Investigating thermal stability based on the structural changes of lactase enzyme by several orthogonal methods

• Main Authors: Márton Király, Borbála Dalmadi Kiss, Péter Horváth, László Drahos, Arash Mirzahosseini, Gyula Pálfy, István Antal, Krisztina Ludányi
• Format: Article
• Language: English
• Published: Elsevier 2021-06-01
• Series: Biotechnology Reports
• Subjects: NMR; Proteomics; β-Galactosidase; Aggregation; Mass spectrometry
• Online Access: http://www.sciencedirect.com/science/article/pii/S2215017X21000539

Thermal stability of lactase (β-galactosidase) enzyme has been studied by a variety of physico-chemical methods. β-galactosidase is the main active ingredient of medications for lactose intolerance. It is typically produced industrially by the Aspergillus oryzae filamentous fungus. Lactase was used as a model to help understand thermal stability of enzyme-type biopharmaceuticals. Enzyme activity (hydrolyzation of lactose) of β-galactosidase was determined after storing the solid enzyme substance at various temperatures. For a better understanding of the relationship between structure and activity changes we determined the mass and size of the molecules with gel electrophoresis and dynamic light scattering and detected aggregation processes. A bottom-up proteomic procedure was used to determine the primary amino acid sequence and to investigate changes in the N-glycosylation pattern of the protein. NMR and CD spectroscopic methods were used to observe changes in higher order structures and to reveal relationships between structural and functional changes.