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Abstract: 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-nitrophenyl-α-l-arabinoside, respectively. Finally, the characterization of the site-directed mutants Glu157Gly and Glu313Gly confirmed the latter as the nucleophile of the reaction and gave experimental evidence, for the first time in GH42, of the role of Glu157 as the acid/base of the catalyzed reaction. [Copyright &y& Elsevier]