Journal of Biotechnology 111 (2004) 59–65
Hydroperoxide-lyase activity in mint leaves Volatile C6-aldehyde production from hydroperoxy-fatty acids Mohamed Gargouri a,∗ , Philippe Drouet b , Marie-Dominique Legoy b b
a National Institute of Applied Science and Technology (INSAT), Tunis, Tunisia Laboratoire de Génie Protéique et Cellulaire, Université de La Rochelle, La Rochelle, France
Received 1 September 2003; received in revised form 15 March 2004; accepted 19 March 2004
Abstract The extraction of 13-hydroperoxide-lyase activity from mint leaves as well as its use for C6-aldehyde production was studied in this work. The enzyme cleaves 13(S)-hydroperoxy-C18 fatty acids into C6-aldehyde and C12-oxo-acid. Two mint species were tested: Mentha veridis and Mentha pulegium. The headspace injection method coupled to gas chromatography was used for volatile compound analysis. The optimal conditions for temperature and pH were, respectively, 15 and 7 ◦ C. We also studied the specific synthesis of hexanal and hexenals respectively from 13(S)-hydroperoxy-linoleic acid and 13(S)-hydroperoxy-linolenic acid. Considerable quantities of aldehyde (up to 2.58 mol) were produced after 15 min of cleavage reaction in 2 ml stirred at 100 rpm, especially in presence of extract of M. veridis. The conversion yields decreased from 52.5% as maximum to 3.3% when using initial hydroperoxide concentrations between 0.2 and 15 mM. An unsaturated aldehyde, the 3(Z)-hexenal was produced from 13(S)-hydroperoxy-linolenic acid. The 3(Z)-isomer was unstable and isomerized in part to 2(E)-hexenal. In this work, we observed a very limited isomerization of 3(Z)-hexenal to 2(E)-hexenal, since the reaction and the volatile purge were carried out successively in the same flask without delay or any contact with the atmosphere. These aldehydes contribute to the fresh green odor in plants and are widely used in perfumes and in food technology. Their importance increases especially when the starting materials are of natural biological origin as used in this work. GC–MS analysis allowed the identification of the products. © 2004 Elsevier B.V. All rights reserved. Keywords: Hexanal; Hexenal; Hydroperoxy-fatty acid; Hydroperoxide-lyase; Mint leaves; Mentha
1. Introduction Hydroperoxide-lyase (HPLS) cleaves hydroperoxyfatty acid into an aldehyde and an oxo-acid (Hatanaka ∗ Corresponding author. Tel.: +216-71-703-929; fax: +216-71-704-329. E-mail address:
[email protected] (M. Gargouri).
et al., 1978; Sekiya et al., 1983; Hatanaka, 1993; Gardner, 1995; Gargouri, 2001). In plant tissues, two groups of HPLS can be distinguished: the first cleaves 9(S) hydroperoxy-C18 fatty acids into two C9 fragments, and the second cleaves 13(S) hydroperoxy-C18 fatty acids into C6-aldehyde and C12-oxo-acid (Vick and Zimmerman, 1988; Gardner, 1991). These activities were observed, in general, separately in plant
0168-1656/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2004.03.005
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organisms. The first was detected in pear fruit and leaves (Kim and Grosch, 1981; Gargouri and Legoy, 1998) and the second in watermelon seedling (Vick and Zimmerman, 1976), tea leaves (Hatanaka et al., 1979, 1982b), tomato fruit (Matthew and Galliard, 1978), alfalfa seedling (Sekiya et al., 1979), soybean (Matoba et al., 1985) and spinach leaves (Blée and Joyard, 1996). In cucumber fruit both activities were found (Galliard and Phillips, 1976; Gargouri and Legoy, 1998). These activities correspond to two different enzymes, which were separated by Matsui et al. (1989). Transformation reactions using biocatalysts are more and more used in recent years, including commercial applications of enzymes in converting plant oils into added-value products. The plant lipoxygenase pathway, including HPLS, catalyzes the oxidation of lipids producing flavors. Aldehydes can be converted to alcohols by enzymatic hydrogenation. The aldehyde and alcohol products of the lipoxygenase pathway have different odors depending on their structure and then on the enzyme specificities (Gargouri, 2001). The volatile compound mixtures lead to a “green odor” characteristic of the plant species depending on their environment and the season. These compounds are emitted by green tissues in response to various stimuli, and can accomplish a physiological role as insect attraction, communication with other species, repellent agent or bacteriocide (Hatanaka, 1996). The leaf aldehyde: 2(E)-hexenal, and the leaf alcohol: 3(Z)-hexenol are the major contributors