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The thermal stability of enzyme activity is readily measured by isothermal titration calorimetry (ITC). Most protein stability assays currently used measure protein unfolding, but do not provide information about enzymatic activity. ITC enables direct determination of the effect of enzyme modifications on the stability of enzyme activity.
This work demonstrates a new method for measuring the stability of enzyme activity by isothermal titration calorimetry (ITC). The peak heat rate observed after a single injection of the substrate solution into an enzyme solution is correlated with enzyme activity. Multiple injections of the substrate into the same enzyme solution over time show the loss of enzyme activity. The assay is autonomous, requiring very little personnel time, and is applicable to most media and enzymes.
Enzymes are proteins capable of catalyzing a wide array of organic reactions. Most enzymes function in aqueous solution at near neutral pH thus avoiding the use of harsh solvents. Because of their high selectivity, enzyme catalyzed reactions produce fewer (in some cases no byproducts) byproducts than non-selective catalysts such as acids and bases1. This is especially relevant in food manufacturing where all chemical reactions must be done so the final product is safe for human consumption. Currently, enzymes are used to produce high fructose corn syrup2, cheese3, beer4, lactose-free milk5, and other important food products. While this paper focuses on enzyme use in the food industry, there are many other uses for enzymes including in green chemistry and drug synthesis.
The utility of enzymes is limited by the stability of enzyme activity, which depends on maintaining the three-dimensional structure of the enzyme. The enzyme structure can be stabilized by modifications such as PEGylation6, immobilization on a solid support7, genetic modifications8, and formulations. Currently, enzyme stability is typically measured by differential scanning calorimetry (DSC) and endpoint enzyme activity assays9. DSC measures the temperature at which an enzyme unfolds; the higher the temperature, the more stable the structure. However, loss of activity often occurs at a lower temperature than required to unfold the enzyme or domains within the enzyme10. Therefore, DSC is not sufficient to determine whether an enzyme modification increases the stability of enzyme activity. Endpoint enzyme assays are usually time intensive, require multiple samples, and often involve a coupled colorimetric reaction that is not applicable to highly colored or opaque solutions or suspensions.
This work demonstrates a method for direct measurement of the stability of enzyme activity by isothermal titration calorimetry (ITC). ITC measures the rate of heat released or absorbed during the course of a reaction. Since nearly all reactions produce or absorb heat, ITC can be used for most enzyme-catalyzed reactions, including reactions that do not have a coupled reaction or occur in opaque media such as milk. ITC has been used for many decades to measure chemical kinetic parameters for many kinds of reactions, but the protocol presented here focuses on using ITC to measure the peak heat rate of enzyme-catalyzed reactions and demonstrates that enzyme activity is linearly correlated with the peak heat rate. ITC measurements of peak heat rates are mostly autonomous and require very little personnel time to setup and analyze.
1. Preparing samples
2. Performing the experiment
3. Setting up ITCrun
4. Analyzing data
The representative results in Figure 1 and Figure 5 show data from two enzymes, lactase and invertase. Lactase and invertase catalyze the hydrolysis of a disaccharide into two monosaccharides, endothermically and exothermically, respectively. Both enzymatic reactions were run at concentrations that precluded saturation of the enzyme.
The lactase data demonstrate how ITC data can be used to estimate enzyme stability. Four sequent...
A major advantage of the ITC enzyme stability assay described here is automation. Once all the appropriate buffers and solutions are made, the set-up time for each assay is approximately 15 min for the person doing the assay. In contrast, the conventional assays for invertase and lactase activity require about 2 h with continual involvement of the person doing the assay and many enzymatic activity assays take considerably more person-hours. In a previous publication, we have demonstrated how data from the ITC method comp...
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Name | Company | Catalog Number | Comments |
a-Lactose | Fisher Scientific | unknown (too old) | 500g |
Sodium Acetate, Anhydrous 99% min | Alfa Aesar | A13184-30 | 250g |
Lactase | MP Bio | 100780 | 5g |
Hydrocholric Acid Solution, 1N | Fisher Scientific | SA48-500 | 500mL |
Benchtop Meter- pH | VWR | 89231-622 | |
Ethanol 70% | Fisher Scientific | BP8231GAL | 1gallon |
Micro-90 | Fisher Scientific | NC024628 | 1L (cleaning solution) |
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