Theoretical analysis of a translocation-like model with saturable kinetics.

A theoretical analysis of the initial rates of product appearance in both compartments of a specifically designed diffusion cell separated by an asymmetrical enzyme membrane is presented. Variable substrate concentrations and different substrate diffusional limitations were considered. Our analysis shows that, under specific conditions, not only a product accumulation occurs in the compartment opposite to that in which the reaction takes place, but that substrate saturable kinetics can be obtained. These product translocation-like kinetics appear similar to those observed with translocation processes reported for biological situations. For such phenomena, a key role of the diffusion layer surrounding a bioactive surface is proposed.     

Vectorial product concentration obtained with a permeable immobilized enzyme membrane. A new approach to the analysis of biological transport systems.

The theoretical analysis of the distribution on both sides of a flat porous membrane of the product generated by an enzyme covalently bound only on to one side of the membrane separating two compartments of widely different volumes is presented. Contrary to what occurs with heterogeneous symmetric systems, the diffusional limitations at the enzyme level play a prominent role, not only on the apparent enzyme activity, but also on product flux-splitting. The mathematical model developed shows that it is possible to concentrate the reaction product in the compartment opposite to that where the reaction occurs. The influence of the parameters and of the physical characteristics of an asymmetrical system on product distribution is analysed. This theoretical analysis is in excellent agreement with experimental data obtained with glucose oxidase immobilized on a porous collagen membrane.     

Reaction and diffusion in a gel membrane reactor containing immobilized cells

To investigate the effect of diffusional limitations and heterogeneous cell distribution in a gel-immobilized cell system, a gel membrane reactor has been constructed. The reactor consists essentially of a gel layer with immobilized cells, flanked by two well-mixed chambers. Through one chamber substrate is pumped, and this chamber is the equivalent of the outside of a spherical gel bead. The second closed measuring chamber contains a small quantity of liquid that can equilibrate with the inside surface of the membrane, eventually after a long transient. Analysis of the liquid in this chamber can give direct information on substrate and product concentrations at the gel surface, and is and indication of the situation in the center of a gel bead. The gel membrane reactor appears to be an excellent tool to study diffusion and reaction in a gel-containing immobilized cells. A mathematical model with time- and position-dependent cell concentration and diffusion coefficient is described. Experimental data show the effective diffusion coefficient of glucose in an alginate gel to decrease with yeast cell concentration. Moreover, kinetic parameters could be determined, using the mathematical model. Microscopic analysis confirmed the proliferation of the gel-entrapped microorganisms in the outer layer of the matrix, as predicted by the model. Potentially, this type of reactor has a clear potential to study the physiology of gel-immobilized cells. (c) 1992 John Wiley & Sons, Inc.     

A novel reactor concept for the enzymatic reduction of poorly soluble ketones

Reductions of poorly soluble ketones often suffer from low total turnover numbers conferring to the coenzyme and large volumes which are needed for the conversion. The novel emulsion membrane reactor overcomes these limitations. From an emulsion consisting of an organic substrate and an aqueous buffer phase, the aqueous phase is separated selectively by using a hydrophilic ultrafiltration membrane and fed to a subsequent enzyme membrane reactor. The product outflow is recirculated to the emulsion stirred vessel and, due to the partition coefficients, the aqueous phase is recharged with substrate while the product is extracted. This new reactor concept will be compared to the classical enzyme membrane reactor. The latter was operated under the same conditions over a period of 4 months at a space-time yield of 21.2 g l⁻¹ day⁻¹. As a model system the enantioselective reduction of 2-octanone to (S)-2-octanol (ee > 99.5%) is used, carried out by a carbonyl reductase from Candida parapsilosis. NADH is regenerated by formate dehydrogenase from Candida boidinii. In comparison to the classical enzyme membrane reactor the total turnover number could be increased by a factor 9 using the novel emulsion membrane reactor.     

Internal thermodynamics of enzymes determined by equilibrium quench: values of Kint for enolase and creatine kinase

The equilibrium constant (Kint) for the enzyme-bound substrate and product of a one substrate/one product enzyme (enolase) and for those of a two substrate/two product enzyme (creatine kinase) have been determined. The values of Kint were determined by the rapid quenching of equilibrium mixtures of enzyme and radiolabeled substrate and product, under conditions where all of the marker substrate and product are bound. The scope and limitations of this method are discussed. Values of Kint have been collected from the literature, and it is shown that these data are consistent with the theory for kinetically optimized enzymes that is developed in the preceding paper.     

