STRUCTURE OF THE BETA-GALACTOSIDASE GENE FROM THERMUS SP. STRAIN T2 -EXPRESSION IN ESCHERICHIA-COLI AND PURIFICATION IN A SINGLE-STEP OF AN ACTIVE FUSION PROTEIN

The nucleotide sequence of both the bgaA gene, coding for a thermostab le beta-galactosidase of Thermus sp, strain T2, and its flanking regio ns was determined. The deduced amino acid sequence of the enzyme predi cts a polypeptide of 645 amino acids (M-r, 73,595), Comparative analys is of the open reading frames located in the flanking regions of the b gaA gene revealed that they might encode proteins involved in the tran sport and hydrolysis of sugars. The observed homology between the dedu ced amino acid sequences of BgaA and the beta-galactosidase of Bacillu s stearothermophilus allows us to classify the new enzyme within famil y 42 of glycosyl hydrolases, BgaA was overexpressed in its active form in Escherichia coli, but more interestingly, an active chimeric beta- galactosidase was constructed by fusing the BgaA protein to the cholin e-binding domain of the major pneumococcal autolysin, This chimera ill ustrates a novel approach for producing an active and thermostable hyb rid enzyme that can be purified in a single step by affinity chromatog raphy on DEAE-cellulose, retaining the catalytic properties of the nat ive enzyme. The chimeric enzyme showed a specific activity of 191,000 U/mg at 70 degrees C and a K-m value of 1.6 mM with o-nitrophenyl-beta -D-galactopyranoside as a substrate, and it retained 50% of its initia l activity after 1 h of incubation at 70 degrees C.