Deactivation enzyme

Pectic enzymes are inactivated by pasteurization. Citms juices require higher temperatures for enzyme deactivation than for pasteurization. Heat treatment at 85—94°C for 30 s inactivates pectic enzymes (9) and is more than adequate for pasteurization. 

Enzymatic reactions frequently undergo a phenomenon referred to as substrate inhibition. Here, the reaction rate reaches a maximum and subsequently falls as shown in Eigure 11-lb. Enzymatic reactions can also exhibit substrate activation as depicted by the sigmoidal type rate dependence in Eigure 11-lc. Biochemical reactions are limited by mass transfer where a substrate has to cross cell walls. Enzymatic reactions that depend on temperature are modeled with the Arrhenius equation. Most enzymes deactivate rapidly at temperatures of 50°C-100°C, and deactivation is an irreversible process. 

Over the past few years there have been an increasing number of reports of diseases that are becoming resistant to previously effective drug treatments. This resistance is often due to the presence of enzymes that bring about chemical modification of the drug to an inactive form, e.g. /S-lactamase enzymes deactivate (6-lactam antibiotics by their conversion to penicillanic acid. 

A model developed by Leksawasdi et al. [11,12] for the enzymatic production of PAC (P) from benzaldehyde (B) and pyruvate (A) in an aqueous phase system is based on equations given in Figure 2. The model also includes the production of by-products acetaldehyde (Q) and acetoin (R). The rate of deactivation of PDC (E) was shown to exhibit a first order dependency on benzaldehyde concentration and exposure time as well as an initial time lag [8]. Following detailed kinetic studies, the model including the equation for enzyme deactivation was shown to provide acceptable fitting of the kinetic data for the ranges 50-150 mM benzaldehyde, 60-180 mM pyruvate and 1.1-3.4 U mf PDC carboligase activity [10]. 

A remaining crucial technological milestone to pass for an implanted device remains the stability of the biocatalytic fuel cell, which should be expressed in months or years rather than days or weeks. Recent reports on the use of BOD biocatalytic electrodes in serum have, for example, highlighted instabilities associated with the presence of 02, urate or metal ions [99, 100], and enzyme deactivation in its oxidized state [101]. Strategies to be considered include the use of new biocatalysts with improved thermal properties, or stability towards interferences and inhibitors, the use of nanostructured electrode surfaces and chemical coupling of films to such surfaces, to improve film stability, and the design of redox mediator libraries tailored towards both mediation and immobilization. 

It was postulated that the differences in enzyme activity observed primarily result from interactions between enzyme-bound water and solvent, rather than enzyme and solvent. As enzyme-associated water is noncovalently attached, with some molecules more tightly bound than others, enzyme hydration is a dynamic process for which there will be competition between enzyme and solvent. Solvents of greater hydrophihcity will strip more water from the enzyme, decreasing enzyme mobility and ultimately resulting in reversible enzyme deactivation. Each enzyme, having a unique sequence (and in some cases covalently or noncovalently attached cofactors and/or carbohydrates), will also have different affinities for water, so that in the case of PPL the enzyme is sufficiently hydrophilic to retain water in all but the most hydrophilic solvents. 

Sadana and collaborators (Sadana, Raju and Shahin, 1989) have proposed an empirical stability index (SI) for enzyme deactivation, which makes more quantitative the effect of different variables on enzyme stability. 

Sadana, A., Raju, R.R. and Shahin, E. (1989) A stability index for enzyme deactivation. 

When the enzyme is incubated at 4 °C with aqueous buffer, a very small deactivation constant is found. In the presence of a second phase of MTBE, deactivation is 30% higher (4 °C). The highest increase in enzyme deactivation is due to the temperature the deactivation constants are 22-fold (1.3 x 17) or 255-fold (15 X 17) higher when incubating the enzyme at 20 °C in pure buffer or in a two-phase system respectively. In the presence of the substrate benzaldehyde almost the entire enzyme is deactivated within 1 h. This deactivation in a two-phase system is to some extent dependent on the size of the phase boundary and can... 

One method is to run the reaction in an aqueous buffer/organic solvent biphasic system. This makes it possible to work at high substrate and product concentrations and at the pH-optimum of the enzyme. In addition, in water-immiscible solvents the non-enzymahc addition of HCN to the carbonyl group is non-existent or extremely slow. Possible disadvantages are enzyme deactivation at the interface and the presence of organic solvent dissolved in the aqueous phase [15, 17, 18]. 

Ionic liquids are generally regarded as highly stable, and the widely used dial-kylimidazolium ionic liquids are indeed thermostable up to 300 °C [4]. The propensity of the [BF4] and [PF6] anions to hydrolyze with liberation of HF [37], which deactivates many enzymes, has already been mentioned. The [TfO] and [ Tf2N] anions, in contrast, are hydrolytically stable. Dialkylimidazolium cations have a tendency to deprotonate at C-2, with ylide (heterocarbene) formation. Such ylides are strong nucleophiles and have been used as transesterification catalysts, for example [38]. These could cause enzyme deactivation as well as background transesterification when formed in small amounts from anhydrous ionic liquids and basic buffer salts, for example. 