Influence of substrate and product diffusion on the heterogeneous kinetics of enzymic reversible reactions

When a reversible reaction is catalyzed by a surface bound enzyme, the diffusion of both substrate and product can considerably modify the kinetic properties of the reaction. According to this theoretical analysis, the enzyme activity is decreased due to the presence of substrate and product concentration gradients in the enzyme microenvironment, and the relative kinetic importance of the two diffusion steps mainly depend on the value of a dimensionless criterion inversely proportional to the equilibrium constant. Moreover diffusional effects increase with increasing bound enzyme activity, but decrease with increasing substrate and product concentration. Analytical expressions are established for the limiting values of substrate and product concentrations in the enzyme microenvironment, as well as for the increase in half-maximal-activity substrate concentration in the presence of substrate and product diffusional limitations.     

Enzyme product blot for nondestructive assay of protein catalytic function in polyacrylamide gels

A new method that permits rapid, sensitive, and specific enzymatic assay of proteins in polyacrylamide gels is described. The enzyme product blot described in this report involves percolation of the reaction mixture through a gel containing native enzyme which converts the labeled substrate to a labeled product with differing chemical properties. A permeable membrane with specific ligand-binding properties overlies the gel and binds the enzyme product, but not the substrate, as reaction mixture is blotted vertically. This membrane is washed free of substrate and the location of the product is identified by autoradiography. The autoradiogram is compared with the stained gel in order to recover the enzyme for amino acid sequence analysis. The enzyme product blot is demonstrated using glycerol kinase and hexokinase.     

Determination of the distribution of catalyst activity across a permeable membrane containing an immobilized enzyme. Indeterminacy of a functional approach to a structural problem.

Porous membranes were fabricated from collodion and impregnated with papain, inhomogeneously through the thickness of the membrane. These membranes were placed between reservoirs containing N-alpha-benzoyl arginineamide, a substrate for the enzyme papain. The progress of the reaction was monitored by sampling the reservoirs on each side for ammonia, a reaction product. From these data the diffusion coefficient, enzyme activity, and distribution of enzyme activity of the membrane were estimated. The limitations of this approach are discussed in the context of the analysis of biological transport systems.     

Enzyme reactions at the surface of living cells. I. Electric repulsion of charged ligands and recognition of signals from the external milieu

The dynamic behaviour of a polyelectrolyte-bound enzyme is studied when diffusion of substrate or diffusion of product is coupled to electric repulsion and to Michaelis-Menten enzyme reaction. The definition of the classical concepts of electric partition coefficients and Donnan potential of a polyelectrolyte membrane has been extended under global non-equilibrium conditions. This extension is permissible when a strong repulsion exists of substrate and product by the fixed negative charges of the membrane. Coupling between product diffusion, electric repulsion and enzyme reaction at constant advancement may result in a hysteresis loop of the partition coefficient as the product concentration is increased in the reservoir. This hysteresis loop vanishes as the rate of product diffusion increases. No hysteresis loop may occur when electric repulsion effects are coupled to substrate diffusion and reaction. The existence of multiple values of the partition coefficient for a fixed concentration of product implies that the membrane may store short-term memory of the former product concentration present in the external milieu. The occurrence of hysteresis generated by coupling enzyme reaction, product diffusion, electric partition effects at constant advancement of the reaction may be viewed as a sensing device of product concentration in the external milieu. Surprisingly, non-linearities required to generate this sensing device come from electrostatic effects and not from enzyme kinetics.     

Critical limitations in biological production of chemicals: process or genetic solutions?

Abstract: Production of bulk chemicals by biological processes is presently limited by failure of contemporary biological and bioreactor technology to deliver high product concentrations in high space-time yields in fluids of sufficiently low water content for subsequent down-stream processing operations. Limitations in the bioreactor portion of the process can arise due to failure to process sufficient substrate, substrate inhibition, inadequate rates or yields, and product inhibition. Various process approaches for addressing many of these limitations have been demonstrated or conceptualized. Less developed but potentially effective are genetic strategies addressing these process limitations. Ideally, the most effective combination of genetic and process approaches should be integrated in a synergistic fashion to maximize the economic potential of biological production of chemicals.     

