Optimal control of an enzyme reaction subject to enzyme deactivation. I. Batch process

This paper is concerned with the study of an enzymatic system in a repeated batch process where the enzyme is subject to deactivation. The particular system studied was the enzymatic hydrolysis of Penicillin G to 6-aminopenicillanic acid. Utilizing standard optimization techniques, pH and temperature control policies were determined that would maximize the product yield.     

Oscillatory enzyme reactions and Michaelis–Menten kinetics

Oscillations occur in a number of enzymatic systems as a result of feedback regulation. How Michaelis–Menten kinetics influences oscillatory behavior in enzyme systems is investigated in models for oscillations in the activity of phosphofructokinase (PFK) in glycolysis and of cyclin-dependent kinases in the cell cycle. The model for the PFK reaction is based on a product-activated allosteric enzyme reaction coupled to enzymatic degradation of the reaction product. The Michaelian nature of the product decay term markedly influences the period, amplitude and waveform of the oscillations. Likewise, a model for oscillations of Cdc2 kinase in embryonic cell cycles based on Michaelis–Menten phosphorylation–dephosphorylation kinetics shows that the occurrence and amplitude of the oscillations strongly depend on the ultrasensitivity of the enzymatic cascade that controls the activity of the cyclin-dependent kinase.     

Mechanisms of metal-salt methods in enzyme cytochemistry with special reference to acid phosphatase

Summary This review is concerned with theoretical and experimental aspects of the factors governing the localizing potentialities of cytochemical enzyme reactions that are based on the metal-salt principle, that is, the precipitation of the primary product of the enzymatic reaction by a heavy-metal ion at the enzymatic site. Special attention is given to the lead phosphate precipitation process in acid phosphatase cytochemistry. The various model systems developed for the study of the factors involved in precipitation are described and their advantages and disadvantages discussed. Furthermore, the various cytochemical methods so far used for the demonstration of acid phosphatase activity are critically evaluated in the light of the results obtained with the model systems.     

Application of the enzyme thermistor to the direct estimation of intrinsic kinetics using the saccharose-immobilized invertase system.

The possibility of using the enzyme thermistor (ET) for the direct determination of kinetic parameters (Km, Ki, Vm) of immobilized enzyme (IME) was evaluated using different preparations of invertase conjugated to bead celluloses. Two different ET columns packed with IME were operated in the mode of a differential enzyme reactor (short length, low substrate conversion). Kinetic parameters of the above IME reactor were computed by a nonlinear curve-fitting procedure. The obtained kinetic parameters were superverified by means of an independent differential reactor (DR) system. This system utilized an indirect postcolumn analytical method based on determination of glucose concentration in the stirred reservoir. Best agreement between the data acquired by direct (ET) and indirect (DR) methods was obtained if the ET column was operated at flow rates within the range of 1.0-1.5 ml min-1 using invertase-cellulose chlorotriazine conjugate. Influence of heat loss and flow nonideality is discussed. The proposed ET method offers a rapid, convenient, and general approach to determination of kinetic constants of IME preparations by omitting postcolumn analytical methods.     

Determination of total and active immobilized enzyme distribution in porous solid supports

The total and active immobilized enzyme (IME) distributions in porous supports are studied both theoretically and experimentally. In order to determine experimentally the enzyme distribution profiles within a single particle, we construct a diffusion cell containing controlled-pore glass particles such that the cell would mimic a large pellet support. Our purpose is to study the interplay between the diffusion process within the interparticle void space and immobilization process in the controlled-pore glass particles onto the evolution of the (total and active) enzyme distributions. A mathematical model is developed to describe the interaction of various processes within the diffusion cell. The immobilized enzymes are determined for a system of trypsin and controlled-pore glass particles. The total amount of enzymes are determined by the amino acid analysis, and the active fraction is obtained by an active-site titration. The experimentally measured total IME profiles compare very well with that predicted by the model. The determined active enzyme profile is found to be nonuniform one, and it represents about 40% of the total enzyme immobilized in the support particles.     

