Transient response of encapsulated enzymes in hollow-fiber reactor

A mathematical model for the transient response of encapsulated enzymes is developed showing the effects of the outer boundary layer, the encapsulating membrane, the partition coefficient, and diffusion with reaction within the encapsulating medium. The model incorporates both first-order kinetics and Michaelis-Menten kinetics for the reaction rate. Using typical hollow-fiber or microcapsule parameters, the model shows that (a) the partition coefficient affects the overall rate only when the rate-limiting step is diffusion through the membrane, (b) the transient overall effectiveness factor rises sharply with time and approaches an asymptotic value for most situations, and (c) the first-order approximation to Michaelis-Menten kinetics is not valid when the initial outside bulk concentration is higher than the Michaelis constant and the overall rate is reaction limited. The model is compared with experimental data using uricase in a hollow-fiber enzyme reactor configuration. Batch assay and CSTUER (continuous-stirred ultrafiltration enzyme reactor) studies were conducted on the free enzyme to provide some of the parameters used in the model. The CSTUER data fit the case of substrate inhibition kinetics with the apparent Michaelis constant approaching zero. The hollow-fiber reactor was conducted with uricase dissolved in both a buffer solution and a concentrated hemoglobin solution. Diffusivities of the solute were measured in both solutions as was the osmotic pressure of the hemoglobin solution. While experimental data for uricase in buffer solution could easily be matched by the model, that in the concentrated hemoglobin solution could not.     

On the performance of a hollow-fiber bioreactor for acidolysis catalyzed by immobilized lipase

The present communication describes the chemical modification of anhydrous butterfat by interesterification with oleic acid catalyzed by a lipase of Mucor javanicus. Two reactor configurations were tested, a batch-stirred tank reactor containing suspended lipase and a batch-stirred tank reactor in combination with a hollow-fiber membrane module containing adsorbed lipase. The goal of this research was to assess the advantage of using a (hydrophobic) porous support to immobilize the lipase in attempts to engineer butterfat with increased levels of unsaturated fatty acid residues (oleic acid) at the expense of medium-to-long chain saturated fatty acids (myristic and palmitic acids). Reactions were carried out at 40 degrees C in the absence of solvent under controlled water activity, and were monitored by chromatographic assays for free fatty acids. The results obtained indicate that the rate of interesterification using the proposed reactor configuration is enhanced by a factor above 100 relative to that using suspended lipase, for the same protein mass basis. Although hydrolysis of butterfat occurred to some degree, the enzymatic process that uses the hollow-fiber reactor was technically superior to the stirred tank system. Copyright 1998 John Wiley & Sons, Inc.     

New Evidence for Dimerization-Dependence of the Activity of Candida Rugosa Lipase

The specific activity of the lipase of Cundidu rugosu decreases with increasing enzyme concentration even in the presence of soluble substrates. Data about the hydrolysis of 2-chloropropionic acid ethyl ester (CPEE) had suggested that this phenomenon may be caused either by dimerization of the lipase or by adsorption onto the reactor wall. In order to distinguish between both models, experiments were carried out by changing not only the enzyme concentration but also the wetted surface area of the reaction vessel. These novel data reveal that wetted glass surfaces are of only minor importance - if any. Thus, the decrease of activity seems to be caused by some kind of dimerization of the lipase. In addition, it is shown that adsorption onto hydrophobic surfaces can have a dramatic effect on the specific activity. In the presence of large hydrophobic surface areas the specific activity is found to be almost as high as that observed in the presence of insoluble substrate. The analysis of a commonly used test system for lipase activity measurements based on triacetin hydrolysis exhibits a similar activity-enzyme concentration dependence.     

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.     

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.     

An ultrafiltration membrane bioreactor for the lipolysis of olive oil in reversed micellar media

The enzymatic hydrolysis of olive oil using Chromobacterium viscosum lipase B encapsulated in reversed micelles of dioctyl sodium sulfosuccinate (AOT) in isooctane was investigated in an ultrafiltration ceramic membrane reactor of tubular type, operating in a batch mode. Water concentration was found to be a critical parameter in the enzyme kinetics and hydrolysis yield of the reaction. The size of micelles, recirculation rate, and substrate concentration were found to be the major factors affecting the separation process. A correlation that enables the prediction of final conversion degrees in this bioreactor from the initial reaction conditions was established. (c) 1993 Wiley & Sons, Inc.     

