Production of precursors for anti-Alzheimer drugs: Asymmetric bioreduction in a packed-bed bioreactor using immobilized D. carota cells

(S)-1-Phenylethanol derivatives, which are the precursors of many pharmacological products, have also been used as anti-Alzheimer drugs. Bioreduction experiments were performed in a batch and packed-bed bioreactor. Then, the kinetics constants were determined by examining the reaction kinetics in the batch system with free and immobilized carrot cells. Also, the effective diffusion coefficient (D_e) of acetophenone in calcium alginate-immobilized carrot cells was investigated. Kinetics constants for free cells, which are intrinsic values, are reaction rate V_max?=?0.052?mmol?L^?1?min^?1, and constants of the Michaelis–Menten K_M?=?2.31?mmol?L^?1. Kinetics constants for immobilized cells, which are considered apparent values, are V_{max, app}?=?0.0407?mmol?L^?1 min^?1, K_{M, app}?=?3.0472?mmol?L^?1 for 2?mm bead diameter, and V_{max, app}?=?0.0453?mmol?L^?1 min^?1, K_{M, app}?=?4.9383?mmol?L^?1 for 3?mm bead diameter. Average value of effective diffusion coefficient of acetophenone in immobilized beads was determined as 1.97?×?10^?6?cm²?s^?1. Using immobilized carrot cells in an up-flow packed-bed reactor, continuous production of (S)-1-phenylethanol through asymmetric bioreduction of acetophenone was performed. The effects of the residence time and concentrations of substrate were investigated at pH 7.6 and 33°C. Enantiomerically pure (S)-1-phenylethanol (ee?>?99%) was produced with 75% conversion at 4-hr residence time.     

Influences of nonuniform activity distributions on the apparent maximum reaction rate and apparent Michaelis constant of immobilized enzyme reactions

The influences of nonuniform activity distribution within a porous solid support on the apparent kinetic parameters, V_m^app and K_m^app, of immobilized enzyme reactions following the Michaelis-Menten kinetics were theoretically investigated. As the enzyme is distributed to the neighborhood of the external surface of the support, V_m^app and K_m^app approach their respective intrinsic values over a wide range of substrate concentration. There is a close relationship between the nonuniform distribution and internal diffusional resistance. Changes in these two factors provide similar effects on V_m^app and K_m^app. As long as the immobilized enzyme reaction follows Michaelis-Menten kinetics, the nonuniform activity distribution never makes K_m^app less than its intrinsic value.     

Probing a functional role of Glu87 and Trp89 in the lid ofHumicola lanuginosa lipase through transesterification reactions in organic solvent

To reveal the functional role of Glu87 and Trp89 in the lid ofHumicola lanuginosa lipase, site-directed mutagenesis at Glu87 and Trp89 was carried out. The catalytic performance of wild-type and mutated lipases was studied in transesterification reactions in cyclohexane at a controlled water activity. Two different acyl donors were used in the investigation: tributyrin, a natural substrate for a lipase, and vinyl butyrate, an activated ester suitable for fast and efficient lipase-catalyzed transformations in preparative organic synthesis. As acyl acceptor 1-heptanol was used. The Glu87Ala mutation decreased theV _max,app value with tributyrin and vinyl butyrate by a factor of 1.5 and 2, respectively. TheK _m,app for tributyrin was not affected by the Glu87Ala mutation, but theK _m,app for vinyl butyrate increased twofold compared to the wild-type lipase. Changing Trp89 into a Phe residue afforded an enzyme with a 2.7- and 2-fold decreasedV _max,app with the substrates tributyrin and vinyl butyrate, respectively, compared to the wild-type lipase. No significant effects on theK _m,app values for tributyrin or vinyl butyrate were seen as a result of the Trp89Phe mutation. However, the introduction of a Glu residue at position 89 in the lid increased theK _m,app for tributyrin and vinyl butyrate by a factor of >5 and 2, respectively. The Trp89Glu mutated lipase could not be saturated with tributyrin within the experimental conditions (0–680 mM) studied here. With vinyl butyrate as a substrate theV _max,app was only 6% of that obtained with wild-type enzyme.     

