Table 2.
Purification steps of LacA-WT and mutants from JM109(DE3)
| Mutant LacA | Steps of purification | Total activity (U) | Total protein (mg) | Specific activity (U/mg) | Purification factor | Yield (%) |
|---|---|---|---|---|---|---|
| WT | Crude β-galactosidase | 5.13 | 9.04 | 0.57 | 1.0 | 100 |
| Probond™resin | 4.12 | 1.66 | 2.48 | 4.53 | 80.49 | |
| E62V | Crude β-galactosidase | 4.53 | 8.18 | 0.55 | 1.0 | 100 |
| Probond™resin | 3.81 | 1.59 | 2.39 | 4.35 | 84.1 | |
| R77W | Crude β-galactosidase | 5.02 | 9.25 | 0.54 | 1.0 | 100 |
| Probond™resin | 3.56 | 1.39 | 2.56 | 4.74 | 71 | |
| A191V | Crude β-galactosidase | 4.32 | 8.72 | 0.50 | 1.0 | 100 |
| Probond™resin | 3.54 | 1.42 | 2.49 | 4.98 | 81.94 | |
| A301V | Crude β-galactosidase | 1.15 | 8.66 | 0.13 | 1.0 | 100 |
| Probond™resin | 0.89 | 1.51 | 0.59 | 4.54 | 77.1 | |
| F361Y | Crude β-galactosidase | 2.06 | 9.11 | 0.24 | 1.0 | 100 |
| Probond™resin | 1.72 | 1.55 | 1.11 | 4.63 | 83.5 | |
| A524T | Crude β-galactosidase | 5.11 | 8.25 | 0.62 | 1.0 | 100 |
| Probond™resin | 4.1 | 1.62 | 2.53 | 4.08 | 80.23 |
Wild-type and mutants of LacA were purified to homogeneityby affinity chromatography Ni2+ ProBond™ resin. The relative activity of β-galactosidase was determined in 100 mM buffer Z (pH 7.0) at 55 °C with substrate oNPG (4 mg/ml)