Transient method for proposing the mechanisms of reactions over immobilized enzymes

The transient response method is introduced to elucidate the mechanism of reaction over immobilized enzyme. Glucose oxidation over the glucose oxidase that was immobilized on ion-exchange resin using glutaraldehyde as a linking agent is selected as an example here. The transient responses of a fixed-bed reactor to step increases and decreases in glucose, oxygen, and gluconolactone feed concentrations have been monitored and interpreted. From some responses, we have found that gluconolactone is formed in the reaction of glucose with adsorbed oxygen, while hydrogen peroxide is formed in the reaction of oxygen with adsorbed glucose. Combining all information from interpreting the responses with the literature, a mechanistic picture can be obtained as follows: \documentclass{article}\pagestyle{empty}\begin{document}$$ \begin{array}{*{20}c} {E_{{\rm ox}} + G \to E_{{\rm red}} GL} \\ {E_{{\rm red}} GL \to E_{{\rm red}} + GL} \\ {E_{{\rm red}} + {\rm O}_2 \to E_{{\rm ox}} {\rm H}_2 {\rm O}_2 } \\ {E_{{\rm ox}} {\rm H}_2 {\rm O}_2 \to E_{{\rm ox}} + {\rm H}_2 {\rm O}_2 } \\ \end{array} $$\end{document}.     

A simple method for the determination of individual rate constants for substrate hydrolysis by serine proteases

A simple method is presented for the determination of individual rate constants for substrate hydrolysis by serine proteases and other enzymes with similar catalytic mechanism. The method does not require solvent perturbation like viscosity changes, or solvent isotope effects, that often compromise nonspecifically the activity of substrate and enzyme. The rates of substrate diffusion into the active site (k1), substrate dissociation (k-1), acylation (k2), and deacylation (k3) in the accepted mechanism of substrate hydrolysis by serine proteases are derived from the temperature dependence of the Michaelis-Menten parameters kcat/Km and kcat. The method also yields the activation energies for these molecular events. Application to wild-type and mutant thrombins reveals how the various steps of the catalytic mechanism are affected by Na+-binding and site-directed mutations of the important residues Y225 in the Na+ binding environment and L99 in the S2 specificity site. Extension of this method to other proteases should enable the derivation of detailed information on the kinetic and energetic determinants of protease function.     

Measurement of rate constants for actin filament elongation in solution

This paper describes a simple method to measure the rate constants for actin filament elongation using pyrene-actin fluorescence as a measure of the polymer concentration and unlabeled actin filaments as nuclei. With careful selection of conditions, the initial rate of polymerization is directly proportional to the actin monomer concentration above the critical concentration. Plots of initial rate versus actin concentration give the critical concentration (x intercept), the association rate constant, k+ (slope), and the dissociation rate constant, k-(y intercept). By calibrating the system under conditions where the absolute values of these rate constants are known from previous electron microscopic experiments [T. D. Pollard and M. S. Mooseker (1981) J. Cell Biol. 88, 654-659; J. A. Cooper, S. B. Walker, and T. D. Pollard (1983) J. Muscle Res. Cell Motil. 4, 253-262], one can calculate the absolute values of the rate constants under other conditions as well as the length of the filaments used as a nuclei. This approach has proven useful for evaluating the effect of actin-binding proteins on the polymerization process.     

Preparative production of hydroquinone from benzoquinone catalysed by immobilized d-glucose oxidase

Benzoquinone can replace O₂ as an electron acceptor in the oxidation of d-glucose catalysed by A. niger d-glucose oxidase. As a result, a useful chemical, hydroquinone, is formed in nearly 100% yield. A column packed with d-glucose oxidase immobilized onto alumina was operated for two weeks with no measurable decline in its catalytic efficiency and produced more than one hundred grams of hydroquinone.     

Transient response of the human-clothing system

1. 1. A transient clothing model which considers the effects of adsorption and thermal capacitance on the dynamic thermal response of clothing was developed.

2. 2. Moisture adsorption and desorption by the fabric are the major factors that affect the transient response of clothing.

3. 3. This moisture can come from evaporated sweat or from the environment.

4. 4. The clothing model was combined with a modified version of the two-node thermal model of the human body.

5. 5. The combined model shows that, during transients, the mix of latent and sensible heat flow from the skin may differ considerably from the corresponding heat flows from the clothing surface to the environment.

6. 6. The alteration of the heat flows can have a significant impact on the thermal response of the body by changing the sweat rate required to achieve the heat loss necessary to maintain thermal balance.