Multiple continuous stirred tank reactors VS. Batch reactors — some design considerations

Summary A comparison of the performance of single CSTR and 2-CSTR systems against a stirred batch immobilized enzyme (IME) reactor has been made. When the downtimes in a batch reactor are not too small, the 2-CSTR system may become attractive and therefore a proper consideration should be given to the 2-CSTR system particularly when the application of a plug flow reactor is not practicable.NCL Communication No-2415     

Regulation and activation of phytoene synthase, a key enzyme in carotenoid biosynthesis, during photomorphogenesis

 During photomorphogenesis in higher plants, a coordinated increase occurs in the chlorophyll and carotenoid contents. The carotenoid level is under phytochrome control, as reflected by the light regulation of the mRNA level of phytoene synthase (PSY), the first enzyme in the carotenoid biosynthetic pathway. We investigated PSY protein levels, enzymatic activity and topological localization during photomorphogenesis. The results revealed that PSY protein levels and enzymatic activity increase during de-etiolation and that the enzyme is localized at thylakoid membranes in mature chloroplasts. However, under certain light conditions (e.g., far-red light) the increases in PSY mRNA and protein levels are not accompanied by an increase in enzymatic activity. Under those conditions, PSY is localized in the prolamellar body fraction in a mostly enzymatically inactive form. Subsequent illumination of dark-grown and/or in far-red light grown seedlings with white light causes the decay of these structures and a topological relocalization of PSY to developing thylakoids which results in its enzymatic activation. This light-dependent mechanism of enzymatic activation of PSY in carotenoid biosynthesis shares common features with the regulation of the NADPH:protochlorophyllide oxidoreductase, the first light-regulated enzyme in chlorophyll biosynthesis. The mechanism of regulation described here may contribute to ensuring a spatially and temporally coordinated increase in both carotenoid and chlorophyll contents. Received: 14 February 2000 / Accepted: 15 March 2000     

Model film studies in enzyme histochemistry with special reference to glucose-6-phosphate dehydrogenase

Summary This paper deals with the progress made over the last few years in our understanding of enzyme cytochemical staining methods as studied using a fundamental approach with the aid of a model system of thin gel films. Although model films with a matrix of polyacrylamide have been mostly used, the properties and possible applications of other matrices are also reviewed. The chemical aspects of the entrapment of enzyme molecules into a matrix are summarized. Special attention has been paid in model film studies to the principles of the trapping reaction of a diffusable precursor resulting from the enzymatic conversion of a substrate. They are considered here as they concern the cytochemical demonstration of acid phosphatase activity with a lead salt. The effect of fixatives on different enzyme activities, the diffusion rate of substrates and chromogenic compounds to the enzyme site, and enzyme kinetics under cytochemical conditions are also discussed, since they are factors which influence the final results of the staining procedures. The advantage of model film studies in enabling the direct correlation of cytochemical and biochemical results is outlined with special reference to the cytochemical determination of glucose-6-phosphate dehydrogenase with Tetra Nitro BT. A method for determining enzyme activities in the soluble fraction of isolated cells after incorporation in model films is described for the first time. This method has proved to be highly appropriate for microscopical observations of glucose-6-phosphate dehydrogenase activity in single cells, because it results in a good morphology and no formazan precipitaties outside the cells. On the other hand, this type of model film forms a bridge between fundamental model film studies using purified enzyme and quantitative enzyme cytochemistry performedin situ.     

The kinetics of penicillin-V deacylation on an immobilized enzyme

An immobilized Penicillin-V-acylase (commercial name, Novozym 217) with high specificity for the phenoxyacetyl-(V)- side chain was investigated in a recycle reactor and in a batch reactor to find the enzymatic reaction rate as a function of conversion, x, substrate concentration, c(A) (0) and pH. The reaction rate depends strongly on pH, and both products, phenoxy-acetic acid and 6-APA, inhibit the reaction. Nonspecific side reactions amount to only a few per cent when c(A) (0) <150mM and pH& gt; 6.5. The effectiveness factor for commercial-size particles is found to be about 0.65, and a value of 1.3mM is obtained for the equilibrium constant, K(eq), of the deacylation reaction. A kinetic model for the deacylation process which includes the effect of pH and of the reverse (acylation) reaction is proposed. Rate data for particles of different size are fitted to the nonlinear model. Five kinetic parameters and an effective diffusivity for the immobilized enzyme particles are determined.     

