A. Pinsky

Biochemistry of lipoxygenase in relation to food quality

A renewed interest in lipoxygenase has led to detailed studies of its isoenzymes, substrate specificity, and the nature of its reaction products. Lipoxygenase is highly specific for cis,cis-1,4-pentadiene systems such as linoleic, linolenic, and arachidonic acid (or ester) and catalyzes the formation of the corresponding hydroperoxides with a cis,-trans-conjugated diene system. The hydroperoxides can then undergo enzymic or spontaneous degradation, producing a range of carbonyl compounds. This review will discuss the biochemical properties of this enzyme and its contribution to the quality of raw and processed food products. An attempt has been made to discuss both the desirable and undesirable effects associated with the action of lipoxygenase, citing specific food examples where appropriate.

Pigment bleaching, carbonyl production and antioxidant effects during the anaerobic lipoxygenase reaction

Abstract Bleaching of the plant pigments β-carotene and chlorophyll a occurs during the lipoxygenase-mediated oxidation of unsaturated fatty acids. The type-1 lipoxygenase from soybeans is a poor pigment-bleacher under aerobic (air or oxygen-saturated) conditions. However, under anaerobic (argon) conditions, lipoxygenase-1 is a powerful pigment bleaching agent in the presence of substrate fatty acid and product hydroperoxide. When β-carotene or chlorophyll a was bleached, absorbance at 285 nm increased, indicating the formation of keto- or oxodienes. Simultaneously, the conjugated diene hydroperoxide present in the reaction mixture disappeared. The rate of bleaching of carotene was faster than for chlorophyll. The addition of antioxidants (butylated hydroxyanisole, butylated hydroxytoluene, α-tocopherol, propyl gallate, or tertiary butylated hydroxyquinone) affected the rates of pigment bleaching and carbonyl production. Butylated hydroxyanisole, butylated hydroxytoluene and α-tocopherol were slightly more effective than propyl gallate in inhibiting pigment bleaching, while tertiary butylated hydroxyquinone was completely inhibiting. The production of carbonyl compounds continued essentially unchanged in the presence of butylated hydroxyanisole, butylated hydroxytoluene and α-tocopherol, while tertiary butylated hydroxyquinone and propyl gallate reduced the rate to zero. Based on these results, a scheme for the anaerobic bleaching of plant pigments during the lipoxygenase-catalyzed reaction is proposed. The formation of an enzyme-fatty acid radical complex which reacts with the plant pigment appears to be necessary for bleaching to occur. Certain antioxidants (tertiary butylated hydroxyquinone) preferentially react at this point in the pathway, thus effectively blocking chlorophyll and carotene bleaching.

Egg plant lipoxygenase: Isolation and partial characterization

Egg plant lipoxygenase EC 1.13.1.13 when purified on Ecteola cellulose was resolved into two active fractions with most of the activity in the first fraction (A). This fraction when further purified on Sephadex G200-120 had 20 times the specific activity of the crude material. It proved to be a single substance by electrophoresis and immunological technique. The pH optimum was 6.5. Its activity was specific for thecis,cis-1,4 pentadiene structure. There was no inhibition by cyanide, azide, EDTA or fluoride. Nordihydroguaretic acid, on the other hand, exhibited strong inhibition at 3 × 10−3M concentration. The specific antibody caused 50% inhibition.

Pigment bleaching by soybean lipoxygenase type-2 and the effect of specific chemical modifications

Abstract In contrast to lipoxygenase-1 of the soybean, the lipoxygenase-2 isomer had very little anaerobic activity as determined by diene formation, carbonyl production and pigment bleaching. Chlorophyll and carotene inhibited the aerobic activity of this isomer. The inhibition at low concentrations appeared to be noncompetitive and at higher concentrations, competitive. Increasing pigment concentrations over 20 μg/ml for carotene and 3.75 μg/ml for chlorophyll decreased the rate of bleaching. When chlorophyll was added after the lipoxygenase had begun to react, diene formation was inhibited but pigment bleaching was even greater than when the chlorophyll was added together with the lipoxygenase linoleic acid mixture. This increased bleaching is most probably due to the presence of free radicals. Antioxidants reduced diene and carbonyl formation as well as chlorophyll and carotene bleaching through their action as free radical scavengers. Bromosuccinimide and iodoacetic acid caused a pronounced decrease in activity. The former indicates the involvement of tryptophan in the active center as shown as well by the change in spectrum caused by this reagent. The latter indicates that methionine is most probably involved in the lipoxygenase action. Methyl mercuric iodide and tetranitromethane had very little effect on the lipoxygenase activity, indicating for the former that no sulphydryl groups are involved and for the latter that tyrosine is not in the active center. This is in contrast to the effects of these reagents on lipoxygenase-1. A scheme is proposed to show the pathways of lipoxygenase-2 activities.