Glucose oxidase immobilized electrode for potentiometric estimation of glucose

Glucose oxidase has been immobilized onto a thin platinum strip, by co-crosslinking with bovine serum albumin and glutaraldehyde. The retention of redox characteristics of glucose oxidase has been verified by cyclic voltammetry. The activity of the immobilized enzyme reduces to a quarter of its value when the enzyme is in solution but improves when coimmobilized with 1 urea. The potentiometric response builds up and remains stable after 100 s. It is sensitive to the thickness of the immobilizing matrix, pH and temperature. An improvement in the performance of the electrode has been achieved by coimmobilizing 2 urea and metal ions such as Mg²⁺ and Mn²⁺. The presence of Cu has been proved to be detrimental. The electrode has been calibrated in the 0.1–5.0 mM glucose concentration range. It gives a stable response for more than 50 independent assays and can be stored for 60 days without significant loss of function.     

Quantifying the inactivation rate constants for the molecular species comprising the catalytic cycle of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase

When an unstable enzyme is incubated with its substrate(s), catalysis may cease before chemical equilibrium is attained. The residual substrate concentrations depend on their initial concentrations, the initial enzymic activity, and the inactivation rate constants for each molecular species that comprise the catalytic cycle. The underlying theory has been elaborated previously for single-substrate reactions and here it is extended to bi-substrate reactions. The theory is illustrated by application to glucose 6-phosphate dehydrogenase, which is unstable when exposed to a low concentration of sodium dodecyl sulphate. It is shown that the ternary complex containing both substrates is resistant to inactivation while each of the remaining complexes undergoes first-order decay. Rate constants for the inactivation of each complex are calculated.     

Determination of equilibrium and individual rate constants for subtilisin-catalyzed transesterification in anhydrous environments

We report here the first determinations of individual rate constants and equilibrium constants for enzymatic reactions in essentially anhydrous organic solvents. Using the added nucleophile method we have measured the effect of changing solvent on the binding and catalytic steps for subtilisin-catalyzed transesterification of N-protected amino acid esters. The detailed information generated indicates that once the substrate has bound to the enzyme, the catalytic machinery can work at rates equivalent to those in water. The decreased overall rates for subtilisin suspended in anhydrous solvents are merely the result of extremely high values for K(s), in most cases, coupled with low concentrations of nucleophile ( approximately 1.0M in organic solvents, and 55M in water). The method described, which is generally applicable, and straightforward experimentally, will, we believe, enable a clearer understanding of how changing solvent can predictably affect the activity and specificity of the enzyme. (c) 1992 John Wiley & Sons, Inc.     

Reaction rate constants of superoxide scavenging by plant antioxidants

Plant phenols may exert protective effects by scavenging superoxide, which is implicated in tissue damage and accelerated inactivation of vasorelaxing nitric oxide. Preventing the interaction of superoxide with tissue biomolecules depends not only on the extent of superoxide scavenging but also on scavenging velocity. However, information on superoxide scavenging kinetics of plant phenols is scarce. We describe an improved lucigenin-based chemiluminescence assay for kinetic analysis. The use of potassium superoxide (KO₂) as a nonenzymatic superoxide source allowed simple and reliable determination of the second-order reaction rate constants between superoxide and plant antioxidants at physiologically relevant conditions, avoiding unspecific effects of other reactive oxygen species or superoxide-generating enzymes. We calculated the rate constants for phenols of different structures, ranging from 2.9 × 10³ mol⁻¹ l s⁻¹ for morin to 2.9 × 10⁷ mol⁻¹ l s⁻¹ for proanthocyanidins. Compounds with pyrogallol or catechol moieties were revealed as the most rapid superoxide scavengers, and the gallate moiety was found to be the minimal essential structure for maximal reaction rate constants with superoxide.     

New method of determining kinetic constants for immobilized enzymes in a packet-bed reactor system.

A new graphical method of determining the kinetic constants for immobilized-enzyme systems is proposed and illustrated with some examples. The advantages of using this method are that these kinetic constants can be determined accurately and conveniently from the conversion data of a packed-column enzyme-reactor system.     

Kinetic constants determination for an alkaline protease from Bacillus mojavensis using response surface methodology

The kinetic constants for an alkaline protease from Bacillus mojavensis were determined using a central composite circumscribed design (CCCD) where concentration of substrate (casein) and the assay temperature were varied around their center point. The K(m),V(max), K(cat), activation energy (E(a)) and temperature coefficient (q(10)) were determined and the values of these kinetic constants obtained were found comparable to that obtained with conventional methods. The Michaelis-Menten constant (K(m)) for casein decreased with corresponding increase in V(max), as reaction temperature was raised from 45-60 degrees C. The protease exhibited K(m) of 0.0357 mg/ml, 0.0270 mg/ml, 0.0259 mg/ml, and 0.0250 mg/ml at 45, 50, 55, and 60 degrees C, respectively, whereas V(max) values at these temperatures were 74.07, 99.01, 116.28, and 120.48 microg/ml/min, respectively, as determined by response surface methodology. The Arrhenius plot suggested that the enzyme undergoes thermal activation above 45 degrees C until 60-65 degrees C followed by thermal inactivation. Likewise, the energy of activation (E(a)) was more between 45-55 degrees C (9747 cal/mol) compared to E(a) between 50-60 degrees C (4162 cal/mol).     

