Immobilized glucose oxidase–catalase and their deactivation in a differential-bed loop reactor

Glucose oxidase containing catalase was immobilized with a copolymer of phenylenediamine and glutaraldehyde on pumice and titania carrier to study the enzymatic oxidation of glucose in a differential-bed loop reactor. The reaction rate was found to be first order with respect to the concentration of limiting oxygen substrate, suggesting a strong external mass-transfer resistance for all the flow rates used. The partial pressure of oxygen was varied from 21.3 up to 202.6 kPa. The use of a differential-bed loop reactor for the determination of the active enzyme concentration in the catalyst with negligible internal pore diffusion resistance is shown. Catalyst deactivation was studied, especially with respect to the presence of catalase. It is believed that the hydrogen peroxide formed in the oxidation reaction deactivates catalase first; if an excess of catalase is present, the deactivation of glucose oxidase remains small. The mathematical model subsequently developed adequately describes the experimental results.     

Deactivation of immobilized beef liver catalase by hydrogen peroxide

Immobilized beef liver catalase has been used in a flow reactor to decompose hydrogen peroxide; at the same time the catalase is inactivated by its substrate. A model has been developed which predicts this rate of decomposition of peroxide and inactivation of catalase. First order dependence on peroxide concentration is assumed. The model was verified by experiment for a range of operating conditions and then used to predict the effects of a change in operating variables.     

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.     

Continuous steroid biotransformations in microchannel reactors

The use of microchannel reactor based technologies within the scope of bioprocesses as process intensification and production platforms is gaining momentum. Such trend can be ascribed a particular set of characteristics of microchannel reactors, namely the enhanced mass and heat transfer, combined with easier handling and smaller volumes required, as compared to traditional reactors. In the present work, a continuous production process of 4-cholesten-3-one by the enzymatic oxidation of cholesterol without the formation of any by-product was assessed. The production was carried out within Y-shaped microchannel reactors in an aqueous-organic two-phase system. Substrate was delivered from the organic phase to aqueous phase containing cholesterol oxidase and the product formed partitions back to the organic phase. The aqueous phase was then forced through a plug-flow reactor, containing immobilized catalase. This step aimed at the reduction of hydrogen peroxide formed as a by-product during cholesterol oxidation, to avoid cholesterol oxidase deactivation due to said by-product. This setup was compared with traditional reactors and modes of operation. The results showed that microchannel reactor geometry outperformed traditional stirred tank and plug-flow reactors reaching similar conversion yields at reduced residence time. Coupling the plug-flow reactor containing catalase enabled aqueous phase reuse with maintenance of 30% catalytic activity of cholesterol oxidase while eliminating hydrogen peroxide. A final production of 36 m of cholestenone was reached after 300 hours of operation.     

Energy characteristics of beam-plasma interaction in a closed volume

Energy exchange between an electron beam and plasma during a beam-plasma discharge in a closed cavity excited by the electron beam is analyzed using computer simulations by the KARAT code. A method allowing one to analyze the beam-plasma interaction in the quasi-steady stage of the discharge is proposed. Qualitative characteristics of energy exchange (such as beam energy losses and the energy distributions of beam electrons and plasma particles leaving the discharge) both during spontaneous discharge excitation and in the presence of initial beam modulation by regular or noiselike signals are determined. The results obtained enable one to estimate the energy characteristics of a plasma processing reactor based on a beam-plasma discharge.     

Inactivation of immobilized catalase by hydrogen peroxide in continuous reactors

It is shown theoretically that in continuous reactions the rate of catalase inactivation by hydrogen peroxide depends on the type of reactor and the order of the chemical reaction.     

