kinetic behavior of soybean lipoxygenase: a comparative study of the free enzyme and the enzyme...

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KINETIC BEHAVIOR OF SOYBEAN LIPOXYGENASE: A COMPARATIVE STUDY OF THE FREE ENZYME AND THE ENZYME IMMOBILIZED IN AN ALGINATE SILICA SOL-GEL MATRIX' AN-FEI HSU', EMILY WU, THOMAS A FOGLIA and GEORGE J. PIAZZA U.S. Department of Agriculture Agricultural Research Service Eastern Regional Research Center 600 East Mermaid Lane Wyndmoor, PA 19038 Received for Publication September 10,1998 Accepted for Publication February 16, 1999 ABSTRACT Lipoxygenase (LOX) is an enzyme that regioselectively introduces a hydroperoxide into polyunsaturated fatty acids (PUFA). We recently reported a procedure that immobilizes soybean LOX within an alginate sol-gel matrix. In this study, the kinetic profile of fiee LOX was compared with that of the sol-gel immobilized LOX. The temperature dependent activity profile offree LOX was optimal at 25C whereas immobilized LOX had optimal activity over the temperature range of 25-392. Enzyme activity, measured in aqueous bufer, for both the free and immobilized LOXpreparations had K, values of 2.5 and 1.40 mmoles/L, respectively, and V,, values of 0.056 and 0.02 pmol/min, respectively. The relutive rates of oxidation of linoleic acid and acylglycerols containing linoleoyl residues catalyzed by free and immobilized LOX also were determined The results showed that both free and immobilized LOX favor linoleic acid as a substrate. Relative substrate preference for fiee LOX was linoleic acid >1- monolinolein > 1,3-dilinolein >trilinolein, and for immobilized LOX was linoleic acid >1,3-dilinolein I-monolinolein >trilinolein. In general, LOX immobilized in alginate silica sol-gel matrix retained the physical and chemical characteristics of free LOX. 'Mention of brand or firm name does not constitute an endorsement by the US Department of Agriculture over others of a similar nature not mentioned. 'Author for correspondence: Dr. An-Fei Hsu, Hides, Lipids and Wool Research Unit, Eastem Regional Research Center, ARS, USDA, 600 E. Mermaid Lane, Wyndmoor, PA 19038. Tel: 2 15 233-6410. Fax: 215 233-6559, E-mail: [email protected] Journal of Food Biochemistry 24 (2000) 21 -3 I. AN Rights Reserved. "Copyright 2000 by Food & Nutrition Press, Inc.. Trumbull, Connecticut. 21

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Page 1: KINETIC BEHAVIOR OF SOYBEAN LIPOXYGENASE: A COMPARATIVE STUDY OF THE FREE ENZYME AND THE ENZYME IMMOBILIZED IN AN ALGINATE SILICA SOL-GEL MATRIX

KINETIC BEHAVIOR OF SOYBEAN LIPOXYGENASE: A COMPARATIVE STUDY OF THE FREE ENZYME AND THE ENZYME IMMOBILIZED IN AN ALGINATE SILICA SOL-GEL

MATRIX'

AN-FEI HSU', EMILY WU, THOMAS A FOGLIA and GEORGE J. PIAZZA

U.S. Department of Agriculture Agricultural Research Service

Eastern Regional Research Center 600 East Mermaid Lane

Wyndmoor, PA 19038

Received for Publication September 10,1998 Accepted for Publication February 16, 1999

ABSTRACT

Lipoxygenase (LOX) is an enzyme that regioselectively introduces a hydroperoxide into polyunsaturated fatty acids (PUFA). We recently reported a procedure that immobilizes soybean LOX within an alginate sol-gel matrix. In this study, the kinetic profile of f iee LOX was compared with that of the sol-gel immobilized LOX. The temperature dependent activity profile offree LOX was optimal at 25C whereas immobilized LOX had optimal activity over the temperature range of 25-392. Enzyme activity, measured in aqueous bufer, for both the free and immobilized LOXpreparations had K, values of 2.5 and 1.40 mmoles/L, respectively, and V,, values of 0.056 and 0.02 pmol/min, respectively. The relutive rates of oxidation of linoleic acid and acylglycerols containing linoleoyl residues catalyzed by free and immobilized LOX also were determined The results showed that both free and immobilized LOX favor linoleic acid as a substrate. Relative substrate preference for f iee LOX was linoleic acid >1- monolinolein > 1,3-dilinolein >trilinolein, and for immobilized LOX was linoleic acid >1,3-dilinolein I-monolinolein >trilinolein. In general, LOX immobilized in alginate silica sol-gel matrix retained the physical and chemical characteristics of free LOX.

