A comparative study of immobilized amyloglucosidase in a packed bed reactor and a continuous feed stirred tank reactor

The catalytic activity of amyloglucosidase covalently attached to DEAE-cellulose was studied in a packed bed reactor and a continuous feed stirred tank reactor (CSTR) for the reaction maltose → glucose. At low flow rates mass-transfer limitations in the bed reactor lead to lower conversions for this reactor compared to the CSTR. Simple theoretical expressions for these reactors were compared with the experimental results. There are significant differences between the kinetic parameters and pH profile of the immobilized and free enzyme. The immobilized enzyme also showed greater stability at 50°C than did free amyloglucosidase. The temperature dependence of the reaction rate was the same for immobilized and free enzyme.     

Design of immobilized enzyme reactors for the continuous production of fructose syrup from whey permeate

Biocatalyst inactivation is inherent to continuous operation of immobilized enzyme reactors, meaning that a strategy must exist to ensure a production of uniform quality and constant throughput. Flow rate can be profiled to compensate for enzyme inactivation maintaining substrate conversion constant. Throughput can be maintained within specified margins of variation by using several reactors operating in parallel but displaced in time. Enzyme inactivation has been usually modeled under non-reactive conditions, leaving aside the effect of substrate and products on enzyme stability. Results are presented for the design of enzyme reactors under the above operational strategy, considering first-order biocatalyst inactivation kinetics modulated by substrate and products. The continuous production of hydrolyzed-isomerized whey permeate with immobilized lactase and glucose isomerase in sequential packed-bed reactors is used as a case study. Kinetic and inactivation parameters for immobilized lactase have been determined by the authors; those for glucose isomerase were taken from the literature. Except for lactose, all other substrates and products were positive modulators of enzyme stability. Reactor design was done by iteration since it depends on enzyme inactivation kinetics. Reactor performance was determined based on a preliminary design considering non-modulated first-order inactivation kinetics and confronted to such pattern. The new pattern of inactivation was then used to redesign the reactor and the process repeated until reactor performance (considering modulation) matched the assumed pattern of inactivation. Convergence was very fast and only two iterations were needed.     

A new process combining adsorption and enzyme reaction for producing higher-fructose syrup

A process for producing a higher-fructose syrup containing more than 50% fructose was developed that involves a new system combining selective adsorption of fructose and an immobilized glucose isomerase reaction. Continuous countercurrent contact of the liquid stream with the solid adsorbent is simulated by advancing adsorption columns against the fixed inlets and outlet of liquid streams without actual movement of the solid adsorbent, while the immobilized enzyme reactors are stationary. Two mathematical models, an intermittent moving-bed and a continuous moving-bed model, are presented for calculation the concentration profiles of glucose and fructose in the system. The validity of the models is experimentally confirmed, and a criterion for good production in the system is presented. This system requires less desorbent than a process using a fixed-bed adsorber and a simulated moving-bed process to produce the syrup with 45-65% fructose content, the level desired in food manufacture.     

Scale-up of flat-plate reactors for enzymatic hydrolysis of fats and oils

To determine the feasibility of continuous enzymatic fat-splitting, immobilized lipase reactors were constructed from alternating layers of enzyme support material and separator screens. Partially purified lipase from Thermomyces lanuginosus was loaded onto the support material at pH 5.5 by irreversible adsorption. Melted edible tallow at 51°C was pumped through the immobilized enzyme layers and swept from the downstream separator screens by buffer recycled from a continuous oil/water separator. Results from continuous operation of 10-layer reactors were compared with data from single-layer reactors. The activity per square centimeter of 10-layer reactors was nearly as much as that of single-layer reactors at the same enzyme loading and oil feed rate. Data were fitted to an empirical mathematical model.     

On-line determination of glucose and lactate concentrations in animal cell culture based on fibre optic detection of oxygen in flow-injection analysis.

A flow-injection analysis (FIA) system based on fibre optic detection of oxygen consumption using immobilized glucose oxidase (GOD) and lactate oxidase (LOD) is described for the on-line monitoring of glucose and lactate concentrations in animal cell cultures. The consumption of oxygen was determined via dynamic quenching by molecular oxygen of the fluorescence of an indicator. GOD and LOD were immobilized on controlled pore glass (CPG) in enzyme reactors which were directly linked to a specially designed fibre optic flow-through cell covering the oxygen optrode. The system is linear for 0-30 mM glucose, with an r.s.d. of 5% at 30 mM (five measurements) and for 0-30 mM lactate, with an r.s.d. of 5% at 30 mM (five measurements). The enzyme reactors used were stable for more than 4 weeks in continuous operation, and it was possible to analyse up to 20 samples per hour. The system has been successfully applied to the on-line monitoring of glucose and lactate concentrations of an animal cell culture designed for the production of recombinant human antithrombine III (AT-III). Results of the on-line measurement obtained by the FIA system were compared with the off-line results obtained by a glucose and lactate analyser from Yellow Springs Instrument Company (YSI).     

