9076
M.-C. Bontoux, M. Gelo-Pujic / Tetrahedron Letters 47 (2006) 9073–9076
hydroxylase nor its gene was described for Aeromonas
sp. However, it was not possible to optimize the produc-
tivity or to develop large scale fermentation due to the
poor growth of A. caviae, the lack of data concerning
the expression and the regulation of proline hydroxylase
gene. To optimize the enzyme production, our efforts are
oriented towards genetic engineering and recombinant
strain construction of a high density biomass and a tight
control of gene expression.
ferrous sulfate (1 mM). Reaction mixtures were incu-
bated at 25 °C and 130 rpm for 24 h. Samples (50 ll)
were withdrawn and following reactants were added:
0.1 ml of 2% NaHCO3, 1 ml of dry ethanol and 0.3 ml
of a 0.25% solution of 2,4-dinitrofluorobenzene in etha-
nol. The mixtures were shaken at 43 °C and 1000 rpm
for 2 h. Reactions were stopped by adding 50 ll of
1 M HCl and the samples were analyzed by HPLC with-
out further treatment.
Typical experimental procedure: PDA (potato-dextros-
agar) was supplied by Difco. TES (N-tris[hydroxy-
methyl]methyl-2-aminoethane sulfonic acid) was pur-
chased from Sigma. All chemicals and solvents were
used without any further purification if not stated other-
wise. NMR spectra were recorded in DMSO-d6 solu-
tions on a Bruecker instrument operating at 27 °C and
300 MHz for a proton NMR and 75.5 MHz for the car-
bon NMR. Chemical shifts (d) are expressed in ppm rela-
tive to HMDS (proton NMR) and TMS (carbon
NMR). Reactions were followed by HPLC/UV/MS on
a Watres Aliance 2790 separation module equipped with
996 PDA detector and micromasse ZQ using RP18 col-
umn (100 mm, 5 lm particle size) under the following
conditions: flow rate 1 ml/min; solvents: A: H2O/0.1%
HCOOH and B: acetonitrile/0.1% HCOOH. Gradient
was programmed from 100% A to 80% B over 20 min.
Retention times (tr in minutes) of DNP-derivatives were:
6.51 (4-trans-hydroxy-L-proline), 9.29 (L-proline) and
References and notes
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9.33
(2,4-dinitrofluorobenzene).
2,4-Dinitrophenyl
derivative of L-proline (DNP-L-Pro) was synthesized,
purified and a calibration curve was obtained for a con-
centration range between 1 and 50 mg/l. The conversion
was measured based on the transformation of the start-
ing substrate.
General screening conditions: Microorganisms were
grown on sterile PDA medium for 48–72 h. Two plugs
of 5 mm diameter (pieces of medium covered with
microbial culture) were cut and transferred to 96-deep
well screening plates. A sterile buffer (TES, 1 ml, pH
7) was added, and thus the prepared biocatalysts were
stored frozen until used. For the activity assay, bio-
catalysts were de-frozen by incubation at 37 °C step,
followed by the addition of L-proline (10 mM), 2-keto-
glutaric acid (10 mM), L-ascorbic acid (5 mM) and