Kinetics of bilirubin oxidase and modeling of an immobilized bilirubin oxidase reactor for bilirubin detoxification

The unbound bilirubin concentration and the enzymatic rate of bilirubin degradation by bilirubin oxidase in bilirubin-serum albumin solutions have been investigated experimentally and theoretically. A stoichiometric bilirubin-serum albumin binding analysis shows that the unbound bilirubin concentration depends only on the molar ratio of the total bilirubin concentration to the total serum albumin concentration. From the theoretical analysis and the measured unbound bilirubin concentrations, serum albumin may be modelled as a molecule having two binding sites, primary and secondary, with stoichiometric equilibrium constants of K(1) = 6 x 10(7)M(-1) and K(2) = 4.5 x 10(6)M(-1), respectively. The rate of total bilirubin degradation in bilirubin-serum albumin mixtures is zero order. An immobilized bilirubin oxidase reactor model, which shows good agreement with experimental bilirubin conversions, is presented. At a flow rate of 1 mL/min with a 8-mL reactor volume, a 50% bilirubin conversion per pass was observed with an inlet bilirubin concentration of 350muM and a serum albumin concentration of 500muM.     

Nonisothermal immobilized enzyme reaction in a packed-bed reactor

Summary This work investigates the reaction behavior of immobilized enzymes in a packed-bed reactor. The effect of heat generation due to exothermic enzyme reaction is considered. Conservations of substrate and energy constitute two coupled nonlinear partial differential equations which are simultaneously solved by a numerical method. It is found that substrate conversion is generally increased at higher temperature. However, the extent of temperature heavily depends on the magnitude of the dimensionless Michaelis constant which is defined as the ratio of Michaelis constant to inlet substrate concentration. At low dimensionless Michaelis constant, substrate conversion is considerably affected by temperature, but at high dimensionless Michaelis constant, the temperature effect is negligibly small. It is also found that maximum bulk temperature of reaction mixtures occurs around a dimensionless reactor length of 1.3 for the case with high substrate conversion.     

Maltodextrin hydrolysis in a fluidized-bed immobilized enzyme reactor

The present work deals with maltodextrin hydrolysis by glucoamylase immobilized onto corn stover in a fluidized bed reactor. An industrial enzyme preparation was covalently grafted onto corn stover, yielding an activity of up to 372 U/g and 1700 U/g for support particle sizes of 0.8 and 0.2 mm, respectively. A detailed kinetic study, using a differential reactor, allowed the characterization of the influence of mass transfer resistance on the reaction catalyzed by immobilized glucoamylase. A simple and general mathematical model was then developed to describe the experimental conversion data and found to be valid.     

Studies on the kinetics of immobilized enzyme using a recycling enzyme reactor system

A method of measuring kinetic parameters of immobilized enzyme with a recycling enzyme reactor system is described. By analyzing the plot of the dimensionless variable Ln(1-X)/X versus the time needed for a unit conversion, t/X, the mechanism of enzymatic reaction can be recognized and then its basic parameters can be evaluated. On the basis of the experimental data measured by P.R. Coulet et al., it has been proposed that the successive degradation of maltodextrins by the collagen membrane-bound amyloglucosidase was a product glucose inhibition reaction and their corresponding constants have been found with this method.     

An immobilized alpha-galactosidase continuous flow reactor

An α-galactosidase which will hydrolyze the oligosaccharides melibiose, raffinose, and stachyose has been immobilized on nylon microfibrils suitable for use in large flow-through reactors. This catalyst system is stable for many months, both under use and storage conditions. The immobilized enzyme behaves similarly to the enzyme in solution, characteristically exhibiting both product and substrate inhibition. The catalyst is prepared in situ and a large, 8-liter reactor has been made. The catalyst has been used to reduce the raffinose concentration in beet sugar molasses.     

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.     

Development of a stable continuous flow immobilized enzyme reactor for the hydrolysis of inulin

A 23.5-fold purified exoinulinase with a specific activity of 413 IU/mg and covalently immobilized on Duolite A568 has been used for the development of a continuous flow immobilized enzyme reactor for the hydrolysis of inulin. In a packed bed reactor containing 72 IU of exoinulinase from Kluyveromyces marxianus YS-1, inulin solution (5%, pH 5.5) with a flow rate of 4 mL/h was completely hydrolyzed at 55 degrees C. The reactor was run continuously for 75 days and its experimental half-life was 72 days under the optimized operational conditions. The volumetric productivity and fructose yield of the reactor were 44.5 g reducing sugars/L/h and 53.3 g/L, respectively. The hydrolyzed product was a mixture of fructose (95.8%) and glucose (4.2%) having an average fructose/glucose ratio of 24. An attempt has also been made to substitute pure inulin with raw Asparagus racemosus inulin to determine the operational stability of the developed reactor. The system remained operational only for 11 days, where 85.9% hydrolysis of raw inulin was achieved.     

