Enzymes play a pivotal role in a wide variety of industries. They not only speed manufacturing, they can improve quality, reduce waste, and optimize product yield—ensuring more cost-efficiency and higher profitability. The enzyme and substrate fit each other like lock and key, and only substrates with the right shape will be transformed by the enzyme. This is what makes enzymes specific in their actions.
Enzymes help make chemical reactions happen. They either catalyze (cause) or speed up chemical reactions that help convert raw materials into other products, like fermentation for bio-pharmaceutical, fuel ethanol, wine, and beer production. Enzymes are incorporated in detergents and animal feed formulation. Regardless of the reaction type or finished product, enzyme quality, activity, and stability are critical to achieving the desired outcome.
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Enzyme activity is the rate of enzyme reaction—generally expressed as units of substrate converted (or product formed) per time unit. Enzyme activity depends on numerous factors. Therefore, measuring enzyme activity is a precise undertaking that requires technical knowledge, skill, and technique, as well as modern laboratory instruments.
Enzyme assays are laboratory methods that measure the rate of enzyme reactions (enzyme activity). These assays also help to develop analysis technologies and to study enzyme catalytic mechanisms. They are vital for the study of enzyme kinetics and enzyme inhibition. Usually the assay is carried out by determining the enzyme activity with and without activation by an added coenzyme. The activity can be monitored by measuring changes in concentration of substrates or products during the reaction.
Enzyme kinetics is the study of the chemical reactions that are catalyzed by enzymes. In enzyme kinetics, technicians measure reaction rates and investigate the effects of the varying conditions of the reaction. Studying an enzyme’s kinetics can reveal the catalytic mechanism of this enzyme, its role in metabolism, how its activity is controlled, and how a drug or an agonist might inhibit the enzyme.
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The enzymatic assay can be direct, indirect, or coupled. In the case of direct assay, substrate is added to the sample, the end-product is determined, and the substrate or assay reagent is directly modified by the enzyme. The signal is generated by interaction with another reagent or another reaction.
Developing a reliable analytical method for enzyme assay or enzyme activity involves many different steps. Overall method development is tedious and time consuming. Method development starts with identifying the key method variables through design of experiment (DoE), which requires many sets of samples to be tested for enzyme activities. Flexible method parameters for each enzyme type: measuring wavelength, blank measurement, buffer addition, reagents additions, substrate addition, enzyme specific incubation temperature, enzyme specific incubation time and data collection duration all make the enzyme assay method development and method transfer much more simple and reliable from research and development (R&D) to QA/QC labs.
Depending on the type of enzymes and their sensitivity, many different analytical methods are used. Most enzyme assays are based on spectroscopic techniques, with the two most used being absorption and fluorescence.
Enzyme assays based on photometry, fluorometry, 96-, 384-, or even 1536-well format microplate offer a high-throughput alternative to the traditional spectrophotometers. The microplate format is convenient for high-throughput analysis using a 200μL assay volume and is commonly used in life science applications. However, the microplate method suffers from temperature stabilization, absorption correction, and edge effect.
Measuring enzyme parameters using UV-VIS spectrophotometry can be slow-going. But an exciting evolution in efficiency has arrived. Break free from the limitations of UV-VIS spectrophotometric enzyme assay. The Gallery Enzyme Master systems—the first automated enzyme analyzers designed specifically for enzyme assay applications, combine robust hardware and new custom-designed software. These best-in-class solutions streamline enzyme method development and reliable routine enzyme assay/activity testing—for walkaway efficiency day in and day out. You’ll appreciate fully-automated reagents, incubations and measurements to ensure your method and results can be easily duplicated in other labs and the time- and labor-savings speak for themselves.
Measuring enzyme assays is a precise job and can be influenced by many variables. Results accuracy is highly dependent pH and temperature stability.
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Automated discrete analyzers offer a promising solution to the challenges of enzyme standardization. Learn more from this article.
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