Single molecule assays of beta-galactosidase from two wild-type strains of E. coli: effects of protease inhibitors on microheterogeneity and different relative activities with differing substrates.

Beta-galactosidase was induced in E. coli wild type strains ATCC 8677 and 35321 in the presence of various protease inhibitors. Single enzyme molecule assays were performed using a capillary electrophoresis based protocol. The presence of the protease inhibitors had a minimal effect on the average and distribution of single molecule activities. Two novel capillary electrophoresis based single enzyme molecule assays for beta-galactosidase were developed using DDAO-beta-D-galactopyranoside and fluorescein-beta-D-digalactopyranoside as substrates. Double incubations were performed on individual enzyme molecules to demonstrate the reproducibility of the assays. Assays performed on beta-galactosidase from strains 8677 and 35321 demonstrated that the relative activities of the enzyme for the different substrates differed between the strains. Sequencing showed that these two strains differ in their primary sequence by a single amino acid substitution in position 280, which is in the region of the active site.