β-Galactosidase isolated from Ranunculus arvensis seeds to synthesize trisaccharide: Kinetics and thermodynamic properties

• Published in: Food bioscience 2024-06, Vol.59, p.103943, Article 103943
• Main Authors: Alghamdi, Suad A., Rehman, Khalil ur, Zaman, Umber, Alshareef, Sohad Abdulkaleg, Zghab, Imen, Alanazi, Amal N., Nasr, Samia, Khan, Shahid Ullah, Alissa, Mohammed, Alqasem, Abdullah A.
• Format: Article
• Language: English
• Subjects: Galacto-oligosaccharide; Lactose hydrolysis; Transgalactosylation
• ISSN: 2212-4292
• DOI: 10.1016/j.fbio.2024.103943

β-Galactosidase was isolated from Ranunculus arvensis seeds using DEAE-cellulose, Sephadex G-100, and Con A sepharose-4B chromatography. The enzyme was purified to electrophoretic homogeneity with a specific activity of 50 U/mg of protein and a yield of 7.1%. The molecular mass of the isolated β-galactosidase, as estimated by SDS-PAGE, was 18 kDa, indicating that it was a monomeric form The purified β-galactosidase has a glycoproteinic nature when applied to Con-A-Sepharose-4B chromatography. An activation energy (Ea) of 11 kcal/mol of lactose, pH 5.0, and 50 °C were found to be the optimum parameters to purify β-galactosidase from R. arvensis seeds. The residual activity test was carried at 55–75 °C, allowing calculating the half-lives of 533–48 min, enthalpy (ΔH° = 110.38–110.21 kJ/mol), free energy (ΔG° = 109.88–109.77 kJ/mol), and entropy (ΔS° = 1.52–1.26 J/mol·K). The β-galactosidase produced from this species is better than the previously described enzyme due to its kinetic and thermodynamic properties, and it could be used in various industrial applications. Purified β-galactosidase, when incubated with high lactose concentration, showed transgalactosylation activity, leading to trisaccharides as a major product of total galactooligosaccharide (GOS). Therefore, the purified β-galactosidase could be used as a potential alternative in the food industry and would be further explained for trisaccharide synthesis. Ranunculus arvensis β-galactosidase: insights into its purification, kinetics, thermodynamic characterization, and trisaccharide synthesis. [Display omitted] •β-Galactosidase from Ranunculus arvensis was purified 185 fold with 7.1% yield.•The enzyme was found thermostable upto 50 °C.•The maximum turnover Vmax for ortho-NPG is 96 μmol/min/mg of protein and a Km of 0.4 mM.•β-Galactosidase is used for the synthesis of galacto-oligosaccharides.•It is a highly thermophilic enzyme that could be suitable for biotechnological applications.