Isolation and properties of free and immobilized β‐galactosidase from the psychrotrophic enterobacterium Buttiauxella agrestis (strain NC4)

Abstract

A study of the beta-galactosidase produced by the psychrotrophic bacterium Buttiauxella agrestis has been carried out. This micro-organism was isolated from raw milk and the enzyme isolated using standard methods. Molecular mass was estimated to be 515 kDa. The isoelectric point was close to 4.45. Optimum pH was 7.25. Maximal activity was observed at 50 degrees C and activation energy was estimated to be 39.1 kJ mol-1. Lactose enhanced thermal stability. Using p-nitrophenyl-beta-D-galactopyranoside as the substrate, the Km was 11 mumol l-1 and Vmax was 85 U mg-1 protein. beta-Mercaptoethanol and ethanol were inhibitors; glycerol acted as a complex effector. The enzyme required divalent cations for activity while it was inhibited by EDTA. When the enzyme was immobilized in diethyl aminoethylcellulose the optimum pH of activity was 8. Km was 47 mumol l-1 and Vmax was 96 U mg-1 protein.

Effect of chlorination on β-D-galactosidase activity of sewage bacteria and Escherichia coli

“…However, without NB in the assay medium the enzyme activity of sewage samples did not increase. These results indicate that the NB supported an enzyme activator and enhanced thermal stability of b-D-galactosidase, as previously shown for b-D-galactosidase from the psychrotrophic enterobacterium Buttiauxella agrestis (Amarita et al 1995). Lack of increase in sewage enzyme activity when temperature was increased may be explained by the presence of b-D-galactosidases that were unstable without activators and stabilizers in the assay medium (Wallenfels and Weil 1972 ; Amarita et al 1995), whereas components of the NB increased the thermal stability of the enzyme.…”

“…These results indicate that the NB supported an enzyme activator and enhanced thermal stability of b-D-galactosidase, as previously shown for b-D-galactosidase from the psychrotrophic enterobacterium Buttiauxella agrestis (Amarita et al 1995). Lack of increase in sewage enzyme activity when temperature was increased may be explained by the presence of b-D-galactosidases that were unstable without activators and stabilizers in the assay medium (Wallenfels and Weil 1972 ; Amarita et al 1995), whereas components of the NB increased the thermal stability of the enzyme. Furthermore, different target and non-target bacteria in sewage may have different b-D-galactosidase temperature characteristics and the temperature optimization curve of sewage presented in this study represented the combined temperature dependencies of the different species.…”