THESE Maryse Bonnin Jusserand - Université de Bourgogne

Résultats
reaction was started by adding 50µL ornithine decarboxylase (1mg mL -1 protein) and the
mixture was incubated for 1h at 35°C. The reaction was stopped by adding 1mL of the
reaction sample to 1.75mL borate buffer (1 M, pH 9) and 750 µL methanol. The samples were
derivatized for HPLC analysis. One unit corresponds to 1 µmol putrescine min -1 mg -1
proteins.
The activity of the ornithine decarboxylase from O. oeni was tested towards, L-citrulline, L-
2,4-diaminobutyric acid, D-ornithine, L-arginine, L-glutamine, L-histidine, 6-aminocaproic
acid, 2,6-diaminopimelic acid and L-lysine at 2 mM concentration using the assay conditions
described above.
Reaction of Derivatization and HPLC Analysis.
Aminoenone derivatives were obtained by adding 40 µl of internal standard (2,4,6-
trimethylphenethylamine hydrochloride 2 mg mL -1 ), and 30 µL of DEEMM (diethyl
ethoxymethylenemalonate) to the sample containing borate buffer and methanol. After 30 min
in an ultrasound bath, the sample was then heated to 70°C for 2 h to allow complete
degradation of excess DEEMM and reagent byproducts.
The analyses were performed according to Gomez-Alonso et al. (5) on a Varian HPLC
(Varian Inc., Walnut Creek, CA) using an Alltech (Grace) HPLC column (C18-HL) particle
size 5 µm (250 mm X 4.6 mm) thermostatized at 16°C through the binary gradient (phase A,
25 mM acetate buffer pH 5.8 with 0.02% sodium azide; phase B, 80:20 mixture of acetonitrile
and methanol) and a flow rate 0.9 mL min -1 . For detection, a photodiode array detector
monitored at 280 nm was used. The target compounds were identified according to the
retention times and UV-vis spectral characteristics of the derivatives of the corresponding
standards and were quantified using the internal standard method.
Results and Discussion
Screening for the ornithine decarboxylase gene
PCR amplification was used to screen for the ornithine decarboxylase (odc) gene among 263
O. oeni strains collected from wine and cider all over the world (Fig 1). Five of the 263 strains
gave the expected size amplicon (1,900 bp) which reflected the presence of the odc gene (Fig.
2). Among these five strains, three were isolated from wine and came from South Africa
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