An immobilised catalase peroxidase from the alkalothermophilic Bacillus SF for the treatment of textile-bleaching effluents

A catalase peroxidase (CP) from the newly isolated Bacillus SF was used to treat textile-bleaching effluents. The enzyme was stable at high pH values and temperatures, but was more sensitive to deactivation by hydrogen peroxide than monofunctional catalases. Based on the Michaelis-Menten kinetics of the CP, a model was developed to describe its deactivation characteristics. The enzyme was immobilised on various alumina-based carrier materials with different shapes and the specific activity increased with the porosity of the carrier. The shape of the carrier had an important influence on the release of oxygen formed during the catalase reaction from the packed-bed reactor and Novalox saddles were found to be the most suitable shape. Bleaching effluent was treated in a horizontal packed-bed reactor containing 10 kg of the immobilised CP at a textile-finishing company. The treated liquid (500 l) was reused within the company for dyeing fabrics with various dyes, resulting in acceptable colour differences of below Delta E*=1.0 for all dyes.     

Continuous conversion of sucrose to fructose and gluconic acid by immobilized yeast cell multienzyme complex

A multienzyme complex consisting of invertase, glucose oxidase, and catalase was reconstituted by binding glucose oxidase using concanavalin A (Con A) to the cell wall of Sacchararomyces cerevisiae, previously induced for maximal activities of invertase and catalase. The cell flocculate obtained was stabilized by entrapment in polyacrylamide using γ irradiation at 100 kR. This complex showed a shortening of the lag period and enhancement in gluconic acid production as compared to a similar mixture of soluble enzymes. The efficacy of the multienzyme complex has been compared with that of mixed multienzyme system composed of individually immobilized enzymes. The immobilized multienzyme complex in a continuous-flow stirred-tank reactor system could be operated for continuous conversion of sucrose to fructose and gluconic acid. The reactor system did not show any loss in efficiency in a continuous operation over 20 days.     

Increasing the efficiency of the enzymatic hydrolysis of potato starch in a membrane reactor

The aim of the work was to define the physicochemical parameters of a reaction system that alter the effectiveness of a continuous recycle membrane reactor during potato starch hydrolysis. The enzymatic hydrolysis of starch in an ultrafiltration reaction system proceeded with a continuous decrease in the permeate flux, accompanied by an increase in dry substance content in both the permeate and retentate fractions. The decrease in the permeate flux was caused by an increase in feed viscosity. If a prehydrolysis process was conducted, it was possible to enzymatically hydrolyse potato starch in solutions with concentrations up to 20%. A quasi-steady state of starch enzymatic hydrolysis was reached in the ultrafiltration reaction system by alternately supplementing it with starch solution and water.     

Characterization and properties of catalase immobilized onto controlled pore glass and its application in batch and plug-flow type reactors

Bovine liver catalase was covalently immobilized onto controlled pore glass (CPG) beads modified with 3-aminopropyltriethoxysilane (3-APTES) followed by treatment with glutaraldehyde. Coupling of catalase onto CPG was optimized to improve the efficiency of the overall immobilization procedure. The optimum coupling conditions: pore diameter of CPG, pH, buffer concentration, temperature, coupling time and initial catalase amount per grams of carrier were determined as 70 nm, 6.0, 75 mM, 5 °C, 7 h and 6 mg catalase, respectively. Catalytic efficiencies (k_cat/K_m) and thermal inactivation rate constants (k_i) of ICPG1 were determined and compared with that of free catalase. Suitability of ICPG1 was also investigated by using it in batch and plug-flow type reactors. When the remaining activity of ICPG1 retained was about 50% of its initial activity the highest total productivity of ICPG1 was determined as 7.6 × 10⁶ U g immobilized catalase⁻¹ in plug-flow type reactor. However, the highest total productivity of ICPG1 was 6.2 × 10⁵ U g immobilized catalase⁻¹ in batch type reactor. ICPG1 may have great potentials as biocatalyst for the application in decomposition of hydrogen peroxide in plug-flow type reactor.     

