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.     

Improved immobilized enzyme systems using spherical micro silica sol-gel enzyme beads

Spherical micro silica sol-gel immobilized enzyme beads were prepared in an emulsion system using cyclohexanone and Triton-X 114. The beads were used for thein situ immobilization of transaminase, trypsin, and lipase. Immobilization during the sol to gel phase transition was investigated to determine the effect of the emulsifying solvents, surfactants, and mixing process on the formation of spherical micro sol-gel enzyme beads and their catalytic activity. The different combinations of sol-gel precursors affected both activity and the stability of the enzymes, which suggests that each enzyme has a unique preference for the silica gel matrix dependent upon the characteristics of the precursors. The resulting enzyme-entrapped micronsized beads were characterized and utilized for several enzyme reaction cycles. These results indicated improved stability compared to the conventional crushed form silica sol-gel immobilized enzyme systems.     

Diffusion and the limiting substrate in two-substrate immobilized enzyme systems

The effects of mass transport resistances on two-substrate immobilized enzyme systems are investigated theoretically. It is shown that the effects of mass transport resistances on the overall reaction rate are related mainly to the transport of the limiting substrate. In the absence of external mass transport resistances, the limiting substrate can be identified by knowing only the ratio of the bulk substrate concentrations, the permeability of the support to the two substrates, and the stoichiometry of the reaction. However, a combination of internal and external mass transport resistances may result in the other substrate becoming limiting. These effects are most significant when the mass transport resistances are high. Applications in the design of enzyme electrodes and chemical reactors are discussed.     

Hysteresis,oscillations, and pattern formation in realistic immobilized enzyme systems

Summary Hysteresis, oscillations, and pattern formation in realistic biochemical systems governed by P.D.E.s are considered from both numerical and mathematical points of view. Analysis of multiple steady states in the case of hysteresis, and bifurcation theory in the cases of oscillations and pattern formation, account for the observed numerical results. The possibility to realize these systems experimentally is their main interest, thus bringing further arguments in favor of theories explaining basic biological phenomena by diffusion and reaction.     

A general solution for the steady-state kinetics of immobilized enzyme systems

A power series solution is presented which describes the steady-state concentration profiles for substrate and product molecules in immobilized enzyme systems. Diffusional effects and product inhibition are incorporated into this model. The kinetic consequences of diffusion limitation and product inhibition for immobilized enzymes are discussed and are compared to kinetic behavior characteristic of other types of effects, such as substrate inhibition and substrate activation.     

The influence of bond chemistry on immobilized enzyme systems for ex vivo use

The ideal derivatized support for the clinical use of an immobilized enzyme system should irreversibly bind active enzyme. We have investigated the behavior of heparinase and bilirubin oxidase immobilized via cyanogen bromide, tresyl chloride, epoxide, or carbodiimidazole activated natural and synthetic matrices. The protein bound to each activated support was 90% for cyanogen bromide (CNBr) activated agarose, 50-80% for tresyl chloride activated agarose, and 50% for oxirane activated acrylic (Eupergit C). The activity retention of immobilized heparinase was greatest (50%) with CNBr activated agarose while for the immobilization of bilirubin oxidase, the activity retention was greatest (25-30%) with tresyl chloride activated agarose and oxirane activated acrylic.The stability of the different covalent bonds was studied in vitro with radioiodinated enzymes. The leaching profiles showed the same trends for each support and chemistry. A plateau in portein leaching was reached after a few hours of incubatttion and the transient leaching period was well represented byu a logarithimic function of time. The amount of enzyme released from the least stable support (CNBr activated agarose) in 24 h was injected intravenously in New Zealand white rabbits. Using an indirect enzyme-linked immunnosorbant assay (ELISA), no immune responce was detected. The transient leaching profile was shortenend by washingthe enzyme-support conjugate with 1M hydroxylamine, pH8.5 intermolecular cross-linking with glutaraldehyde also improves the enzyme-support stability. Tresyl chloride and oxirane activated supports produce bonds with improved stability without adversely affecting enzymatic activity.     

Stabilization of enzyme immobilized in temperature-sensitive hydrogels

-Galactosidase was immobilized in a crosslinked poly(N-isopropylacrylamide-co-acrylamide) hydrogel which exhibits an LCST(lower critical solution temperature) behavior. The hydrogel collapses above the LCST, and expands below the LCST. The temperature-dependent phase transition was around 37 °C. The stability of immobilized enzyme was investigated at different temperatures which allow different degrees of collapse in the hydrogel matrix. It was hypothesized that the immobilzed enzyme is more stable in the collapsed matrix due to the physical restraint imposed on the enzyme entrapped.     