Influence of intraparticle diffusional limitation on the observed kinetics of immobilized enzymes and on catalyst design

Numerical solutions to the equations describing simultaneous mass transfer and enzymic reaction within porous spherical particles have been used to examine the effect of enzyme content and other parameters on the kinetic behavior of immobilized enzymes. These solutions have also been compared with experimental data for enzymes immobilized to DEAE-cellulose particles. The influence of particle size and enzyme content on catalyst design is discussed.     

Diffusion and chemical reaction rates with nonuniform enzyme distribution: an experimental approach

The study of the effects of nonuniform distributions of immobilized beta-galactosidase on the overall reaction rate of the hydrolysis of lactose are presented. Diffusion inside the particles has been characterized by measuring the diffusion rates of two beta-galactosidase substrates: lactose and ONPG in a commercial silica-alumina support. Effective diffusivities have been determined by the chromatographic method under inert conditions. The results obtained for tortuosity can be explained assuming that the transport only takes place in the macropores. The distribution of the immobilized enzyme has been measured by means of confocal microscopy technique. The enzyme has been tagged with FITC and immobilized in particles of different diameters, the internal local concentrations of the enzyme have been determined with the aid of an image computer program. As expected, a more nonuniform internal profile of the enzyme was found when the particle diameter was bigger. Experiments under reaction conditions were carried out in batch reactors using lactose and ONPG as substrates and particles of the immobilized beta-galactosidase of different diameter (1 x 10(-4) to 5 x 10(-3) m) as catalyst, employing a temperature of 40 degrees C for lactose and 25 and 40 degrees C for ONPG, respectively. The mass balance inside the particle for the substrates has been solved for the internal profiles of the immobilized enzyme inside particles of different size and the enzymatic reactions considered. The calculated and the experimental effectiveness factor values were similar when particles under 2.75 x 10(-3) m in diameter were employed. For the same Thiele modulus, a particle with nonuniform distribution of enzyme showed a higher effectiveness as a catalyst than particles with a more uniform distribution.     

Probing a functional role of Glu87 and Trp89 in the lid ofHumicola lanuginosa lipase through transesterification reactions in organic solvent

To reveal the functional role of Glu87 and Trp89 in the lid ofHumicola lanuginosa lipase, site-directed mutagenesis at Glu87 and Trp89 was carried out. The catalytic performance of wild-type and mutated lipases was studied in transesterification reactions in cyclohexane at a controlled water activity. Two different acyl donors were used in the investigation: tributyrin, a natural substrate for a lipase, and vinyl butyrate, an activated ester suitable for fast and efficient lipase-catalyzed transformations in preparative organic synthesis. As acyl acceptor 1-heptanol was used. The Glu87Ala mutation decreased theV _max,app value with tributyrin and vinyl butyrate by a factor of 1.5 and 2, respectively. TheK _m,app for tributyrin was not affected by the Glu87Ala mutation, but theK _m,app for vinyl butyrate increased twofold compared to the wild-type lipase. Changing Trp89 into a Phe residue afforded an enzyme with a 2.7- and 2-fold decreasedV _max,app with the substrates tributyrin and vinyl butyrate, respectively, compared to the wild-type lipase. No significant effects on theK _m,app values for tributyrin or vinyl butyrate were seen as a result of the Trp89Phe mutation. However, the introduction of a Glu residue at position 89 in the lid increased theK _m,app for tributyrin and vinyl butyrate by a factor of >5 and 2, respectively. The Trp89Glu mutated lipase could not be saturated with tributyrin within the experimental conditions (0–680 mM) studied here. With vinyl butyrate as a substrate theV _max,app was only 6% of that obtained with wild-type enzyme.     

