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Enzymology Assays

Creative Enzymes is a renowned service provider, supporting a majority of the enzyme testing market. Our unique advantages are highlighted by an extremely broad range of assays, from basic activity quantification to the most complicated mechanistic studies, which cover almost every step in fundamental research and product development. We are able to perform a single test at the most precise level, as well as design and complete a whole package of services as the solution to a thematic project. We offer a wide spectrum of reagents and methods and will help you to choose the tool that is most suitable for your need of enzyme tests.

Enzymes are the one-of-a-kind biological catalysts that are indispensable for the vast majority of reactions in a living system. They perform the most important transformations in the processes of metabolism, cellular signaling, and hormone regulations. For all applications of enzymes, proper determination of the enzyme activity is the core component and, in most cases, the very first result to obtain before starting the entire chain of research or development activities. This is the reason that Creative Enzymes endeavors to perfect the enzymology assays. Depending on the specific enzyme, application purpose, and specification requirement, the desired assay could be simple as single activity measurement or complex as catalytic mechanism elucidation, testing several representative enzymes or high-throughput screening of thousands of candidates, detection of an existing enzyme or determination of enzymatic kinetics in great details; Creative Enzymes is happy to help to solve the challenge in any event. Based on the resources that are required for the task, we list the enzymology assays below in the order of increasing complexity:

Activity measurement

Enzyme characterization and enzymology assays could be complex. However, in many practices, simple activity measurement is used to quickly determine the effective level of an enzyme or to detect the presence of a class of enzymes. In industrial manufacturing and quality control processes, this test could reflect the actual contents of an enzyme in the product. This is especially important to maintain consistent product performance because enzymes are sensitive to all types of environmental changes, including pH, temperature, and impurities, and could lose their activity quickly over the period of manufacturing, transportation, and storage. In the scenario of pharmaceutical and biotechnical research, single-point catalytic activity measurement, indicated by the reaction velocity, is also used in the early stage to rapidly determine activity levels against known substrates in large batches, which enables high-throughput screening and target identification at an acceptable cost. Therefore, accurate activity measurement takes an important place in many applications.

The principle of enzyme activity measurement is based on the fact that a substrate is converted into a product under the catalysis of an enzyme. Thus, monitoring the formation of the product over time will give the catalysis efficiency of the enzyme. Alternatively, the time-dependent decrease in the level of the substrate is equally useful in determining the enzyme activity. When two or more substrates or products are involved in the enzymatic reaction, measuring one product or substrate is sufficient to achieve the goal, and the one with the easiest detection and least interference will be chosen in most cases.

Although activity measurement may seem easy compared to other enzymology assays, researchers still need to watch for several limitations. It is to be noted that all enzymes reach their optimum activity under different conditions, whereas in a high-throughput screening, one single reaction condition is often used. As a result, the differences in the activity levels of enzymes may be true only in the current condition and cannot be used for quantitative comparison. Another challenge that frequently troubles researchers is that detection of formation of the product or the consumption of the substrate could be strongly disturbed by other ingredients in the reaction matrix, such as inactive solutes or other enzymes. In such cases, the signal to noise ratio must be considered for accurate measurement. Proper selection of the detection method and parameters often gives solutions to such problems.

Creative Enzymes has been providing the activity measurement service for numerous customers helping with quick development in various applications. We have exhibited an exceptional capability of designing the most suitable tests for each customer, which is powered by the largest collection of enzymes, cofactors, and substrates, and the unique technology platform. As mentioned above, each enzyme may have distinct optimal working conditions. Please share us your specific requirement and the target enzyme as detailed as possible, so that we could perform the most appropriate assay.

• Development of analysis methods

For a developed process or commercialized product, routine validation is required as part of the quality control. A valid and reliable activity assay is, therefore, necessary for an enzyme product. In other cases, a research group may desire to obtain an accurate assay method to move the research activity forward. Hence, a new assay often needs to be developed, for activity determination under an unprecedented condition, or for a newly discovered enzyme. For either case, it is important to consider the general rules valid for all enzyme assays. The main factors include solvent, temperature, pH, ionic strength, type of ions, surface active agents, and the proper concentrations of the essential components such as substrates and enzymes.

