Table 3.
Catalytic rates of GV-hybrids.
| Substrate | Clone | Mutational composition | kcat(min-1) | Km(μM) |
kcat/Km (×106M-1min-1) |
Fold increase to G0 | Fold increase to V0 | Fold increase to GV0 |
|
| ||||||||
| Paraoxon | G0 | (rePON1-4E9) | 9.1 ± 0.01 | 29.4 ± 0.6 | 0.3 ± 0.01 | 1.0 | - | - |
| V0 | (OPH-11M) | 705.3 ± 16.59 | 220.4 ± 1.1 | 3.2 ± 0.06 | 10.4 | 1.0 | - | |
| GV0 | G0-V0 hybrid | 146.1 ± 8.26 | 27.7 ± 4.7 | 5.3 ± 0.59 | 17.2 | 1.7 | 1.0 | |
| GV1 | G0-V1 hybrid | 3024.7 ± 99.40 | 214.4 ± 9.0 | 14.1 ± 0.13 | 45.7 | 4.4 | 2.7 | |
| GV2 | G9-V2 hybrid | 3271.3 ± 47.17 | 247.9 ± 4.7 | 13.2 ± 0.06 | 42.7 | 4.1 | 2.5 | |
| GV3 | G9-V3 hybrid | 2366.3 ± 35.38 | 219.3 ± 6.7 | 10.8 ± 0.49 | 35.0 | 3.4 | 2.0 | |
| GV4 | G10-V2 hybrid | 3061.6 ± 29.20 | 184.9 ± 12.5 | 16.6 ± 0.96 | 53.7 | 5.2 | 3.1 | |
| GV5 | G10-V3 hybrid | 1541.1 ± 25.27 | 105.3 ± 4.7 | 14.7 ± 0.42 | 47.5 | 4.6 | 2.8 | |
| GV6 | G11-V2 hybrid | 1676.6 ± 59.53 | 105.3 ± 13.0 | 16.0 ± 1.41 | 51.8 | 5.0 | 3.0 | |
| GV7 | G11-V3 hybrid | 3258.6 ± 51.67 | 54.5 ± 0.9 | 59.8 ± 0.06 | 193.7 | 18.7 | 11.3 | |
| PNPDPP | G0 | (rePON1-4E9) | 4.0 ± 0.2 | 23.0 ± 2.5 | 0.2 ± 0.01 | 1.0 | - | - |
| V0 | (OPH-11M) | 29.0 ± 2.3 | 31.1 ± 5.3 | 0.9 ± 0.09 | 5.4 | 1.0 | - | |
| GV0 | G0-V0 hybrid | 17.0 ± 0.3 | 3.0 ± 0.1 | 5.7 ± 0.20 | 33.0 | 6.2 | 1.0 | |
| GV1 | G0-V1 hybrid | 73.0 ± 1.0 | 16.7 ± 2.1 | 4.4 ± 0.48 | 25.0 | 4.7 | 0.8 | |
| GV2 | G9-V2 hybrid | 96.3 ± 3.0 | 29.2 ± 0.8 | 3.3 ± 0.20 | 18.8 | 3.5 | 0.6 | |
| GV3 | G9-V3 hybrid | 68.6 ± 2.2 | 37.4 ± 2.5 | 2.1 ± 0.88 | 12.1 | 2.3 | 0.4 | |
| GV4 | G10-V2 hybrid | 81.9 ± 1.6 | 21.7 ± 5.4 | 3.8 ± 0.47 | 21.8 | 4.1 | 0.7 | |
| GV5 | G10-V3 hybrid | 65.3 ± 4.6 | 16.0 ± 5.6 | 4.3 ± 1.22 | 24.4 | 4.6 | 0.8 | |
| GV6 | G11-V2 hybrid | 98.0 ± 7.2 | 21.6 ± 3.6 | 4.6 ± 0.42 | 26.0 | 4.9 | 0.8 | |
| GV7 | G11-V3 hybrid | 155.5 ± 3.7 | 29.7 ± 0.6 | 5.2 ± 0.02 | 29.8 | 5.6 | 0.9 | |
| DFP | G0 | (rePON1-4E9) | 176.8 ± 17.3 | 86.5 ± 30.1 | 2.1 ± 0.5 | 1.0 | - | - |
| V0 | (OPH-11M) | 556.2 ± 9.1 | 31.1 ± 1.9 | 17.9 ± 0.8 | 8.4 | 1.0 | - | |
| GV0 | G0-V0 hybrid | 621.5 ± 6.8 | 519.5 ± 31.7 | 1.2 ± 0.1 | 0.6 | 0.1 | 1.0 | |
| GV1 | G0-V1 hybrid | 882.4 ± 27.0 | 29.6 ± 4.6 | 30.1 ± 3.7 | 14.1 | 1.7 | 25.1 | |
| GV2 | G9-V2 hybrid | 825.2 ± 13.5 | 22.3 ± 4.9 | 37.9 ± 7.8 | 17.8 | 2.1 | 31.6 | |
