Background: β-Glucosidase assay is performed with purified or semipurified enzymes extracted from cell lysis.
However, in screening studies, to find bacteria with β-glucosidase activity among many tested bacteria, a fast
method without cell lysis is desirable. In that objective, we report an
in vivo β-glucosidase assay as a fast
method to find a β-glucosidase producer strain.
Results: The method consists in growing the strains for testing in a medium supplemented with the artificial
substrate
p-nitrophenyl-β-glucopyranoside (
pNPG). The presence of β-glucosidases converts the substrate to
p-nitrophenol (pNP), a molecule that can be easily measured in the supernatant spectrophotometrically at
405 nm. The assay was evaluated using two
Bifidobacterium
strains:
Bifidobacterium longum
B7254 strain that
lacks β-glucosidase activity and
Bifidobacterium pseudocatenulatum
B7003 strain that shows β-glucosidase
activity. The addition of sodium carbonate during
pNP measurement increases the sensitivity of
pNP detection
and avoids the masking of absorbance by the culture medium. Furthermore, we show that
pNP is a stable
enzymatic product, not metabolized by bacteria, but with an inhibitory effect on cell growth. The
β-glucosidase activity was measured as units of enzyme per gram per minute per dry cell weight. This method
also allowed the identification of
Lactobacillus
strains with higher β-glucosidase activity among several
lactobacillus species.
Conclusion: This
in vivo β-glucosidase assay can be used as an enzymatic test on living cells without cell
disruption. The method is simple, quantitative, and recommended, especially in studies screening for bacteria
not only with β-glucosidase activity but also with high β-glucosidase activity.
