Lipoxygenase-1 Activity of Soybean Genotypes Grown in Argentina

Abstract

The lipoxygenase-1 (LOX-1) activity of 19 soybean genotypes was quantified in two consecutive years. The LOX-1 activity produced by any cultivar was essentially the same in both, 1995 and 1996 crop years. The lowest values of LOX-1 activity were found in NK 555 cultivar whereas Asgrow 5409 cultivar had the highest values.

Introduction

Lipoxygenase (LOX) was first reported in soybeans almost 67 years ago [1]. LOX catalyzes the hydroperoxidation of polyunsaturated fatty acids containing a cis, cis-1, 4-pentadiene system, but structures other than fatty acids are known to be oxidised [2,3]. The cleavage of fatty hydroperoxides into short-chain aldehydes and alcohols has been studied, suggesting that lipoxygenase could be used as a versatile biocatalyst [4].

Normal soybean seeds contain three lipoxygenase isozymes, named LOX-1, LOX-2 and LOX-3 which differ in substrate specificity, optimum pH for catalytic activity, isoelectric point and thermal stability [5,6]. LOX-1, an enzyme with a pH optimum of 9 to 10, represents a large class of other less-studied LOX of this type. For the biocatalytic production of a natural aroma compound, lipoxygenase is needed on a large scale and the alkaline isozyme (LOX-1) seems to be the most suitable for this purpose [7]. The objectives of this work were to screen and compare the soybean LOX-1 activity in some genotypes cultivated in Argentine in two consecutive years.

Results and Discussion

The biosynthesis of LOX isozymes in soybean is under genetic control [8,9]. Furthermore, weather conditions have been found to play a considerable role in influencing the activities of the LOX isozymes [7]. In the present work, the differences in activity among the different cultivars from the same year were larger than those between the generations (crop years) of a cultivar. The LOX-1 activity produced by any cultivar was essentially the same in both, 1995 and 1996 crop years (

Table 1. Lipoxygenase-1 activity (ΔOD.mg prot^-1.min^-1) in 19 soybean cultivars during 1995 and 1996 crop years. Mean values ± standard deviations (n = 3).

Cultivar	Crop year
	1995	1996
NK 555 Forrest	7.44 ± 0.14a 8.53 ± 0.13b	7.81 ± 0.16 a 8.98 ± 0.17b
NK 641 Tancacha	8.75 ± 0.15bc 8.85 ± 0.10bcd	8.96 ± 0.16b 8.66 ± 0.14c
Copetona 53 Prata	9.02 ± 0.15cd 9.24 ± 0.14d	9.22 ± 0.19b 9.51 ± 0.11d
Asgrow 5308 Federada Casilda RA 587	9.83 ± 0.30e 10.0 ± 0.20ef 10.3 ± 0.10f	9.96 ± 0.24e 10.8 ± 0.14f 10.1 ± 0.13e
Federada 1 INTA Granera 73	10.9 ± 0.10g 11.4 ± 0.12h	12.3 ± 0.12g 11.2 ± 0.16h
Asgrow 6404 Tacuarí	11.5 ± 0.15h 12.2 ± 0.20i	11.6 ± 0.11i 12.7 ± 0.18ij
Montera 74 RA 702	12.8 ± 0.10j 13.1 ± 0.15j	13.2 ± 0.12k 12.5 ± 0.15j
Torcaza 63 Charata 76 Torcacita 58	13.1 ± 0.10j 13.9 ± 0.15k 14.5 ± 0.20m	13.3 ± 0.13k 13.4 ± 0.12k 14.8 ± 0.16m
Asgrow 5409	16.0 ± 0.20n	16.5 ± 0.23n

^aMean values within each column followed by the same letter do not differ statistically P=0.05.

). These results are not in agreement with the data obtained by Márczy et al. [7] suggesting that, in the samples studied, genetic is stronger than environmental influences.

Statistically significant variations were found among genotypes in both, 1995 and 1996 crop years. A total of 13 groups with different enzymatic activity were observed. In 1995, the LOX-1 activity ranged from 7.44 (NK 555) to 16.0 (Asgrow 5409) ΔOD.mg prot^-1.min^-1; whereas in 1996 it varied between 7.81 (NK 555) and 15.5 (Asgrow 5409) ΔOD.mg prot^-1.min^-1. In general, mean values from 1996 were higher than those from 1995, with exception of Tancacha, RA 587, Granera 73, RA 702 and Charata 76 cultivars.

In the last decade, many attempts to improve the flavours of soybean products have centered around the genetic elimination of LOX from the seeds [8,9,10]. More recently some works [4,6] focus on the potential of LOX for the efficient production of useful compounds. Hence, cultivars with high LOX activity, such as Torcacita 58 and Asgrow 5409, could be used as a source of the Lox-1 isozyme.

Experimental

Plant material

Nineteen soybean genotypes were chosen. The experiment was conducted at the Estación Experimental Agropecuaria (EEA-INTA) of Manfredi, Córdoba, Argentina. Seeds were harvested, in the crop years 1995 and 1996, by hand at maturity when seed moisture was reduced to 10% or less. One hundred seeds (taken randomly from each seed sample) were powdered by grinding and soybean flour of each cultivar was extracted according to Pignata et al. [11].

Lipoxygenase assay

The method of Axelrod et al. [5] was followed with a slight modification. The activity of LOX-1 isozyme was determined via the increase in absorbance at 234 nm after addition of linoleic acid in 0.1M phosphate buffer (pH 9.0). Lipoxygenase-1 activity was expressed as an optical density increase per mg protein^-1 per minute^-1 (ΔOD.mg prot^-1.min^-1).

Protein content

Protein determinations were performed by the method of Kalckar [12].

Statistical analysis

Lipoxygenase-1 determinations were conducted in triplicate. Statistical differences among geno-types from each crop year were estimated from ANOVA test at the 5% level (P=0.05). Whenever ANOVA indicated significant difference, a pairwise comparison of means by least significant difference (LSD) was carried out.