| GV3 | G9-V3 hybrid | 685.6 ± 60.7 | 140.7 ± 42.6 | 5.0 ± 1.1 | 2.4 | 0.3 | 4.2 | |
| GV4 | G10-V2 hybrid | 908.6 ± 2.2 | 31.5 ± 3.2 | 29.0 ± 3.0 | 13.6 | 1.6 | 24.2 | |
| GV5 | G10-V3 hybrid | 899.4 ± 1.7 | 19.4 ± 5.9 | 48.7 ± 14.9 | 22.8 | 2.7 | 40.6 | |
| GV6 | G11-V2 hybrid | 838.3 ± 32.0 | 35.6 ± 9.1 | 24.2 ± 5.3 | 11.3 | 1.4 | 20.2 | |
| GV7 | G11-V3 hybrid | 1039.2 ± 2.8 | 22.3 ± 2.9 | 47.0 ± 6.0 | 22.0 | 2.6 | 39.2 | |
|
| ||||||||
| Substrate | Clone | Mutational composition | kcat(min-1) | Km(μM) |
kcat/Km (M-1min-1) |
Fold increase to G0 | Fold increase to V0 | Fold increase to GV0 |
|
| ||||||||
| DSM | G0 | (rePON1-4E9) | 0.02 ± 0.01 | 1054.6 ± 934.1 | 14.6 ± 1.9 | 1.0 | - | - |
| V0 | (OPH-11M) | 0.13 ± 0.01 | 1637.5 ± 252.4 | 77.5 ± 9.8 | 5.3 | 1.0 | - | |
| GV0 | G0-V0 hybrid | 0.06 ± 0.09 | 1210.6 ± 612.9 | 5.0 ± 1.1 | 0.3 | 0.1 | 1.0 | |
| GV1 | G0-V1 hybrid | 0.22 ± 0.01 | 748.9 ± 181.4 | 295.6 ± 52.9 | 20.3 | 3.8 | 59.1 | |
| GV2 | G9-V2 hybrid | 0.17 ± 0.02 | 832.2 ± 236.8 | 214.0 ± 38.4 | 14.7 | 2.8 | 42.8 | |
| GV3 | G9-V3 hybrid | 0.12 ± 0.01 | 680.1 ± 354.4 | 206.6 ± 90.1 | 14.2 | 2.7 | 41.3 | |
| GV4 | G10-V2 hybrid | 0.23 ± 0.01 | 708.6 ± 99.5 | 324.0 ± 28.9 | 22.2 | 4.2 | 64.8 | |
| GV5 | G10-V3 hybrid | 0.29 ± 0.12 | 10964.5 ± 5736.8 | 28.1 ± 5.1 | 1.9 | 0.4 | 5.6 | |
| GV6 | G11-V2 hybrid | 0.14 ± 0.02 | 593.8 ± 159.8 | 234.8 ± 37.9 | 16.1 | 3.0 | 46.9 | |
| GV7 | G11-V3 hybrid | 0.22 ± 0.01 | 432.5 ± 45.7 | 504.3 ± 48.5 | 34.6 | 6.5 | 100.9 | |
| Malathion | G0 | (rePON1-4E9) | N.D. | N.D. | N.D. | - | - | - |
| V0 | (OPH-11M) | 0.71 ± 0.04 | 2211.5 ± 160.5 | 319.4 ± 4.8 | - | 1.0 | - | |
| GV0 | G0-V0 hybrid | 0.21 ± 0.01 | 994.2 ± 33.7 | 212.6 ± 1.1 | - | 0.7 | 1.0 | |
| GV1 | G0-V1 hybrid | 1.90 ± 0.04 | 2736.5 ± 14.8 | 695.3 ± 17.5 | - | 2.2 | 3.3 | |
| GV2 | G9-V2 hybrid | 5.14 ± 0.29 | 6395.5 ± 450.4 | 803.8 ± 11.0 | - | 2.5 | 3.8 | |
| GV3 | G9-V3 hybrid | 3.75 ± 0.05 | 7980.0 ± 43.8 | 470.4 ± 3.7 | - | 1.5 | 2.2 | |
| GV4 | G10-V2 hybrid | 2.72 ± 0.40 | 7642.5 ± 139.5 | 356.9 ± 12.8 | - | 1.1 | 1.7 | |
| GV5 | G10-V3 hybrid | 12.68 ± 3.19 | 27460.0 ± 7891.1 | 464.1 ± 17.2 | - | 1.5 | 2.2 | |
| GV6 | G11-V2 hybrid | 1.27 ± 0.25 | 3274.0 ± 924.9 | 393.9 ± 34.6 | - | 1.2 | 1.9 | |
| GV7 | G11-V3 hybrid | 4.15 ± 0.02 | 3133.5 ± 95.4 | 1324.0± 47.5 | - | 4.2 | 6.2 | |
N.D., not determined.