Soybean, isoenzymes

Grosch, W. and Laskawy, G., Co-oxidation of carotenes requires one soybean lipoxygenase isoenzyme, Biochim. Biophys. Acta, 575, 439, 1979. 

Although LOX from soybean seed is the best characterized of plant LOXs, this enzyme is present in a wide variety of plant and animal tissues (Liavonchanka and Feussner, 2006). The enzyme occurs in a variety of isoenzymes, which often vary in their optimum pH and in product and substrate specificity. Given the occurrence of multiple LOX isoenzymes in soybean leaves and the proposed roles of these enzymes in the plant metabolism, it is possible that individual isoenzymes play specific functions (Feussner and Wasternack 2002). The molecular structure of soybean LOX is the most reported, and four isoenzymes have been isolated (Baysal and Demirdoven 2007). Soy isoenzyme 1 produces 9- and 13-hydroperoxides (1 9) when the enzyme acts on free PUFA at pH 9.0, its optimum pH (Lopez-Nicolas and others 1999). Soy isoenzyme 2 acts on triglycerides as well as free PUFA leading to 9- and 13-hydroperoxide... 

It is supposed that the catalytic mechanism proceeds through a free-radical intermediate which reacts directly with oxygen or an organic iron intermediate [63]. The three-dimensional protein structure of the native form of LOX isoenzyme L-1 from soybean has already been described [64, 65]. 

However, it should be noted that isoenzymes, i.e., different enzymes catalyzing identical reactions, would have the same four-digit classification. The classification system provides only the basis for a unique identification of an enzyme the particular isoenzyme and its source still have to be specified. For example, peroxidases isolated from soybeans and horseradishes have the same classification, i.e., EC 1.11.1.7. Currently, there are approximately 3200 enzymes that have been listed and assigned classification numbers. 

Although HRP is commercially available, it is not currently available in bulk quantities or at a cost that that is sufficiently low to make full-scale applications of enzymatic treatment feasible. It has been suggested that the use of a less expensive and more ubiquitous source of peroxidase could circumvent the problem of enzyme cost. For example, in 1991, the seed coat of the soybean was identified as a rich source of a single peroxidase isoenzyme [84]. Because the seed coat of the soybean is a waste product of the soybean industry, soybean shells could provide an inexpensive and abundant source of peroxidase. Thus, soybean peroxidase (SBP) has the potential of being a cost-effective alternative to HRP for wastewater treatment. 

Considerable purification of lipoxygenase from soybeans was achieved by Balls et al (15), They used conventional protein purification methods and the carotene oxidase reaction to follow the enzyme activity. Theorell et al, (16), in 1947, crystallized the enzyme, or more correctly, one of the several isoenzymes in soybean. We believe this was lipoxygenase-1 as described later. 

Isoenzymes. Kies (90), who questioned the identity of lipoxygenase and carotene oxidase, thought that the lipoxygenase activity of soybeans was caused by more than one enzyme. She discovered that a partially... 

Engeseth, N.J. B.P. Klein K. Warner. Lipoxygenase isoenzymes in soybeans effect on crude oil quality. /. Food. Sci. 1987,53, 1015-1019. 

Indications that soybeans contained more than one form of LOX came from the apparent loss of carotene oxidase activity in partially purified LOX (Kies, 1947), from studies on the relative activities on triacylglycerol or free fatty acid substrates (Koch et al., 1958), and from the effects, of purification (Dolev et al., 1967) or pH (Galliard and Phillips, 1971) on the proportions of the 9- and 13-hydroperoxides formed. Guss et al. (1967, 1968a) found several electrophoretically distinguishable bands of LOX in soybean extracts. Characterization of different forms of LOX followed the isolation by Christopher et al. (1970) of an isoenzyme different from the Theorell type. 

Two main types in soybean are now known (Table II). The classic Theorell enzyme is LOX-1. Some confusion exists in the nomenclature, because Verhue and Francke (1972) called this form the acid enzyme (active on free fatty acids only) and Grosch et al. (1977) referred to it as the alkaline enzyme (the pH optimum is 9). The second type, LOX-2, is optimally active at pH 6.5 this is the ester enzyme of Verhue and Francke (1972), the neutral enzyme of Grosch ef al. (1977), and the b enzyme of Yamo-moto et al. (1970). The type-1 and type-2 nomenclature is now used by most workers and should be retained to avoid ambiguities. Both types are further separated into isoenzymic forms Grosch et al. (1977) refer to LOX-1,1 and LOX-1,2 isoenzymes from type 1, and LOX-2,1 and LOX-2,2 isoenzymes from type 2. Christopher et al. (1972) and Weber et al. (1974) describe a type-3 enzyme, but results (Grosch et al., 1977) indicate that this may be a mixture of isoenzymes. The major differences between types 1 and 2 are summarized in Table II. 

Chan (1972,1973) and Roza and Francke (1973a) demonstrated that soybean LOX contained one atom of nonheme iron per molecule, and this has been confirmed for the type-1 enzyme (Finazzi-Agro et al., 1973) and for both types 1 and 2 (Pistorius and Axelrod, 1974). The same value of one atom of iron per molecule has been demonstrated for two isoenzymes of LOX from bush bean (Hurt and Axelrod, 1977) and for pea LOX (Spaapen et al., 1977). 

Amino acid compositions for soybean and pea enzymes have been studied by several groups (see Veldink et al., 1977). Although there is general agreement for most amino acids, various values have been reported for the cysteine and cystine contents, ranging, for the soybean enzyme, from 4 (Mitsuda et al., 1967a) to 12 (Axelrod, 1974) half-cystine residues. Amino acid analyses of two isoenzymes from pea have been obtained by Eriksson and Svensson (1970) and Arens et al. (1974). 

Soybean mixed isoenzymes 9.0 Oxygen 0 0-30 70-100 Several groups (see text)... 

Soybean purified acid isoenzyme Soybean purified acid isoenzyme 7.0 9.0 Oxygen Oxygen Ambient 0 ca. 50 50 5 95 Roza and Francke (1973b)... 

Lipoxygenases from different sources differ in their cooxidation activity. Enzymes from peas and beans (Phaseolus sp.) and the LOX-2 from soybean have a high cooxidation potential potato LOX is intermediate, whereas wheat, flax, and soybean LOX-1 have poor cooxidation activity (Grosch et al., 1976, 1977). Thus, the carotene oxidase activity of soybeans is associated with the LOX-2 isoenzyme, and this explains the observed selective loss of carotenoid bleaching activity during the purification and heat treatment of the classic (i.e., LOX-1) enzyme from soybean (Kies et al., 1969). 

Several isoenzymes of lipoxygenase are known, including L-1 and L-2 from soybeans, with pH optima of 9.0 and 6.5, respectively. Isoenzymes L-1 with high pH optima prefer anionic free fatty acids, and L-2 with pH optima near neutral prefer neutral substrates such as triacylglycerols. L-1 produces the 13-hydroperoxide stereoselectively from linoleic acid, while L-2 produces a... 

In the rate-limiting step of catalysis, the isoenzyme LOX 1 from soybeans abstracts the pro-(S)-hydrogen from the n-8 methylene... 

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