Front Matter

16.3.2 Other lipoxygenases

Lipoxygenases 5- or 9-specific

Beside n-6 LOXs with SBLOX-1 as an archetype, 5- or 9-LOXs (depending on

whether arachidonic or linoleic acids are used as substrates) are the second most

studied LOXs. To obtain access to both 9-HPODE and 5-HPETE, a vegetal source

of LOX was the only viable alternative since, to our knowledge, no such LOXs are

available commercially at a reasonable price. The most common source of 9-LOX is

potato (Mulliez et al., 1987), tomato (Regdel et al., 1994), barley seeds (Van Aarle et

al., 1991) and wheat (Ku ̈hn et al., 1987), and corn germs (Veldink et al., 1972). With

LA as substrate, such vegetal LOXs display a rather high selectivity in 9(S)-HPODE

formation. However, as in the case of AA, various proportions of 8(S)-HPETE and

11(S)-HPETE have been detected, as well as 5(S)-HPETE (Mulliez et al., 1987;

Regdel et al., 1994). Matthew and co-workers reported a rather simple method to

produce 9(S)-HPODE. The flesh (225 g) of tomatoes and ammonium linoleate

were first mixed in 750 mL of 0.1 M acetate buffer pH 5.5 (final substrate concen-

tration 1 mM) and then incubated at 25 8 C with 15 s oxygen flushing every 2 min. At

the end of the reaction the yield in HPODs was 69 % with a 9- to 13-HPODE ratio of

96 : 4, and a high optical purity for the formed 9(S)-HPODE (Matthew et al., 1977).

Following the success of the ‘under-pressure lipoxygenation’ methodology (see

Section 16.2.3), we were interested in using 9-LOXs to generate 9(S)-HPODE and

5(S)-HPETE to extend our procedure. We anticipated that the critical point of such a

synthesis would be the optimum pH of the LOX used. Indeed, unlike SBLOX-1, 9-

LOXs have acidic or neutral optimum pHs. A survey of the literature revealed that

barley seed LOX combined several advantages, some of which were an optimum pH

centered around 7.5, and a good disposability of the vegetal source. In order to reach

a sufficient specific activity and to eliminate a hydroperoxide-degrading activity

present in the seeds, the partial purification of barley seed LOX (BSLOX) was under-

taken according to an existing purification procedure (Van Aarle et al., 1991). This

involved only classical methods of purification which could be easily performed, and

led to a BSLOX solution of 45Umg–1of protein, a specific activity which was

comparable to that of commercially available SBLOX-1. Using the same methodol-

ogy developed for SBLOX-1, we found the following values to be optimal using LA

as substrate: borate buffer 0.1 M, pH 8, oxygen pressure 2.5 bar, temperature 5 8 C,

enzyme concentration 400 U per 30 mL, and substrate concentration 0.05 M. By

using all these optimized values in a single experiment we were able to produce

from LA and LnA, 9(S)-HPODE and 9(S)-HPOTE respectively (Figure 13) in

40–50 % yields, with high selectivity (more than 95 %) and very high enantiomeric

excess (more than 98 % ee). The unreacted substrate could be recovered at the end of

the reaction (Martini et al., 1996b).

Despite the lower substrate concentration used and a lower yield as compared with

SBLOX-1, BSLOX is a useful enzyme to generate both 9(S)-HPODE and 9(S)-

HPOTE with high selectivities. Unfortunately, with AA as substrate, very low yields

in 5-HPETE were obtained (less than 5 %). Further work is needed to understand this

failure, as 5-HPETE is a highly valuable compound, especially in human physiology,

being the precursor of leukotrienes.

350 16 Properties and Applications of Lipoxygenases