title: An efficient two step purification and molecular characterization of beta-galactosidases from Aspergillus oryzae.
abstract: Beta-galactosidases (beta-D-galactoside-galactohydrolases (EC 3.2.1.23), lactases) are important industrial enzymes used for the hydrolysis of lactose from milk and milk whey. These enzymes are produced by different organisms and purified by multi-step procedures. The multi-step purification schemes are cost and time ineffective which can also lead to poor yield, denaturation and loss of enzymatic activity. In our study, extracellular beta-galactosidase from mutant strain Aspergillus oryzaeH26-10-7 was purified by a two step procedure, Metal-ion Affinity Chromatography (IMAC) followed by size-exclusion separation. Purified enzyme was characterized by sodium dodecyl Sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and zymographic analysis. This fungal beta-galactosidase was characterized as a protein corresponding to 113 kDa. Enzyme from mutant strain was found to have five times higher catalytic activity on the synthetic substrate o-nitrophenyl-beta-D-galactopyranoside (ONPG) compared to the wild type enzyme. Moreover, the mutant enzyme was more thermo resistant compared to the wild type. This highly important technological characteristic can be exploited in food industry. Moreover, based on the IMAC patterns of wild type and mutant enzymes, similarities in their His topography were supposed.
authors.affiliation: Laboratoire d'Interactions Moléculaires et de Technologies de Séparations (LIMTechS), Centre de Recherche de Royallieu, Université de Technologie de Compiègne (UTC), Compiègne, France.
authors.forename: Daniela
authors.initials: D
authors.lastname: Todorova-Balvay
authors.forename: Ivanka
authors.initials: I
authors.lastname: Stoilova
authors.forename: Stoyanka
authors.initials: S
authors.lastname: Gargova
authors.forename: Mookambeswaran A
authors.initials: MA
authors.lastname: Vijayalakshmi
mesh_terms: D001236:Aspergillus oryzae / Q000201:enzymology*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D009154:Mutation / Q000235:genetics; D013696:Temperature; D001616:beta-Galactosidase / Q000737:chemistry* / Q000302:isolation & purification*
chemical_list: D001616:beta-Galactosidase