Diffusional and electrostatic effects on apparent kinetic parameters of reactions catalyzed by enzyme immobilized on the external surface of a support

Diffusional and electrostatic effects on the apparent maximum reaction rate V_m^app and the apparent Michaelis constant K_m^app were investigated theoretically for a system in which an enzyme immobilized on the external surface of a solid support catalyzes a reaction according to Michaelis-Menten kinetics. In such a system, the dependence of V_m^app and K_m^app on the substrate concentration can be expressed analytically. When the support and substrate carry charges of the same sign, resulting in a repulsive force between them, both V_m^app and K_m^app decrease with increasing substrate concentration, but they never decrease below the respective intrinsic values. On the other hand, when the support and substrate carry charges of opposite sign and therefore an attractive force occurs, V_m^app decreases towards its intrinsic value, while K_m^app decreases to values below its intrinsic value in the region of high substrate concentration.     

Gel entrapped enzymes: kinetic studies of immobilized beta-galactosidase

β-galactosidase from E. coli (β-D -galactose galactohydrolase, EC 3.2.1.23) has been entrapped in a crosslinked 2-hydroxyethyl methacrylate gel with a 35% retention of activity. The kinetic behavior of the gel-entrapped enzyme has been studied in a recirculation reactor system, the substrate being o-nitrophenyl-βhyphen;D - galactopyranoside. Kinetic constants were determined for particle sizes ranging from 69 to 231 μm in diameter and compared to those of the free enzyme. External diffusion effects were eliminated by operating at high recirculation flow rates. A fourfold increase in K_m(app) was observed for the 231 μm particles, consistent with existing theoretical treatments for internal diffusion effects. An Arrhenius plot of rate data showed significant curvature at higher temperatures, which was attributed to the effects of internal diffusion. The pH–activity profile of the gel-entrapped enzyme was bell-shaped at high substrate concentration and, in contrast to the free enzyme, could be fitted to the titration curve of two ionizable groups, a basic group having a pK of 8.6. The gel-entrapped enzyme had a higher pH optimum and retained a larger percentage of its maximal activity at alkaline pH than the free enzyme; its pH stability at high pH was also much better. The thermal stability of the gel-entrapped enzyme was studied and found to be 14 days at 22°C and 65 min at 45°C.     

Operational effectiveness factors of immobilized enzyme systems

The steady-state and operational effectiveness factors for hydrolytic enzymes immobilized in spherical gel particles have been calculated by the collocation method for a wide range of microenvironmental conditions (given by the Thiele modulus) and macroenvironmental conditions (given by the Sherwood number and the relative substrate content). The operational effectiveness factor is a measure of the ratio of the times required to convert a defined amount of substrate with the same amount of free and immobilized enzyme, respectively. Calculations were made for reactors where the diffusion layers of the different enzyme-containing gel particles do not overlap. The theoretical values were compared with experimental values for stirred reactors with chymotrypsin and trypsin immobilized in spherical particles (Sepharose and Sephadex). Low molecular weight substrates were used. The theoretical and experimental values were found to agree within the experimental error. This demonstrates the predictive capacity of the collocation method in estimating steady-state and operational effectiveness factors for enzyme reactors. The microenvironment and macroenvironment were both found to influence the effectiveness over a wide range of substrate concentrations. However, the macroenvironmental influence is negligible when the Sherwood number of the reactor is larger than ～50. Then, the diffusion layer thickness is small compared with the dimensions of the enzyme-containing particles. The effectiveness factors calculated here can also be used to predict the performance of continuous stirred tank and plug-flow reactors.     

