tion of the various isomers was determined (Figure 3), 13(S)-HPODE being the
major product of the reaction (96 %, 95 % ee).
Four years later the use of a much more sophisticated stainless steel reactor
(Scheme 1) was described (Martini et al., 1994). The reactor (MU 4004), which
was provided by SOTELEM (Rueil Malmaison, FRANCE), was originally designed
as a chemical reactor, and had a working capacity of 100 mL, a working temperature
up to 150 8 C, and a working pressure range of 0 to 200 bar.
The vessel proved to be particularly suited to carry out lipoxygenation reactions,
and allowed detailed studies to be made of the influence of different parameters on
the reaction, especially oxygen or air pressure, temperature, pH and substrate and
enzyme concentrations. LA was chosen as substrate, the agitation speed was set at
maximum (1600 rpm), and 30 mL of borate buffer (0.1 M) was used as solvent.
Each parameter was studied separately, and in each case the kinetics of HPOD
formation were followed by UV spectroscopy. At the end of the reaction the HPODs
formed were reduced and the distribution of each isomers determined by normal-
phase HPLC. The enantiomeric excess of the major product of the reaction
(13(S)-HPODE) was also determined by chiral-phase HPLC. From this study, the
following optimal values were determined: oxygen pressure: 2.5 bar (1, 2.5, 5,
10, 25, 50, 100 bar), temperature 5 8 C (5, 15, 25, 35 8 C), pH: 11 (7, 8, 9, 10, 11),
substrate concentration: 0.1 M (0.05, 0.1, 0.2 M), and enzyme concentration 4
mg mL–1(0.5, 1, 2, 4 mg mL–1). It should also be noted that in each case, the
best yields in HPOD was correlated with the highest selectivity in 13(S)-
HPODE. Thus, it could be concluded that deviation from the optimal catalytic ac-
tivity of the enzyme resulted in a lower selectivity. All optimal values of each para-
meters were then grouped in a single experiment. After 30 min of reaction, a max-
imum yield of 99 % in HPOD (UV determination) was reached, with 13(S)-HPODE
being the major isomer formed (95.5 %, 98 % ee).
16.2 How to use lipoxygenases 341
Scheme 1 SOTELEM MU 4004 stainless steel reactor used for lipoxygenation (Martini et al., 1994).