to the fresh green odor. These compounds are widely used in perfumes and in food technology. They are used to impart a green character as well as an impression of freshness. The separated use of one or several of these flavors is highly desirable. This can only be offered by the enzymatic pathway that allows producing selectively the desired volatile product. The use of enzymes is promoted by the increasing demand for all types of natural products, including natural flavors perceived as being better than synthetics. The enzymes of the lipoxygenase pathway can be efficiently used as technological tools in the conversion of large quantities of vegetable oils into flavors. That needs the identification of adequate enzyme activities. Despite its presence in many organisms, the HPLS activity is generally low and limits the aldehyde
and alcohol production in vivo (Gargouri, 2001). This is why the biotechnological pathway using separated enzyme activity appears as an interesting alternative to the extraction of the products from plants, especially for selective and high yield production. As mentioned above, the presence of enzymatic system cleaving hydroperoxy-fatty acids was reported for several plants, still there is no work describing any HPLS activity in mint. The extraction of such an activity from mint leaves as well as its use for C6-aldehyde production is reported in this work.
2. Material and methods 2.1. Preparation of hydroperoxy-fatty acids The 13(S)-hydroperoxy-fatty acids: 13(S)-hydroperoxy-linoleic acid and 13(S)-hydroperoxy-linolenic acid, used as substrates of HPLS, were prepared enzymatically by the oxidation of the corresponding fatty acids as follows: In 1 l flask, 500 mg fatty acid (linoleic or linolenic acids, Sigma, France) were mixed with 15 mg soybean lipoxygenase-1 (Sigma, France) in 200 ml borate buffer pH 9.6. After 1 h under 25 ◦ C, 300 rpm and 30 ml/min oxygen, the enzyme was inactivated by adding H2 SO4 (Normapur Prolabo, France). The total conversion of fatty acid to hydroperoxy-fatty acid was checked by measuring the absorbance at 234 nm on a Lambda 16 Prekin-Elmer UV spectrophotometer, assuming a molar extinction coefficient value of ε = 25,000 cm−1 M−1 (Gargouri et al., 1996). After production, hydroperoxy-fatty acid was extracted with diethyl ether (3× the same volume, Sigma, France). The organic phase was dried with MgSO4 (Sigma, France), and diethyl ether was evaporated under vacuum. Hydroperoxide was then diluted in ethanol and stored at −20 ◦ C. The major products were 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid (95%) and 13(S)-hydroperoxy-9(Z), 11(E),15(Z)-octadecatrienoic acid (95%) obtained, respectively, from linoleic and linolenic acids in presence of soybean lipoxygenase. The regioselectivity was analyzed by HPLC with a Hypersil-10 Shandon silica column (4.6 mm × 300 mm). The mobile phase was composed of hexane/diethyl ether/acetic acid (85/14.9/0.1) with a flow of 1.5 ml/min and UV detection at 234 nm.
M. Gargouri et al. / Journal of Biotechnology 111 (2004) 59–65
2.2. Extraction of HPLS activity The mint leaves and top part of the stems (from local market) were used as source of the lyase activity. The plant material was first washed and cut into small pieces. The 30 g were then mixed with 100 ml phosphate buffer 0.2 M, pH 7 adding 1% Tween 20 (Sigma, France). The mixture was homogenized twice during 30 s in a blender and centrifuged 30 min at 15,000 rpm. The supernatant was recovered and used as source of HPLS. Two mint species were used: Mentha veridis and Mentha pulegium. 2.3. Protein determination Protein concentration was analyzed in the enzyme preparation using the BCA method with bovine serum albumin as standard (Smith et al., 1985). The mint extract used contained 21 mg protein/ml. 2.4. Reaction of hydroperoxy-fatty acid cleavage The cleavage of hydroperoxy-fatty acids in presence of HPLS produces volatile compounds. In order to analyze these compounds with precision, the reaction was conducted in the same flask that will be used for analysis on the automatic sampler of headspace injector coupled to gas chromatography (Gargouri and Legoy, 1998). The substrate of the reaction was used after evaporating the solvent. It was mixed to 1.8 ml aqueous extract of mint in a 2 ml final volume completed with phosphate buffer 0.2 M, pH 7. After 15 min of reaction under 13–20 ◦ C and 100 rpm in the sealed flask, the conversion was stopped by adding H2 SO4 before connection to the injection system in order to begin the analysis.