Flow kinetics of nylon open-tubular and beaded immobilized glucose oxidase reactors

Equations describing the flow kinetics of immobilized glucose oxidase in open nylon tubes and in tubes filled with solid glass spheres were experimentally determined. Reactors of three different tube (dt) and bead (db) diameters were tested using various linear flow rates (vf) and glucose concentrations ([S]). The kinetics of the open-tubular reactors were described by [P] = 1.5.10(-3) [S] L0.86 vf-0.78 dt-0.9 and the kinetics of the beaded-tubular reactors by [P] = 5.10(-3) [S] L0.98 vf-0.75 dt-1.0 (db/dt)2.7, where [P] equals the concentration of H2O2 formed. The aspect ratio, db/dt, is the critical design factor for beaded reactors.     

Preparation of some immobilized linked enzyme systems and their use in the automated determination of disaccharides

1. Glucose oxidase (EC 1.1.3.4), amyloglucosidase (EC 3.2.1.3), invertase (EC 3.2.1.26) and beta-galactosidase (EC 3.2.1.23) were covalently attached via glutaraldehyde to the inside surface of nylon tube. 2. The linked enzyme system, comprising invertase immobilized within a nylon tube acting in series with glucose oxidase immobilized in a similar way, was used for the automated determination of sucrose. 3. The linked enzyme system, comprising beta-galactosidase immobilized within a nylon tube acting in series with glucose oxidase immobilized in a similar way, was used for the automated determination of lactose. 4. The linked enzyme system, comprising amyloglucosidase immobilized within a nylon tube acting in series with glucose oxidase immobilized in a similar way, was used for the automated determination of maltose. 5. Mixtures of glucose oxidase and amyloglucosidase were immobilized within the same piece of nylon tube and used for the automated determination of maltose. 6. Mixtures of glucose oxidase and invertase were immobilized within the same piece of nylon tube and used for the automated determination of sucrose.     

Polyaniline nanofiber coated monolith reactor for enzymatic bioconversion

The monolith reactor has many advantages over other reactors including good mass transfer, low-pressure drop and high enzyme stability. In this study, an enzyme-coated monolith with high enzyme stability and activity was developed. The monolith surface was coated with polyaniline nanofibers containing glucose oxidase immobilized on its surface. The developed enzyme-coated monolith was used for the bioconversion of glucose to glucolactone at different residence times. The conversion ratio increased up to 83% at increasing residence time. In addition, the polyaniline-coated monolith showed excellent enzyme activity and long-term stability. The stability of the enzyme-coated monolith containing the polyaniline nanofibers showed almost 100% residual activities after 8 days operation, while rapid enzyme deactivation and detachment was observed when the enzymes were immobilized without the polyaniline nanofibers.     

Effects of immobilization on the kinetics of enzyme-catalyzed reactions. I. Glucose oxidase in a recirculation reactor system.

Glucose oxidase from Aspergillus niger was immobilized on nonporous glass beads by covalent bonding and its kinetics were studied in a packed-column recycle reactor. The optimum pH of the immobilized enzyme was the same as that of soluble enzyme; however, immobilized glucose oxidase showed a sharper pH-activity profile than that of the soluble enzyme. The kinetic behavior of immobilized glucose oxidase at optimum pH and 25 degrees C was similar to that of the soluble enzyme, but the immobilized material showed increased temperature sensitivity. Immobilized glucose oxidase showed no loss in activity on storage at 4 degrees C for nearly ten weeks. On continuous use for 60 hr, the immobilized enzyme showed about a 40% loss in activity but no change in the kinetic constant.     

Hydrolysis of particulate tributyrin in a fluidized lipase reactor.