'Mention of brand or firm name does not constitute an endorsement by the US Department of Agriculture over others of a similar nature not mentioned.

'Author for correspondence: Dr. An-Fei Hsu, Hides, Lipids and Wool Research Unit, Eastem Regional Research Center, ARS, USDA, 600 E. Mermaid Lane, Wyndmoor, PA 19038. Tel: 2 15 233-6410. Fax: 215 233-6559, E-mail: [email protected]

Journal of Food Biochemistry 24 (2000) 21 -3 I . AN Rights Reserved. "Copyright 2000 by Food & Nutrition Press, Inc.. Trumbull, Connecticut. 21

Page 2: KINETIC BEHAVIOR OF SOYBEAN LIPOXYGENASE: A COMPARATIVE STUDY OF THE FREE ENZYME AND THE ENZYME IMMOBILIZED IN AN ALGINATE SILICA SOL-GEL MATRIX

22 A.-F . HSU ETAL.

INTRODUCTION

Soybean lipoxygenase (EC1.13.11.12) (LOX) selectively oxidizes linoleic acid to form 13(s)-hydroperoxy-9(Z), 1 1(E)-octadecadienoic acid (HPOD) (Siedow 1991). This oxidation reaction is of interest because hydroperoxide derivatives of polyunsaturated fatty acids (PUFA) are receiving industrial interest (Kato et al. 1992, 1993; Vaugh and Gardner 1993). To enhance the commercial potential of this reaction, several methods for immobilizing LOX have been described in the literature (Cuperus et al. 1995; Pinto and Maci’as 1996; Parra-Diaz et al. 1993). In general, these LOX preparations differed in their relative stability and immobilization efficiency and did not provide an immobilized LOX with improved temperature stability. Recently, Hsu et al. (1997) entrapped LOX within an alginate-silicate sol-gel matrix, and this preparation exhibited good thermal stability. Moreover, this form of immobilized LOX could be reused for the enzyme-catalyzed oxidation of PUFA. In a parallel study, Shen et al. (1998) intercalated LOX into delaminated phyllosilicate layers cross-linked with silicates. The LOX-phyllosilicate composite prepared by this procedure had high enzyme activity and good storage stability at room temperature but only retained moderate enzyme activity upon recycling.

The process of enzyme immobilization can change the kinetic parameters of an enzyme. Generally, when an enzyme is immobilized to a support matrix by covalent chemical bonding, larger changes in its kinetic behavior are expected compared to when the enzyme is physically entrapped in a polymer matrix. Although a physically entrapped enzyme may retain its native conformation, the presence of a surrounding matrix may still influence its kinetic parameters. Accordingly, the purpose of this study was to compare the kinetic parameters of free LOX with those of LOX immobilized by entrapment in an alginate-silicate sol- gel matrix. All assays were conducted in the presence of deoxycholate because prior work has shown that this bile salt enhances the activity of LOX on esterified linoleic acid (Piazza et al. 1994).

MATERIALS AND METHODS

Materials

Soybean lipoxygenase (Type I-B, LOX), linoleic acid (LA), cumene hydroperoxide, 1,3-dilinolein, linoleic acid, I-monolinolein, sodium alginate, sodium deoxycholate and trilinolein were obtained from Sigma (St. Louis, MO). Tetramethoxy orthosilicate (TMOS) and the sodium salt of xylenol orange were purchased from Aldrich Chemical (Milwaukee, WI). All other reagents (analytical grade) were obtained from commercial suppliers.