The use of a single-fiber reactor for the enzymatic removal of amino acids from solutions

In this article we describe the use of bench-scale single-fiber dialyzers for the development and testing of an immobilized enzyme reactor for the treatment of leukemia. The treatment is based on the enzymatic removal of specific amino acids from the blood of leukemia patients. L-Lysine alpha-oxidase and catalase were coimmobilized within the void space of the porous region of asymmetric hollow-fiber membranes for the removal of L-lysine from simulated human plasma solutions. Hollow-fiber reactor performance was evaluated using a small single-fiber dialyzer (SFD) consisting of a single fiber encased in a protective glass shell. This small reactor affords ease of use, requires small amounts of chemicals and biochemicals, and gives useful reactor performance data. Single-fiber dialyzers were constructed using polyamide fibers with a molecular weight cutoff of 10,000 (PA10 fibers); these fibers demonstrated the best compatibility with and retention of the enzymes. The SFD performance in removing L-lysine from solution was evaluated under both steady and pulsatile flow operation. Pulsatile flow was tested for two reasons: (1) to enhance the radial mass transfer of lysine within the SFD and (2) to simulate the pulsatile flow of blood in dialysis treatment. The use of pulsatile flow increased lysine conversion by 15% over the steady-flow case. Approximately 40% of the lysine was removed from simulated plasma by the SFD in a 4-h experiment using pulsatile flow in the recycle mode.     

Microflora of nuclear research reactor pool water

In the course of research done it was concluded that circulation of pool water through the nuclear reactor core produces a bactericidal effect on microflora due to influence of radiation of various types. Contents of microbes returns to the initial level after 2-4 months after circulation was stopped. Microflora of pool water comprises big amount of coccus, G-positive rods and fungi and a lower content of G-negative rods if compared to water which had been used to fill reactor pool. There is an increased number of radioresistant forms with intensified production of catalase and nuclease. Supposedly, presence of these enzymes gives to the microbes certain advances to survive in high-radiation zones.     

Evaluation of D-amino acid oxidase from Rhodotorula gracilis for the production of alpha-keto acids: a reactor system

The study reports on the development of a bioreactor for the production of alpha-keto acids from D,L- or D-amino acids using Rhodotorula gracilis D-amino acid oxidase. D-Amino acid oxidase was co-immobilized with catalase on Affi-Gel 10 matrix, and the reactor was operated as a continuous-stirred tank reactor (CSTR) or stirred tank with medium recycling conditions. The optimum substrate concentration and quantity of biocatalyst were determined (5 mM and 1.2 mg/L, respectively). Under optimum operating conditions, product formation was linearly related to both substrate and enzyme concentration, showing the system to be highly flexible. Under these conditions, in a stirred tank, over 90% conversion was achieved in 30 min with a maximum production of 0.23 g of pyruvic acid/day/enzyme units. Product was recovered by ion exchange chromatography. The operational stability of the reactor was high (up to 9.5 h of operation without loss of activity) and the inactivation half-life was not reached even after 18 h or 36 bioconversion cycles. This represents the first case of a reactor developed successfully with a D-amino acid oxidase. (c) 1994 John Wiley & Sons, Inc.     

Recycling of textile bleaching effluents for dyeing using immobilized catalase

Catalase was immobilized on alumina carrier and crosslinked with glutaraldehyde. Storing stability, temperature and pH profiles of enzyme activity were studied in a column reactor with recirculation and in a batch stirred-tank reactor. The immobilized enzyme retained 44% of its activity at pH 11, 30 °C and 90% at 80 °C, pH 7. The half-life time of the immobilized catalase was increased to 2 h at pH 12, and 60 °C. Acceptable results were achieved when the residual water from the washing process of H₂O₂-bleached fabrics was treated with the immobilized enzyme and then reused for dyeing.     

An on-line method for the collection and analysis of quasi-steady-state kinetic data for enzyme-catalyzed reactions.

