What is an Enzyme Assay?
An Enzyme assay aims to determine whether a particular enzyme can reduce or increase a specific chemical compound in a sample. Enzyme assays are performed using steps ranging from pre-treatment to incubation and measurement. Here are some examples of Enzyme assays. Once you have a general idea of the steps involved, it’s time to try them out for yourself.
An enzyme assay is an enzymatic test performed to measure the enzyme’s activity. It measures changes in enzyme activity as a function of time and temperature. The results can be used to derive the required value. The main factors that affect enzyme activity include temperature, pH, buffer type, and ionic strength. It is also important to maintain strict controls on these parameters. This book describes the basic requirements for enzyme assays.
Optical methods allow the measurement of changes in concentration over time. UV-Vis spectrophotometry is one such technique. The absorbance is measured continuously over time, at defined time intervals. The continuous measurements allow the experimenter to monitor the behavior of the assay as it proceeds. In this way, deviations in the reaction rate can be detected, and enzyme activity can be calculated. Enzyme assays differ in their sensitivity.
A reliable enzyme assay requires multiple steps and extensive work. The overall method development process can take four to six weeks for highly trained personnel. In addition to identifying key variables, the analysis also requires numerous samples to determine the optimal conditions of the assay. Luckily, automated solutions are available to overcome the major challenges of enzyme analysis. The main advantages are:
Enzyme assay Techniques
There are several types of enzyme assay techniques. The first type, known as spectrophotometry, measures the amount of substrate consumed by the enzyme. This measure is directly proportional to the concentration of the enzyme. Another type, known as a “continuous” enzyme assay, measures changes in the substrate or products over time. In this type, enzyme concentration is determined by comparing a single sample to the average of several samples taken over a period of time.
The second ELISA technique employs polyclonal antibodies raised against various cellulase enzymes. It has been used to quantitatively analyze the target enzymes in both culture filtrates and commercial enzyme preparations. It is sensitive enough to detect enzyme concentrations even at very low concentrations. In contrast, a high concentration of an enzyme can interfere with the assay. Moreover, the absorption profile of the cellulase monocomponents was studied using ELISA. Accellerase 1000 and Celluclast contain different cellulase enzymes, and the ELISA assay showed differences between them.
Initial-rate kinetic assays are performed by coupling the reaction of interest to a chromogenic substrate. The chromogenic substrate is monitored with a spectrophotometer, and the initial rate is approximated by fitting a tangent line to the first few data points. On the other hand, a discontinuous assay involves coupling the enzyme with a cyclical pseudo-zero order reaction. This is highly sensitive and can be automated using a robotic platform.
Enzyme assay Protocols
An enzyme assay protocol outlines the steps necessary to determine the activity of an enzyme. These steps include setting up the assay, specifying the active enzyme concentration and fraction, and preparing the buffer and inhibitor. Afterward, the protocol describes the data analysis steps. Each protocol includes a typical example experiment and possible pitfalls to avoid. For instance, you should note that it is possible to overestimate the activity of an enzyme if the concentration of the substrate is too high.
The essential requirements for an enzyme assay are discussed, and some of the common errors and pitfalls. The most important parameters that affect enzyme activity are temperature, pH, ionic strength, and the proper concentration of essential components. There are no standardized conditions for all enzymes, but many are optimum in the visible spectrum. Therefore, body temperature can be used as the assay temperature, and it’s usually between 37 and 25 degrees Celsius.
Examples of Enzymes assay
A common method to measure enzyme activity is to conduct an assay in a well-defined condition, where several variables are closely controlled. Temperature is one of these factors, which can affect results by as much as four percent. For this reason, the temperature of the assay solution is of utmost importance. Additionally, the pH and ionic strength of the buffer solution are crucial parameters to control during the assay. Therefore, this parameter is critical in the development and routine measurement of enzyme assays.
These measurements should be done in a linear range since a nonlinear behavior of an enzyme can result in inaccurate results. Nevertheless, there are some oddities to this rule. In some cases, the data points may not lie within the linear range and must be adjusted to achieve the desired results. The enzyme concentration is measured in units per volume and corresponds to how much of the substrate is converted in a minute. The reaction speed and concentration can be determined by using different assay methods.