Effect of temperature and enzyme origin on the enzymatic synthesis of oligosaccharides

The aim of this research is to quantify the effect of temperature and enzyme origin on the enzymatic synthesis of oligosaccharides. Quantification of these effects is important because temperature and enzyme origin are important process parameters. A kinetic model was used to describe the concentrations in time. The kinetic parameters were determined by using data obtained in batch experiments at various temperatures (20, 30, 40, and 50 degrees C) and by using beta-galactosidases from Bacillus circulans, Aspergillus oryzae, Kluyveromyces lactis, and Kluyveromyces fragilis. The effect of temperature on the kinetic parameters could be described with the Arrhenius equation, except for the inhibition parameter. Slightly higher oligosaccharide yields were found at higher temperatures. However, the influence of the initial lactose concentration was much larger. The higher yield at higher temperatures is an additional advantage when operating at high initial lactose concentrations and consequently elevated temperatures. Clear differences between the beta-galactosidases were found concerning amount, size, and type of oligosaccharides produced. The beta-galactosidase from B. circulans produced the most abundant amount, the most different, and largest-sized oligosaccharides. The beta-galactosidases from Kluyveromyces spp. produced mainly trisaccharides. The kinetic parameters for the different enzymes were determined and differences were discussed.     

An approach in mathematical modeling of an upflow packed-bed reactor for enzymatic hydrolysis of wheat straw

The behavior of a packed-bed reactor for enzymatic hydrolysis of pretreated wheat straw has been described by means of a mathematical model. The flow pattern has been evaluated by residence time distribution experiments. Small deviations from ideal plug flow behavior were found using the dispersion model. The kinetic model proposed for enzymatic hydrolysis of cellulosic fraction of pretreated wheat straw has been derived from batch experimental data. Variations of enzyme concentration throughout the straw bed have been approximately described using a ramp variation of adsorbed enzyme. The final explains qualitatively the experimental results.     

Determination of intrinsic parameters for immobilization reactions of catalase and amyloglucosidase in porous glass supports

Experiments have been carried out for immobilizing enzyme-catalase and amyloglucosidase in controlled-pore glass particles of two different pore sizes. The experimental results have been analyzed, initial-stage analysis for the rate parameters of immobilization reactions and long-time analysis for determining the evolution of the immobilization process. These investigations suggest that the overall process of immobilization is controlled by the restricted diffusion of enzymes into the pores of the support. As a result, immobilized enzyme (IME) can penetrate only up to a certain distance into the support. The penetration depth of IME for the support-enzyme system mentioned have been evaluated from the experimental bulk enzyme concentration data in a batch recirculation reactor.     

Replacement of immobilised cell bioreactors by smaller immobilised enzyme bioreactors: unique-outcome predictability for cytochromes P450 isoforms?

Both immobilized enzymes (IME) and immobilized cells (IMC) are acceptable as the biocatalysts essential for the attainment of rapid rates of bioconversion in bioreactors. IMC can display higher than expected cellular permeability whilst IME can exhibit high catalytic constant (k_cat/K_m) despite limitations on substrate utilisation due to an unstired diffusion layer of solvent. Scale-down switching from IMC to IME involves the replacement of high-volume biotechnology by low-volume biotechnology, sometimes using IME mimics in partially non-aqueous solvent systems. Highly purified IME systems covalently immobilised to particles of, for instance, microcrystalline cellulose or porous glass, can retain both the hydrophilic and hydrophobic intermediate products in situ of the chosen sequence of enzyme reactions. These bioconversions, therefore, are as efficient as those with IMC where enzymes are often particle- or membrane-bound so that even hydrophilic intermediates are not released rapidly into solution. This mimicry of in vivo biosynthetic pathways that are compartmentalised in vivo (e.g. of lysosomes, mitochondria and endoplasmic reticulum) can replace larger IMC by IME especially in application of up to 2700 cytochromes P450 isoforms in bioprocessing. In silico investigation of appropriate model IME systems, in comparison with IMC systems, will be needed to define the optimal bioreactor configuration and parameters of operation, such as pH, T and oxygen mass transfer rate (OTR). The application solely of hazop (applied hazard and operability concepts) may, nevertheless, not be recommended to replace fully the in silico and real-lab pilot-scale and scale studies. Here, food-safe bioprocessing has to be achieved without incorporation of recognised biohazards; especially in the form of unacceptable levels of toxic metals that promote a risk-analysis uncertainty.     