Selection of CalB immobilization method to be used in continuous oil transesterification: analysis of the economical impact

Enzymatic transesterification of triglycerides in a continuous way is always a great challenge with a large field of applications for biodiesel, bio-lubricant, bio-surfactant, etc. productions. The lipase B from Candida antarctica (CalB) is the most appreciated enzyme because of its high activity and its non-regio-selectivity toward positions of fatty acid residues on glycerol backbone of triglycerides. Nevertheless, in the field of heterogeneous catalysis, we demonstrated that the medium hydrophilic nature of the support used for its commercial form (Lewatit VPOC1600) is a limitation. Glycerol is adsorbed onto support inducing drastic decrease in enzyme activity. Glycerol would form a hydrophilic layer around the enzyme resulting in diffusional limitations during triglyceride transfer to the enzyme. Accurel MP, a very hydrophobic macroporous polymer of propylene, was found not to adsorb glycerol. Immobilization conditions using this support were optimized. The best support was Accurel MP1001 (particle size<1000 μm) and a pre-treatment of the support with acetone instead of ethanol enables the adsorption rate and the immobilized enzyme quantity to be maximized. An economical approach (maximization of the process net present value) was expanded in order to explore the impact of immobilization on development of an industrial packed bed reactor. The crucial ratio between the quantity of lipase and the quantity of support, taking into account enzyme, support and equipped packed bed reactor costs was optimized in this sense. The biocatalyst cost was found as largely the main cost centre (2-10 times higher than the investments for the reactor vessel). In consequence, optimal conditions for immobilization were a compromise between this immobilization yield (90% of lipase immobilized), biocatalyst activity, reactor volume and total investments.     

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.     

Hollow-fiber membrane bioreactors using immobilized E. coli for protein synthesis

Actively growing Escherichia coli C600(pBR322), immobilized within the macroporous matrix of asymmetric-wall hollow-fiber membranes, has been propagated to extremely high densities, typically more than 10(12) cells/mL of accessible void volume, in some regions cells accounting for nearly 100% of the available macrovoid volume forming a tissue-like mass. Production rates of beta-lactamase, an enzyme used as an indicator of the culture's biosynthetic potential, remained at high and relatively stable levels for more than three weeks of continuous operation, and effluent supernatant enzyme activities attained 25% of the accumulated level measured in a 24-h shaker-flask culture. Based on the accessible void volume within the fiber wall, the beta-lactamase productivity was independent of the specific asymmetric membrane used. On a per cell basis, however, cells cultured using hollow-fiber membranes were only 10% as productive as those in the shaker-flask culture, possibly due to the high packing density or culture aging. By contrast, the hollow-fiber bioreactor was 100 times more productive than the shaker-flask culture on a reactor-volume basis, primarily as a consequence of the high cell densities. Reactor productivity was dependent on the number of cells in the reactor, suggesting that reactor performance was kinetically controlled and not mass transport limited.     

Properties of the lipid body lipase of Pinus eduUs and electrophoretic purification of its 64 kDa subunit

Lipase (triacyiglycerol acylhydrolase, EC 3.1.1.3) in oilseeds can be associated with either the lipid body or glyoxysomal membrane and can have various pH optima and substrate specificities. There is conflicting evidence for the subcellular location of lipase in gymnosperms, and little information exists on its activity characteristics. In this report, Pinus edulis (pinyon) was found to have an acid lipase, which was associated with the lipid body fraction and the activity of which increased during germination. Active lipase from the solubilized lipid body membrane was determined by gel permeation chromatography to have a molecular weight of 260 000. Further attempts to purify the active enzyme were unsuccessful. A lipid body membrane protein of 64 kDa which increased in parallel with lipase activity during germination was isolated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. excised, and polyclonal antibodies were made against it. Using these antibodies, active lipase was immunoprecipitated from solution, thus indicating that the 64 kDa protein is a subunit of the lipase. Pinus edulis lipase had a pH optimum of ca 4.5. and it exhibited little specificity for triacyiglycerol substrates in vitro. The lipase was specific in activity against fluorometric substrates, with the highest activity against methyl-umbelliferyl laurale. Lipase activity was inhibited by high concentrations of non-ionic detergent. This lipid body acid lipase appears to be primarily responsible for lipid hydrolysis during pinyon germination.     