Characterization of membrane domains by frap experiments at variable observation areas

In this paper we show that FRAP experiments at variable beam radii provide an experimental approach for investigating membrane organization and dynamics, with great potential for identifying micrometer-sized domains and determining their size and the diffusion coefficient of the lipid and protein molecules they contain. Monte Carlo simulations of FRAP experiments at variable beam radii R on models of compartmentalized membranes have allowed us to establish the relationships (i) between the mobile fraction M of a diffusing particle and the size r of the domains, and (ii) between the apparent diffusion coefficient D_app and the real diffusion coefficient D₀ of this particle inside the domains. Furthermore, in its present stage of development, this approach allows us to specify whether these domains are strictly closed or not. This approach was first validated on an experimental model of a strictly compartmentalized membrane consisting of a monolayer of apposed spherical phospholipid bilayers supported by silica beads of known radius (0.83 μm). To prevent fusion between the spherical bilayers 5 mol% of a polymer-grafted phospholipid was added to the lipids. Analysis of the M versus R data yielded a radius r of 0.92±0.09 μm for the spherical bilayers, close to that of the supporting silica beads. When applied to the experimental data available for lipids and proteins in the plasma membrane of living cells, this approach suggests the existence of domains within these membranes with a radius of about 0.4 – 0.7 μm for the lipids and 0.25 μm for the proteins. These domains are not strictly closed and they are believed to be delineated by fluctuating barriers which are more or less permeable to lipid and protein molecules. Received: 4 September 1997 / Revised version: 19 January 1998 / Accepted: 19 January 1998     

Detection of non-Brownian diffusion in the cell membrane in single molecule tracking

Molecules undergo non-Brownian diffusion in the plasma membrane, but the mechanism behind this anomalous diffusion is controversial. To characterize the anomalous diffusion in the complex system of the plasma membrane and to understand its underlying mechanism, single-molecule/particle methods that allow researchers to avoid ensemble averaging have turned out to be highly effective. However, the intrinsic problems of time-averaging (resolution) and the frequency of the observations have not been explored. These would not matter for the observations of simple Brownian particles, but they do strongly affect the observation of molecules undergoing anomalous diffusion. We examined these effects on the apparent motion of molecules undergoing simple, totally confined, or hop diffusion, using Monte Carlo simulations of particles undergoing short-term confined diffusion within a compartment and long-term hop diffusion between these compartments, explicitly including the effects of time-averaging during a single frame of the camera (exposure time) and the frequency of observations (frame rate). The intricate relationships of these time-related experimental parameters with the intrinsic diffusion parameters have been clarified, which indicated that by systematically varying the frame time and rate, the anomalous diffusion can be clearly detected and characterized. Based on these results, single-particle tracking of transferrin receptor in the plasma membrane of live PtK2 cells were carried out, varying the frame time between 0.025 and 33 ms (0.03-40 kHz), which revealed the hop diffusion of the receptor between 47-nm (average) compartments with an average residency time of 1.7 ms, with the aid of single fluorescent-molecule video imaging.     

Effect of internal diffusion in heterogeneous enzyme systems: Evaluation of true kinetic parameters and substrate diffusivity

The effect of internal diffusion on the overall reaction rate in spherical particles and membranes containing immobilized enzymes has been investigated theoretically. Since they represent open systems, the MichaelisMenten kinetics is obeyed in the absence of diffusional effects at steady state even at high enzyme concentrations. When internal diffusion perturbs the reaction, the system can not be described any more by K_M and V_max? alone, but is conveniently characterized by the modulus. Assuming that only internal diffusion interferes with the enzyme reaction, the effect of the modulus on the overall rate of reaction is illustrated by the results of computer calculations. Plots of the overall reaction rate against the substrate concentration are hyperbolas at various moduli for both membranes and spherical particles and no sigmoidal curves are obtained with immobilized enzyme systems. Since the conventional plots of enzyme kinetics do not yield straight lines under such conditions, a graphical method is proposed to determine K_M and V_max? as well as the substrate diffusivity in the enzymic medium.     