Pancreatic lipase has been immobilized onto stainless steel beads by adsorption followed by crosslinking, and onto polyacrylamide by covalent bonding. The activities of the two types of immobilized enzyme toward the particulate substrate, tributyrin emulsion droplets, were determined experimentally, and rate constants based on Michaelis-Menten kinetics were calculated. The activity of the stainless steel-lipase was determined for various flow conditions and for various support sizes by the use of a differential fluidized bed recycle reactor. The rate constants calculated indicate that the experimental reaction rate is free from mass transfer influences, since the observed Michaelis constant does not vary with the fluidization velocity or with the support particle size. In addition, the Michaelis constant of the stainless steel-lipase was found to be equal to that of the free enzyme, suggesting that adsorption and subsequent crosslinking does not alter the enzyme-substrate affinity. The emulsion substrate mass transfer rates, calculated from the filtration theory, indicate that each substrate particle which contact the immobilized enzyme is hydrolyzed to a significant extent. The experimentally determined kinetic rate constants may be used directly to predict the size of integral fluidized bed reactors.     

Immobilization of glucoamylase and glucose oxidase in activated carbon: effects of particle size and immobilization conditions on enzyme activity and effectiveness

Glucoamylase and glucose oxidase have been immobilized on carbodiimide-treated activated carbon particles of various sizes. Loading data indicate nonuniform distribution of immobilized enzyme within the porous support particles. Catalysts with different enzyme loading and overall activities have been prepared by varying enzyme concentration in the immobilizing solution. Analysis of these results by a new method based entirely upon experimentally observable catalyst properties indicates that intrinsic catalytic activity is reduced by immobilization of both enzymes. Immobilized glucoamylase intrinsic activity decreases with increasing enzyme loading, and similar behavior is suggested by immobilized glucose oxidase data analysis. The overall activity data interpretation method should prove useful in other immobilized enzyme characterization research, especially in situations where the intraparticle distribution of immobilized enzyme is nonuniform and unknown.     

Limited modifications of soya proteins by immobilized subtilisin: comparison of products from different reactor types

A comparison of different immobilized enzyme reactors has been made for the limited modification of soya storage proteins and the products compared with those from action of the soluble enzyme. Clarified total water extracts of soya protein were subjected to the action of subtilisin in a soluble and immobilized form. The sodium dodecyl sulfate (SDS) electrophoresis patterns of soya proteins modified by enzyme in the two forms differed for unbuffered soya protein at the same pH of 8.0. However, identical patterns could be obtained by a downward adjustment of the pH of soya protein treated with immobilized enzyme. The same SDS electrophoresis pattern could be obtained for a packed column of immobilized enzyme and a well-mixed vessel by buffering. Operation of the column reactor at higher superficial linear velocities (above 1.47 cm/min), higher protein concentrations (8.8% w/v), and prolonged periods (24 h) led to a bed compression attributed to the protein coating of the support.     

Characterization of CIM monoliths as enzyme reactors

The immobilization of the enzymes citrate lyase, malate dehydrogenase, isocitrate dehydrogenase and lactate dehydrogenase to CIM monolithic supports was performed. The long-term stability, reproducibility, and linear response range of the immobilized enzyme reactors were investigated along with the determination of the kinetic behavior of the enzymes immobilized on the CIM monoliths. The Michaelis-Menten constant K(m) and the turnover number k(3) of the immobilized enzymes were found to be flow-unaffected. Furthermore, the K(m) values of the soluble and immobilized enzyme were found to be comparable. Both facts indicate the absence of a diffusional limitation in immobilized CIM enzyme reactors.     

Simultaneous determination of glucose, ethanol and lactate in alcoholic beverages and serum by amperometric flow injection analysis with immobilized enzyme reactors.

Glucose, ethanol and lactate were determined simultaneously in a flow injection system by using a parallel configuration of immobilized enzyme reactors. Hydrogen peroxide produced was monitored amperometrically at the potential of +0.65 V vs. Ag/AgCl. Linear relations between sensor responses and each species were observed in the ranges of 0.02-10 mM (glucose), 5 x 10(-4)-0.1% (v/v) (ethanol) and 0.005-1 mM (lactate) with correlation coefficients larger than 0.999 for each species. The relative standard deviations for 10 successive injections were 1.4, 0.5 and 1.1% for glucose (1 mM), ethanol (5 x 10(-3)% (v/v] and lactate (0.05 mM), respectively. Analysis of serum samples was performed with urate-eliminating reactors which were set just before each immobilized enzyme reactor. Interference of ascorbate in a serum sample was completely eliminated by using an ascorbate-eliminating reactor which was set before the sample injection valve. Application of the system to alcoholic beverages and control serum was described and the results were compared with those of free enzymatic, spectrophotometric analysis (F-kit or C-test method).     

