Analysis of enzyme activity

Current data indicate that the analysis of cellulase enzyme activities may be the best method for determining the projected cellulose conversion of the overall system, and therefore the hydrolytic power of the system under evaluation. With development, the analysis of enzyme activities may also serve as a "real time" method of monitoring the stability of the system, with radical changes in enzyme activities indicative of potential process upset. 

UV/Vis-spectroscopy is the classical method of analysis of enzyme activity. The principle is the change in absorption behavior of a substrate during the reaction process, for example by modification or Hberation of a chromophoric function. A number of enzymes from different classes can be assayed spectrophoto-metrically using their natural substrates or cofactors. In this way, activity of acetyltransferases can be estimated by measurement of absorption of acetyl coenzyme A at 232 nm [33]. Oxidoreductases which require a cofactor, e.g., NAD/NADH, to carry out the transfer of hydrogen can be characterized by measuring the absorption of this cofactor depending on its oxidation stage [33]. 

The discussion to this point has focused on the isolation of intact mitochondria. By various chemical and physical treatments, mitochondria may be separated into their four components. This allows biochemists to study the biological functions of each component. For example, by measuring enzyme activities in each fraction, one can assign the presence of a particular enzyme to a specific region of the mitochondria. Studies of mitochondrial subfractions have resulted in a distribution analysis of enzyme activities in the four locations (Table E10.1). This type of study is often referred to as an enzyme profile or enzyme activity pattern and the enzyme may be considered a marker enzyme. For example, cytochrome oxidase, which is involved in electron transport, is a marker enzyme for the inner membrane. 

Yuan, Z., Zhao, J. and Wang, Z. X. (2003). Flexibility analysis of enzyme active sites by crystallographic temperature factors. Protein Eng. 16(2), 109-114. 

Flow cytometry is well suited for the analysis of enzyme activity and kinetics at the single cell level (Watson and Dive, 1994). Flow cytometric assays for numerous enzymes including esterases, proteases, peroxidases, lipases, and oxidoreductases3 are available and are widely used in research and clinical practice. To date, flow cytometry has not been widely exploited as a screening tool for enzyme engineering purposes, but this is rapidly changing. 

As evident from the above discussion, ILs have emerged as alternative solvent systems for biocatalysis which has already established with a range of different class of enzymes performing better or at least comparable to conventional orgattic solvents. The added advantages of easy work up procedure, possibility of recycling the solvent and multiple uses of enzymes were also noted. However, the complexity in the nature of ILs has resulted variation in enzyme performance in terms of reaction rate and enantioselectivity. A brief comparative analysis of enzyme activity versus nature of the component of ILs would be helpful for better understanding of the subject and careful selection of ILs for a desired reaction. 

ABPP strategies for the in vivo Analysis of Enzyme Activities... 

The in vivo imaging studies carried out with cysteine protease-directed probes [50] underscored the need for a generally applicable methodology for in vivo analysis of enzyme activities. Indeed, as exemplified by many protease families, most enzymes are subject to multiple mechanisms for... 

U. Schagerlof, H. Schagerlof, D. Momcilovic, G. Brinkmalm, and F. Tjemeld, Endoglucanase sensitivity for substituents in methyl cellulose hydrolysis studied using MALDI-TOFMS for oligosaccharide analysis and stmctural analysis of enzyme active sites. Biomacromolecules, 8 (2007) 2358-2365. 

Medes observed further that the excretion of this compound paralleled the patient s protein or tyrosine intake and that administration of homogentisic acid did not affect urine composition. These findings suggested that the disease is due to a block of the transformation of tyrosine to homogentisic acid. Although this is the most probable interpretation, it has not yet received direct confirmation by analysis of enzyme activity in the liver. Furthermore, determination of the abnormal metabolite in the blood has not been included in the studies of tyrosinosis. The possibility of an abnormal excretion of the compound at the level of the renal tubules cannot be excluded entirely. 

Interaction factors (/) will have values in the range / < 1 for positive cooperativity and / > 1 for negative cooperativity. It is very difQcult to obtain accurate estimates of individual interaction factors, or intrinsic dissociation constants, from steady-state kinetic analysis of enzyme activity. 

Since such unpredictable effects should be avoided during analysis of enzyme activities, as a rule the initial reaction rate, vq, is measured as soon as possible after the start of the reaction. The basics of the kinetic properties of enzymes in the steady state were given by Briggs and... 

Identification and quantification of copper in MCO enzymes is essential for accurate analysis of enzyme activity and function, particularly because of the issues delineated above (incomplete complements of copper atoms and formation of different multimers). There are a few methods that are eommonly used for copper... 

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