Page 3: KINETIC BEHAVIOR OF SOYBEAN LIPOXYGENASE: A COMPARATIVE STUDY OF THE FREE ENZYME AND THE ENZYME IMMOBILIZED IN AN ALGINATE SILICA SOL-GEL MATRIX

CHARACTERIZATION OF LIPOXYGENASE 23

Immobilized Lipoxygenase

Lipoxygenase (LOX) was immobilized into an alginate-silicate sol-gel matrix using the procedure of Hsu et al. ( 1997). Briefly, equal volumes of LOX in borate buffer pH 9.0 (10 mg/mL) and aqueous sodium alginate (4% w/v) were mixed and the alginate was allowed to form glass beads in CaCl, solution (0.2 M, 100 mL). The beads subsequently were cross-linked by the controlled hydrolysis of tetramethoxy orthosilicate (TMOS) at room temperature. The beads were isolated by filtration, washed with borate buffer, and air-dried at room temperature for a few hours. For storage, air-dried beads were soaked with 75% glycerol for 6 h, filtered, air dried again and stored at 4C.

Assay for Lipoxygenase Activity

LOX activity was determined by measuring the extent of hydroperoxide formed from linoleic acid, as described previously (Piazza et al. 1994). In a typical assay, linoleic acid (5 pmol) in methylene chloride was placed into an Erlenmeyer flask (10 mL) and the solvent evaporated under a stream of nitrogen. To the residue was added 0.2 mL deoxycholate (DOC, 100 mM), 1.8 mL sodium borate buffer (0.2 M, pH 9.0) and immobilized LOX (1.5 g, 0.5 mg protein). Oxidation was carried out at 25C with agitation at 250 rpm for 2 h. The reaction mixture was quenched with 400 pL of 1 M citic acid, and linoleic acid hydroperoxide (HPOD) was extracted (2 mL x 2) with chloroform:methanoI (2:1, v/v). The extracts were combined and dned over anhydrous Na,SO, and evaporated under a stream of nitrogen. The dry residue was redissolved in 3 mL of absolute ethanol and assayed for hydroperoxide (HPOD) formation. Control experiments for determining the extent autooxidation of immobilized LOX or linoleic acid were conducted by measuring HOPD formation in the absence of presence of linoleic acid and using sol-gel beads without entrapped LOX. In all controls only minor amounts of hydroperoxides were detected.

HPOD Determination

HPOD levels were estimated spectrophotometrically using the xylenol orange procedure (Jiang et al. 1991). The xylenol orange reagent consisted of 100 pM xylenol orange, 250 pM ammonium ferrous sulfate, 25 mM H,SO,, and 4 mM 2,6- di-t-butyl-4-methylphenol in methanoVwater (90: 10, vh). The reagent (2 mL) was added to the HPOD sample (10-50 pL) and the volume was adjusted to 2.1 mL with ethanol. The mixture was incubated at room temperature for 45 min, and the absorbence at 560 nm was measured versus a blank that was a mixture of 2.0 mL xylenol orange reagent and 100 pL ethanol. Freshly diluted cumene hydroperoxide (80%) was used for the daily preparation of the xylenol orange reagent calibration curve.

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24 A.-F . HSU ETAL.

Protein Determination

Protein content was determined by a modified Lowry assay using bovine serum albumin (BSA) as the standard (Bensadoun and Weinstein 1976).

Kinetic Procedure

The enzymatic activity of immobilized LOX and free LOX was measured as described above. In most of the experiments, the amount of linoleic acid used was 5 pmoles unless indicated otherwise in the figure legends. To compare kinetic parameters, such as substrate dependence, temperature effect, or solvent participation, the amount of protein in immobilized Lox and free LOX were identical (0.3 mg of protein). All experiments were conducted in triplicate. The standard error was 5%.

RESULTS

Relationship Between LOX Activity and Amount of Enzyme

A comparative study was made on the extent of hydroperoxide (HPOD) formed as a function of the amount of free or immobilized LOX. Comparative studies were made at fixed substrate concentration (linoleic acid, 5 pmole). The reaction buffer medium was presaturated with oxygen to eliminate the limitation by oxygen. Figure 1 shows the rate of oxidation of linoleic acid with increasing concentrations of free and immobilized LOX. For free LOX maximal reaction was determined when the protein concentration reached 200 pg, after which HPOD formation declined sharply. On the other hand, formation of HPOD catalyzed by immobilized LOX showed an initial lag up to 550 pg of protein and slowly increased up to 870 pg. Further increasing immobilized-LOX concentrations resulted in a decrease in HPOD formation. Figure 1 indicated that the optimal amount of protein for HPOD production using immobilized LOX was between 700- 1000 pg.

