Effect of phenolic compounds on the rapid direct enzymatic detection of β-D-galactosidase and β-D-glucuronidase

Abstract

For the detection of indicator bacteria such as total coliforms or Escherichia coli (E. coli), microbial enzyme profiles are a preferred option compared to classical methods, because the reactions are more sensitive and rapid to perform and bacteria can be detected and enumerated through specific enzyme activities (Romprẻ et al., 2002). Chromogenic or fluorogenic enzyme substrates are used to detect the enzymes b-D-galactosidase (β-GAL) and b-D-glucuronidase (β-GUD). The chromogenic enzyme substrates are phenol-based, for example the enzyme substrates for β-GUD are p-nitrophenyl-b-D-glucuronide (PNPG) and 5-bromo-4-chloro-3-indolyl-b-D-glucuronide (XGLU), where PNPG produces ρ-nitrophenol (a yellow colour) and XGLU produces a blue indoxyl product. The fluorogenic substrate used to detect β-GUD is 4-methylumbelliferyl-b- D-glucuronide (MUG) (Manafi, 1996; 2000). β-GAL is detected by using the chromogenic substrates chlorophenol red- β-galactopyranoside (CPRG) and o-nitrophenyl-b-D-galactopyranoside (ONPG) (Edberg et al., 1988; Cheng et al., 2002). As phenolic compounds have extensive industrial applications, they are often found to be present in the aquatic environment (Llompart et al., 2002; Asan and Isildak, 2003). Several
major sources are responsible for the presence of these phenolic compounds in the environment, e.g. pesticides, bactericides, wood preservatives and dyes. Phenolic compounds are also present in pulp processing, petroleum refining, leather tannery, textiles and plastics (Lee et al., 1996; Angelino and Gennaro, 1997; Pẽnalver et al., 2002; Asan and Isildak, 2003; Lupetti et al., 2004). Furthermore, phenols may also be found in fertilizers and explosives (Aktas et al., 2006).

Keywords: coliforms, E. coli, faecal, b-D-galactosidase, b-D-glucuronidase, inhibition, wastewater