Lipoxygenases

Beans and pulses in glass jars
Beans and legumes which are sources of lipoxygenases. Photo by ulleo, c/o of Pixabay.

Lipoxygenases (linoleate:oxygen oxidoreductase, EC 1.13.11.12) are found throughout animal and plant species. They are most abundant in grain legume seeds and potato tubers but they are found in all higher organisms especially their organs and tissues (Eskin et al., 1977). You might see the enzyme referenced as lipoxidase and ‘carotene oxidase’. They are also often abbreviated to LOX and have been known about since the early 1960s. The enzymes are commonly called dioxygenases because of their ability to activate oxygen.

It is an enzyme with a non-haem and non-sulphur-iron but has an iron-bearing active site and is monomeric. That means there are no sub-unit structures. It does not actually need a metal or a prosthetic group for activity although there is a clear need for a site to generate hydroperoxyl radicals which are extremely reactive species.  

The enzyme catalyses the dioxygenation of fatty acids containing the (1Z,4Z)-pentadiene structure (Schilstra et al., 1994). Polyunsaturated fatty acids (PUFAs) containing these moieties usually produce 9- or 13-hydroxyperoxy octadecadienoic acid. These compounds often go onto to produce the range of volatile derivatives found in a all sorts of plant products – olives are a classic example.

Lipoxygenases are the major class of initiators of lipid oxidation although the enzyme itself is not the actual initiator ! It produces reservoirs of lipid hydroperoxides (LOOH) that decompose to generate LO and OH radicals when exposed to light and heat. The enzyme works in conjunction with peroxidases which are also notorious for generating off-flavours and browning compounds too. 

Lipoxygenases are a serious issue for post-harvest and storage of produce especially legumes and pulses. The enzyme catalyzes the formation of off-flavours by hydroperoxidation of lipids. This always seems to occur in seeds following their harvesting and is responsible for a host of off-notes described as beany, viney etc.

The presence of the enzyme is not entirely clear. In physiological sense they are probably present to help with degradation of large molecules prior to senescence such as the oxidation of plant pigments, carotenoids and chlorophyll, or of cholesterol. They could also be involved in pest resistance, pathogen defence, reducing biotic and abiotic stress and as a response to wounding by initiating or being part of the healing process. They also help in the production of regulatory molecules and attractants for insects and animals to fruit. Regulatory molecules include jasmonic acid and traumatin in plants (Nguyen et al., 2013).

One study on a novel lipoxygenase in pea roots found that it was important in responses to wounding and biotic stress (Veronico et al., 2006). 

The hydroperoxide products cause feedback inhibition of the lipoxygenase activity. The enzyme activity rises when the cell walls are broken as in processing and there are no longer any biochemical controls in place to regulate the enzyme (Sessa, 1979).

Soybean LOX And Other Lipoxygenases

LOX from soybean is probably the most studied because it is so abundant in seeds and relatively easy to purify. Other sources include:-

asparagus (Asparagus officinalis L.) Ganthavorn & Powers, 1989)

avocado (Jacobo-Velazquez et al., 2010),

banana leaf (Kuo et al., 2006)

barley (Lulai and Baker, 1976; Lulai et al., 1981),

chickpea (Sanz et al., 1992a, b)

haricot beans (Phaseolus vulgaris) (Kermasha and Metche, 1986; Adams & Ongley, 1989).

hazelnut seeds (Santino et al., 2003). 

olives (Lorenzi et al., 2006), olive callus cultures (Williams and Harwood, 2008).

lettuce (L. sativa), (Altunkaya and Gokmen, 2011)

pea seeds (Pisum sativum var. TelephoneL.) (Szymanowska et al., 2009),

sesame seeds (Marvian-Hosseini and Asoodeh, 2017) 

sweet corn germ (Theerakulkait and Barrett, 1995)

tomatoes (Anthon and Barrett, 2003)

wheat germ (Nicholas et al., 1982; Bhirud & Sosulski, 1993), durum wheat (Gokmen et al., 2007)

The lipoxygenases in seeds have been thoroughly reviewed (Loiseau et al., 2001).

Soybean lipoxygenase (LOX-1) is similar in behaviour to mammalian 15-lipoxygenase.

Lipoxygenase In Seeds 

Lipoxygenase activity is generally distributed in the germ (embryo) of seeds and not so much in the kernel. This is certainly the situation for sweet corn (Wagenknecht, 1959; Lee, 1981; Lee et al., 1989). In an inbred yellow dent corn, the activity of lipoxygenase was generally higher in germ than in endosperm tissues even throughout kernel development (Belefant and Fong, 1991). gardner (1970) thought lipoxygenase was primrily sited in the mature tissue of mature hybrid corn.