农药残留检测关键用酶固定化研究进展

为保障消费者食用安全,迫切需要研发农产品和食品中的农药残留快速检测技术.酶抑制法检测是目前农药残留快速检测技术中的主要研究方向之一,而酶的固定化是用基于酶抑制法原理对农药残留检测研究中的重要步骤.通过物理或化学的方法高效地将酶固定于载体上,同时保持酶的催化活性是开发各类基于酶抑制法检测农药残留传感器的关键.本文将从固定...     

Response surface optimization for the continuous glucose isomerization process

Production of fructose via a continuous glucose isomerization process was optimized using response surface methodology. Glucose isomerization was performed using immobilized glucose isomerase in a flow-through tubular reactor. Process factors eg pH (7.0–7.8), temperature (50–60°C), flow rate (5–17 ml min^–1) and glucose content (30–50% w/w) of the feedstock solution were simultaneously tested according to a central composite experimental design. Measured responses such as % isomerization, and fructose yield (gh^–1) has an excellent correlation with tested factors. The highest desirability,D, (geometric mean of % isomerization and fructose yield) was obtained when the feedstock (56–60°C) had 34–36% glucose, a pH of 7.4–7.8 and was pumped at 15 ml min^–1.     

Agitation rate effects on plasmid stability in immobilized and free-cell continuous cultures of recombinant E. coli.

Escherichia coli B/pTG201 recombinant cells were immobilized by entrapment in a carrageenan gel and cultivated in nonselective media to investigate the effect of agitation rate on plasmid stability, biomass concentration, and enzyme productivity. These parameters were studied in continuous cultures for free and immobilized cells, respectively. Immobilized recombinant cells exhibit an increase in the stability of the plasmid pTG201 compared to free cells, even under conditions where the tendency of plasmid stability for free cells decreased generally more rapidly under a higher agitation rate. Intensive agitation, resulting also in a strong shear stress, greatly reduced cell concentration within gel beads throughout the course of growth. Higher enzyme expression of catechol 2–3, dioxygenase was also obtained in leaked cells due to better maintenance of plasmid stability and higher plasmid copy number with regard to free cells. Enzyme productivity of leaked and free cells in minimal medium decreased with the increase in agitation rate, due to decreased plasmid stability; however, in LB medium, it increased in the presence of higher agitation rate related to important cell concentration.     

Agitation rate effects on plasmid stability in immobilized and free-cell continuous cultures of recombinant E. coli

Escherichia coli B/pTG201 recombinant cells were immobilized by entrapment in a carrageenan gel and cultivated in nonselective media to investigate the effect of agitation rate on plasmid stability, biomass concentration, and enzyme productivity. These parameters were studied in continuous cultures for free and immobilized cells, respectively. Immobilized recombinant cells exhibit an increase in the stability of the plasmid pTG201 compared to free cells, even under conditions where the tendency of plasmid stability for free cells decreased generally more rapidly under a higher agitation rate. Intensive agitation, resulting also in a strong shear stress, greatly reduced cell concentration within gel beads throughout the course of growth. Higher enzyme expression of catechol 2–3, dioxygenase was also obtained in leaked cells due to better maintenance of plasmid stability and higher plasmid copy number with regard to free cells. Enzyme productivity of leaked and free cells in minimal medium decreased with the increase in agitation rate, due to decreased plasmid stability; however, in LB medium, it increased in the presence of higher agitation rate related to important cell concentration.     

