Purification and characterization of α-galactosidase isolated from Klebsiella pneumoniae in the human oral cavity

• Published in: International journal of biological macromolecules Vol. 261; no. Pt 1; p. 129550
• Main Authors: Abdul Kareem, Zainab G., Yasser Al-Zamily, Oda M., Al-Khafaji, Noor S.K.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.03.2024
• Subjects: Affinity chromatography; alpha-Galactosidase - chemistry; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Enzymes purification; Humans; Hydrogen-Ion Concentration; Ion exchange chromatography; Kinetic studies; Kinetics; Klebsiella pneumoniae; Klebsiella pneumoniae - metabolism; Molecular Weight; Renal Dialysis; Temperature; α-Galactosidase; α-Galactosidase; Enzymes purification; Affinity chromatography; Klebsiella pneumoniae; Kinetic studies; Ion exchange chromatography
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2024.129550

The enzyme α-Galactosidase (α-D-galactoside galactohydrolase [EC 3.2.1.22]) is an exoglycosidase that hydrolyzes the terminal α-galactosyl moieties of glycolipids and glycoproteins. It is ubiquitous in nature and possesses extensive applications in the food, pharma, and biotechnology industries. The present study aimed to purify α-galactosidase from Klebsiella pneumoniae, a bacterium isolated from the human oral cavity. The purification steps involved ammonium sulfate precipitation (70 %), dialysis, ion exchange chromatography using a DEAE-cellulose column, and affinity monolith chromatography. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis was used to determine the molecular weight of the purified enzyme. The kinetic constants, Michaelis constant (Km) and maximal velocity (Vmax), for this enzyme were determined by using p-nitrophenyl-α-D-galactopyranoside as substrate. The results showed that the purification fold, specific activity, and yield were 126.52, 138.58 units/mg, and 21.5 %, respectively. The SDS-PAGE showed that the molecular weight of the purified enzyme was 75 kDa. The optimum pH and temperature of the purified α-galactosidase were detected at pH 6.0 and 50 °C, respectively. The kinetic constants, Michaelis constant (Km) and maximal velocity (Vmax), for this enzyme were 4.6 mM and 769.23 U/ml, respectively. α-galactosidase from Klebsiella pneumoniae was purified and characterized. (SDS-PAGE) analysis showed that the purified enzyme appeared as single band with a molecular weight of 75 kDa.