Covalent immobilization of lipase from Candida rugosa onto poly(acrylonitrile-co-2-hydroxyethyl methacrylate) electrospun fibrous membranes for potential bioreactor application

A simple way of fabricating enzymatic membrane reactor with high enzyme loading and activity retention from the conjugation between nanofibrous membrane and lipase was devised. Poly(acrylonitrile-co-2-hydroxyethyl methacrylate) (PANCHEMA) was electrospun into fibrous membrane and used as support for enzyme immobilization. The hydroxyl groups on the fibrous membrane surface were activated with epichlorohydrin, cyanuric chloride or p-benzoquinone, respectively. Lipase from Candida rugosa was covalently immobilized on these fibrous membranes. The resulted bioactive fibrous membranes were examined in catalytic efficiency and activity for hydrolysis. The observed enzyme loading on the fibrous membrane with fiber diameter of 80–150 nm was up to 1.6% (wt/wt), which was as thrice as that on the fibrous membrane with fiber diameter of 800–1000 nm. Activity retention for the immobilized lipase varied between 32.5% and 40.6% with the activation methods of hydroxyl groups. Stabilities of the immobilized lipase were obviously improved. In addition, continuous hydrolysis was carried out with an enzyme-immobilized fibrous membrane bioreactor and a steady hydrolysis conversion (3.6%) was obtained at a 0.23 mL/min flow rate under optimum condition.     

Effect of pH on the production of lipolyzed butter oil by a calf pregastric esterase immobilized in a hollow-fiber reactor: II. Multiresponse kinetics

The lipolysis of butter oil in a hollow-fiber reactor containing an immobilized calf pregastric esterase was studied at 40 degrees C and at pH values of 4.0, 5.0, 6.0, and 7.0. The concentrations of ten fatty acid species in the lipolyzed product were determined using high-performance liquid chromatography (HPLC). The relative specificity of this esterase depended on pH. Three mathematical models derived from a generalized Michaelis-Menten mechanism were tested for their ability to describe the rates of release of individual specific fatty acids. Loss of enzyme activity was modeled using first order kinetics. The models for deactivation and reaction kinetics were fit simultaneously to the data. The parameters of the model were also tested for dependence on pH. The model was successful in describing the rates of release of all ten fatty acid species for a range of space times and pH values.     

Electrospun polyacrylonitrile nanofibrous membranes for lipase immobilization

Polyacrylonitrile (PAN) nanofibers could be fabricated by electrospinning with fiber diameter in the range of 150–300 nm, providing huge surface area for enzyme immobilization and catalytic reactions. Lipase from Candida rugosa was covalently immobilized onto PAN nanofibers by amidination reaction. Aggregates of enzyme molecules were found on nanofiber surface from field emission scanning electron microscopy and covalent bond formation between enzyme molecule and the nanofiber was confirmed from FTIR measurements. After 5 min activation and 60 min reaction with enzyme-containing solution, the protein loading efficiency was quantitative and the activity retention of the immobilized lipase was 81% that of free enzyme. The mechanical strength of the NFM improved after lipase immobilization where tensile stress at break and Young's modulus were almost doubled. The immobilized lipase retained >95% of its initial activity when stored in buffer at 30 °C for 20 days, whereas free lipase lost 80% of its initial activity. The immobilized lipase still retained 70% of its specific activity after 10 repeated batches of reaction. This lipase immobilization method shows the best performance among various immobilized lipase systems using the same source of lipase and substrate when considering protein loading, activity retention, and kinetic parameters.     

Alcoholysis catalyzed by Candida antarctica lipase B in a gas/solid system obeys a Ping Pong Bi Bi mechanism with competitive inhibition by the alcohol substrate and water.