Compositions and compositional-behavioural relationships of enzymes immobilized on porous inorganic supports via titanium(IV) species

The compositions and compositional-behavioural relationships of glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.1.3) immobilized on titanium(IV)-activated porous inorganic supports have been investigated for several transition metal activation techniques based on the metal-link/chelation method developed by our group. The highest activity (239 Ug^?1 matrix) of immobilized glucoamylase was obtained with the hydrous titanium(IV) oxide derivative of the support when this and a 15% w/v TiCl₄ solution were dried at 45°C in vacuum for 30 h. However, the immobilized enzyme preparation displayed a very unstable behaviour, as did also the preparation which was obtained by drying the mixture of support and transition metal solution at atmospheric pressure. This was mainly due to an enzyme deactivation by titanium inhibition instead of enzyme loss in substrate solution. When amination and carbonylation steps were included in the immobilization technique much more stable preparations were obtained, mainly when the support was activated by drying at 45°C with a 15% w/v TiCl₄ solution ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1495}\text{h}$) although with a lower initial activity (35.6 Ug^?1 matrix). The pure TiCl₄ support activation rather than TiCl₄/HCl solution support activation led to less stable immobilized enzyme preparations (washing and amination solvent chloroform, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 365}\text{h}$; washing and amination solvent water, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 276}\text{h}$) than the preparation obtained with the dried titanium(IV)-activated support. This was due to loss of enzyme-titanium(IV) complex in solution, as the interactions between the titanium(IV) and the silanol groups of the porous silica are weak. However, the amination (with 1,6-diaminohexane) and carbonylation (with glutaraldehyde) steps always led to immobilized enzyme preparations with constant specific activities and protein/titanium(IV) ratio. This suggests that the spacing effect introduced by these reactions removes the titanium(IV) inhibition of glucoamylase.     

New enzymatic immobilized biocatalysts for detoxification of organophosphorus compounds

New immobilized biocatalysts based on phosphotriesterase and porous fabric materials impregnated with chemically cross-linked chitosan and sulphate chitosan gels were investigated. Analysis of the rheological characteristics of enzyme-containing gels confirmed their high plasticity and mechanical strength, while scanning electron microscopy verified their macroporous structure. The fabric matrix could absorb and retain a large amount of liquid thereby increasing its own weight 3.5-4.5 fold. The catalytic characteristics of the immobilized biocatalyst hydrolyzing Paraoxon, Coumaphos, Chlorpyrifos and Diisopropyl fluorophosphate were investigated. The catalytic efficacy of the soluble enzyme was 3.0-5.5-times higher compared to the immobilized form mainly due to the lower K_m values. With constant 55-60% humidity the biocatalyst retained 77% and 67-70% activity after 50-day storage at 4°C and 23°C, respectively. Benzalkonium chloride appeared to be an appropriate preservative for long-term storage of immobilized biocatalyst in a wet state.     

New enzymatic immobilized biocatalysts for detoxification of organophosphorus compounds

New immobilized biocatalysts based on phosphotriesterase and porous fabric materials impregnated with chemically cross-linked chitosan and sulphate chitosan gels were investigated. Analysis of the rheological characteristics of enzyme-containing gels confirmed their high plasticity and mechanical strength, while scanning electron microscopy verified their macroporous structure. The fabric matrix could absorb and retain a large amount of liquid thereby increasing its own weight 3.5–4.5 fold. The catalytic characteristics of the immobilized biocatalyst hydrolyzing Paraoxon, Coumaphos, Chlorpyrifos and Diisopropyl fluorophosphate were investigated. The catalytic efficacy of the soluble enzyme was 3.0–5.5-times higher compared to the immobilized form mainly due to the lower K_m values. With constant 55–60% humidity the biocatalyst retained 77% and 67–70% activity after 50-day storage at 4°C and 23°C, respectively. Benzalkonium chloride appeared to be an appropriate preservative for long-term storage of immobilized biocatalyst in a wet state.     

A “parallel plate” electrostatic model for bimolecular rate constants applied to electron transfer proteins

A “parallel plate” model describing the electrostatic potential energy of protein-protein interactions is presented that provides an analytical representation of the effect of ionic strength on a bimolecular rate constant. The model takes into account the asymmetric distribution of charge on the surface of the protein and localized charges at the site of electron transfer that are modeled as elements of a parallel plate condenser. Both monopolar and dipolar interactions are included. Examples of simple (monophasic) and complex (biphasic) ionic strength dependencies obtained from experiments with several electron transfer protein systems are presented, all of which can be accommodated by the model. The simple cases do not require the use of both monopolar and dipolar terms (i.e., they can be fit well by either alone). The biphasic dependencies can be fit only by using dipolar and monopolar terms of opposite sign, which is physically unreasonable for the molecules considered. Alternatively, the high ionic strength portion of the complex dependencies can be fit using either the monopolar term alone or the complete equation; this assumes a model in which such behavior is a consequence of electron transfer mechanisms involving changes in orientation or site of reaction as the ionic strength is varied. Based on these analyses, we conclude that the principal applications of the model presented here are to provide information about the structural properties of intermediate electron transfer complexes and to quantify comparisons between related proteins or site-specific mutants. We also conclude that the relative contributions of monopolar and dipolar effects to protein electron transfer kinetics cannot be evaluated from experimental data by present approximations.