Production of immobilized penicillin acylase using aqueous polymer systems for enzyme purification and in situ immobilization

A novel approach for the isolation and purification of penicillin acylase (PA), which couples aqueous two-phase partitioning and enzyme immobilization has been investigated.A PA yield of 90% was achieved by treating E. coli cells with 4% butyl acetate, freeze-thawing step, and pressure homogenization. PA purification (93% recovery) was achieved by (1) removing cell debris via precipitation with polyethylene glycol (PEG 2000); (2) aqueous two-phase partitioning using a PEG 2000 + phosphate system (87% recovery).An in situ enzyme immobilization approach, using oxirane acrylic or aldehyde-agarose beads dispersed in the PEG-rich phase, was explored for the conversion of penicillin G to 6-aminopenicillanic acid. An appropriate immobilization reaction time was found. The catalytic performance of the enzyme, when immobilized, was found not to be affected by recycling of the phase-forming components.     

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.     

Flow dynamics of immobilized enzyme reactors

Summary Enzymic conversion of glucose to fructose was carried out in a packed bed and in a fluidized bed reactor. The flow dynamics of these two flow systems, loaded with two different types of immobilized loaded with two different types of immobilized glucose isomerase particles, were studied. The theoretical RTD curve calculated from the axial dispersed plug flow model equation was matched to the experimental RTD curve by an optimization technique. The effect of fluid velocity on the extent of liquid dispersion was established. Theoretical predictions on the conversion of glucose to fructose were calculated using three mathematical models, namely, a plug flow model, a continuous stirred tank reactor (CSTR) model and an axial dispersed plug flow model. The experimental results showed that the axial dispersed plug flow model was superior in predicting the performance of both the packed bed and fluidized bed reactor.Abbreviations C Dimensionless concentration - D Dispersion coefficient [cm²/sec] - d _p Mean particle diameter [cm] - E Enzyme concentration [mol/gm] - F Fructose concentration [mol/cm³] - F _e Equilibrium fructose concentration [mol/cm³] - G Glucose concentration [mol/cm³] - G _e Equílibrium glucose concentration [mol/cm³] - G _o Initial glucose concentration [mol/cm³] - Reduced glucose concentration [mol/cm³] - K Equilibrium constant - K _mf Forward reaction rate constant [mol/cm³] - K _mr Reserve reaction rate constant [mol/cm³] - K _m Rate constant [mol/cm³] - L Total length of the reactor bed [cm] - l Length [cm] - Q Flow rate [cm³/s] - r Rate of reaction based on volume of substrate - u Superficial liquid velocity [cm/s] - v Interstitial liquid velocity [cm/s] - V Reactor bed volume [cm³] - V _mf Forward reaction rate constant [mol/s·g enzyme] - V _mr Reserve reaction rate constant [mol/s·g enzyme] - z Dimensionless distance along the reactor - Density [g/cm²]     

Biosensors and enzyme immobilized electrodes.

A biosensor is a device which consists of a biological sensing element connected to a transducer. The transducer can be electronic, optical, electrical, etc. This emerging technology offers us a powerful tool which is radically altering our approach to analytical methods. It was realised that enzymes are natural sensors on account of their highly selective nature. Much of the impetus to the work has come from medical requirements. Instant analysis of clinical samples has an obvious appeal to physicians and patients alike. Of particular interest is the possibility of continuous 'in-vivo' monitoring of metabolites, drugs and proteins using miniature, portable systems. In recent years, there has been a growing demand for biosensors in the fields of veterinary science, animal husbandry, the food industry and environmental monitoring. However, the possibility of successful application rests upon future developments. Increasing attention will have to be paid to the engineering of both the basic components and the device on the whole. New biochemical reactions will either have to be discovered or engineered through genetic manipulation or chemical techniques. Optimization of response time, selectivity, stability and low costs should receive priority considerations.     

On-line synthesis utilizing immobilized enzyme reactors based upon immobilized dopamine beta-hydroxylase

Immobilized enzyme reactors (IMERs) based upon dopamine beta-hydroxylase (DBH) have been developed. Immobilized artificial membrane (IAM) and glutaraldehyde-P (Glut-P) stationary phases have been used to immobilize DBH. When DBH is immobilized on the Glut-P interphase the enzyme is outside the stationary phase whereas with the IAM interphase the enzyme is embedded within the interphase surroundings. The activity of each IMER and their ability for on-line hydroxylation has been investigated. The resulting IMERs are enzymatically active and reproducible. The IMERs can be utilized through the use of coupled chromatography to characterize the cytosolic (DBH-Glut-P-IMER) and membrane-bound (DBH-IAM-IMER) forms of the enzyme. The substrate is injected onto the individual IMERs and the reactants and products are eluted onto a phenylboronic acid column for on-line extraction. The substrates and products are then transported via a switching valve to coupled analytical columns. The results demonstrate that enzyme-substrate and enzyme-inhibitor interactions can be investigated with the on-line system. These IMERs can be utilized for the discovery and characterization of new drug candidates specific for the soluble form and membrane-bound form of DBH. The effects of flow-rate, contact time, pH and temperature have also been investigated.     

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.     

