Table 1.
Purification table and kinetic parameters for hydrolysis of p-nitrophenyl esters by recombinant AxeA and AxeB.
| Enzyme | Fraction | Proteina(Mg) | Activityb(U) | Spec Activity (U mg−1) | Purification fold | Yield (%) | KM(mM) | Vmax(U mg−1) | kcatc(s−1) | kcat/KM(s−1/mM−1) |
|---|---|---|---|---|---|---|---|---|---|---|
| AxeA | Crude | 33 (±0.83) | 341 (±2.7) | 10.4 | 1.0 | 100 | ND | ND | ND | ND |
| Purified | 7.2 (0.1) | 566 (±3.7) | 80.9 | 7.7 | 62.5 | 0.1 | 28.8 | 4.5×10−11 | 4.28E−10 | |
| AxeB | Crude | 105 (±0.4) | 209 (±2.6) | 1.9 | 1.0 | 100 | ND | ND | ND | ND |
| Purified | 55 (±0.5) | 544 (±5.7) | 9.98 | 5.25 | 38.5 | 0.23 | 12.78 | 1.82×10−11 | 7.94E−11 |
a
Protein concentration was estimated using Bradford Assay.
b
Activity was assayed using p-nitrophenyl acetate as a substrate.
c
Kcat was calculated assuming (i) a molecular weight of 40 kDa for both AxeA and AxeB as estimated by SDS-PAGE analysis (The calculation also assumed a single active site per monomeric protein), Final Enzyme amount in all the kinetic assays=3 μg.