Use of an enzyme thermistor in continuous measurements and enzyme reactor control.

The enzyme thermistor measures the heat produced by the action of an immobilized enzyme on a substrate present in the sample. Its application in analysis of discrete samples, e.g., in clinical chemistry, is well documented, but it has not been used so far for continuous measurements. We decribe here the application of the enzyme thermistor for continuous monitoring and control of enzyme reactors. An enzyme thermistor filled with coimmobilized glucose oxidase and catalase was used to measure the amount of glucose in the outflow from a column reactor containing immobilized lactase acting on a lactose solution pumped through the reactor. The lactose conversion was kept on a constant level, irrespective of the actual enzymatic activity in the reactor, by regulating the flow through the reactor. The experiments were carried out with aqueous solutions of lactose as well as with whey from cow's milk.     

Ethanol production by immobilized cells of Zymomonas mobilis

Summary Columnar reactors containing immobilized cells of Zymomonas mobilis were utilized for the continuous production of ethanol from glucose. Two different immobilization strategies were investigated. In one case, cells were entrapped in borosilicate glass fiber pads, while in the other, cells were immobilized via flocculation. The reactors were operated in both the fixed-bed and expanded-bed manner. Ethanol productivities as high as 132 g/l·h were achieved. Data obtained from studies employing 5.0 and 10.0% glucose concentrations are presented. Problems encountered during the operation of the continuous, immobilized cell reactors are discussed.Operated by Union Carbide Corporation under contract W-7405-eng-26 with the U.S. Department of Energy.     

Glucose isomerase immobolized on porous glass

Partially purified glucose isomerase from a Streptomyces species was immobilized on porous glass particles and studied for various characteristics concerning its use as an industrial catalyst. The activities were investigated in relation to the reaction parameters and the enzyme deactivation was studied systematically under various reaction conditions. The half-life of the immobilized enzyme was found to exceed 200 days at 50°C. The rate equation of the reversible glucose ? fructose reaction was derived and the kinetic constants were determined. The rate equation was found to be in good agreement with experimental data for both forward and reverse reactions. The degree of diffusional effects was experimentally measured and theoretically analyzed.     

Continuous conversion of starch to glucose with immobilized glucoamylase

Partially purified glucoamylase from Aspergillus awamori NRRL 3112 was immobilized on diethylaminoethyl cellulose in the presence of low ionic-strength acetate buffers at pH 4.2. The active enzyme–cellulose complex was used to convert starch substrates continuously to glucose in stirred reactors. Substrate concentrations as high as 30% could be quantitatively converted to glucose at a rate of more than 25 mg/min/liter at 55°C for periods of 3 to 4 weeks in a 4-liter reactor. Shutdowns were due to mechanical problems and not to loss of enzymes, which could be recovered with no appreciable loss of specific activity. Transfer products, such as isomaltose and panose, were present in immobilized enzyme-produced syrups but to no greater degree than in soluble glucoamylase digests of starch.     

An experimental method to determine the substrate protection of enzyme against deactivation in a reversible reaction.

The substrate protection effect on an enzyme in a reversible reaction was studied by using glucose isomerase immobilized in small particles (radius less than 100 micron). Deactivation of the enzyme at various substrate concentrations in Tris buffer, pH 8.25, at 62.1 degrees C was studied in eight-column reactor sets. At set times the immobilized enzyme in one of the eight reactors was taken out and rinsed thoroughly, and then its residual activity was determined. The conclusions are, first, that the protection by the reactant is equal to the protection by the product, and, secondly, that the half-life of the enzyme increases slowly at high sugar concentrations. Thus the experimental method described here appears to be a useful one for the determination of substrate protection of enzyme deactivation in reversible reactions.     

Conversion of cellobiose to glucose using immobilized β-glucosidase reactors

Cellobiose is an intermediate in the enzymatic hydrolysis of cellulose to glucose and acts as an inhibitor for the cellulase enzymes. The conversion of cellobiose to glucose was studied with β-glucosidase adsorbed on Amberlite DP-1, a cation-exchange resin. The best overall pH for adsorption and reactor operation was near 5.0. The K_m values increased with increasing enzyme loading due to competitive inhibition. The maximum practical enzyme loading was about 28 units/g resin. The immobilized enzyme was operated continously in both packed bed and fluidized bed reactors, giving half-lives between 200 and 375 h.