The enzyme can be found in both the cytosol of the cell or attached to the membrane. Soybean leaf lipoxygenases have been studied for their differing levels of stability (Baracat-Pereira et al., 2001).

Formation Of Off-Flavours

The level of off flavours formed depends on the type of substrate and its concentration in the legume seed. Most off-flavours are derived from volatile short-chain aldehydes, ketones and alcohols (Kobayashi et al., 1995; Yuan and Chang, 2007). The rate and specificity of oxidation reactions catalysed by lipoxygenase depends on the type of fatty acid and their concentration (briefly referenced earlier), pH, enzyme source and isozyme form.

The preferred substrate is linoleic acid, linolenic acid and arachidonic acids although the latter is oxidised more slowly (Gardner, 1986). We know of at least three different isoenzyme variants based on work with soybean lipoxygenase which differ in their specificity for C9 and C13 on linoleic acid, their pH optima and their phase preferences for three types of reaction:-

LPOx-1: optimum pH of activity is 9, prefers charged substrates such fat peroxide radicals (LOO•). Has no phase specificity and does not mind whether it reacts in aqueous or organic phases.

LPOx-2: optimum pH of activity is 6.8, prefers un-ionized acids, esters and organic phases; reactions are depressed in emulsions. Has strong co-oxidation capacity.

LPOx-3: optimum pH is around 6.8 and prefers emulsions and strong co-oxidation capabilities.

Isozymes Of Lipoxygenase

As well as these general preferences, isozymes have their own specificities and ‘quirks’. The garden pea has four lipoxygnase isoenzymes which were isolated from the cv. Little Marvel. The main isoenzymes are called PL I and PL II which have a molecular weight of 95 kDal and an optimum activity between pH’s 7 and 7.5 but no activity above pH 8. The other two isoenzymes occur in very small quantities.

Soybean has many isoenzymes and its not entirely certain how many there really are (Christopher et al., 1970; 1972). Olive callus cultures produced 2 isoforms (Williams and Harwood, 2008) and there are two isoenzymes known for chickpea called CL-1 and CL-2 (Sanz et al., 1992).

These isoenzymes exhibit distinct features for preference of substrate, kinetic parameters, and positional specificity of substrate oxygenation (Feussner and Wasternack, 2002) and are tightly regulated according to recent research into their gene transcription.

Extraction Of Lipoxygenases

The general method for extraction of lipoxygenases involves ammonium sulphate fractionation (25-50% or 35-65% saturation), gel filtration and then ion-exchange chromatography (Yoon and Klein, 1979) or hydrophobic chromatorgraphy (Sanz et al., 1992). Isolelectric focussing in a granulated gel can then refine the lipoxygenases further.

In one example with olive callus cultures, acetone was added to suspensions of LOX to stabilise activity and insolubilise lipids (Williams and Harwood, 2008). The enzyme suspension was than salt precipitated followed by ion-exchange chromatography to isolate the enzyme.

In some examples such as the isolation of LOX from olive fruit (Olea europaea L.), a series of differential centrifugations coupled to hydrophobic chromatography was needed (Lorenzi et al., 2006).

Assay Of Activity    

Lipoxygenase activity is analysed in pulse flours by this typical method adapted from Shariati-Levari et al., (2016). The crude protein is extracted by adding 100 mL of 0.2 M sodium phosphate (Na3PO4) buffer (pH 6.6) to pulse samples (5 g). The solution is then slowly stirred for 1 hour at 4 °C. One milliliter of the mixture is centrifuged at 9500 × g for 10 min.

To assay the activity in this flour supernatant, linoleic acid (10 μL, 99.5%) is mixed with 10 μL of Tween‐20 (polyethylene sorbitan monolaurate) which is an emulsifier in a 1‐mL tube. Potassium hydroxide (0.5 mL, 0.1 M) is added until the solution became clear. A  subsample (6 μL) of the substrate mixture is added to 994 μL of sodium phosphate buffer (pH 6.6) to create a 0.37 mM linoleate solution. To prepare for the reaction, 2.7 mL of sodium phosphate buffer, 5 μL of the crude protein extraction, and 0.3 mL of the 0.37 mM linoleate are mixed in a 5 mL centrifuge tube. A total of 300 μL is then aliquoted into a UV plate micro well and read at an absorbance of 234 nm with a spectrophotometer. The activity is measured at various time intervals to 90 minutes.  One unit of LOX activity was defined as an increase of 0.1 A234/min (Sosulski and Gadan, 1988). Measurement is taken on 3 different flour samples.

