Table 4.
GC‐MS analysis of GLC and its reaction products 2‐keto‐GLC, 3‐keto‐GLC and 2,3‐diketo‐GLC. The reaction mixture contained 1 mL of 10 mm potassium phosphate buffer (pH 7.0), 1 U of AmPDH or its respective variant, 45 mm 1,4‐benzoquinone as electron acceptor and, initially, 16.7 mm GLC. Samples (200 μL) were taken after 3, 8 and 120 min of reaction time, respectively. After deproteinization through a 10 kDa ultrafiltration membrane, the filtrate was used for subsequent GC‐MS analysis. For GLC and 2‐keto‐GLC, an external calibration was possible with commercially available standards, allowing for a quantitative determination of their concentrations (mm) with a detection limit of 0.2 μm. Because no standards were commercially available for 3‐keto‐GLC and 2,3‐diketo‐GLC, only semiquantitative results (relative concentrations in mm) could be calculated, based on the peak‐area‐ratios of the respective analyte to 2‐keto‐GLC at a given mass to charge ratio. To obtain the ‘Ratio’, the percentage of each compound with respect to the sum of (relative) concentrations was calculated for each variant
| Variant | Reaction time (min) | Concentration (mm) | Relative concentration (mm) | Ratio (%) | |||||
|---|---|---|---|---|---|---|---|---|---|
| GLC | 2‐keto‐GLC | 3‐keto‐GLC | 2,3‐diketo‐GLC | GLC | 2‐keto‐GLC | 3‐keto‐GLC | 2,3‐diketo‐GLC | ||
| AmPDH | 3 | 2.69 | 1.72 | 0.000 | 0.061 | 60.2 | 38.5 | 0.0 | 1.4 |
| 8 | 2.43 | 1.69 | 0.001 | 0.273 | 55.4 | 38.4 | 0.0 | 6.2 | |
| 120 | 0.43 | 1.33 | 0.026 | 1.271 | 14.0 | 43.6 | 0.9 | 41.6 | |
| H103A | 3 | 3.42 | 0.597 | 0.000 | 0.100 | 83.1 | 14.5 | 0.0 | 2.4 |
| 8 | 2.91 | 1.65 | 0.004 | 0.342 | 59.3 | 33.7 | 0.1 | 7.0 | |
| 120 | 1.62 | 1.49 | 0.024 | 1.108 | 38.2 | 35.1 | 0.6 | 26.1 | |
| Q392A | 3 | 3.07 | 1.50 | 0.002 | 0.186 | 64.5 | 31.6 | 0.0 | 3.9 |
| 8 | 2.63 | 1.87 | 0.002 | 0.724 | 50.3 | 35.8 | 0.0 | 13.9 | |
| 120 | 1.04 | 1.49 | 0.032 | 1.540 | 25.4 | 36.2 | 0.8 | 37.6 | |
| Y510A | 3 | 3.35 | 0.126 | 0.000 | 0.002 | 96.3 | 3.6 | 0.0 | 0.1 |
| 8 | 3.40 | 0.386 | 0.000 | 0.034 | 89.0 | 10.1 | 0.0 | 0.9 | |
| 120 | 2.73 | 1.59 | 0.010 | 0.401 | 57.7 | 33.6 | 0.2 | 8.5 | |
| V511F | 3 | 3.62 | 0.311 | 0.001 | 0.005 | 91.9 | 7.9 | 0.0 | 0.1 |
| 8 | 3.60 | 1.09 | 0.001 | 0.030 | 76.3 | 23.1 | 0.0 | 0.6 | |
| 120 | 2.42 | 2.46 | 0.025 | 0.387 | 45.7 | 46.5 | 0.5 | 7.3 | |
| V511W | 3 | 3.34 | 1.70 | 0.001 | 0.058 | 65.5 | 33.4 | 0.0 | 1.1 |
| 8 | 3.04 | 2.46 | 0.010 | 0.331 | 52.0 | 42.1 | 0.2 | 5.7 | |
| 120 | 0.275 | 1.84 | 0.150 | 1.564 | 7.2 | 48.0 | 3.9 | 40.9 | |
| H556A | 3 | 3.86 | 0.293 | 0.001 | 0.018 | 92.5 | 7.0 | 0.0 | 0.4 |
| 8 | 3.46 | 0.811 | 0.000 | 0.075 | 79.6 | 18.6 | 0.0 | 1.7 | |
| 120 | 3.54 | 2.80 | 0.001 | 0.466 | 52.0 | 41.2 | 0.0 | 6.8 | |
| H556N | 3 | 3.92 | 0.090 | 0.000 | 0.003 | 97.7 | 2.2 | 0.0 | 0.1 |
| 8 | 3.66 | 0.187 | 0.000 | 0.012 | 94.8 | 4.9 | 0.0 | 0.3 | |
| 120 | 3.45 | 1.17 | 0.001 | 0.110 | 72.9 | 24.7 | 0.0 | 2.3 | |