A special mixing device for initiating enzyme-catalyzed reactions is used to rapidly achieve an unperturbed quasi-steady state. An on-line computer is employed to sample the initial conditions, the mixing time, and concentrations that change as a function of time during this quasi-steady state phase. A statistical method for estimating initial, quasi-steady state rates from the time course of the enzyme-catalyzed reaction is described. Practical considerations for using this parameter estimation system lead to the conclusion that for the enzyme-catalyzed reaction tested, the extent overall reaction should be above .2% for high initial substrate concentrations, and above 1% for initial substrate concentrations in the range of the Michaelis constant. Application of this method to a typical enzyme-catalyzed reaction suggests that objective estimates of initial rates from a given set of concentrations and corresponding times can be obtained with a standard error in the range of 2–3%, but that reproducibility is not better than about 10%. When this procedure was used to estimate initial rates for the glycerol dehydrogenase-catalyzed oxidation of glycerol by NAD, it was found that this enzyme did not behave according to the classical “Michaelis-Menten” mechanism of enzyme action.     

Highly sensitive enzyme thermistor determination of ADP and ATP by multiple recycling enzyme systems

An enzyme thermistor method for the determination of ADP and/or ATP with signal amplification by recycling procedures is described. Pyruvate kinase (PK) and hexokinase (HK) coimmobilised on aminopropyl-controlled pore glass were applied in a column reactor. Addition of an excess of phosphoenolpyruvate (PEP) and glucose leads to cofactor recycling and production of glucose-6-phosphate and pyruvate. In presence of PEP an amplification of the sensitivity up to 30 times was reached as compared with the HK-catalysed reaction alone. An additional signal amplification was accomplished by recycling the pyruvate leaving the first enzyme reactor in a second reactor containing L-lactate dehydrogenase, lactate oxidase and catalase. In the presence of NADH an overall amplification of the sensitivity for ATP or ADP up to 1700 times was found. The limits of detection were 6 × 10⁻⁵ M cofactor without recycling at all, 2 × 10⁻⁶M with recycling in the kinase bienzyme reactor and 1 × 10⁻⁸M with the dual recycling system.     

Experimental operation of immobilized multienzyme systems in optimal and suboptimal configurations

Two mixed immobilized enzyme systems, glucoamylase–glucose isomerase and glucose isomerase–glucose oxidase–catalase, were operated to verify theoretical predictions that optimal bifunctional catalyst configutations could exist superior to those where the catalysts were mixed uniformly or arranged sequentially in a tubular reactor. The experimental results for all three configurations conformed to the theoretical values sufficiently closely to support of optimal catalyst profiles.     

Prediction of dissolved oxygen and carbon dioxide concentration profiles in tubular photobioreactors for microalgal culture

A model is developed for prediction of axial concentration profiles of dissolved oxygen and carbon dioxide in tubular photobioreactors used for culturing microalgae. Experimental data are used to verify the model for continuous outdoor culture of Porphyridium cruentum grown in a 200-L reactor with 100-m long tubular solar receiver. The culture was carried out at a dilution rate of 0.05 h-1 applied only during a 10-h daylight period. The quasi-steady state biomass concentration achieved was 3.0 g. L-1, corresponding to a biomass productivity of 1.5 g. L-1. d-1. The model could predict the dissolved oxygen level in both gas disengagement zone of the reactor and at the end of the loop, the exhaust gas composition, the amount of carbon dioxide injected, and the pH of the culture at each hour. In predicting the various parameters, the model took into account the length of the solar receiver tube, the rate of photosynthesis, the velocity of flow, the degree of mixing, and gas-liquid mass transfer. Because the model simulated the system behavior as a function of tube length and operational variables (superficial gas velocity in the riser, composition of carbon dioxide in the gas injected in the solar receiver and its injection rate), it could potentially be applied to rational design and scale-up of photobioreactors. Copyright 1999 John Wiley & Sons, Inc.     

Enhanced catalase synthesis by a novel combined system of photocatalytic reactor and fermentor

A novel combined system of a photocatalytic reactor, with UV and titanium dioxide as photocatalyst, and a fermentor with Bacillus sp. F26 as catalase producer was developed. Production of catalase was enhanced by 14% to 18.5 U ml⁻¹ without affecting cell growth.Nomenclature q_s: specific glucose consumption rate; μ: specific growth rate; q_p: specific CAT production rate