Production of penicillins and cephalosporins in an immobilized enzyme reactor

Summary Four enzymes required for the biosynthesis of pencillins and cephalosporins by Streptomyces clavuligerus have been immobilized on an anion exchange resin. The capabilities of the system have been studied by circulation of reaction mixtures through the immobilized enzyme reactor. Within 30 min, all of the substrate -(l--aminoadipyl)-l-cysteinyl-d-valine is consumed and converted to a mixture of penicillins and cephalosporins. After 60 min the major antibiotic products are (iso)penicillin N and desacetylcephalosporin C. The activity of the immobilized enzyme reactor activity is stable to storage at temperatures below 4°C but activity is lost on repeated use.     

An airlift loop reactor for the transformation of steroids by immobilized cells

Summary The flow behaviour of calcium alginate beads in an airlift reactor (ALR) with external loop was dependent on the airflow rate into and the amount of beads in the reactor. The performance of immobilizedArthrobacter simplex for the ¹-dehydrogenation of hydrocortisone in the ALR compared favourably to that in a stirred tank reactor. The physical stability of the calcium alginate beads was significantly greater in the ALR.     

Study of an enzyme membrane reactor with immobilized fumarase for production of L-malic acid

The conversion of fumaric acid into L-malic acid by fumarase immobilized in a membrane reactor was analyzed experimentally. The enzyme was entrapped in asymmetric capillary membranes made of polysulfone. The performance of the reactor was evaluated in terms of conversion degree, reaction rate, and stability. The influence of operating conditions, such as amount of immobilized enzyme, substrate concentration, residence time, and axial flow rate, were investigated. The kinetic parameters K(m), V(max), and k(+2) were also measured. The stability of the immobilized enzyme was very good, showing no activity decay during more than 2 weeks of continuous operation.     

Parameter evaluation and performance studies in a fluidized-bed immobilized enzyme reactor

Dispersion and mass-transfer characteristics and fluidization parameters influencing the performance of a small pilot-plant immobilized enzyme reactor are evaluated. The suitability of a dispersed plug-flow model to predict the conversions obtained in the enzymatic reaction (starch → glucose) catalyzed by amyloglucosidase immobilized to solid and porous carriers is assessed. The performance of a fluidized-bed reactor is compared on the basis of a normalized residence time with that of a fixed bed and found to be superior.     

New enzymatic immobilized biocatalysts for detoxification of organophosphorus compounds

New immobilized biocatalysts based on phosphotriesterase and porous fabric materials impregnated with chemically cross-linked chitosan and sulphate chitosan gels were investigated. Analysis of the rheological characteristics of enzyme-containing gels confirmed their high plasticity and mechanical strength, while scanning electron microscopy verified their macroporous structure. The fabric matrix could absorb and retain a large amount of liquid thereby increasing its own weight 3.5-4.5 fold. The catalytic characteristics of the immobilized biocatalyst hydrolyzing Paraoxon, Coumaphos, Chlorpyrifos and Diisopropyl fluorophosphate were investigated. The catalytic efficacy of the soluble enzyme was 3.0-5.5-times higher compared to the immobilized form mainly due to the lower K_m values. With constant 55-60% humidity the biocatalyst retained 77% and 67-70% activity after 50-day storage at 4°C and 23°C, respectively. Benzalkonium chloride appeared to be an appropriate preservative for long-term storage of immobilized biocatalyst in a wet state.     

New enzymatic immobilized biocatalysts for detoxification of organophosphorus compounds

New immobilized biocatalysts based on phosphotriesterase and porous fabric materials impregnated with chemically cross-linked chitosan and sulphate chitosan gels were investigated. Analysis of the rheological characteristics of enzyme-containing gels confirmed their high plasticity and mechanical strength, while scanning electron microscopy verified their macroporous structure. The fabric matrix could absorb and retain a large amount of liquid thereby increasing its own weight 3.5–4.5 fold. The catalytic characteristics of the immobilized biocatalyst hydrolyzing Paraoxon, Coumaphos, Chlorpyrifos and Diisopropyl fluorophosphate were investigated. The catalytic efficacy of the soluble enzyme was 3.0–5.5-times higher compared to the immobilized form mainly due to the lower K_m values. With constant 55–60% humidity the biocatalyst retained 77% and 67–70% activity after 50-day storage at 4°C and 23°C, respectively. Benzalkonium chloride appeared to be an appropriate preservative for long-term storage of immobilized biocatalyst in a wet state.     

