dedicated specifically to the enzymatic synthesis of PUFA-HPODs and, as stated by
the authors, this work represented a major improvement since it was the first time
that hundreds of milligram of such an HPOD had been synthesized, even if the yields
were modest and the substrate concentration quite low.
Seven years later, Laakso described the continuous production of 13(S)-HPODE
and 15(S)-HPETE using immobilized SBLOX-1 covalently coupled to agarose
(Laakso, 1982). A buffered solution of substrates (10–4M) was passed through a
reactor column filled with the linked enzyme at a flow rate of 11 mL h–1. Produc-
tivity of the system was about 0.6 mg of HPOD mL–1of wet gel per hour, and when
operating at 25 8 C for a period of 125 h, no loss of productivity was observed.
Despite the value of these two examples, lipoxygenation at that time still had
problems of substrate solubility, and this led to a need for high reaction volumes.
However, following these pioneer studies, various methods have been developed
in order to allow the use of higher substrate concentrations.
16.2.3 Oxygen pressure
In, 1990, Iacazio et al. described the use of SBLOX-1 under positive oxygen pressure
at high LA concentration, using a Schlenck tube as a reaction vessel (Iacazio et al.,
1990). To 10 mL of borate buffer (Na 2 B 4 O 7 10H 2 O; 0.1M; pH 9.5) were added
280 mg of LA (1 mmol, 10–1M) and 20 mg of enzyme. The temperature was
kept at 0 48 C, and the reaction started by pressurization of the reactor (4 bar
of pure oxygen) and vigorous stirring of the reactants. At the beginning of the reac-
tion the substrate was mainly undissolved, but as the reaction proceeded, the reaction
mixture cleared and was perfectly limpid after 10 min. This indicated a rapid solu-
bilization of the substrate and after 1 h, an 80 % yield of HPOD was achieved. After
reduction (using NaBH 4 ) and normal and chiral-phase HPLC analysis, the distribu-
340 16 Properties and Applications of Lipoxygenases
Figure 3. Isomeric distribution of linoleic acid HPODs shown by SBLOX-1 at high substrate concen-
tration under oxygen pressure (Iacazio et al., 1990).