Enzyme Reaction Performance Parameters

Links between Enzyme Reaction Performance Parameters 2.3.3.1 Rate Acceleration... 

In the past ten years, our group studied chemometrics for kinetic analysis of reaction curve to estimate parameters of enzyme reaction systems the following results were foimd. (a) In terms of reliability and performance for estimating parameters, the use of the integrated rate equations with the predictor variable of reaction time is superior to the use of the integrated rate equations with predictor variables other than reaction time (Liao, et al., 2005a) (b) the integration of kinetic analysis of reaction curve with other methods to quantify initial rates... 

On the main window to perform kinetic analysis of reaction curve, original data are listed and plotted for eyesight-checking of data for steady-state reaction. Text boxes are used to input some common parameters like fC, and most parameters are read from the text file for the reaction curve. Subprogram for an enzyme reaction system is called for running results are displayed on the main window and may be saved in text file for further analysis. 

The efficiency of solution-phase (two aqueous phase) enzymatic reaction in microreactor was demonstrated by laccase-catalyzed l-DOPA oxidation in an oxygen-saturated water solution, and analyzed in a Y-shaped microreactor at different residence times (Figure 10.24) [142]. Up to 87% conversions of l-DOPA were achieved at residence times below 2 min. A two-dimensional mathematical model composed of convection, diffusion, and enzyme reaction terms was developed. Enzyme kinetics was described with the double substrate Michaelis-Menten equation, where kinetic parameters from previously performed batch experiments were used. Model simulations, obtained by a nonequidistant finite differences numerical solution of a complex equation system, were proved and verified in a set of experiments performed in a microreactor. Based on the developed model, further microreactor design and process optimization are feasible. 

The immobilization procedure may alter the behavior of the enzyme (compared to its behavior in homogeneous solution). For example, the apparent parameters of an enzyme-catalyzed reaction (optimum temperature or pH, maximum velocity, etc.) may all be changed when an enzyme is immobilized. Improved stability may also accrue from the minimization of enzyme unfolding associated with the immobilization step. Overall, careful engineering of the enzyme microenvironment (on the surface) can be used to greatly enhance the sensor performance. More information on enzyme immobilization schemes can be found in several reviews (7,8). 

The performance of a biotreatment system ultimately depends on optimization of the activity of microbes and the ability to control the process parameters of the treatment system [157]. In this respect, the ability to monitor gene copy numbers and gene expression is highly useful for real time optimization of the efficiency of a biotreatment system. Advanced molecular techniques as well as low cost methods (e.g., antibody detection of enzymes based on color reaction strips fluorescence i.e., GFP marked organisms with UV light detection) can also be applied to monitor the microbial community structure, persistence of the added bacteria, and their interactions with indigenous populations. 

Prior to being able to study the function and mechanism of an enzyme, it is essential that suitable assays be available to monitor enzyme activity toward different substrates and to determine the kinetic parameters kcat and Km for the reactions. A brief overview of the known assays for the evaluation of PLCB(. activity is thus appropriate. The ideal assay for a phospholipase C would utilize a phospholipid substrate, not an analogue with a modified headgroup or side chains. Such an assay should be sensitive to minimize the quantities of enzyme and substrates that would be required, and it should be convenient to implement so that analyses may be readily performed. 

It can be seen from Eq. (5) that the maximum possible concentration on the surface, c, influences significantly the transport rate. This parameter is a function of the available surface area as well as of the density of the reactive sites. Because of that, the matrix structure plays a very important role in such adsorp-tion/desorption processes. In the case of biological reactions, where the chemical conversion is performed by immobilized enzymes, the immobilization also plays an important role in order to achieve an optimal enzyme density on the reactive surface. 

The program is reported to carry out simple Hiickel molecular orbital calculations to determine the relative sensitivity of aromatic carbon atoms to oxidation and the relative stability of keto and enol tautomers. Klopman et al. (1999) have reported that for polycyclic aromatic hydrocarbons, adequate reactivity is an essential but not sufficient condition for enzyme catalyzed reaction. The accessibility of the reactive site (i.e., the absence of steric hindrance) was also found to be important. Genetic algorithms have been used to optimize the performance of the biotransformation dictionary by treating the initial priority scores set by expert assessment as adjustable parameters (Klopman et al., 1997). 

Kinetics of Immobilized Enzymes. Another major factor in the performance of immobilized enzymes is the effect of the matrix on mass transport of substrates and products. Hindered access to the active site of an immobilized enzyme can affect the kinetic parameters in several ways. The effective concentration of substrates and products is also affected by the chemistry of the matrix especially with regard to the respective partition coefficients between the bulk solution and the matrix. In order to understand the effects of immobilization upon the rate of an enzyme-catalyzed reaction one must first consider the relationship between the velocity of an enzyme-catalyzed reaction and the... 

The development of enzyme-catalyzed processes in organic solvents makes it possible to perform enzymatic analysis in organic solvents. Earlier work involved the addition of moderate amounts of solvents to improve substrate solubility, but the new trend is to operate in almost water-free conditions. The selection of reaction parameters is important. Thus, it is necessary to optimize the solvent (118,119) as well as the enzyme support (120). The polarity of the solvent is also important the more polar the solvent, the less stable the enzyme (119). Thus, extremely hydrophobic solvents are useful, provided the substrates and products are soluble. The choice of support is governed by its tendency to attract minute amounts of water present in the system. The supports are characterized with regard to their aquaphilicity There is an inverse correlation between aquaphilidty and catalytic activity of the adsorbed enzyme (121). 

---