A recombinant Rhixobium meliloti beta -galactosidase was purified to homogeneity from an Escherichia coli expression system. The gene for the enzyme was cloned into a pKK223-3 plasmid which was then used to transform E. coli JM109 cells. The enzyme was purified 35-fold with a yield of 34% by a combination of DEAE-cellulose (pH 8.0) and two sequential Mono Q steps (at pH 8.0 and 6.0, respectively). The purified enzyme had an apparent molecular moss of 174 kDa and a subunit molecular weight of kDa indicating that it is a dimer. It was active with both synthetic substrates p-nitrophenyl beta -D-galactopyranoside (PNPG) and o-nitrophenyl beta -D-galactopyranoside (ONPG) with K-m(PNPG) and K-m(ONPG) of 1 mM at 25 degreesC. The k(cat)/K-m ratios for both substrates were approximately 70 mM(-1) sec(-1) indicating no clear preference for either PNPG or ONPG, unlike E. coli beta -galactosidase. After non-denaturing electrophoresis, active beta -galactosidase bands were identified using 5-bromo-4-chloro-3-indolyl beta -D-galactopyranoside (X-gal-) or 6-homo-2-naphthyl beta -D-galactopyranoside (BNG) and diazo blue B.