Lipoxygenase of Thermoactinomyces vulgaris, purification and characterization of reaction products

Abstract 1. 1. A lipoxygenase preparation was obtained from Thermoactinomyces vulgaris and was purified by affinity chromatography on a linoleyl aminoethyl sepharose column. 2. 2. Two active fractions were obtained. 3. 3. The fraction obtained by elution with 100 mM borate buffer pH 9.0 was used in the subsequent work. 4. 4. Th. vulgaris lipoxygenase oxidized linoleic acid into two products: 13-HPOD and 9-HPOD at a ratio of 44 to 56, respectively. 5. 5. The identification and characterization of the isomers was done by HPLC, I.R. and mass spectrometry. 6. 6. When arachidonic acid was used as substrate, 15-HPETE and 15-HETE were found to be the main enzymatic products.

Lipoxygenase of the thermophilic bacteria thermoactinomyces vulgaris—properties and study on the active site

Abstract 1. 1. A lipoxygenase activity was purified from Thermoactinomyces vulgaris and some of its properties were characterized. 2. 2. The enzyme showed a temperature activity range of 40–55°C with still significant activity over 60°C. 3. 3. The pH of activity on linoleic acid had a broad range with an optimum at pH 6.0 and a weaker one at pH 11.0. 4. 4. On arachidonic acid the pattern was narrow bell-shaped with an optimum at pH 6.5. 5. 5. The purified lipoxygenase from Th. vulgaris showed an apparent K m of 1 mM and V max of 0.84 μmol diene/min/mg protein. 6. 6. It was inhibited by the oxidation products, 9-HPOD and 13-HPOD. 7. 7. A 160,000 Da molecular weight of the enzyme was determined by molecular filtration. Methionine, tyrosine, tryptophan and cysteine are apparently involved in its activity.

Methylmercuric iodide modification of lipoxygenase-1: Effects on the anaerobic reaction and pigment bleaching

The effect of methylmercuric iodide modification of sulfhydryl groups in soybean lipoxygenase-1 on linoleate oxidation, carbonyl production and beta-carotene and chlorophyll alpha bleaching were determined under aerobic and anaerobic conditions. Linoleate oxidation at pH 9.0 was strongly inhibited by modification of the enzyme. On the other hand, pigment bleaching was enhanced with the modified enzyme. Unmodified lipoxygenase-1 was not sensitive to chlorophyll inhibition, but activity of modified lipoxygenase-1 was affected. Linoleate oxidation was inhibited up to 70% when 2.2 microM chlorophyll was present in the reaction mixture. Chlorophyll inhibition was similar with affinity chromatography-purified lipoxygenase-2 and modified lipoxygenase-1. Unmodified lipoxygenase-1 exhibited high bleaching activity under anaerobic conditions and relatively low activity under aerobic (oxygen or air) conditions. Modified lipoxygenase-1 showed a significant increase in carotene and chlorophyll bleaching under both anaerobic and aerobic conditions. Under anaerobic conditions in the presence of either pigment, both modified and unmodified lipoxygenase-1 exhibited high 285 nm absorbing material production. Antioxidants (butylated hydroxyanisole, butylated hydroxytoluene, alpha-tocopherol, propyl gallate and tertiary butylated hydroxyquinone ) were powerful inhibitors of pigment bleaching by modified lipoxygenase-1. However, only tertiary butylated hydroxyquinone and propyl gallate blocked the increase in the rate of absorbance at 285 nm.

Lipoxygenase isoenzymes from Solanum tuberosum

Abstract Two lipoxygenase isoenzymes were separated from potato tubers ( Solanum tuberosum ). Experiments with chemical modifications showed that tryptophan is essential for enzyme activity and that one or more tryosine residues was involved. On the other hand, no lysine or sulfhydryl groups were necessary. Both enzymes had an optimum pH of 5·5. They were not affected by calcium ions but were inhibited by cysteine.

Isolation of soybean lipoxygenase-2 by affinity chromatography

Abstract Isoenzyme lipoxygenase-2 from soybean was isolated by affinity chromatography. Gel electrophoresis showed it to be a single protein. Its pH optimum of 6.5, range of 5.0–8.0 and activity which increased when Ca2+ was added identified the isolated enzyme as lipoxygenase-2.

A convenient method for lipoxygenase isoenzyme determination

Abstract The red-brown color obtained when peroxides react with ferrous ammonium thiocyanate permits identification and determination of lipoxygenase isoenzymes separated in cellulose acetate gel electrophoresis. The reaction is much more sensitive than the Coomassive red protein staining reaction.