title: Purification and analysis of an extremely halophilic beta-galactosidase from Haloferax alicantei.
abstract: As a first step in the development of a reporter system for gene expression in halophilic archaea, a beta-galactosidase was purified 140-fold from Haloferax alicantei (previously phenon K, strain Aa2.2). An overproducing mutant was first isolated by UV mutagenesis and screening on agar plates containing X-Gal substrate. Cytoplasmic extracts of the mutant contained 25-fold higher enzyme levels than the parent. Purification of the active enzyme was greatly facilitated by the ability of sorbitol to stabilise enzyme activity in the absence of salt, which allowed conventional purification methods (e.g., ion-exchange chromatography) to be utilised. The enzyme was optimally active at 4 M NaCl and was estimated to be 180 +/- 20 kDa in size, consisting of two monomers (each 78 +/- 3 kDa). It cleaves several different beta-galactoside substrates such as ONP-Gal, X-Gal and lactulose, but not lactose, and also has beta-D-fucosidase activity. No beta-glucosidase, beta-arabinosidase or beta-xylosidase activity could be detected. The amino-acid sequence at the N-terminus and of four proteolytic products has been determined.
authors.affiliation: Department of Microbiology, University of Melbourne, Parkville, Australia.
authors.forename: M L
authors.initials: ML
authors.lastname: Holmes
authors.forename: R K
authors.initials: RK
authors.lastname: Scopes
authors.forename: R L
authors.initials: RL
authors.lastname: Moritz
authors.forename: R J
authors.initials: RJ
authors.lastname: Simpson
authors.forename: C
authors.initials: C
authors.lastname: Englert
authors.forename: F
authors.initials: F
authors.lastname: Pfeifer
authors.forename: M L
authors.initials: ML
authors.lastname: Dyall-Smith
mesh_terms: D000595:Amino Acid Sequence; D004795:Enzyme Stability; D005798:Genes, Bacterial; D017930:Genes, Reporter; D006216:Halobacteriaceae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D001616:beta-Galactosidase