The evaluation of linoleic acid autooxidation and the effect of calcium alginate sol-gel (without entrapped LOX) on HPOD production also were conducted in this study. Results showed that hydroperoxide formation was insignificant compared to reactions containing enzyme and linoleic acid. Products generated from the oxidation of linoleic acid by free and immobilized LOX were analyzed by the normal phase HPLC (Piazza et al. 1994) (data not shown). Hydrperoxy products after reduction to their hydroxy analogs, were methylated and analyzed by HPLC. Five major products were resolved and characterized as 0x0 fatty acids, and various double bond and hydroxy substituted isomers of reduced HPOD. Under our incubation conditions (pH 9.0, borate buffer) the major hydroperoxy derivative of

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CHARACTERIZATION OF LIPOXYGENASE 25

linoleic acid synthesized by both the free and immobilized LOX was 13- hydroperoxyoctadeca-9,ll (Z,E)-dienoic acid.

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FIG. I . EFFECT OF LOX (0-1200 pg) ON HPOD FORMATION CATALYZED BY FREE AND IMMOBILIZED LOX

Reactions were carried out as described under “Materials and Methods”. HPOD formation was measured by the xylenol orange method. (0-0, free LOX; 0-0, immobilized LOX)

Effect of Substrate Concentration

The rate of HPOD formation was measured at substrate concentrations ranging from 0-10 pmoles. Higher concentrations of substrate were not used to avoid product inactivation of LOX. Figure 2 shows that HPOD production catalyzed by free and immobilized LOX increased with increasing substrate concentrations. Lineweaver-Burk plots (Fig. 2 insert) were linear, which indicated that under these conditions the reactions apparently follow Michaelis-Menten kinetics. Best fit of the data in Fig. 2 was obtained by nonlinear regression analysis. This gave K,,, and V,, values of 2.50 mmole/L and 0.056 pmole/min, respectively for free poxygenase. For oxidation reactions catalyzed by immobilized LOX, the K,,, and V, values were 1.40 mmolek and 0.02 pmole/min, respectively. Because of poor substrate solubility concentrations above 10 mM, the values for V, are considered as best estimates of the data.

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Page 7: KINETIC BEHAVIOR OF SOYBEAN LIPOXYGENASE: A COMPARATIVE STUDY OF THE FREE ENZYME AND THE ENZYME IMMOBILIZED IN AN ALGINATE SILICA SOL-GEL MATRIX

CHARACTERIZATION OF LIPOXYGENASE 27

Catalytic Activity of LOX in the Presence of Organic Solvent

The enzymatic activity of immobilized LOX at 25C was examined in mixtures of isooctane and aqueous buffer that were presaturated with oxygen. Figure 3 shows that the maximal rate of HPOD formation occurred in 10% by volume isooctane. At this level of isooctane the rate of HPOD formation was 60% greater than it was in purely aqueous medium. The oxidation rate diminished at isooctane concentrations higher than 10%. The effect of different concentrations of isooctane in the enzyme activity of free LOX also was studied (data not shown). Results indicated that lower concentrations of isooctane (less than 30%) did not affect LOX activity, however, higher amounts of isooctane decreased LOX activity.

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FIG. 3. EFFECT OF ISOOCTANE ON HPOD FORMATION FORM LlNOLElC ACID AS CATALYZED BY IMMOBILIZED LOX

Assay mixtures contained 1.5 g of immobilized LOX (500 pg of protein), 5 pmol of linoleic acid in 2 mM sodium borate buffer, pH 9.0 and various YO by volumes of isooctane as indicated. Assays were

conducted for 2 h at 15C. The data are means for five determinations.(o-O, immobilized LOX)

Temperature Dependence of Free and Immobilized LOX

The enzymatic activity of free and immobilized LOX was examined over the temperature range of 5-60C. Figure 4 shows that the temperature activity profile of free LOX was optimal at 25C. For immobilized LOX, the optimal activity occurred over the temperature range of 25-35C. HPOD formation using both free

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28 A.-F . HSU ETAL.

