M. Leahy et al., Purification and some characteristics of a recombinant dimeric Rhizobium melioloti beta-galactosidase expressed in Escherichia coli, ENZYME MICR, 28(7-8), 2001, pp. 682-688
A recombinant Rhixobium meliloti beta -galactosidase was purified to homoge
neity from an Escherichia coli expression system. The gene for the enzyme w
as 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 combi
nation 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 mos
s 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-g
alactopyranoside (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 clea
r preference for either PNPG or ONPG, unlike E. coli beta -galactosidase. A
fter non-denaturing electrophoresis, active beta -galactosidase bands were
identified using 5-bromo-4-chloro-3-indolyl beta -D-galactopyranoside (X-ga
l-) or 6-homo-2-naphthyl beta -D-galactopyranoside (BNG) and diazo blue B.
(C) 2001 Elsevier Science Inc. All rights reserved.