Measuring ß-galactosidase activity in opaque dairy solutions under optimum conditions for galactooligosaccharide synthesis by isothermal titration calorimetry.

The dairy industry uses enzymes to make cheese, alter product flavor, and eliminate lactose. The activities of these enzymes have been measured in clear buffered solutions, but because of the limitations of spectrophotometric methods, enzyme activities have not been measured in opaque or colored dairy products where they are used. Isothermal titration calorimetry (ITC) can be used to determine reaction kinetics in opaque and colored solutions by measuring the heat rate (thermal power) from enzyme-catalyzed reactions as a function of time. This study used ITC to measure ß-galactosidase activity in opaque solutions of milk, sweet whey, sweet whey permeate, acid whey, and acid whey permeate with 2 ß-galactosidase (Enzyme Commission number 3.2.1.23) isozymes derived from Aspergillus oryzae and Kluyveromyces lactis. The components of the dairy fluids alter the enzyme kinetics and reaction thermodynamics, and the reactions catalyzed by the 2 homologues differ as shown by differing thermodynamic profiles. The study demonstrates that ITC can be used to measure enzyme activity in opaque and colored dairy fluids and identify reactions by their thermodynamic properties.

Activity, stability, and binding capacity of ß-galactosidase immobilized on electrospun nylon-6 fiber membrane.

In this research, we explored various immobilized enzyme support materials, including the novel nylon-6 fiber membrane (NFM), and evaluated the increase in surface area and its effect on enzyme binding potential. We also manipulated incubation and reaction conditions and assessed the subsequent effects on activity and stability of ß-galactosidase, with comparisons between various solid support materials and free (dissolved) enzyme. Nylon-6 fiber membranes were created by electrospinning and were compared with other materials as solid supports for enzyme binding. The other materials included polyvinylidene fluoride 5-kDa nanofiltration dairy membranes, nylon-6 pellets, and silica glass beads. Scanning electron microscopy revealed the large surface area of NFM, which correlated with greater enzyme activity compared with the relatively flatter surfaces of the other solid support materials. Enzyme activity was measured spectrophotometrically with the color-changing substrate o-nitrophenyl-ß-d-galactopyranoside. Compared with the other solid supports, NFM had greater maximum enzyme binding potential. Across pH conditions ranging from 3.5 to 6.0 (including the optimal pH of 4.0-5.0), enzyme activity was maintained on the membrane-immobilized samples, whereas free enzyme did not maintain activity. Altering the storage temperature (4, 22, and 50°C) affected enzyme stability (i.e., the ability of the enzyme to maintain activity over time) of free and polyvinylidene fluoride membrane samples. However, NFM samples maintained stability across the varying storage temperatures. Increasing the immobilization solution enzyme concentration above the maximum enzyme binding capacity had no significant effect on enzyme stability for membrane-immobilized samples; however, both had lower mean stability than free enzyme by approximately 74%. With further development, ß-galactosidase immobilized on NFM or other membranes could be used in continuous processing in the dairy industry for a combination of filtration and lactose hydrolysis-creating products that are reduced in lactose and increased in sweetness, with no requirement for "added sugars" on the nutrition label and no enzyme listed as final product ingredient.