A kinetic model for energy spilling-associated product formation in substrate-sufficient continuous culture

It has been demonstrated that excess substrate can cause uncoupling between anabolism and catabolism, which leads to energy spilling. However, the Luedeking-Piret equation for product formation does not account for the energy spilling-associated product formation due to substrate excess. Based on the growth yield and energy uncoupling models proposed earlier, a kinetic model describing energy spilling-associated product formation in relation to residual substrate concentration was developed for substrate-sufficient continuous culture and was further verified with literature data. The parameters in the proposed model are well defined and have their own physical meanings. From this model, the specific productivity of unit energy spilling-associated substrate consumption, and the maximum product yield coefficient, can be determined. Results show that the majority of energy spilling-associated substrate consumption was converted to carbon dioxide and less than 6% was fluxed into the metabolites, while it was found that the maximum product yield coefficients varied markedly under different nutrient limitations. The results from this research can be used to develop the optimized bioprocess for maximizing valuable product formation.     

Application of ultrafiltration for enzyme retention during continuous enzymatic reaction

In most enzymatic reactions, batch or continuous, separation of the enzyme for reuse is difficult if not impossible. A process will be presented in which an Ultrafiltration membrane serves to separate the reaction products from the enzyme and the substrate. In this manner the enzyme may be retained and re-used. Furthermore, under these conditions, the enzyme need only be present in catalytic amounts regardless of the amount of product produced. Under proper operating conditions and proper ultrafiltration membrane selection, a pure solution of α-amylase from Bacillus subtilis may be retained with no loss in enzyme activity over a test period of 30 hr after steadystate has been achieved. In the presence of substrate, the membrane support and ultrafiltration cell serve as the reaction vessel for the hydrolysis of starch. The substrate is continuously pumped into the cell under constant ultrafiltration pressure. The di-, oligo-, and polysaccharides formed from the enzyme reaction then either pass through the membrane as products or are retained. The molecular weight distribution of the products is dependent on the nominal molecular weight cut-off of the membrane, absolute ultrafiltration pressure, enzyme-to-substrate ratio, temperature, and residence time of the substrate in the reactor. In addition to the partial hydrolysis of starch by α-amylase, some preliminary findings on the complete hydrolysis of starch by glucoamylase will also be presented. In these latter studies, the substrate may be completely hydrolyzed to glucose units.     

Enzyme reactions at the surface of living cells. II. Destabilization in the membranes and conduction of signals

The dynamics of a bound-enzyme reaction is studied when the diffusion of both the substrate and the product is coupled to their electric repulsion and to enzyme reaction. Contrary to what is occurring when substrate diffusion is uncoupled with electric repulsion and enzyme reaction, no hysteresis loop of the partition coefficient exists. The electric partition coefficient monotonically declines as substrate or product concentration is increased in the reservoir. The random perturbation of a steady state may generate a localized destabilization of substrate and product concentration. This destabilization must propagate in the membrane and may be viewed as the conduction of a signal. These conduction phenomena are entirely due to electric effects. In the absence of these effects, the system is homeostatic, that is it returns back to its initial steady state after a perturbation. Obviously under these conditions conduction of signals cannot occur. Increasing the ionic strength of the external milieu tends to stabilize the system and to suppress conduction effects in the membrane.     

Preparative scale Baeyer-Villiger biooxidation at high concentration using recombinant Escherichia coli and in situ substrate feeding and product removal process

An efficient biocatalytic process based on the use of adsorbent resin (in situ substrate feeding and product removal) makes experiments at high substrate concentration possible by overcoming limitations due to substrate and product inhibition. This process was successfully applied to the preparative scale Baeyer-Villiger biooxidation of (-)-(1S,5R)-bicyclo[3.2.0]hept-2-en-6-one (25 g). Whole cells of recombinant E. coli (1 liter) overexpressing cyclohexanone monooxygenase were used as a biocatalyst and the substrate was preloaded onto the adsorbent resin. The corresponding lactone was obtained in 75-80% yield. Time for cell growth and biotransformation is about 24 h each and oxygen supply can be improved by using a tailor-made bubble column.     

Packed-Bed Bioreactors Under the Influence of Chemical Modulators

This report considers the behaviour of packed-bed immobilized enzyme reactors operating in the presence of substrate and/or product sequestrators. In some cases, enzyme inhibition by the reaction product and presence of chemical modulators are also considered. For each case, an appropriate analytical model is developed. Using numerical simulations, it is shown that reactor performance is impaired by substrate sequestration. This effect can be partially reversed when competitive sequestration by product or modulator is operational.

