Isolation of a β-Galactosidase-Encoding Gene from Bacillus licheniformis: Purification and Characterization of the Recombinant Enzyme Expressed in Escherichia coli

Abstract.

The Bacillus licheniformisβ-galactosidase gene, lacBl, was cloned on a 5.8-kb HindIII fragment into pBR322 and expressed by its own promoter in Escherichia coli. Deletion and complementation analysis showed that the enzyme-encoding region was located on a 4.1-kb HindIII-ClaI fragment. The transcription region for the lacBl was identified on this fragment with promoter- and terminator-probe plasmids. The deduced sequence of 149 aa of the N-terminal part of lacBl showed aa sequence homology with β-Gal from B. stearothermophilus, B. circulans, Haloferax alicantei, Clostridium perfringens, Arthrobacter sp.. No significant homology was shared with those found in the lacZ and lacS families. The recombinant β-galactosidase (LacB1) was purified by FPLC. The molecular mass of the enzyme (80 kDa) and its optimal pH (5.7) and temperature (45°C) were determined.