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activity assays_extracts
recombinant enzyme assays
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| Activites were measured in cell free extracts, prepared from three biological replicates with at least three independent measurements per replicate | |||||||||||
| Control reactions without substrate, cofactor or enzyme did not show any activity | |||||||||||
| Abbreviations: KDF: 2-Keto-3-deoxy-L-fuconate; DOP: 2,5-dioxopentanoate | |||||||||||
| Dehydrogenase activity was determined by following the formation of NADH/NADPH at 340 nm | |||||||||||
| Aldolase and dehydratase activity were determined by following KDF formation using the thiobarbituric acid assay | |||||||||||
| L-Fucose dehydrogenase (L-Fucose <-> L-Fuconolactone) | L-Fuconate dehydratase (L-Fuconate <-> KDF) | ||||||||||
| L-fucose extracts | D-glucose extracts | L-fucose extracts | D-glucose extracts | ||||||||
| substrate | activity [mU/mg] | deviation | activity [mU/mg] | deviation | substrate | activity [mU/mg] | deviation | activity [mU/mg] | deviation | ||
| L-fucose | 142.19 | 23.31 | 6.1 | 2.06 | L-fuconate | 0.65 | 0.17 | 0.027 | 0.023 | ||
| D-arabinose | 95.13 | 3.01 | 3.63 | 0.39 | D-arabinonate | 1.17 | 0.25 | 0.035 | 0.017 | ||
| KDF aldolase (KDF <-> Pyruvate + L-Lactaldehyde) | Lactaldehyde deyhdrogenase (L-Lactaldehyde <-> Lactate and DOP <-> oxoglutarate) | ||||||||||
| L-fucose extracts | D-glucose extracts | L-fucose extracts | D-glucose extracts | ||||||||
| substrate | activity [mU/mg] | deviation | activity [mU/mg] | deviation | substrate | activity [mU/mg] | deviation | activity [mU/mg] | deviation | ||
| L-lactaldehyde (+ pyruvate) | 11.3 | 0.1 | 12.8 | 1.38 | DOP | 139.9 | 14.2 | 70 | 7.5 | ||
| D-lactaldehyde (+pyruvate) | 2.6 | 0.94 | 3.7 | 0.01 | L-lactaldehyde | 10.6 | 3.1 | 2 | 0.1 | ||
| D-lactaldehyde | 3.1 | 0.4 | 3.1 | 0.1 | |||||||
| Lactate dehydrogenase (Lactate <-> Pyruvate) | |||||||||||
| L-fucose extracts | D-glucose extracts | ||||||||||
| substrate | activity [mU/mg] | deviation | activity [mU/mg] | deviation | |||||||
| pyruvate | 5.15 | 1.34 | 4.28 | 1.44 | |||||||
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| Activites were measured with recombinante enzymes , prepared from three biological replicates with at least three independent measurements per replicate | |||||||||||||||
| Control reactions without substrate, cofactor or enzyme did not show any activity | |||||||||||||||
| Abbreviations: KDF: 2-Keto-3-deoxy-L-fuconate | |||||||||||||||
| Dehydrogenase activity was determined by following the formation of NADH/NADPH at 340 nm | |||||||||||||||
| Aldolase and dehydratase activity were determined by following KDF formation using the thiobarbituric acid assay | |||||||||||||||
| L-Fucose dehydrogenase (L-Fucose <-> L-Fuconolactone) | L-Fuconate dehydratase (L-Fuconate <-> KDF) | ||||||||||||||
| SSO1300 | SSO3124 | ||||||||||||||
| substrate | Vmax [U/mg] | deviation | Km [mM] | deviation | kcat [s-1] | kcat/Km [mM-1s-1] | substrate | Vmax [U/mg] | deviation | Km [mM] | deviation | kcat [s-1] | kcat/Km [mM-1s-1] | ||
| L-fucose (+ NADP+) | 26.7 | 0.89 | 0.12 | 0.02 | 19.4 | 161.9 | L-fuconate | 0.018 | 0.002 | n.d. | n.d. | 0.013 | n.d. | ||
| D-arabinose (+ NADP+) | 21.5 | 0.81 | 0.22 | 0.03 | 12.8 | 58.2 | D-arabinonate | 0.078 | 0.002 | n.d. | n.d. | 0.055 | n.d. | ||
| KDF aldolase (KDF <-> Pyruvate + L-Lactaldehyde) | |||||||||||||||
| SSO3197 | |||||||||||||||
| substrate | Vmax [U/mg] | deviation | Km [mM] | deviation | kcat [s-1] | kcat/Km [mM-1s-1] | |||||||||
| L-lactaldehyde (+ pyruvate) | 1.9 | 0.13 | 8.8 | 1.15 | 0.9 | 0.1 | |||||||||
| 2-keto-3-deoxy-D-gluconate | 33.8 | 3.6 | 10.7 | 1.8 | 27.5 | 2.6 | |||||||||
| Lactaldehyde deyhdrogenase (L-Lactaldehyde <-> Lactate ) | |||||||||||||||
| SSO3117 | |||||||||||||||
| substrate | Vmax [U/mg] | deviation | Km [mM] | deviation | kcat [s-1] | kcat/Km [mM-1s-1] | |||||||||
| L-lactaldehyde | 0.17 | 0.001 | n.d. | n.d. | 0.15 | n.d. | |||||||||
