Cloning and analysis of the β-galactosidase-encoding gene from Clostridium thermosulfurogenes EM1

Abstract

Clostridium thermosulfurogenes EM1 produced a thermostable (up to 70 °C) β-galactosidase (βGal) with a pH optimum of 7 during growth on lactose. The gene ( lacZ) encoding this enzyme was cloned and expressed in Escherichia coli using pUC18 as a vector. The nucleotide sequence of a 2.7-kb PstI fragment carrying the lacZ gene was determined. The open reading frame for lacZ, which encoded a protein of 716 amino acids with a calculated M r of 83 728, was confirmed by the identity of its deduced aa sequence with the chemically determined N-terminal aa sequence of the purified βGal of C. thermosulfurogenes EM1. The structural gene was preceded by a possible promoter sequence, 5'-TTGTAG (-35), 5'-TAATAT (-10); and a ribosome-binding site, 5'-AGGAGG. The cloned βGal was found to be indistinguishable from the native enzyme. The M r of the active βGal was 170000, as determined by Superose 12HR gel filtration and gradient gel electrophoresis. This indicated that this enzyme is composed of two identical subunits. Comparison of the aa sequences of different βGal revealed that five large regions of similarity with the enzymes from E. coli ( lacZ, ebgA), Klebsiella pneumoniae ( lacZ), and Lactobacillus bulgaricus are present in the βGal of C. thermosulfurogenes EM1 and that the putative active site residues (Glu 461 and Tyr 503 in the E. coli lac Z-encoded βGal) are conserved (Glu 389 and Tyr 429). Therefore, the thermostable βGal of C. thermosulfurogenes EM1 is more closely related to the enzyme of E. coli than to the likewise thermostable one of Bacillus stearothermophilus. The lacZ gene of C. thermosulfurogenes might be useful as a reporter gene in genetic systems where a low G + C content of the gene or thermostability of the enzyme is desirable.