Hydrolysis of the soluble fluorescent molecule carboxyumbelliferyl-beta- d-glucuronide by E. coli beta-glucuronidase as applied in a rugged, in situ optical sensor

Abstract

Techniques utilizing β-glucuronidase (GUS) activity as an indicator of Escherichia coli ( E. coli) presence use labeled glucuronides to produce optical signals. Carboxyumbelliferyl-β- d-glucuronide (CUGlcU) is a fluorescent labeled glucuronide that is soluble and highly fluorescent at natural water pHs and temperatures and, therefore, may be an ideal reagent for use in an in situ optical sensor. This paper reports for the first time the Michaelis–Menten kinetic parameters for the binding of E. coli GUS with CUGlcU as K m = 910 μM, V max = 41.0 μM min −1, V max/ K m 45.0 μmol L −1 min −1, the optimal pH as 6.5 ± 1.0, optimal temperature as 38 °C, and the Gibb's free energy of activation as 61.40 kJ mol −1. Additionally, it was found CUGlcU hydrolysis is not significantly affected by heavy solvents suggesting proton transfer and solvent addition that occur during hydrolysis are not limiting steps. Comparison studies were made with the more common fluorescent molecule methylumbelliferyl-β- d-glucuronide (MUGlcU). Experiments showed GUS preferentially binds to MUGlcU in comparison to CUGlcU. CUGlcU was also demonstrated in a prototype optical sensor for the detection of E. coli. Initial bench testing of the sensor produced detection of low concentrations of E. coli (1.00 × 10 3 CFU/100 mL) in 230 ± 15.1 min and high concentrations (1.05 × 10 5 CFU/100 mL) in 8.00 ± 1.01 min.