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3.7.3.5 Production of β-galactosidase
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Chapter III Material and methods
The o-nitrophenyl-β-D-galactopyranoside (ONPG) substrate was used to determine β-
galactosidase activity as described by Zárate et al. (2000), with modifications as described
below. The strains which were able to grow in M17 medium (Merck) (which contains lactose
as only carbon source), were harvested by centrifugation and washed twice in phosphate
buffered saline (PBS) at pH 7.4. Strains growing on M17 were thought to have β-
galactosidase activity, which enables utilization of lactose and hence growth on this type of
medium. The samples were adjusted to an A580 nm of 1.0. Aliquots (100 μl) of each of the
bacterial suspensions were incubated in the presence of 2 mM ONPG for 40 min in a water
bath at 37°C. The reaction was terminated by addition of 1 ml 0.25 M Na2CO3. The samples
were centrifuged at 7,200 x g for 10 min at 4°C and the supernatants were recovered to
measure the absorbance at 420 nm. A standard curve was obtained with o-nitro-phenol (ONP,
Sigma) (concentrations of 0.05 to 0.5 μmol/ml in 0.05 μmol/ml increments). In order to
compare the activity of those strains able to hydrolyze ONPG, cell-free extracts were
prepared by disruption using a French pressure cell (SLM Aminco, SLM Instruments Inc.,
Lorch, Germany). Briefly, 10 ml of overnight cultures grown in M17 broth were harvested by
centrifugation (7,200 × g at 4°C for 10 min) washed twice with buffer KH2PO4/Na2HPO4 (50
mM, pH 7.25) and resuspended in the same buffer.
The cell suspension was passed through the French pressure cell at 1,000 psi for at
least two times. Cell debris was separated by centrifugation at 10,000 × g at 4°C for 10 min.
Cell extracts were kept on ice until incubation with the substrate as previously described.
Protein contents were determined by the method of Bradford (1976), using bovine serum
albumin (Roche, Mannheim) as standard. One enzymatic unit was defined as the micromoles
of ONP liberated from ONPG per milligram of protein and per min.