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FIG. 4. EFFECT OF TEMPERATURES ON HPOD FORMATION CATALYZED BY FREE LOX (0.3 MG PROTEIN) AND IMMOBILIZED LOX (1.5 G, 0.3 MG PROTEIN)

Both free and immobilized LOX were incubated with 10 mg of linoleic acid for 2 h at various temperatures. Data represent the average of two experiments, each with three replicates. (0-0 free

LOX; 0-0, immobilized LOX)

and immobilized LOX decreased below and above these temperatures. This result was in contrast to those of Piazza et al. (1994) and Parra-Diaz et al. (1993) who reported maximum production of HPOD was at 15C for covalently immobilized LOX. They indicated that decreased HPOD production at temperatures higher than 15C was due to either decomposition of HPOD andor anaerobic by-product formation caused by decreased oxygen solubility at higher temperatures.

Oxidation Rate of Linoleie Acid and its Derivatives

Table 1 lists the relative rate of free and immobilized LOX catalyzed oxidation of linoleic acid and acylglycerol containing linoleoyl residues. The extent of oxidation was measured using the xylenol orange method, which measures hydroperoxide formation. Using free LOX, the substrate preference suggested that linoleic acid was the best substrate. However, 1-monolinolein oxidized at a rate comparable to linoleic acid (94%), whereas 1,3 dilinolein had much slower reactivity than linoleic acid (63% of linoleic acid). The oxidation of trilinolein by free LOX was only 18%. Substituting free LOX with immobilized LOX, linoleic acid was still the best oxidation substrate, and 1-monolinolein was oxidized at a much slower rate (73% of linoleic acid). In contrast to free LOX, 1,3-dilinolein

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CHARACTERIZATION OF LIPOXYGENASE 29

was a good substrate (93% of linoleic acid) for immobilized LOX. The reason for the higher activity of immobilized LOX toward 1,3-dilinolein is not clear and remains to be investigated.

TABLE 1. INFLUENCE OF VARIOUS SUBSTRATES UPON HPOD FORMATION CATALYZED BY

FREE AND IMMOBILIZED-LOX

Substrates Immobilized LOX Free LOX

Linoleic Acid 100% f 4 100% f 4 1 -monolinolein 73% f 3 94% f 3 1,3 -dilinolein 94% f 4 63% f 4 trilinolein 19%*2 18% f 2

Assays contained 6 pmoles of substrate, 400 pL of 2% deoxycholate, and 1.6 mL of sodium borate buffer (pH 9.0, saturated with oxygen) and I .5 mg of immobilized LOX or 500 pg of free LOX. Reactions were camed out for 2 h as described under “Materials and Methods”. HPOD formation was followed using the xylenol orange method. The data shown are normalized to linoleic acid.

DISCUSSION

In this study, it is apparent that oxidation reactions catalyzed by free LOX are more sensitive to product inhibition than oxidations with immobilized LOX. Free lipoxygenase is typically inactivated by HPOD accumulation and partial anaerobic conditions that develop during the reaction (Siedow et al. 1991). The initial lag in HPOD production using immobilized LOX possibly resulted from diffusion limitation of the substrate, linoleic acid, into the sol-gel matrix. The deactivation of immobilized LOX by HPOD accumulation is slower than free LOX, since hydroperoxy derivatives produced during the reaction can dif€bse out of the sol-gel matrix. Thus, contact of immobilized enzyme with peroxy products is limited. This further suggests that the activity of lipoxygenase in an immobilized state may remain active for longer reaction time periods. Both K,,, and V,, values, though only best estimates, strongly indicated that LOX activity decreased significantly with immobilization. These results demonstrate that the immobilization affects the substrate affinity of LOX, but there was no effect on the kinetic behavior of LOX.

Previously, Piazza et al. (1 994) reported that when 35% by volume of isooctane was added to a reaction mixture, the oxidation of linoleic acid catalyzed by immobilized LOX increased three fold. In that study, LOX was immobilized by covalent binding to a carbonyl di-imidazole activated polymer. In this study, we found that isooctane only slightly increased the oxidation rate of linoleic acid using LOX entrapped in a sol-gel matrix. It appeared that a larger amount of HPOD was produced in the reaction catalyzed by the covalently immobilized LOX in aqueous mixtures containing organic solvent. Piazza et al. (1 994) also studied the rate of oxidation of linoleic acid catalyzed by immobilized LOX in the aqueous buffer

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30 A.-F . HSU E T A L .

containing other organic solvents. They found that ethers and ketones inhibited the oxidation reaction and polar organic solvents were detrimental to LOX activity.