The kinetics of alcoholysis of methyl propionate and n-propanol catalyzed by Candida antarctica lipase B supported onto silanized Chromosorb P was studied in a continuous solid/gas reactor. In this system the solid phase is composed of a packed enzymatic sample and is percolated by nitrogen as carrier gas, which simultaneously carries substrates to the enzyme while removing reaction products. In this reactor the thermodynamic activity of substrates and effectors can be perfectly adjusted allowing kinetic studies to be performed under different operating conditions. The kinetics obtained for alcoholysis were suggested to fit a Ping Pong Bi Bi mechanism with dead-end inhibition by the alcohol. The values of all apparent kinetic parameters were calculated and the apparent dissociation constant of enzyme for gaseous ester was found very low compared with the one obtained for liquid ester in organic medium, certainly due to the more efficient diffusion in the gaseous phase. The effect of water thermodynamic activity was also investigated. Water was found to act as a competitive inhibitor, with a higher inhibition constant than n-propanol. Thus alcoholysis of gaseous methyl propionate and n-propanol catalyzed by C. antarctica lipase B was found to obey the same kinetic mechanism as in other non-conventional media such as organic liquid media and supercritical carbon dioxide, but with much higher affinity for the substrates.     

The lipoprotein lipase of white adipose tissue. Changes in the adipocyte cell-surface content of enzyme in response to extracellular effectors in vitro.

An indirect labelled-second-antibody cellular immunoassay for adipocyte surface lipoprotein lipase was used to assess the changes that occurred during the incubation of cells in the presence and absence of effectors. In the absence of any specific effectors, the amount of immunodetectable lipoprotein lipase present at the surface of adipocytes remained constant throughout the 4 h incubation period at 37 degrees C. Under such conditions total cellular enzyme activity also remained constant, with no activity appearing in the medium. In the presence of heparin, cell-surface immunodetectable lipoprotein lipase increased by up to 20%, whereas in the presence of cycloheximide they decreased by up to 60%. Thus the obvious turnover of enzyme from this cell-surface site was found to be relatively rapid and dependent for its replenishment, at least in part, on protein synthesis. In the presence of insulin alone, a substantial increase in cell-surface lipoprotein lipase protein occurred, only part of which was dependent on protein synthesis. The total cellular activity of lipoprotein lipase was unaffected by the presence of insulin. The insulin-dependent increase in cell-surface enzyme was potentiated somewhat in the presence of dexamethasone, which was not shown to exert any independent effect. Glucagon, adrenaline and theophylline all produced a significant decline in the cell-surface immunodetectable lipoprotein lipase, which in the case examined (adrenaline) was partially additive with regard to the independent effect of cycloheximide. Cell-surface immunodetectable lipoprotein lipase amounts were decreased significantly when cells were incubated in the presence of either colchicine or tunicamycin. The concerted way in which cell-surface lipoprotein lipase altered during the incubations of adipocytes in the presence of effectors suggested that the translocation of enzyme to and from this cellular site was dependent on hormonal action and the integrity of intracellular protein-transport mechanisms.     

Integration of purification with immobilization of Candida rugosa lipase for kinetic resolution of racemic ketoprofen

The two processes for the partial purification and for the immobilization of a crude lipase preparation (Candida rugosa Lipase OF) have been successfully integrated into one by simple adsorption of the enzyme onto a cation ion exchanger resin (SP-Sephadex C-50) at pH 3.5. Due to selective removal of the unfavorable lipase isoenzyme (L1), the enzyme components (mainly L2 and L3) that are tightly fixed on the resin displayed a significantly improved enantioselectivity (E value: 50 versus 13 with addition of Tween-80) in the biocatalytic hydrolysis of 2-chloroethyl ester of rac-ketoprofen. The activity yields of the immobilized lipase were 48 and 70%, respectively when emulsified and non-emulsified substrates were employed for enzyme assay. Moreover, the concentration of Tween-80 was found to be a factor affecting the lipase enantioselectivity. By using such an immobilized enzyme as biocatalyst, the process for preparing enantiopure (S)-ketoprofen becomes simpler and more practical as compared with the previously reported procedures and the product was obtained with >94% ee at 22.3% conversion in the presence of an optimal concentration (0.5 mg/ml) of Tween-80 at pH 3.5. Furthermore, the operational stability of the immobilized biocatalyst was examined in different types of reactors. In an air-bubbled column reactor, the productivity was much higher than that in a packed-bed column reactor, in spite of a slightly lower stability. Under optimal conditions, the air-bubbled column reactor could be operated smoothly for at least 350 h, remaining nearly 50% activity.     