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with He (40 ml min−1 for 10 min). The isolated headspace volatile compounds were then adsorbed onto Tenax trap column (28 cm, Tekmar Concentrator 3000, USA) and water was removed with dry He (50 ◦ C, 1 min). The volatile compounds were thermally desorbed at 200 ◦ C for 4 min, and were condensed in a cold trap (MFA 815, Fison Instruments, France) maintained at −150 ◦ C with liquid nitrogen. The extracted sample was automatically injected into GC 8000 (Fison Instruments, France) by heating the cryofocusing unit 90 s at 200 ◦ C. Compounds were eluted onto a CP-Wax-57 fused silica capillary column (50 m × 0.32 mm, Chrompack, The Netherlands). Oven temperature was initially 70 ◦ C, increased after injection at 13 ◦ C min−1 up to 220 ◦ C and then maintained 8 min. Helium carrier gas was used with a flow rate of 1 ml min−1 and FID detector was used at 250 ◦ C. Retention times were 6.7 min (hexanal), 7.7 min (3(Z)-hexenal), 8 min (2(E)-hexenal), 9 min (hexanol) and 9.5 min (3(Z)-hexenol). (ii) Static headspace: The sealed vial containing the reaction medium was heated in the automatic sampler (Hewlett-Packard 19395A, USA) from 25 to 80 ◦ C in order to reach equilibrium between the liquid and the gas phases. The medium was pressurized 12 s and the gas phase through the transfer line (95 ◦ C) was removed from the vial during 1 min (helium flow rate 30 ml min−1 ). The sample containing the volatile compounds was then injected during 1 min in the GC (Hewlett-Packard 5890, USA). The instrument was equipped with a cross-linked silica capillary column (30 m × 0.25 mm, Hewlett-Packard HP-5). The analytical conditions were similar to those described in the previous method. Pure hexanal, 2(E)-hexenal and 3(Z)-hexenol (Sigma, France) were used as standards.
2.5. Analysis of volatile compounds 2.6. GC–MS The headspace injector coupled to gas chromatography is a precision instrument for volatile compound analysis. Two different apparatus were used in this work to quantify and identify the products; a dynamic headspace and a static headspace instruments. (i) Dynamic headspace: At the end of the reaction, the tube was connected to Purge & Trap autosampler (Tekmar 2016, USA) and then pressurized with helium (He, 50 kPa). The tube was purged
The reaction products were identified by coupling the headspace GC (Hewlett-Packard 5890) to a quadrupole MS (Hewlett-Packard, USA). Helium flow rate was 1.2 ml min−1 . MS conditions were as follows: mass scan range m/z 20–550, source temperature 150 ◦ C, ionization voltage 70 eV. NBS 75 K.L library was used to confirm compound identification.
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2.7. 1-Hexanal Produced from 13(S)-hydroperoxy-linoleic acid in the presence of M. veridis extract using the protocol described above. It was immediately analyzed by GC–MS, m/z: 100 (M+ , 1%), 82 ([M − H2 O]+ , 22), 72 (32), 71 (17), 67 (21), 57 (75), 56 (91), 53 (9), 44 (87), 41 (100), 29 (73) and 27 (74). 2.8. 3(Z)-Hexenal Prepared from 13(S)-hydroperoxy-linolenic acid in the presence of M. veridis extract using the protocol described above. The m/z 98 (M+ , 5%), 97 (3), 83 (14), 80 (8), 69 (33), 55 (28), 42 (20), 41 (100), 39 (36), 29 (19) and 27 (17). 2.9. 2(E)-Hexenal The product of isomerization of the previous compound: m/z 98 (M+ , 4%), 97 (5), 83 (32), 80 (7), 69 (32), 57 (27), 55 (52), 42 (50), 41 (99), 39 (100), 29 (79) and 27 (66).
