyme and of the heat denaturation of proteins in general. The shear effect on the cogelled enzyme was also studied at different temperatures and Reynolds numbers. The results indicated that the cogelled enzyme is a more convenient form for continuous operation in the tubular membrane reactor (TMR), a reactor configuration particularly suitable for industrial applications.     

A deactivation model involving pH for immobilized and soluble enzymes

A pH-dependent deactivation model is developed for immobilized and soluble enzymes in which the rate of enzyme decay is assumed proportional to the concentration of theactive enzyme. The model developed applies reasonably well to pH-stability data of Johnson and coworkers (1977 a,b,c; 1978 a,b,c).     

Enzyme activity maintenance in packed-bed reactors via continuous enzyme addition

An operational scheme for using immobilized enzymes in packed-bed reactors that permits operation at a constant throughput rate and constant product quality is described. The scheme used columns operated in series with continuous enzyme addition to compensate for enzyme decay. A mathematical technique was developed to determine the enzyme addition rate, enzyme usage, and enzyme volume in the column system. Operation of columns in series is compared to operation where the flow rate is decreased to compensate for a loss of enzyme activity for both zero-and first-order decay. The analyses indicated that columns in series resulted in better enzyme utilization but larger reactor volumes than parallel reactors with decreasing flow rate.