Comparison of a batch, fed-batch and continuously operated stirred-tank reactor for the enzymatic synthesis of (R)-mandelonitrile by using a process model

The suitability of a batch, fed-batch and continuously operated stirred-tank reactor for the enzymatic production of (R)-mandelonitrile in an aqueous-organic biphasic system was investigated by using a process model. The considered biphasic system is 10-50% (v/v) 100 mM sodium citrate buffer of pH 5.5 dispersed in methyl tert-butyl ether. The constraints were that 750 moles of benzaldehyde per cubic meter should react towards (R)-mandelonitrile with an enantiomeric excess of 99% and a conversion of 98%. A continuously operated stirred-tank reactor could not meet the constraints, but the production in a batch or fed-batch reactor was feasible. The choice for a batch or fed-batch reactor is dependent on the influence of the costs for reactor operation and for the enzyme on the product costs. The choice for operating at a small or large aqueous-phase volume fraction is dependent on the costs and reusability of the enzyme. The volumetric productivity is maximal when operating as batch. The enzymatic productivity and turnover are maximal when operating as fed batch. In the fed-batch mode, the enzymatic productivity increased by 24-37%, the turnover increased by 50-60% and the volumetric productivity decreased by 33-71% as compared to a batch reactor. By enhancement of mass transfer both the volumetric and enzymatic productivity can be increased considerably, while the turnover is only slightly decreased.     

Optimization of batch processes involving simultaneous enzymatic and microbial reactions

Two general models for batch simultaneous enzymatic and microbial reaction (SEMR) processes are presented, the second derived from and simpler than the first and accounting for enzyme denaturation. Using the second model and parameter values from the literature, simulation was used to examine a range of enzyme addition rate strategies (in which the rate was a linear function of time) for a relatively fast ethanol fermentation and for a longer duration citric acid fermentation, both using cellulose as the substrate. For the ethanol process it is optimal (for a specific objective function which accounts for product value and enzyme cost) to add all the enzyme at the beginning of the process. But for the citric acid process a linearly decreasing enzyme addition rate, coupled with the addition of a small fraction of the enzyme at time zero, is better than pure batch operation or operation with the best constant enzyme feed rate.     

Kinetic behavior of slowly equilibrating association-dissociation enzyme systems]

The shape of the plots of product accumulation versus time (t) has been analysed for slowly equilibrating association-dissociation enzyme systems of the types 2p in equilibrium P (P is enzyme oligomer which is able to dissociate reversibly forming two identical halves p) and M in equilibrium M2 in equilibrium M2 in equilibrium... (M is monomer which has two association sites overlapping with active sites). It is assumed that the rate of equilibration between oligomeric forms is comparable with the rate of over-all enzymatic reaction and that substrate-oligomer complexes are in rapid equilibrium with free components. It has been shown that characteristic feature of kinetic behavior of slowly equilibrating association-dissociation enzyme systems is that the value of tau depends on enzyme concentration (tau is the intercept on t-axis for linear asymptota of the curve of product concentration versus time at t leads to infinity).     

Effects of mass-transfer resistance on apparent stability and performance of fixed-bed immobilized enzyme reactors: theory and experiments with immobilized invertase

Taking the hydrolysis of sucrose by invertase immobilized on anion-exchange resin as an example, the effects of mass-transfer resistance on the apparent stability of immobilized enzyme (IME) and the optimal policy for an IME reaction in a fixed-bed reactor have been studied theoretically and experimentally. The following results were obtained: (1) The effect of mass-transfer resistance on the effective deactivation rate of IME is summarized in two parameters concerning the intraparticle diffusion alpha(p) and the interparticle alpha(f). (2) At a constant processed amount of raw materials, there exists an optimal flow rate of reaction fluid to enhance the reactor performance while the mass-transfer resistance shifts the optimal point. (3) The intrinsic deactivation rate of IME has been estimated from the relationship between the fractional conversion at the reactor outlet and the operation time.     