Characterisation Of Activity

Lipoxygenase from olive fruit has a molecular mass of 98 kDa (Lorenzi et al., 2006). The pH of maximal activity is pH 6. Other types have a mass of 95 kDa (olive), 93kDa (pea seed).

The Vmax of LOX from pea seed (Szymanowska et al., 2009) was 151.5 U/min with a Km for linoleic acid of 0.44 mM which seems very high.

In terms of substrates, lipoxygenase has a stronger affinity for linoleic acid (Km = 82 μM) than for linoleic acid (Km = 306μM) in . Oleic acid is a poor substrate.

Soybean lipoxygenase (LOX-1) is inhibited by endocannabinoids such as N-arachidonoyl dopamine and N-oleoyl dopamine when it is assayed by catalysing the oxygenation of linoleic acid (Nguyen et al., 2013). The Ki values are  3.7 μM and 6.2 μM, respectively. None of the other cannabinoids showed any inhibitory activity

Generally, thermal inactivation follows .

The presence of more than one isoform often complicates assay and activity measurements. For example, thermal inactivation curves for wheat germ lipoxygenase were analysed (Bhirud & Sosulski, 1993).  

Inhibitors Of Activity

 – nordihydroguaiaretic acid (NDGA), caffeic acid and propyl gallate. To a lesser extent ferulic acid, benzoic acid, catechin, quercetin.  Kaempferol was the weakest inhibitor of this aforementioned series (Szymanowska et al., 2009). Sulphites and sulphur dioxide are powerful antioxidants which reduce the formation of peroxides and hydroperoxides. In this regard, the very effect of bleaching helps to minimise LOX activity.

Cyanide in its undissociated acid form is a weak competitive inhibitor of green bean lipoxygenase (Adams, 1989).

Processing Considerations

Lipoxygenase is generally inactivated at temperatures above 60 C and any treatments that have succeeded in this have usually extended the quality and shelf-life of food (Anthon and Barrett, 2003). In their studies on tomato LOX inactivation, they found the enzyme didn’t obey first order kinetics. They were able to fit their data on the basis that three isoenzymes existed.Unfortunately, extended heating also encourages non-enzymic browning reactions too.

LOX inactivation has been studied with a range of processing techniques. Blanching is generally the best method for preserving both colour and flavour in vegetables. Improvements in texture and a reduction in microbial load which causes spoilage is also possible.  The technique is particularly suited to legumes. Lipoxygenases are usually incativated by thermal treatment above 60 C. Unfortunately, too much heating causes non-enzymatic browning and can exceed the level of oxidation by the lipoxygenase in the first place. Most processors blanch to end point in lipoxygenase activity which is suitable for a shelf-life of 18 months at -18 °C (Sheu & Chen, 1991). Green beans for example are treated this way (Adams and Ongley, 1989).

Ideally, excluding oxygen during processing and subsequent packaging is probably the most effective way of eradicating off-flavour formation. The expense however can be considerable.

The Genetics Of Lipoxygenases

LOXs are encoded by gene families (LOX) in most, if not all, of the plant species studied so far (Royo et al., 1996). The transcription of each gene member is under tight developmental control. More than one member is often active at a specific developmental stage, accounting for the occurrence of multiple LOX isoforms. 

General References

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Adams, J.B. and Ongley, M.H. (1989) The behavior of green bean lipoxygenase on chromatography and isoelectric focusing. Food Chem. 31 pp. 57-71 https://doi.org/10.1016/0308-8146(89)90151-9

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Anthon, G.E.Barrett. D.M. (2003) Thermal inactivation of lipoxygenase and hydroperoxytrienoic acid lyase in tomatoesFood Chem. 81(2) pp. 2759.

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Baracat-Pereira, M. C., de Almeida Oliveira, M. G., de Barros, E. G., Moreira, M. A., & Santoro, M. M. (2001). Biochemical properties of soybean leaf lipoxygenases: Presence of soluble and membrane-bound forms. Plant Physiology and Biochemistry39(2), pp. 91-98

Bhirud, P.R., Sosulski, F.W. (1993) Thermal Inactivation Kinetics of Wheat germ Lipoxygenase. J. Fd. Sci., 58(5) pp. 1095-1098   https://doi.org/10.1111/j.1365-2621.1993.tb06122.x

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