In vivo assay to identify bacteria with β-glucosidase activity

Abstract

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.