In temperature dependent studies, we eliminated oxygen as a limiting factor by presaturating the reaction buffer medium with oxygen. This treatment resulted in a shift in the temperature optima to higher temperatures. Furthermore, TLC analysis of the products generated from each experiment showed that no significant decomposition products were obtained when using either free or immobilized LOX at temperatures above 35C.

In summary, entrapment of LOX within an alginate sol-gel matrix gives an immobilized LOX preparation that has similar physical and chemical characteristics as free LOX. In addition, the immobilized enzyme has increased activity over the free enzyme for the oxidation of dilinolein. Other linoleoyl esters are equally oxidized by free and immobilized LOX. In addition, HPOD product inhibition of the enzyme is less in reactions catalyzed by the immobilized LOX. Because of its reusability and hgh retention of enzymatic activity, the immobilized LOX prepared in this study may have potential as a biocatalysis for fatty acid oxygenations. Accordingly, the kinetic properties of this immobilized LOX preparation reported in this study gives the information needed for prospective industrial use of this biocatalysis.

REFERENCES

BENSADOUN, A. and WEINSTEIN, D. 1976. Assay of proteins in the presence of interfering materials. Anal. Biochem. 70, 24 1-250.

CUPERUS, F.P., KRAMER, G.F.H., DERKSEN, J.T.P. and BOUWER, S.T. 1995. Activity of immobilized lipoxygenase used for the formation of perhydroxyacids. Catalysis Today 25,441-445.

HSU, A-F., FOGLIA, T.A. and PIAZZA, G.J. 1997. Immobilization of lipoxygenase in an alginate-silicate sol-gel matrix: Formation of fatty acid hydroperoxides. Biotech. Letters 19( I), 7 1-74.

JIANG, T.Y., WOLLARD, C.S. and WOLFF, S.P. 1991. Lipid hydroperoxide measurement by Fez' in the presence of xylenol orange. Comparison with TBA assay and an iodometsic method. Lipids 26,853-856.

KATO, T., MAEDA, Y., HIRUKAWA, T., NAMAI, T. and YOSHIKOWA, Y. 1992. Lipoxygenase activity increment in infected tomato leaves and oxidation product of linoleic acid by its in vitro enzyme reaction. Biosci. Biotech. Biochem. 56(3), 373-375.

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CHARACTERIZATION OF LIPOXYGENASE 31

KATO, T., YAMAGUCHI, Y . , NAMAI, T. and HIRUKAWA, T. 1993. Oxygenated fatty acids with anti-rice blast fungus activity in rice plants. Biosci. Biotech. Biochem. 57,283-287.

PARRA-DIAZ, D., BROWER, D.P., MEDINA, M.B. and PIAZZA, G.J. 1993. A method for immobilization of lipoxygenase. Biotech. Appl. Biochem. 18,

PIAZZA, G.J., BROWER, D.P. and PARRA-DIAZ, D. 1994. Synthesis of fatty acid hydroperoxide in the presence of organic solvent using immobilized lipoxygenase. Biotechnol. Appl. Biochem. 19,243-252.

PINTO, M.C. and MACI’AS, P. 1996. Synthesis of linoleic acid hydroperoxide using immobilized lipoxygenase in polyacrylamide gel. Appl. Biochem. and Biotech. 59, 309-3 18.

SHEN, S., HSU. A-F., FOGLIA, T.A. and TU, S-I. 1998. Effectiveness of cross-linked phyllosilicates for immobilized of soybean lipoxygenase. Appl. Biochem. and Biotech. 69,79-90.

SIEDOW, J.N. 1991. Plant lipoxygenase: structure and function. Annu. Rev. Plant Physiol. 42, 145-188.

VAUGHN, S.F. and GARDNER, H.W. 1993. Lipoxygenase-derived aldehydes inhibit h g i pathogenic on soybean. J. Chem. Ecol. 19,2337-2345.

359-367.

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