Time enzyme deactivation

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]. 

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. 

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. 

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. 

For a high functional stability an excess of the analyte converting enzyme is generally fixed in front of the sensor. In this way, in spite of the time-dependent deactivation of the immobilized biocatalyst, complete analyte conversion is maintained over a long operation time. Under these conditions the sensitivity and the response time are determined by the rate of mass transfer both outside and inside the enzyme layer. 

Although some efforts are being made to develop EMRs based on the immobilization of peroxidases onto the membrane [106, 112], the most applied configurations correspond to the use of direct contact reactors for wastewater treatment, with SBP and MnP applied to effluents containing both phenols [74] and dyes [8, 85]. Some attempts were made to apply an EMR for the synthesis of oxindole from indole by CPO [86]. However, the reactor was only stable for a short period (10 residence times). Afterward, the polymerization of oxindole yielded a solid substance, which blocked the membrane, causing enzyme deactivation and the reduction of the total turnover numbers compared to those obtained in batch assays. 

Fig. 25.2. Analysis of the catalytic activity and the inactivation of a-chymotrypsin at the single-molecule level, (a) Detection of single enzymatic turnover events of a-chymotrpysin. The fluorogenic substrate (suc-AAPF)2-rhodamine 110 is hydrolyzed by a-chymotrypsin, yielding the highly fluorescent dye rhodamine 110. (b) Representative intensity time trace for an individual a-chymotrypsin molecule undergoing spontaneous inactivation imder reaction conditions, (c) Inactivation trace for the intensity time transient in (b), obtained by counting the amount of turnover peaks in (b) in 10 s intervals. After approximately 1000 s, the enzyme deactivates through a transient phase with discrete active and inactive states, (d) Proposed model for the inactivation process. An initial active state is in equilibrium with an inactive state. This inactive state converts to another inactive state irreversibly whereby the corresponding active state has a lower activity than the previous one. All the transitions involved have energy barriers that can be overcome spontaneously at room temperature...

Other hydrolases have been studied in ionic liquid media such as Penicillin G acilase (PGA) [2]. Stability studies on this enzyme have been carried out in organic solvents as well as in ionic liquids. The enzyme deactivation was practically instantaneous in toluene and dichloromethane, and only measurable stability was in 2-propanol. The stability of PGA in ILs was notably improved with respect to 2-propanol. A half-life time of 23 h was obtained in l-ethyl-3-methylimidazolium bis((trifluoromethyl)sulphonyl imide ([emim ][TfNj ]) which was about 2000-fold higher than that in 2-propanol. 

In a previous work, Tardioli et al. [9] have immobilized CGTase from Thermoanaerobacter sp. by covalent attachment into glyoxy 1-agarose particles and obtained an activity recovery of about 32%, that is, five times the highest values obtained in this work (6.94% with sol-gel encapsulation). It is thought that with the latter method, in addition to the causes listed above for immobilized enzyme activity loss, the immobilization conditions and reagents used by the sol-gel method contribute to enzyme deactivation. 

A further aspect that requires some comment is enzyme deactivation (Joly, 1965 Ricca et al, 2009b). A simple model can be used to approximate the deactivation rate of enzymes to a first-order reaction, in order to predict the time of the course of deactivation, as follows ... 

The comparison of TOFs and TONs of different (bio)catalysts should be exercised with great caution, since these numbers only indicate how fast the catalysts act at the onset of the reaction within a limited time span, but they do not tell whether the activity remains at a constant level or if it dropped due to catalyst/ enzyme deactivation. 

The operational conditions, that is, the concentration of substrate and enzyme, the temperature range, and the reactor configuration are summarized in Table 13.2. The activation energy of the reaction, E, was typically obtained for a batch reactor and compared with that calculated for a CSMR. The data obtained in the CSMR at steadystate enabled us, by using a semi-log plot of reaction rate versus time, to identify a first-order mechanism of enzyme deactivation and to determine both its first-order deactivation constant, kj, and the reaction rate at time zero, r, for each substrate and temperature. It was thus possible to compare the effect of the operational parameters on the activity and stability of these two enzymes. From the Arrhenius plot of these Tq, the E,-values were determined for each substrate, and were found to match the values obtained in the batch reactors. 

The most limiting factor for enzymatic PAC production is the inactivation of PDC by the toxic substrate benzaldehyde. The rate of PDC deactivation follows a first order dependency on benzaldehyde concentration and reaction time [8]. Various strategies have been developed to minimize PDC exposure to benzaldehyde including fed-batch operation, immobilization of PDC for continuous operation and more recently an enzymatic aqueous/octanol two-phase process [5,9,10] in which benzaldehyde is continuously fed from the octanol to the enzyme in the aqueous phase. The present study aims at optimal feeding of benzaldehyde in an aqueous batch system. 

Piper and Fenton [10] indicated that extreme acidity or basicity of the gastric juice denaturalize the enzymatic activity of the pepsin, which shows has a higher activity at a pH = 2. At pH = 5 the enzyme starts to deactivate and at pH= 7, the enzyme irreversibly lose its activity. Fig. 3 shows the pepsin UV-visible spectra before and after interaction with the zeolites while Fig 4 shows the enzymatic activity of the denatured hemoglobin proteolysis versus reaction time. 

The same concept of volumetric in situ heating by microwaves was also exploited by Larhed and coworkers in the context of scaling-up a biochemical process such as the polymerase chain reaction (PCR) [25], In PCR technology, strict control of temperature in the heating cycles is essential in order not to deactivate the enzymes involved. With classical heating of a milliliter-scale sample, the time required for heat transfer through the wall of the reaction tube and to obtain an even temperature in the whole sample is still substantial. In practice, the slow distribution of heat... 

During the process described in problem 12-17, it was discovered that the enzyme used to produce gluconic acid was subject to deactivation, with a half-life of 12 days. It appears that the deactivation process is first-order, such that decreases exponentially with time. 

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