Purification, stabilization and characterization of tomato fatty acid hydroperoxide lyase

Abstract

Fatty acid hydroperoxide lyase (HPO-lyase) was puri®ed 300-fold from tomatoes. The enzymatic activity appeared to be very unstable, but addition of Triton X 100 and b-mercaptoethanol to the buer yielded an active enzyme that could be stored for several months at À808C. The enzyme was inhibited by desferoxamine mesylate (desferal), 2-methyl-1,2-di-3-pyridyl-1-propanone (metyrapone), nordihydroguaiaretic acid (NDGA), n-propyl gallate and butylated hydroxyanisole, suggesting the involvement of free radicals in the reaction mechanism and the existence of a prosthetic group in the active center. However, no heme group could be demonstrated with the methods commonly used to identify heme groups in proteins. Only 13-hydroperoxides from linoleic acid (13-HPOD) and a-linolenic acid (a-13-HPOT) were cleaved by the tomato enzyme, with a clear preference for the latter substrate. The pH-optimum was 6.5, and for concentrations lower than 300 mM a typical Michaelis±Menten curve was found with a K m of 77 mM. At higher a-13-HPOT concentrations inhibition of the enzyme was observed, which could (at least in part) be attributed to 2E-hexenal. A curve of the substrate conversion as a function of the enzyme concentration revealed that 1 nkat of enzyme activity converts 0.7 mmol a-13-HPOT before inactivation. Headspace analysis showed that tomato HPO-lyase formed hexanal from 13-HPOD and 3Z-hexenal from a-13-HPOT. A trace of the latter compound was isomerized to 2E-hexenal. In addition to the aldehydes, 12-oxo-9Z-dodecenoic acid was found by GC/MS analysis. To a small extent, isomerization to 12-oxo-10E-dodecenoic acid occurred. #

Molecular cloning, expression, and enzymatic characterization of Solanum tuberosum hydroperoxide lyase

“…Thus far, HPLs have been characterized from more than 10 organisms. The enzymes were mainly characterized after purification through extraction from plant tissues [2,9,[24][25][26]. Recently, researchers have been trying to express the plant HPL gene in E. coli and P. pastoris and characterize the recombinant enzyme [13][14][15][16][17][18].…”

“…HPL is mainly distributed in higher plants, mushrooms, algae, and penicillium. This enzyme is part of the lipoxygenase (LOX) pathway in plants, which is considered to be equivalent to the arachidonic acid cascade in animals [2].…”

Biotechnological Flavour Generation

“…The gene coding for this enzyme was cloned from banana and heterologously expressed in yeast cells to generate a continuous source of active lyase (Muheim et al, 1997). Another plant HPOlyase was purified 300-fold from tomatoes (Suurmeijer et al, 2000). The tomato enzyme cleaved only 13-hydroperoxides from linoleic acid and ␣-linolenic acid, whereas HPO-lyase from alfalfa (Medico sativa L.) also accepted 9-hydroperoxides as substrates to form the respective volatile C 9 -aldehydes (Noordermeer et al, 1999).…”

Role of lipid reactions in quality of oat products

“…Enzymatic oxidation of double bonds in acyl chains consists of series of reactions. In many plant tissues the hydroperoxides formed by the lipoxygenase reaction are further cleaved by hydroperoxide lyase (EC 4.1.2.-), an enzyme which has been extensively studied in cucumber, tomato and beans (Matsui et al 2000, Suurmeijer et al 2000, Noordermeer et al 2001. The presence of hydroperoxide lyase in cereal grains has not been published in the scientific literature, but the presence of typical reaction products of this enzyme suggests that it is abundant also in cereals (Sjövall et al 2000, Parker et al 2000, Sides et al 2001.…”