Cofactor regeneration in immobilized enzyme systems: chemical grafting of functional NAD in the active site of dehydrogenases

One of the limiting steps in the further development of enzyme technology is the regeneration of cofactors, especially the pyridinic nucleotide cofactors. Immobilization of alcohol dehydrogenase and steroid dehydrogenase is described. In the last case stabilized enzymes could work in non aqueous solvents. Co-enzyme molecules are bound in the immediate vicinity of the active site of the enzyme. Cofactor regeneration was performed with an electron carrier (Phenazine methosulfate). Ageing phenomena were observed. The co-immobilization of superoxide dismutase gives rise to an increase of stability.     

Effectiveness factor calculations for immobilized enzyme catalysts

The steady state, nonlinear diffusion equations which describe reactions in constrained enzyme solutions are of great interest in many biological and engineering applications. As in other types of nonlinear differential equations, exact analytical solutions do not exist except in some simplified cases. In this paper, a general procedure is presented for solving numerically for the substrate concentration profile and effectiveness factor utilizing the transformation method suggested by Na and Na. Design correlations for enzyme solutions constrained within spherical membranes are included. The use of a unique definition of the Thiele Modulus in these charts permits the clear illustration of the effects of substrate concentration and external mass transfer resistances on the overall effectiveness factor for the catalyst particle.     

纳米材料固定化酶的研究进展

近年来,纳米技术为酶固定化提供了多种纳米级材料,纳米材料固定化酶不仅具有高的酶负载量,而且具有良好的酶稳定性。本文基于纳米材料固定化酶,对纳米材料的种类进行了总结,分析了纳米材料对固定化酶性能的影响,并介绍了纳米级固定化方法及纳米材料固定化酶在生物转化、生物传感器、生物燃料电池等领域的应用。     

Packed-bed immobilized enzyme reactors for complex processes

Utilization of enzymic reactors for biotechnological-biomedical applications is currently developing at a sustained pace.Our present study concentrates on development of procedures for describing the performance of devices where enzyme-catalyzed reactions between two substrates take place, and for the rational design and optimization of the reactors considered. Within this context, an analytical model was developed for immobilized enzyme packed-bed reactors; it takes into account internal diffusion limitations for the cosubstrates, and hydrodynamic backmixing effects. In order to overcome the complex mathematical problems involved, the compartmental analysis approach was employed.Using this model, performance was simulated for various configurations of the enzymic unit, i.e. from a continuously operated stirred tank reactor (CSTR) to an essentially plug flow type. In addition, an experimental method is described for quantitatively assessing the backmixing effects prevailing in the reactor.The procedures established also provide the ground for further developments, particularly for systems where, in parallel to the enzymic reaction, additional processes (e. g. complexation) take place.List of Symbols C _j,i mM Concentration of substrate j in the pores of stage - iD _j cm²/s Internal (pore) diffusion coefficient of substrate j; defined in Eq. (7) - D _e cm²/s Axial dispersion diffusion coefficient - D _j, cm²/s cm²/s Bulk diffusion coefficient for substrate j - E mM Enzyme concentration inside the catalytic pores - J _j,immol/s/cm² Net flux of substrate j taking place from the bulk of stage i into the corresponding pores; defined in Eq. (6) - K _m,1, K _m,2 mM Michaelis-Menten constants for cosubstrates 1 and 2, respectively - k s ^–1 Catalytic constant - k _s cm/s Catalytic constant - n Total number of elementary stages in the reactor - Q cm³/s Volumetric flow rate throught the reactor - r cm Radius of the pore - R _j,i mM/s Reaction rate of substrate j in stage i, in terms of volumetric units - S cm² Internal surface of a pore - S _j,0 mM Concentration of substrate j in the reactor feed - S _j,i–1, S _j,i mM Concentration of substrate j in the bulk phase leaving stages i — 1 and i, respectivley - V _i cm³ Total volume of stage i (bulk phase + pore phase + inert solid carrier) - V cm³ Total volume of the reactor - V _m ^* mmol/s/cm² Maximal reaction rate in terms of surface units; defined in Eq. (8) - V _m mM/s Maximal reaction rate in terms of volumetric units; defined in Eq. (8) - V _p cm³ Volume of one pore - y cm Axial coordinate of the pores - y ₀ cm Depth of the pores - Z cm Axial coordinate of the reactor - Z ₀ cm Length of the reactor - ₁ Dimensionless parameter; defined in Eq. (27) - ₂ Dimensionless parameter; defined in Eq. (27) - ₁ Dimensionless parameter; defined in Eq. (27) - ₂ Dimensionless parameter; defined in Eq. (27) - Ratio between the radius of the enzyme molecule and the radius of the pore (dimensionless) - V₁ Dimensionless parameter; defined in Eq. (21) - v₂ Dimensionless parameter; defined in Eq. (21) - Q Volumetric packing density of catalytic particles (dimensionless) - Ø Porosity of the catalytic particles (dimensionless) - Ø Dimensionless concentration of substrate j in pores of stage i; defined in Eq. (16) - _j,i-1,_j,i Dimensionless concentration of substrate j in the bulk phase of stage i; defined in Eq. (18) - Dimensionless position; defined in Eq. (16) - ² s² Variance; defined in Eq. (33) - Mean residence time in the reactor; defined in Eq. (33)