HUNGARIAN JOURNAL OF INDUSTRIAL CHEMISTRY VESZPREM Vol. 29. pp. 35- 38 (2001) A NOVEL METHOD FOR ISOLATION OF LIPOXYGENASE-1 ISOENZYME FROM SOYBEAN MEAL A. Sz. NEMETH, B. SZAJA.NI1, J. Sz. MARCZ¥ and M. L. S1MON2 (Research Institute of Chemical and Process Engineering, University of Kaposvar P.O. Box 125, Veszprem, H-8201, HUNGARY 1 ' SATO Ltd, Szel Street 4, Budapest, HUNGARY 2 Department of Biochemistry, Attila J6zsefUniversity, P.O. Box 533, Szeged, H-6701, HUNGARY) Received: January 30, 2001 A novel method has been elaborated for the isolation of lipoxygenase-1 (Lox-1) isoenzyme from soybean meal. The essential step of the procedure is the selective heat treatment of soybean meal extract at 70 "C in a medium of 0.05 ionic strength and pH 5.2 for 5 min. The heat treated extract practically free from lipoxygenase-2 (Lox-2) and lipoxygenase-3 (Lox-3) isoenzymes was pnrified by DEAE-cellulose ion exchange chromatography. The resulted lipoxygenase-1 isoenzyme was homogeneous as judged by PAGE. A tenfold purification and a relatively high yield (39 %) was achieved. Keywords: soybean; Glycine max; lipoxygenase isoenzymes; selective heat treatment Introduction Soybean seeds contain an iron"containing dioxygenase namely lipoxygenase (linoleate : oxygen oxidoreductase, EC 1.13.11.12) catalyzing the hydroperoxidation of polyunsaturated fatty acids and esters containing a cis,cis-1,4-penta:diene system [1]. The hydroperoxides formed are cleaved by hydroperoxide lyase, resulting in C6 aldehydes in plants. There are at least three lipoxygenase isoenzymes in soybean seeds, named lipoxygenase-1 (Lox-1), lipoxygenase-2 (Lox-2) and lipoxygenase-3 (Lox-3), which exhibit differences in substrate and product specificity, optimum pH for catalytic activity, isoelectric point and thermal stability [2-4]. The levels of the lipoxygenase isoenzymes are influenced to different degrees by both cultivar and climatic effects. The weather conditions can exert a greater effect than the cultivar effect £5]. With linoleic acid as substrate, linoleic acid 13- hydroperoxide and 9-hydroperoxide can be formed in the reaction catalyzed by Iipoxygenase. The ratio of isomers derived from the reaction depends on the isoenzyme(s) present and the reaction conditions {6]. Soybean lipoxygenase can be used to produce flavour compounds, but in some cases the presence of Lox-2 and Lox-3 isoenzymes is undesirable because of side reactions. A large number of methods have been suggested for separation of the isoenzymes, e.g. ion exchange chromatography £7-9], chromatofocusing [lO- ll], electrophoresis [12] and isoelectric focusing [7, 10]. We have found [13] that the selective heat treatment of the soybean meal extract makes the elimination of Lox-2 and Lox-3 isoenzymes possible. Based on this finding, a novel method for the isolation of Lox-1 isoenzyme has been elaborated. Materials and methods Reagents Linoleic acid, Tween 20, DEAE-cellulose and soybean lipoxygenase (Type I-B) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Acrylamide, N,N,N' ,N' -tetramethylethylenediamine and Coomassie Brilliant Blue R-250 were supplied by Reanal Factory of Laboratory Chemicals (Budapest, Hungary) and N,N' • methylene-bis(acrylamide) was obtained from Fluka A.G. (Buchs, Switzerland}. The other chemicals were of analytical grade. The sodium linoleate substrate was prepared according to Axelrod et al. f14}. 36 Apparatus A Biochrom 4060 spectrophotometer was used for enzyme activity measurements and a 2117 Multiphor II Electrophoresis System for electrophoresis (Pharmacia LKB, Uppsala, Sweden). Lipoxygenase Assay The method of Axelrod et al. [14] as modified by Marczy et al. [5] was applied, except that 0.2 M sodium borate buffer was used for Lox-1. One unit of activity was defined as the amount of enzyme producing 1 J.llllol of linoleic acid hydroperoxide per min at room temperature at pH 9.0 (Lox-1) or 6.8 (Lox-2 and Lox-3). Protein Content Protein contents were determined according to Lowry et al. [15], with bovine serum albumin as standard or by absorbance at 280 nm. Electrophoresis Polyacrylamide gel electrophoresis (PAGE) was performed in a continuous buffer system of 0.1 M sodium phosphate buffer (pH 7 .5). The total monomer concentration [acrylarnide + N,N'-methylene- bis(acrylarnide)] in the gel was 5 %, the concentration of N,N' -methylene-bis(acrylarnide) being 2.7% of the total monomer concentration. Samples of 15 J.tg protein in 0.01 M sodium phosphate buffer (pH 7 .5) were applied onto