A theoretical study of concentration of profiles of primary cytochemical-enzyme reaction products in membrane-bound cell organelles and its application to lysosomal acid phosphatase

Synopsis A numerical method was developed for computing the steady-state concentration gradient of a diffusible enzyme reaction product in a membrane-limited compartment of a simplified theoretical cell model. In cytochemical enzyme reactions proceeding according to the metal-capture principle, the local concentration of the primary reaction product is an important factor in the onset of the precipitation process and in the distribution of the final reaction product. The following variables were incorporated into the model: enzyme activity, substrate concentration,K _m, diffusion coefficient of substrate and product, particle radius and cell radius.The method was applied to lysosomal acid phosphatase. Numerical values for the variables were estimated from experimental data in the literature. The results show that the calculated phosphate concentrations inside lysosomes are several orders of magnitude lower than the critical concentrations for efficient phosphate capture found in a previous experimental model study. Reasons for this apparent discrepancy are discussed.     

The Combined Structural and Kinetic Characterization of a Bacterial Nitronate Monooxygenase from Pseudomonas aeruginosa PAO1 Establishes NMO Class I and II

Nitronate monooxygenase (NMO) oxidizes the mitochondrial toxin propionate 3-nitronate (P3N) to malonate semialdehyde. The enzyme has been previously characterized biochemically in fungi, but no structural information is available. Based on amino acid similarity 4,985 genes are annotated in the GenBank^TM as NMO. Of these, 4,424 (i.e. 89%) are bacterial genes, including several Pseudomonads that have been shown to use P3N as growth substrate. Here, we have cloned and expressed the gene pa4202 of Pseudomonas aeruginosa PAO1, purified the resulting protein, and characterized it. The enzyme is active on P3N and other alkyl nitronates, but cannot oxidize nitroalkanes. P3N is the best substrate at pH 7.5 and atmospheric oxygen with k_cat^app/K_m^app of 12 × 10⁶ m⁻¹ s⁻¹, k_cat^app of 1300 s⁻¹, and K_m^app of 110 μm. Anerobic reduction of the enzyme with P3N yields a flavosemiquinone, which is formed within 7.5 ms, consistent with this species being a catalytic intermediate. Absorption spectroscopy, mass spectrometry, and x-ray crystallography demonstrate a tightly, non-covalently bound FMN in the active site of the enzyme. Thus, PA4202 is the first NMO identified and characterized in bacteria. The x-ray crystal structure of the enzyme was solved at 1.44 Å, showing a TIM barrel-fold. Four motifs in common with the biochemically characterized NMO from Cyberlindnera saturnus are identified in the structure of bacterial NMO, defining Class I NMO, which includes bacterial, fungal, and two animal NMOs. Notably, the only other NMO from Neurospora crassa for which biochemical evidence is available lacks the four motifs, defining Class II NMO.     

Preparation and kinetic properties of gel entrapped urate oxidase.

Urate oxidase from hog liver (urate: oxygen oxidoreductase, EC 1.7.33) has been entrapped in a crosslinked 2-hydroxyethyl methacrylate gel with a 47% retention of activity. The kinetic behavior of the gel entrapped enzyme has been studied in a slurried tank reactor using uric acid as substrate. Internal diffusion effects were found to be negligible for particle sizes below 128 mum. A threefold increase in Km (app) was observed for the 128 mum particles and attributed to diffusional effects. The pH activity profile of the gel entrapped enzyme was bell-shaped at high substrate concentration and could be fitted to a titration curve of two ionizable groups, a basic group having a pK of 7.9 and an acidic group with a pK of 11.0. The gel entrapped enzyme showed excellent stability between pH 6.5 and 10.5.     

Effectiveness factors for substrate and product inhibition

The transformation technique of Na and Na (Math. Biosci., 6 , 25, 1970) is extended to convert boundary-value problems involving the steady-state diffusion equation for spherical immobilized enzyme particles exhibiting substrate and product inhibition to initial-value problems. This allows a study of the influence of external mass transfer resistances on the effectiveness factors. It also considerably reduces the number of calculations required to investigate the effect of changes in the kinetic parameters on the overall rate of reaction. The existence of multiple steady states for substrate inhibition kinetics in spherical catalyst particles is illustrated and a criterion for uniqueness of steady states is developed. Effectiveness factors for competitive and noncompetitive product inhibition increase with increasing value of the Sherwood number for the substrate and increasing value of the ratio of substrate to product effective diffusivities within the particle.     