Characterization and solubilization of membrane bound diacylglycerol lipases from bovine brain

Bovine brain contains two diacylglycerol lipases. One is localized in purified microsomes and the other is found in the plasma membrane fraction. The microsomal enzyme is markedly stimulated by the non-ionic detergent, Triton X-100, and Ca2+, whereas the plasma membrane diacylglycerol lipase is strongly inhibited by Triton X-100 and Ca2+ has no effect on its enzymic activity. Both enzymes were solubilized using 0.25% Triton X-100. The solubilized enzymes followed Michaelis-Menten kinetics. The apparent Km values for microsomal and plasma membrane enzymes are 30.5 and 12.0 microM respectively. Both lipases are strongly inhibited by RHC 80267, with Ki values for microsomal and plasma membrane diacylglycerol lipases of 70 and 43 microM, respectively. The retention of microsomal diacylglycerol lipase on a concanavalin A-Sepharose column and its elution by methyl alpha-D-mannoside indicates the glycoprotein nature of this enzyme.     

Synthesis of 2-ethyl hexanol fatty acid esters in a packed bed bioreactor using a lipase immobilized on a textile membrane

An enzymatic process using a packed bed bioreactor with recirculation was developed for the scale-up synthesis of 2-ethylhexyl palmitate with a lipase from Candida sp. 99–125 immobilized on a fabric membrane by natural attachment to the membrane surface. Esterification was effectively performed by circulating the reaction mixture between a packed bed column and a substrate container. A maximum esterification yield of 98% was obtained. Adding molecular sieves and drying the immobilized lipase both decreased the water content at the reactor outlet and around the enzyme, which led to an increase in the rate of esterification. The long-term stability of the reactor was tested by continuing the reaction for 30 batches (over 300 h) with an average esterification yield of about 95%. This immobilized lipase bioreactor is scalable and is thus suitable for industrial production of 2-ethylhexyl palmitate.     

Kinetic resolution of chiral amines with omega-transaminase using an enzyme-membrane reactor

A kinetic resolution process for the production of chiral amines was developed using an enzyme-membrane reactor (EMR) and a hollow-fiber membrane contactor with (S)-specific omega-transaminases (omega-TA) from Vibrio fluvialis JS17 and Bacillus thuringiensis JS64. The substrate solution containing racemic amine and pyruvate was recirculated through the EMR and inhibitory ketone product was selectively extracted by the membrane contactor until enantiomeric excess of (R)-amine exceeded 95%. Using the reactor set-up with flat membrane reactor (10-mL working volume), kinetic resolutions of alpha-methylbenzylamine (alpha-MBA) and 1-aminotetralin (200 mM, 50 mL) were carried out. During the operation, concentration of ketone product, i.e., acetophenone or alpha-tetralone, in a substrate reservoir was maintained below 0.1 mM, suggesting efficient removal of the inhibitory ketone by the membrane contactor. After 47 and 32.5 h of operation using 5 U/mL of enzyme, 98.0 and 95.5% ee of (R)-alpha-MBA and (R)-1-aminotetralin were obtained at 49.5 and 48.8% of conversion, respectively. A hollow-fiber membrane reactor (39-mL working volume) was used for a preparative-scale kinetic resolution of 1-aminotetralin (200 mM, 1 L). After 133 h of operation, enantiomeric excess reached 95.6% and 14.3 g of (R)-1-aminotetralin was recovered (97.4% of yield). Mathematical modeling of the EMR process including the membrane contactor was performed to evaluate the effect of residence time. The simulation results suggest that residence time should be short to maintain the concentration of the ketone product in EMR sufficiently low so as to decrease conversion per cycle and, in turn, reduce the inhibition of the omega-TA activity.     

Model-based comparative performance analysis of membrane aerated biofilm reactor configurations

The potential of the membrane aerated biofilm reactor (MABR) for high-rate bio-oxidation was investigated. A reaction-diffusion model was combined with a preliminary hollow-fiber MABR process model to investigate reaction rate-limiting regime and to perform comparative analysis on prospective designs and operational parameters. High oxidation fluxes can be attained in the MABR if the intra-membrane oxygen pressure is sufficiently high, however the volumetric oxidation rate is highly dependent on the membrane specific surface area and therefore the maximum performance, in volumetric terms, was achieved in MABRs with relatively thin fibers. The results show that unless the carbon substrate concentration is particularly high, there does not appear to be an advantage to be gained by designing MABRs on the basis of thick biofilms even if oxygen limitations can be overcome.