Adaptation of an enzymatic cycling assay for NADP(H) measurement to the COBAS-FARA centrifugal analyzer

NADP(H) measurements by enzymatic amplification are described in which the interface step between cycling (glucose-6-phosphate and glutamic dehydrogenases) and indicator (6-phosphogluconic dehydrogenase) enzymes has been reconfigured, permitting the entire operation to run as a continuous assay on a centrifugal fast analyzer. This is accomplished by using the sequential load feature of the analyzer and incorporating either sodium dodecyl sulfate (SDS) or SDS and hydrogen peroxide as kill reagents to replace the thermal step (destruction of cycle enzymes by boiling). The ability of SDS to render a cycle inoperative during the run time of the indicator enzyme depends on the inherent resistivity and absolute amount of its enzyme proteins to this surfactant. Criteria used to judge the efficacy of a potential kill reagent are based on the sample blank time-response curve and the cycle product recovery by the indicator enzyme. Various other enzyme cycling systems which can be fitted to the centrifugal fast analyzer are highlighted.     

Equilibrium modeling of extractive enzymatic hydrolysis of penicillin G with concomitant 6-aminopenicillanic acid crystallization

In the present downstream processing of penicillin G, penicillin G is extracted from the fermentation broth with an organic solvent and purified as a potassium salt via a number of back-extraction and crystallization steps. After purification, penicillin G is hydrolyzed to 6-aminopenicillanic acid, a precursor for many semisynthetic beta-lactam antibiotics. We are studying a reduction in the number of pH shifts involved and hence a large reduction in the waste salt production. To this end, the organic penicillin G extract is directly to be added to an aqueous immobilized enzyme suspension reactor and hydrolyzed by extractive catalysis. We found that this conversion can exceed 90% because crystallization of 6-aminopenicillanic acid shifts the equilibrium to the product side. A model was developed for predicting the equilibrium conversion in batch systems containing both a water and a butyl acetate phase, with either potassium or D-p-hydroxyphenylglycine methyl ester as counter-ion of penicillin G. The model incorporates the partitioning equilibrium of the reactants, the enzymatic reaction equilibrium, and the crystallization equilibrium of 6-aminopenicillanic acid. The model predicted the equilibrium conversion of Pen G quite reasonably for different values of pH, initial penicillin G concentration and phase volume ratio. The model can be used as a tool for optimizing the enzymatic hydrolysis.     

Linear chromosome-generating system of Agrobacterium tumefaciens C58: protelomerase generates and protects hairpin ends

Agrobacterium tumefaciens C58, the pathogenic bacteria that causes crown gall disease in plants, harbors one circular and one linear chromosome and two circular plasmids. The telomeres of its unusual linear chromosome are covalently closed hairpins. The circular and linear chromosomes co-segregate and are stably maintained in the organism. We have determined the sequence of the two ends of the linear chromosome thus completing the previously published genome sequence of A. tumefaciens C58. We found that the telomeres carry nearly identical 25-bp sequences at the hairpin ends that are related by dyad symmetry. We further showed that its Atu2523 gene encodes a protelomerase (resolvase) and that the purified enzyme can generate the linear chromosomal closed hairpin ends in a sequence-specific manner. Agrobacterium protelomerase, whose presence is apparently limited to biovar 1 strains, acts via a cleavage-and-religation mechanism by making a pair of transient staggered nicks invariably at 6-bp spacing as the reaction intermediate. The enzyme can be significantly shortened at both the N and C termini and still maintain its enzymatic activity. Although the full-length enzyme can uniquely bind to its product telomeres, the N-terminal truncations cannot. The target site can also be shortened from the native 50-bp inverted repeat to 26 bp; thus, the Agrobacterium hairpin-generating system represents the most compact activity of all hairpin linear chromosome- and plasmid-generating systems to date. The biochemical analyses of the protelomerase reactions further revealed that the tip of the hairpin telomere may be unusually polymorphically capable of accommodating any nucleotide.