Add time:08/08/2019         Source:sciencedirect.com

A recombinant Rhizobium meliloti β-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 mass of 174 kDa and a subunit molecular weight of 88 kDa, indicating that it is a dimer. It was active with both synthetic substrates p-nitrophenyl β-D-galactopyranoside (PNPG) and o-nitrophenyl β-D-galactopyranoside (ONPG) with KmPNPG and KmONPG of 1 mM at 25°C. The kcat/Km ratios for both substrates were approximately 70 mM−1 sec−1, indicating no clear preference for either PNPG or ONPG, unlike E. coli β-galactosidase. After non-denaturing electrophoresis, active β-galactosidase bands were identified using 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal) or 6-bromo-2-naphthyl β-D-galactopyranoside (BNG) and diazo blue B.

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