Operational stability of immobilized d-glucose isomerase in a continuous feed stirred tank reactor

d-Glucose isomerization has been studied using immobilized cells of Streptomyces phaeochromogenes in a continuous feed stirred tank reactor (CSTR) where the external film diffusion resistance was negligible. Experiments conducted with various sizes of enzyme particles indicated that a strong internal diffusion resistance improved the apparent stability of these particles. The performance equations of the CSTR were constructed by associating the material balances for the inside porous support matrix with the bulk liquid phase, and enzyme deactivation was also taken into consideration. An iterative method together with the orthogonal collocation method is proposed for the evaluation of effectiveness factor and the substrate concentration profile within the enzyme particles. The numerical results offer an alternative analytical proof for the observation that under strong internal diffusion control the apparent operational stability of immobilized enzyme is improved.     

Arsenylation of nucleoside diphosphates in Rhodospirillum rubrum chromatophores

Rhodospirillum rubrum chromatophores catalyze the formation of ADP-arsenate during illumination with ADP, Mg²⁺ and arsenate. The reaction was measured with (1) firefly luciferase, (2) a coupled enzyme assay involving hexokinase and glucose-6-phosphate dehydrogenase, and (3) a glass electrode. ADP-arsenate hydrolyzed spontaneously with rate constants ranging from 14 to 43 min^?1. Magnesium, arsenate and phosphate accelerated hydrolysis of ADP-arsenate. From a comparison of the three methods, with ADP as the substrate, it is estimated that φ_R (i.e., the ratio between the quantum yields of ADP-arsenate and ATP for light emission from luciferase) is 0.19–0.23. Furthermore, arsenylation rates were 46–52% of phosphorylation rates in experiments with 30 μ M ADP and 0.8 mM arsenate or phosphate. Similarly, the V_app for arsenylation of GDP or IDP was 47–59% of the V_app for phosphorylation during measurements in the presence of 1 mM arsenate or phosphate. The K_app(GDP) was higher during arsenylation than during phosphorylation; the K_app(IDP) was about the same during arsenylation as during phosphorylation. It is suggested that a shift in the equilibrium of substrates and products on the enzyme, toward hydrolysis, is the main cause of the relatively low arsenylation rates.     

A new method for cell immobilization.

A new method for producing particles and membranes containing immobilized bacteria is presented. These immobilized bacteria display good stability over time making them well suited for use in a packed-bed reactor. Such a reactor is tested as a function of the different parameters of the system. The results are qualitatively similar to those obtained with purified enzyme reactors, but some discrepancies with the plug-flow model are noted. It is necessary to use a more sophisticated model in order to fit the experimental data.     

Determination of oxygen profiles in biocatalyst particles by means of a combined polarographic oxygen microsensor.

When studying the effect of immobilization of enzymes or whole cells on the conversion of substrate, more information is gained if measurements of substrate inside the biocatalyst particles are possible. With the methods used until now, only measurements outside the particle can be performed. In this article a method for measuring oxygen profiles in a biocatalyst particle under steady state conditions is described. The biocatalyst particle was made of agarose and contained the enzyme L-lactate 2-monooxygenase. This enzyme decarboxylates lactic acid to acetic acid in the presence of oxygen. The experiments were carried out in a flow chamber with the use of a micromanipulator and a stereomicroscope. The data were sampled by means of a computer. Four different profiles were measured using four different enzyme concentrations. The measured oxygen profiles were reproducible and the signal was very stable. It was also possible to measure the boundary layer around the particle. With the use of the oxygen microsensor, measurements in a biocatalyst particle could be performed accurately, giving way for model validation.