In addition, a comparison is made between some of the predicted characteristics of the reactor and experimental data. It reveals the capabilities and limitations of the models employed.     

Kinetic resolution of racemic glycidyl butyrate using a multiphase membrane enzyme reactor: experiments and model verification

A laboratory-scale multiphase hollow fiber membrane reactor was employed to investigate the lipase-catalyzed enzymatic resolution of racemic glycidyl butyrate. A mathematical formulation was feveloped to simulate the performance of this system. Model parameters were determined independently (except the effective rate constant, k(s)) and incorporated in the model simulations. In this study, two modes of operation are considered: subtractive resolution, in which the unreacted substrate is recovered in the organic stream; and product recovery, where the optically pure product of the enzymatic reaction is recovered in the aqueous stream. Good agreement was obtained between theoretical predictions and experimental results under a variety of conditions. The effect of mass transport limitations on the performance of this system was investigated. An increase in enzyme loading resulted in a higher Thiele modulus due to an elevated rate constant as well as a concomitant decrease in the effective diffusivity. Optical purity decreased in both subtractive resolution and product recovery at higher Thiele modulus with the effect being more pronounced in the product recovery mode. Finally, normalized plots were established to describe the effect of enzyme immobilization on both the effective enzymes activity and effective diffusivity. (c) 1993 Wiley & Sons, Inc.     

Kinetics studies in a continuous steady state hollow fiber membrane enzyme reactor

Porous hollow cellulose fibers have been used to separate a nonflowing enzyme solution of alkaline phosphatase from a continuous flow of substrate. The porosity of the hollow fiber membrane allows the substrate and product to diffuse freely through the membrane while restricting the permeation of the enzyme. The resulting “immobilized” enzyme system has been shown to behave as a continuous reactor—converting p-nitrophenylphosphate to p-nitrophenol. By varying the concentrations, flow rate, etc., either diffusion or enzyme kinetics can be studied. The continual influx of product and removal of substrate at steady state allows the study of kinetics of relatively short half-life enzymes and unstable systems.     

Packed-Bed Bioreactors Under the Influence of Chemical Modulators

This report considers the behaviour of packed-bed immobilized enzyme reactors operating in the presence of substrate and/or product sequestrators. In some cases, enzyme inhibition by the reaction product and presence of chemical modulators are also considered. For each case, an appropriate analytical model is developed. Using numerical simulations, it is shown that reactor performance is impaired by substrate sequestration. This effect can be partially reversed when competitive sequestration by product or modulator is operational.

In addition, a comparison is made between some of the predicted characteristics of the reactor and experimental data. It reveals the capabilities and limitations of the models employed.     

Development of coencapsulating technology for the production of chitosanoligosaccharides

To easily separate chitosanoligosaccharides by size exclusion, an coencapsulating technology of substrate and enzyme was developed. The membrane was composed of alginate and a divalent cation such as calcium. Chitosan and chitosanase were enveloped in this membrane and the product released to medium by size exclusion. The capsule was stabilized in a 2% acetic acid solution (pH 5.0) containing 0.145 M CaCO₃. The leakage of substrate caused by the agitation speed was controlled by increasing alginate and CaCO₃ concentrations. The lower limit of the alginate concentration and the agitation speed were 0.5% and 40 rpm, respectively. Membrane thickness and capsule diameter were 10 μm and 2.5 mm, respectively. By TLC analysis, the composition of chitosanoligosaccharides were mainly 3–6 mers. The molecular weight distribution of the released oligosaccharides ranged from 262 to 3624 Da by GPC.     

Ultrastructural localization of guanylate cyclase in bone cells.

Guanylyl imidodiphosphate (GMP-PNP) hydrolyzing enzyme activity as a means of detecting plasma membrane guanylate cyclase was demonstrated in osteoblasts of chicken tibial metaphysis using a lead citrate histochemical method at the electron microscopic level. Activity was not discerned in osteoclasts or osteocytes. The reaction product development was completely abolished when the sections were incubated with substrate-free or MnCl2-free medium. Guanylate-(beta, gamma-methylene) diphosphate (GMP-PCP) was a less effective substrate than GMP-PNP, and Mn++ was a stronger stimulator than Mg++. No reaction product was observed on the plasma membrane of osteoblasts when beta-glycerophosphate or p-nitrophenylphosphate was used as substrate instead of GMP-PNP. The results implicate guanylate cyclase as a significant effector of osteoblast regulation at the site of the plasma membrane.