◦
incubation in an Eppendorf Thermomixer (30 C, 1200 rpm),
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activity assays were performed as described earlier. After the
activity assay, the reaction mixtures were left for 30 minutes
in total. The supernatants were removed and replaced by fresh
reaction buffer and the initial reaction rate was determined for
three additional cycles in a similar way.
Batch reactions. Reaction mixtures with approximately 25 mg
soluble and 25 mg immobilized ADC were prepared in 4.700
mL 50 mM phosphate buffer (pH 7.5) with 0.05% sodium
2
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◦
azide. After incubation for 10 minutes in a stirred 30 C water
2
003, 11, 285–294.
bath, the activity assays were started by adding 300 mL 50
mM L-aspartic acid. Sampling and analyses were performed
as previously described. The total turnover number (TTN) was
calculated by dividing the moles of b-alanine produced by the
moles of ADC spent.
2
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Acknowledgements
We wouldlikethankSenterNovem, which on behalf ofthe Dutch
Ministry of Economic Affairs supported us with a grant of the
Program Energie Onderzoeksstrategie Lange Termijn, project
EOSLT02034. Furthermore, we would like to thank Prof. Alison
Smith and Dr Michael Webb from the Plant Metabolism Group,
Department of Plant Sciences, University of Cambridge, UK,
for kindly providing us with pRSETA plasmids containing the
panD gene and for useful discussions.
3 C. Mateo, R. Torres, G. Fernandez-Lorente, C. Ortiz, M. Fuentes,
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