Part:BBa_K2398558:Experience

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Applications of BBa_K2398558

Results

The mutations were introduced in the context of an experiment, in which it was tried to generate bete-lactamase activity in a beta-glucuronidase. Two of these mutations, T509A and D508G were already published by Matsumura et al. [1]. In the context, the plasmids were used, GUS is provided under a lac-promoter and a His-tag is attached to the C-terminus of the protein. Seven different mutants were generated. After cloning, one colony of each transformation was inoculated as preculture overnight at 37°C and 220 rpm. The main cultures were inoculated with the respective volume of the overnight cultures to a final density of 0.01 at the starting point. The inducer IPTG was added after cultures reached an OD_600nm of 0.6 - 0.8 and were afterwards incubated for 20 hours at 25°C, 220 rpm. Cells were centrifuged and. Lysates were subjected to the Ni-NTA cartridge and affinity purified throughout several washing steps. Purified proteins were obtained after elution with 500 mM Imidazole solution followed by dialysis. The protein concentration was determined using both, the photospectrometer and a Bradford assay. To determine the activity of the different mutants, an assay with p-nitrophenyl-galactopyranoside (PNPG) as substrate was set up. PNPG is a perfect substrate for GAL. The enzyme hydrolyzes the p-nitophenyl group from the galactopyranosid. The yellow color of the product can be measured at 420 nm. The enzyme was added to different concentrations of the substrate, ranging from 0.047 mM to 3 mM final conentration and the reaction was followed over time for 45 minutes at 21 °C.
With the data, generated from this assay, the kinetic parameters according to Michaelis Menten were calculated via nonlinear regression. When the enzymes were cloned and purified, kinetic Assays were performed to reveal their activity. We tested the mutants, as well as both wildtype enzymes, GUS and GAL as controls. As expected GUS showed no enzyme activity at all. No significant product formation could be measured. Among the inactive proteins were two mutants, which were previously published by Matsumura et al. <x-ref>matsumura2001vitro</x-ref>. As the published activity increase was only 1.5-2 fold and our enzyme concentration was relatively low, it is no surprise, the we couldn’t determine these activities. For the wildtype GAL we could determine a Km value of 0.3466 mM and a Kcat value 49.38 1/s.

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Figure 1: Time course experiment for the wiltype beta-galactosidase and the most promising GUS mutant, T509L. Plotted is the Product concentration at different time points (in s) against the initial substrate concentration

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Figure 1: Activity of wiltype beta-galactosidase and the most promising GUS mutant, T509L. Plotted is the Product concentration after 45 min against the initial substrate concentration. Michaelis menten parameters are indicated for both enzymes.

References

[1] Matsumura, I.; Ellington, A. D. (2001): In vitro evolution of beta-glucuronidase into a beta-galactosidase proceeds through non-specific intermediates. In: Journal of molecular biology 305 (2), S. 331–339. DOI: 10.1006/jmbi.2000.4259.

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