a gel slab {12 em x 4 em). Electrophoresis was carried out for 2 h at 200 mA at 5 °C. The gel was stained for protein with 0.25 % of Coomassie Brilliant Blue R-250 dissolved in methanol:water:acetic acid (23:23:4). Experimental Preparation of Crude Extracts Soybean meal was prepared :&om a Chandor cultivar harvested in 1993. kindly provided by the Cereal Research Institute (Szeged, Hungary). Ten grams of soybean meal was defatted with 20 cm3 portions of petroleum ether at 0 <>c. Defatted meal was extracted with 5 volumes (v/w) of 0.05 M acetic acid by mechanical stirring for 1 h at 4 oc. The suspension was then filtered through cheesecloth and centrifuged at 10,000 g for 20 min. The resulting supernatant was used in the further experiments. Selective heat Treatment The crude extract was heated to 70 oc in a water bath under continuous stirring and was kept at this temperature for 5 min [13]. Immediately after heat treatment the suspension was cooled to room temperature in an ice bath and then was centrifuged at 10,000 g for 20 min at room temperature. Ion Exchange Chromatography Anion exchange chromatography of heat treated soybean meal extract was performed according to Axelrod et al. [14] and Weber et al. [16] with modifications on DEAE-cellulose. 14.5 cm3 heat treated soybean meal extract was dialyzed against 3 x 500 cm3 0.01 M sodium phosphate buffer (pH 7.0). The enzymatically inactive precipitate was discarded and the supernatant was loaded on a DEAE-cellulose column (1.85 x 21 em) equilibrated with 0.01 M sodium phosphate buffer (pH 7.0). Elution (22.5 cm31h) was performed with a linear gradient formed from equal volumes of 0.01 M and 0.22 M sodium phosphate buffer (pH 7 .0). Fractions of 6 cm3 were collected and the protein contents were determined by absorbance at 280 nm. Activities were assayed at pH 9.0 (Lox-1) and 6.8 (Lox-2 + Lox-3). The enzymatically active fractions of Lox-1 were pooled and concentrated by ultrafiltration. Results and discussion· Data from a typical Lox-1 isoenzyme isolation and purification experiment are summarized in Table I. The data show that the selective heat treatment of soybean meal extract at 70 oc is a crucial step in the isolation of Lox-1. Though the purification of Lox-I was less than twofold in respect of specific activity as an effect of heat treatment, the amounts of Lox-2 and Lox-3 isoenzymes were markedly reduced by this simple method. The anion exchange chromatography makes the removal of further protein impurities possible (Fig.l}. Tenfold purification and a relatively high yield (39 %) was achieved by the two purification steps. The purified Lox-l isoenzyme was electrophoretically homogeneus as judged by PAGE (Fig.2). 37 Table 1 Isolation oflipoxygenase-1 isoenzyme from soybean meal a Volume TotalLox-1 Lox-1 Total Lox-1 Purification step (cm3) activity yield protein specific activity Activity Purification (units) (%) (mg) (units/mg Erotein) ratio* extraction 17.0 582.6 100.0 317.6 1.83 5.4 1.0 heat treatment 14.5 392.5 67.4 130.8 3.00 20.1 1.6 DEAE-cellulose 30.0 227.3 39.0 chromatography 11.1 20.48 20.8 11.2 •rn the experiment, 5 g soybean meal was processed *Lox-1 activity I (Lox-2+Lox-3 activity) """ 0.8 12 0.8 ] /"".. 1.0 ~ s -w ! 0.6 _§ l:l "" 0.6 0.8 0 :t:: ~:g <7 § 8 .::: ~ '-' 8 !:l - v ~ I !':; 0.6 0 0.4 X 6 0.4 0 !:l ~ 0 .. 0 ....:) ~ 0 ...... .... 0 0.4 ~ ....:) ~ 4 G-i ...... 0.2 ::; 0 ·:~ 0.2 ..0 0 0 ~1 )· * ,;.. .. """' 1 to ' ; ' t lJ ,.,.....,. * '-' -+ '-" Fig. 2 Polyacrylamide gel electrophoretic pattern of purified Lox-1 isoenzyme. Electrophoresis was carried out in 5 % polyacrylamide gel slab (12 x 4 em) for 2 h with 200 rnA at 5 oc. Samples 15 vg protein each. A: Sigma type I-B Iipoxygenase; B: lipoxygenase-lisoenzyme purified with the new method Conclusion We have found earlier {17, 181 that the selective heat treatment of aqueous extracts of animal organs as kidney and pancreas could be advantageous in enzyme isolations. It was supposed that this simple method would be useful in the isolation of plant enzymes too. The difference in the heat stability of soybean lipoxygenase isoenzymes {19] and the practical demand on Lox-1 isoenzyme make the application of heat treatment obvious. The heat treated soybean mea! extract is practically free from Lox-2 and Lox-3 isoenzymes. It can be used immediately for the production of linoleic acid 13-hydroperoxide, or it can be stored frozen without deterioration for a long time. 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