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The thermoacidophilic bacterium Alicyclobacillus acidocaldarius is a rich source of glycoside hydrolases enabling its growth on several di- and polysaccharides. We report here the purification and the characterization of a β-galactosidase from this source, the cloning of its gene, and the expression and the characterization of the recombinant enzyme (Aaβ-gal).The enzyme was purified 46-fold from A. acidocaldarius extracts; the gene for Aaβ-gal encoded a new member of the glycoside hydrolase family 42 (GH42) and it is flanked by a putative AraC/XylS regulator, however, the two genes were transcribed independently. The recombinant Aaβ-gal was characterized in detail revealing that it is optimally active and stable at 65 °C. Aaβ-gal is very specific for glycosides with an axial C4-OH at their non-reducing end, with k_cat/K_M values of 484, 186, and 332 s⁻¹ mM⁻¹ for 2-nitrophenyl-β-d-galactoside, -fucoside, and 4 …