In order to improve this reaction, a proper understanding of all parameters affecting product yield is desired. Clearly, the high enzyme consumption is a major obstacle for an efficient and economically feasible process. A likely cause of the inefficient use of DERA in this conversion is enzyme deactivation resulting from a reaction of the substrates and (by-) products with the enzyme. In general, aldehydes and (z-halo carbonyls tend to denature enzymes because of irreversible reactions with amino acid residues, especially lysine residues. From the three-dimensional structure it is known that DERA contains several solvent-accessible lysine residues [25]. Moreover, the complicated reaction profile as shown in Scheme 6.5 indicates the potential pitfalls of this reaction. 

Optimization of this reaction is a delicate balance between minimizing enzyme deactivation by keeping the concentration of reactants low and a high enzyme activity and productivity by adding high amounts of substrate. In order to increase the concentration of the lactol 1 at the end of the reaction the initial substrate concentration was increased in a range of 100-600 mM ClAA. At the same time... 

However, enzyme deactivation is still observed under these conditions, as is clearly demonstrated in Figure 6.6, which shows a so-called Selwyn test [26]. In this set-up, ISOOmM AA and 600 mM ClAA were allowed to react with various amounts of DERA under identical conditions. According to Selwyn s theory on enzyme inactivation, plotting eot, in which Sq represents the initial total enzyme amount and t the reaction time, against the concentration of the product p, progress curves should be superimposable provided no inactivation occurs. If not, the assumption that the rate of product formation is proportional to the initial total enzyme amount does not hold true, which could point at enzyme deactivation. 

Malhotra, A., and A. Sadana, Effect of Activation Energy Microheterogeneity on First-order Enzyme Deactivation, Bioteck Bioeng., 30,108 (1987). 

As noted earlier, protein structure is stabilised by a series of weak forces which often give rise to the properties which are functionally important (models of active sites and substrate binding are discussed above). On the other hand, because active sites involve a set of subtle molecular interactions involving weak forces, they are vulnerable and can be transformed into less active configurations by small perturbations in environmental conditions. It is therefore not surprising that a multitude of physical and chemical parameters may cause perturbations in native protein-geometry and structure. Thus, enzyme deactivation rates are usually multi-factorial, e.g. enzyme sensitivity to temperature varies with pH and/or ionic strength of the medium. 

Generally, deactivation rates are determined in the absence of substrate, but enzyme deactivation rates can be considerably modified by the presence of substrate and other materials. 

This implies that the rates of enzyme deactivation and of substrate conversion can be linked. If E and ES are deactivated at the same rate, then this rate will be the same as that for the substrate/enzyme preparation. Extending these notions, an enzyme will deactivate at different rates depending on which of the complex forms is present, and the overall deactivation rate will vary according to the proportions of the different forms of the enzyme that are present. 

Typical flash pasteurisation operations for fruit juices and nectars will employ a plate pasteuriser with heat recovery and final product cooling. Typical flash pasteurisation conditions will use temperatures between 85 and 95°C with holding times varying between 15 and 60 s. Selection of the appropriate conditions will depend on the product, including the level of microbial load pre-pasteurising. If enzyme deactivation is required as well as microbial removal then a temperature between 90 and 95°C will normally be used. At these temperatures, holding times are normally reduced to around 15 s. 

Immobilization has other advantages it can slow enzyme deactivation by inhibiting protease attack and minimizing shear, interfacial, temperature, or solvent denaturation. As for the scarcity of some potentially very useful enzymes, it may be only a temporary problem. The development of cloning techniques, and probably the very increase in demand will result in lower prices. One spectacular instance is sialyl aldolase (see Table I). Industrial production of this enzyme by the gene-cloned strain of Escherichia coli has been reported.1,2 Sialylaldolase is now available from Toyobo at a moderate price. 

Various enzymes have been reported to be susceptible to deactivation upon shearing due possibly to the disturbance of their tertiary structure. Several investigators have studied the interfacial deactivation of T. reesei enzymes (Kim et al., 1982 Reese and Mandels, 1980). The addition of a surfactant has been found to substantially reduce enzyme deactivation. The surfactant impedes the migration of enzyme to the air-liquid interface. 

Howell, J. A. and M. Mangat, "Enzyme Deactivation during Cellulose Hydrolysis," Biotechnol. Bioeng. 20 (1978) 847-863. 

The UF membranes, which have been manufactured reproducibly since about 1980, with cut-offs between 5 to 50 kDa (typically 10 kDa), feature a basically perfect retention (> 99.99%) so that in most cases enzyme deactivation effects dominate over losses of enzyme through the UF membrane. 

Discussion of the concepts of resting and operational stability raises the question of their comparability and mutual convertibility. This subject has been discussed by Yamane et al. (Yamane, 1987). The validity of Eq. (5.74) for the description of enzyme deactivation is assumed. 

In industrial reactors in most cases enzyme deactivation is important over the relevant time horizon of the reactor operating life, of the campaign, or the catalyst batch. Therefore, many attempts have been made to come to grips with the problem, especially in continuous reactors, or at least to alleviate its effects, by various addition strategies. 

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