On-line chromatographic screening of matrix metalloproteinase inhibitors using immobilized MMP-9 enzyme reactor

Matrix metalloproteinase 9 (MMP-9) plays an important role in cancer invasion and metastasis and has been an attractive target for therapeutic intervention of cancer metastasis. However, considering the high cost and intricacy associated with the expression, isolation and purification of the recombinant enzyme for the screening of drug candidates, alternative methods that explore the recycling of enzymes become desirable. In this study, a new immobilized enzyme reactor (IMER) containing human recombinant MMP-9 enzyme was developed and characterized for the on-line screening of MMP-9 inhibitors. The MMP-9 IMER containing active unit of the enzyme (U = 0.08 μmol/min) on the disk was inserted into a HPLC system connected to a UV–vis detector for on-line chromatographic screening. The resulting conjugated enzyme was shown to be an active and stable catalyst for the hydrolysis of MMP-9 chromogenic thiopeptide substrate Ac-PLG-[2-mercapto-4-methyl-pentanoyl]-LG-OC₂H₅. The kinetics profile of the enzyme in IMER and free solution were determined and compared. Selected reversible MMP inhibitors, N-isobutyl-N-(4-methoxyphenylsulfonyl)-glycyl hydroxamic acid (NNGH), doxycycline and minocycline were further characterized using the MMP-9 IMER and free enzyme solution assays. Our system demonstrated applicability as a rapid and cost-effective screening tool for inhibitors of the MMP-9 enzyme.     

Dynamic affinity between dissociable coenzyme and immobilized enzyme in an affinity chromatographic reactor with single enzyme

The affinity chromatographic reactor (ACR) is a bioreactor which utilizes the dynamic interaction or the dynamic affinity between a free coenzyme and immobilized enzymes for the highly efficient regeneration of dissociable coenzymes. Dynamic affinity between free NAD and immobilized alcohol dehydrogenase (ADH) in ACR was investigated by three different methods. ADH catalyzed both oxidation and reduction of NAD, consuming propionaldehyde and ethanol. The theoretical model under consideration elucidated a criterion for the expression of the dynamic affinity as a relationship among the affinity constants and the concentrations of a coenzyme and immobilized enzyme. This criterion was confirmed experimentally by the measurements of the retention time of NAD and the half-life period of the reactor activity after one-shot pulse injection of NAD to ACR. In the stability measurement of the immobilized enzyme, it became clear that ADH was more stable at the higher concentration in immobilization. Although the present case of coenzyme cycling by a single enzyme is very special, with limited chance for the direct application, the results obtained here provide a theoretical basis for ACR with multienzymes-which is of more general use.     

An immobilized cell reactor with simultaneous product separation. II. Experimental reactor performance

The simultaneous separation of volatile fermentation products from product-inhibited fermentations can greatly increase the productivity of a bioreactor by reducing the product concentration in the bioreactor, as well as concentrating the product in an output stream free of cells, substrate, or other feed impurities. The Immobilized Cell Reactor-Separator (ICRS) consists of two column reactors: a cocurrent gas-liquid "enricher" followed by a countercurrent "stripper" The columns are four-phase tubular reactors consisting of (1) an inert gas phase, (2) the liquid fermentation broth, (3) the solid column internal packing, and (4) the immobilized biological catalyst or cells. The application of the ICRS to the ethanol-from-whey-lactose fermentation system has been investigated. Operation in the liquid continuous or bubble flow regime allows a high liquid holdup in the reactor and consequent long and controllable liquid residence time but results in a high gas phase pressure drop over the length of the reactor and low gas flow rates. Operation in the gas continuous regime gives high gas flow rates and low pressure drop but also results in short liquid residence time and incomplete column wetting at low liquid loading rates using conventional gas-liquid column packings. Using cells absorbed to conventional ceramic column packing (0.25-in. Intalox saddles), it was found that a good reaction could be obtained in the liquid continuous mode, but little separation, while in the gas continuous mode there was little reaction but good separation. Using cells sorbed to an absorbant matrix allowed operation in the gas continuous regime with a liquid holdup of up to 30% of the total reactor volume. Good reaction rates and product separation were obtained using this matrix. High reaction rates were obtained due to high density cell loading in the reactor. A dry cell density of up to 92 g/L reactor was obtained in the enricher. The enricher ethanol productivity ranged from 50 to 160 g/L h while the stripper productivity varied from 0 to 32 g/L h at different feed rates and concentrations. A separation efficiency of as high as 98% was obtained from the system.     

Flow injection chemiluminescent assays for glycerol and triglycerides using a co‐immobilized enzyme reactor

A flow injection method for the determination of glycerol using a co‐immobilized enzyme reactor containing glycerokinase and glycerol‐3‐phosphate oxidase is described. The hydrogen peroxide produced is monitored by using a luminol chemiluminescence reaction in the presence of catalyst such as Co(II). The detection limit (2.5 × blank noise) for glycerol is 7 × 10^?3 mmol/L with a sample throughput of 40/h. The calibration graph is linear over the range studied (0.2–1.0 mmol/L) with relative standard deviation 1.2–2.4%. The method is applied to the determination of glycerol in blood serum produced off‐line from triglycerides using lipase isolated from bovine pancreas. Copyright © 2003 John Wiley & Sons, Ltd.