title: Strategy for purification of aggregation prone β-glucosidases from the cell wall of yeast: a preparative scale approach.
abstract: Purification of biotechnologically important proteins is of vital interest to the biotech industry. β-Glucosidases, belonging to Family 1 and Family 3 of the glycosylhydrolases, have varied applications as carbohydrate hydrolyzing and synthesizing enzymes. Obtaining high quantities of these enzymes is important for exploring their biosynthetic potential, structural information and catalytic activities. Classical methods for their preparation fail to deliver high yields because of adoption of several/hydroxyapatite chromatography steps. We report here a preparative method for purification of large quantities of two closely related cell bound β-glucosidases (BGL I and BGL II) from Pichia etchellsii that belong to Family 3 glycosylhydrolases. A combination of ion-exchange and gel filtration chromatography was used to process milligram quantities of protein with recoveries of up to 53%. A simple affinity based separation resulted in resolution of BGL I and BGL II with high recovery and high specific activities of 74IU/mg and 32IU/mg protein respectively. Peptide sequences of BGL II indicated it to be a novel member of Family 3. Methods reported here present a successful strategy for obtaining large quantities of these enzymes.
authors.affiliation: Structural Biology, Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India.
authors.forename: Mohammad Asif
authors.initials: MA
authors.lastname: Shah
authors.forename: Tapan Kumar
authors.initials: TK
authors.lastname: Chaudhuri
authors.forename: Saroj
authors.initials: S
authors.lastname: Mishra
mesh_terms: D002850:Chromatography, Gel / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D010843:Pichia / Q000201:enzymology* / Q000254:growth & development; D001617:beta-Glucosidase / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D005656:Fungal Proteins; D001617:beta-Glucosidase