When measuring a new enzyme or an enzyme under a new condition, researchers often follow the assay method established for a similar condition or a similar enzyme, and many irregular results may be found. This is because enzymes are sensitive catalysts present in small amounts. Often times their activity can only be detected only under the optimum conditions, and different enzymes require different optimum conditions. For example, enzymes from thermophilic bacteria are only active at elevated temperatures, which is quite different from the normal physiological conditions.

Creative Enzymes is well aware of all the key factors in developing a proper assay method. We have successfully set up new enzyme assays for hundreds of customers to meet their needs in performing frequent and reliable activity assays. Under particular circumstances, we even develop methods directly to the features of the target enzyme and not to obey some general standards. We also displayed the capability of developing assays that are considered complex and challenging by enzymologists, such as reversible reactions and coupled tests. In any case, Creative Enzymes is confident in the development of reliable enzyme assays from scratch. We are pleased to provide these assays as powerful tools for long-term studies of new enzymes or known enzymes under new reaction conditions.

• Enzyme kinetics

The enzyme activity can be simply quantified by measurement of the reaction rate, most commonly, the increase or decrease of the amount of substance of the product or substrate, respectively, in a unit time. The derived SI unit katal (kat) is the standard unit for enzyme activity, which is defined as the amount of an enzyme that catalyzes a mole of substrate per second under specified conditions. Alternatively, the International Unit (IU) of enzyme is also commonly used by many suppliers. The IU is defined as the amount of enzyme that catalyzes a micromole (μmol) of substrate per minute. Nonetheless, kat or IU is far from enough of describing the catalytic behavior of an enzyme. As mentioned above, enzyme activity fluctuates largely depending on the reaction condition. Therefore, kat or IU is only meaningful at one specific condition, and the enzyme needs to be tested again once the condition changes even in a small scale. This is especially important to remember when comparing the activity of two enzymes, since the optimum conditions for different enzyme are often different. To serve these purposes, other enzymology parameters were introduced. Some parameters are relatively independent of some reaction conditions, such as Vmax. Some parameters are solely determined by the thermodynamics of the reaction, including Kd and KM. While kinetic parameters are related to the rate constants of various steps in an enzyme reaction, including kcat, k1, and k-1. Altogether, these parameters give a bigger picture of the enzyme activity under different circumstances, which makes it possible to predict enzyme activity under different conditions, or to compare the catalytic activity of different enzymes.

Creative Enzymes is particularly advanced in profiling enzyme kinetics, being able to determine virtually all parameters for an enzyme. With the powerful expert team, we always meet the customer’s expectation on enzyme kinetic assays. With accurate measurement of these parameters, we have helped many customers better quantify and predict the enzyme activity under various conditions. Some customers also used these parameters to have a glance of the catalytic mechanism and the mode of action, which enables further studies and applications of the enzyme.

• Enzyme catalytic mechanisms

The study of enzyme catalytic mechanisms could be extremely complicated, yet necessary to achieve the most valuable products. Given the clear catalytic mechanism, enzymes can be engineered, optimized, and stabilized according to specific needs in an application, including catalyzing unique reactions that do not happen naturally. Without knowing the specific mechanism, optimization of an enzymatic reaction could be only performed based on researchers’ experiences through changing the reaction environment. Based on different perspectives of chemistry, enzyme catalytic mechanisms could be described in different ways. Judging by the chemical nature of an enzyme reaction, the catalytic mechanism could be sorted into specific or general acid-base catalysis, covalent catalysis, substrate orientation, and so on. From the kinetic point of view, the mechanism could be mono- or bi-substrate, random or ping-pong order, and dose-dependent or time-dependent kinetics, and could possibly have synergistic effects which make the mechanism even more complex.

Creative Enzymes is proud to offer the mechanism study of many enzymes. We are capable of revealing the catalytic mechanism of an enzyme in great details, using multiple most updated biochemical technologies including crystallography (structural biology), site-directed mutagenesis, reporter group labeling of the substrate or inhibitor, kinetic measurement with the isotope effect etc. With such information, the customers have successfully fine-tuned enzymes for higher performance or for specific catalysis. We have also seen customers that developed new biocatalysts based on the mechanism we provided. Creative Enzymes have demonstrated insightful characterization of the detailed catalysis mechanism and mode of action, and is willing to help potential customers overcome the challenges during enzyme product development.

Creative Enzymes has been a pioneer in the enzymology assay area. We served customers to optimize a product or to set a solid foundation for further research. You can find any level and scale of enzymology assays available at Creative Enzymes.

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