Cellulase Enzyme assay
A cellulase enzyme assay is widely used in industry and plant culture. The enzyme degrades cellulose through hydrolysis. Among other applications, cellulases are used to produce protoplasts. Various strains of E. coli produce cellulase at varying levels. Our cellulase enzyme assay found the highest activity in the strains CBM21 and CSG8.
There are several methods of determining cellulase activity. Firstly, a viscometer can measure the change in the viscosity of a cellulose derivative solution. The decrease in viscosity is directly proportional to the cellulase activity. The only drawback to this method is that it can only determine the activity of endo-cellulase, while exo-cellulase enzymes produce little or no change in the viscosity.
To test the cellulase activity of native microbes, we first selected the colonies with the highest ratio of gram reaction to colony diameter. These were then characterized using morphology, biochemical analysis, and MIU tests. Finally, we tested the cellulase activity of cellulase-producing strains by preparing nutrient media plates where CMC was the sole carbon source.
Amylase Enzyme assay
The ELISA method has been applied to test the activity of amyloglucosidase in bacteria. It consists of two steps: assaying the amount of reducing sugars produced by a bacteria and determining its concentration—this method used amylolytic enzymes from Bacillus licheniformis and Aspergillus niger. Alternatively, amylolytic enzymes can be extracted from other sources.
For the assay, three grams of flour should be placed into a flask with 50 mL of malate buffer pH 5.4. Dilute this solution with water to make it less than half the concentration. Stir the flask vigorously in a water bath at 40 degC. The reaction will take about fifteen minutes. The enzyme activity will be proportional to the concentration of the substrate. The assay can also be carried out by measuring the kinetic mechanisms and concentration of the enzymes in a sample.
BMY1 is an example of a b-amylase enzyme. The other types are BMY7 and BMY9, and a-amylase is a subtype of the latter. The a-amylase enzymes At1g69830 and BMY7 were tested against purified GST b-amylase proteins. However, the polyclonal mouse serum recognized all three proteins. Interestingly, a-amylase levels increased during a heat shock while those of the other two decreased.
Protease Enzyme assay
To perform the assay, a 1% solution of casein (the substrate) was mixed with 50 mM of Tris-HCl buffer. The enzyme preparation was then subjected to ammonium sulphate precipitation at various saturation levels, ranging from 20 to 100%. The fractions were subsequently analyzed for protease activity and protein content. The highest activity was used for further analysis.
To measure protease activity, the assay requires four 15-mL vials, one as a blank and the other three assays for three different dilutions of the enzyme. The three dilutions are important because they allow for comparison purposes. For each set of four vials, add 5 mL of 0.65% casein solution. Let the samples equilibrate for 5 minutes. Next, add three vials containing different volumes of the enzyme solution.
Although there are a number of applications for proteases, most of them have less desirable properties. The alkaline protease, for example, is produced by a thermophilic bacterium. It requires iron and calcium to function effectively. In addition, alkaline hot springs do not significantly affect the activity of the enzyme. If the enzyme activity is low, it may not be suitable for industrial applications.
The conclusion of an enzyme assay is determined by determining the amount of active enzyme per unit of sample. Depending on the enzyme, the concentration of the sample in the assay mixture may be increased or decreased. It is also required to evaluate the impact of temperature on the enzyme’s solubility. Soluble compounds may precipitate if the solution is kept in a cold room. Another important factor in enzyme activity is the proportion of organic solvents in the assay mixture. The volume of the organic solvent should be constant between assays to ensure that the results are comparable.
In a typical enzyme assay, the concentrations of the substrates, components, and enzymes cannot be known with certainty. Nevertheless, general rules for the procedure are described in the literature. Among these, pH, ionic strength, and the concentration of essential components are described. Enzyme assays can also be complex, reversible reactions or coupled tests. There is no universally acceptable assay condition because of the diversity of enzyme features. However, body temperature can be used to estimate enzyme activity and concentration. The biological temperature is between 37 and 25 degrees Celsius.