Table 1.
Apparent Michaelis-Menten kinetic constants for hydrolysis and transglycosylation reactions catalyzed by wild-type VvEG16 and VvEG16(ΔV152).
| Substrate | Enzyme |
kcat,
app (min−1) |
Km,
app (mM) |
kcat,
app/Km,
app (M−1s−1) |
Assay |
|---|---|---|---|---|---|
| bMLG | VvEG16(ΔV152) | 143±7 | 0.34±0.03* | 7.00* | BCA |
| VvEG16 | 163±5 | 0.36±0.02* | 7.55* | BCA | |
| tXyG | VvEG16(ΔV152) | 68±8 | 1.8±0.4* | 0.63* | BCA |
| VvEG16 | 70±6 | 2.1±0.4 | 0.56* | BCA | |
| HEC | VvEG16(ΔV152) | 10.4±0.8 | 1.1±0.2* | 0.16* | BCA |
| CMC | VvEG16(ΔV152) | 17±2.2 | 2.8±0.5* | 0.10* | BCA |
| kGM | VvEG16(ΔV152) | ND | ND | 0.040±5* | BCA |
| Guar Galactomannan | VvEG16(ΔV152) | ND | ND | ND | BCA |
| Wheat Arabinoxylan | VvEG16(ΔV152) | ND | ND | ND | BCA |
| Laminarin | VvEG16(ΔV152) | ND | ND | ND | BCA |
| GG-PNP | VvEG16(ΔV152) | 0.023±0.002 | 5.9±0.6 | 0.065 | PNP |
| GG-CNP | VvEG16(ΔV152) | 0.45±0.02 | 3.5±0.4 | 2.1 | CNP |
| GG-DNP | VvEG16(ΔV152) | 20±1 | 2.0±0.2 | 170 | DNP |
| GGG-PNP | VvEG16(ΔV152) | 0.15±0.006 | 2.8±0.2 | 0.9 | PNP |
| GGG-CNP | VvEG16(ΔV152) | 1.03±0.04 | 1.1±0.1 | 16 | CNP |
| VvEG16 | 0.98±0.03 | 0.8±0.1 | 20 | CNP | |
| GGG-DNP | VvEG16(ΔV152) | 95±2 | 0.80±0.06 | 2000 | DNP |
| XXXG-CNP | VvEG16 | 12.0±0.5 | 1.1±0.3 | 180 | CNP |
| VvEG16(ΔV152) | 13.9±0.6 | 1.2±0.1 | 190 | CNP | |
| GG→2×G | VvEG16(ΔV152) | ND | ND | ND | HPAEC-PAD |
| GGG→GG+G | VvEG16(ΔV152) | 1.44±0.02 | 0.43±0.02 | 56 | HPAEC-PAD |
| GGGG→2×GG | VvEG16(ΔV152) | 66±1 | 0.094±0.005 | 1.2×104 | HPAEC-PAD |
| GGGG→GGG+G | VvEG16(ΔV152) | 70±10 | 0.5±0.2 | 2300 | HPAEC-PAD |
| 2×GGGG→GG+GGGGGG | VvEG16(ΔV152) | 98±6 | 1.3±0.2 | 1250 | HPAEC-PAD |
| GGGGG→GGG+GG | VvEG16(ΔV152) | 62±2 | 0.26±0.04 | 3500 | HPAEC-PAD |
| G3GGG | VvEG16(ΔV152) | ND | ND | ND | HPAEC-PAD |
| GG3GG→2×GG | VvEG16(ΔV152) | ND | ND | 0.3±0.05 | HPAEC-PAD |
| GGG3G→GGG+G | VvEG16(ΔV152) | ND | ND | 28±5 | HPAEC-PAD |
| GGG3G→GG+G3G | VvEG16(ΔV152) | 36±3 | 1.0±0.1 | 600±20 | HPAEC-PAD |
| G3GGG3GGG→ G3GGG+GGG |
VvEG16(ΔV152) | ND | ND | 25±1 | HPAEC-PAD |
| XXXGXXXG→2×XXXG | VvEG16 | 14±1 | 0.055±0.007 | 4200 | HPAEC-PAD |
| XXXGXXXG→2×XXXG | TmNXG1 (Baumann, et al. 2007) |
9.32±0.16 | 0.076±0.007 | 2000 | HPAEC-PAD |
ND = not determinable;
*
Concentrations expressed as (mg/mL).