Measuring Enzymatic Stability by Isothermal Titration Calorimetry.

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.

Calorimetric Methods for Measuring Stability and Reusability of Membrane Immobilized Enzymes.

The aim of this work is to develop calorimetric methods for characterizing the activity and stability of membrane immobilized enzymes. Invertase immobilized on a nylon-6 nanofiber membrane is used as a test case. The stability of both immobilized and free invertase activity was measured by spectrophotometry and isothermal titration calorimetry (ITC). Differential scanning calorimetry was used to measure the thermal stability of the structure and areal concentration of invertase on the membrane. This is the 1st demonstration that ITC can be used to determine activity and stability of an enzyme immobilized on a membrane. ITC and spectrophotometry show maximum activity of free and immobilized invertase at pH 4.5 and 45 to 55 °C. ITC determination of the activity as a function of temperature over an 8-h period shows a similar decline of activity of both free and immobilized invertase at 55 °C. PRACTICAL APPLICATION: Enzyme-catalyzed reactions occur in mild and environmentally friendly conditions, but are usually too costly to use in food manufacturing. When free enzymes are used, they are used once and replaced for each reaction, but enzymes immobilized on a solid support can be reused and have the additional advantage of being removed from the product. In this study, new calorimetric methods that are universally applicable to characterizing immobilized enzymes are used to determine the activity, stability, and reusability of invertase immobilized on a nanofiber support.

Hyphenation of proton transfer reaction mass spectrometry with thermal analysis for monitoring the thermal degradation of retinyl acetate.

RATIONALE: The processing of retinyl acetate, a vitamin and biomarker, at high temperatures causes significant decomposition of the compound and thus loss of its activity. The rate of mass loss can be conveniently studied by thermogravimetry (TG). However, this technique generally fails to reveal which compounds have evolved from the compound. In this work we propose a new hyphenation approach to continuously monitor the thermal decomposition of retinyl acetate and follow the evolution of specific volatile organic compounds (VOCs). METHODS: Thermal degradation of retinyl acetate was followed by TG coupled to a direct injection mass spectrometer based on proton transfer reaction mass spectrometry (PTR-MS) to follow continuously the thermal decomposition of retinyl acetate. The results were also compared with those obtained by a second evolved gas analysis system based on the coupling of TG with FTIR. RESULTS: The TG results showed two main mass losses, at 180°C and 350°C. When the PTR-MS instrument was connected to the outlet of the TG instrument, specific fragment ions (m/z 43, 61, 75, 85 and 97) showed characteristic evolution profiles. The first mass loss was mainly associated with the release of acetic acid (m/z 43 and 61), whereas the second mass loss was connected with the degradation of the molecule backbone (m/z 43, 61, 75, 85 and 97). These results were substantially correlated with those achieved by TG coupled with FTIR, although PTR-MS showed superior performance in terms of the qualitative identification of specific fragments and better sensitivity toward complex organic VOCs. CONCLUSIONS: The proposed TG-PTR-MS technique shows a great potential for following in real time the thermal degradation of ingredients such as retinyl acetate and identifying compounds evolved at specific temperatures.

Enhancement of the bioactive compound content in strawberry fruits grown under iron and phosphorus deficiency.

BACKGROUND: Among berries, strawberry fruits are one of the richest natural sources of health-beneficial components such as micronutrients, antioxidants and phytochemicals. Strawberry quality depends greatly upon genotype, environmental factors, cultivation techniques and nutrient supply. This study aimed to assess the influence of phosphorus and iron deficiency on the bioactive compound content in strawberry fruits grown under hydroponic conditions. RESULTS: Different nutrient supplies clearly influenced the qualitative parameters of strawberry fruits. Principal component analysis (PCA) showed that three homogeneous clusters could be identified. The three treatments (control, iron deficiency and phosphorus deficiency) differed especially because of their phenolic compounds and antioxidant potential, the strawberry fruits grown under Fe and P deficiency being richer in pelargonidin-3-glucoside, benzoic acids and flavonols than the control fruits. CONCLUSION: Nutrient deficiency had a positive effect on nutritional parameters of strawberry fruits without impairing fruit yield and quality parameters such as acidity, firmness and total soluble solid content. The shaping of nutrient availability in the growing medium could thus be of help in producing an edible yield with the desired qualitative aspects and nutritional value.