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Figure 2 | BMC Research Notes

Figure 2

From: Applying neutral drift to the directed molecular evolution of a β-glucuronidase into a β-galactosidase: Two different evolutionary pathways lead to the same variant

Figure 2

Kinetic parameters for selected variants. Names of the variants are as given in Table 1. Glucuronidase activity is shown with black bars, galactosidase activity with grey bars. Error bars are the standard errors from fits of the data to a Michaelis-Menten model. Variant glucuronidases were overexpressed from pBAD derived plasmids in BW25141 cells. The cells were lysed and the protein purified by Ni-NTA sepharose FF chromatography followed by concentration and buffer exchange by ultra filtration. Protein concentration was estimated by the method of Bradford [29]. Enzyme activity was quantified using the substrates p-nitrophenyl-β-D-galactopyranoside (pNP-gal) and p-nitrophenyl-β-D-glucuronide (pNP-glu) in 50 mM Tris HCl buffer pH 7.4. The absorbance at 405 nm was monitored using a Safire2 microplate reader (Tecan). An extinction coefficient of 17 400 M-1 cm-1 was used to calculate p-nitrophenyl-phosphate concentration. The concentration of protein used ranged from 0 to 200 nM in the glucuronidase assays and from 500 nM to 5 μM in the galactosidase assays. The concentration of pNP-gal ranged from 100 μM to 15 mM and that of pNP-glu varied between 10 μM and 5 mM.

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