3. Results and discussion In many cases, enzymes from the same pathway do not have sufficient activities in the same organism at the same stage of development. Unlike classical compound extraction, biosynthesis uses enzyme activities from different sources to carry out successive reactions. In this study we demonstrated the efficiency of an HPLS activity, extracted from mint leaves, in specific conversion of the soybean lipoxygenase product to aldehydes obtained at high concentrations. The extraction method of the HPLS using a surfactant offers an environment approaching the one that the enzyme had in cell, since it is a membrane-bound enzyme. Studies on the HPLS activity in leaves (Hatanaka et al., 1982a, 1987) demonstrated that the enzyme was mainly localized in chloroplast membranes. These studies described a correlation between the HPLS activity from the leaves of various plants and their chlorophyll content (Sekiya et al., 1983). This justifies our choice for the mint leaves and top part of the stems as source of our enzyme activity. The cleavage of 13(S)-hydroperoxy-linoleic acid in pres-
ence of mint HPLS lead to hexanal and C12 -oxo-acid. While the cleavage of 13(S)-hydroperoxy-linolenic acid lead to 3(Z)-hexenal and the same C12 -oxo-acid. The 3(Z)-double bond in aldehyde is unstable and isomerized to 2(E)-isomer (2(E)-hexenal) spontaneously or enzymatically in presence of enal-isomerase (Hatanaka, 1996; Gardner, 1995). According to these references, the second product oxo-9(Z)-dodecenoic acid isomerizes also to the 10(E)-isomer. First, the influence of temperature on the cleavage of 5 mM and 10 mM 13(S)-hydroperoxy-linoleic acid was studied in the presence of 1.8 ml aqueous extract of mint (M. veridis) in 2 ml total volume at pH 7 and 100 rpm, by measuring with the dynamic headspace the hexanal appeared after 15 min. The reaction was carried out in a sealed tube, connected to the analysis system immediately after the reaction finished. The optimal production of hexanal by cleavage reaction was observed at 15 ◦ C; 1.94 and 2.58 mol hexanal, respectively, from 5 and 10 mM hydroperoxy-linoleic acid. The same HPLS activity was detected in this experience in mint leaves without the top part of the stems. Hexanal production decreases relatively 4.7–8.9% when varying temperature 2 ◦ C on the both sides of the optimal temperature. The concentration of hexanal produced from 5 mM substrate at 15 ◦ C was determined by the dynamic headspace and the static headspace methods coupled to GC in order to compare the two methods. We obtained the same product concentration (1.94 mol/2 ml). At this level of detection both of the methods were efficient. The dynamic headspace could be more useful for detection of traces or small concentrations (lower than nmol). It is a more sensitive analytical technique able to detect potential volatile or semi-volatile contaminants in the sample (Gargouri and Legoy, 1998). The static headspace GC technique is more applied for detection, identification and quantification of volatile compounds, due to its ease of use and to requiring no liquid nitrogen for trapping. pH-dependence was examined for hexenal formation from 13(S)-hydroperoxy-linolenic acid in presence of aqueous extract of mint using the static headspace GC method. HPLS activity in the aqueous extract of mint leaves has an optimal pH at 7. This characteristic could be an interesting advantage for further industrial application of the studied bioconversion in water without pH regulation. Optimal pH
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2.5
20
2
15
1.5 10
1
3
60
2.5
50
2
40
1.5
30
1
20 10
0.5
5
0.5
0
0
0
0
(A)
5
10
13S-hydroperoxy-linoleic acid(mM)
15
0 0
(B)
conversion yield (%)
25
hexanal (micromole)
3
3(Z)-hexenal. This compound is unstable and isomerized in part to 2(E)-hexenal. These volatile compounds were analyzed after their production in presence of HPLS at the same conditions as previously (Fig. 2). The analysis of the two compounds after reaction with HPLS from M. veridis and M. pulegium demonstrated that the cleavage reaction was less important than the one in presence of 13(S)-hydroperoxy-linoleic acid (Fig. 2). Since the reaction and the volatile purge were carried out successively in the same flask without delay or any contact with the atmosphere, we observed a very limited isomerization of 3(Z)-hexenal to 2(E)-hexenal. We described in a previous work (Gargouri and Legoy, 1998) an efficient method for biosynthesis and analysis of 3(Z)-aldehyde in a purge and trap system that we applied in this study. This gives a supplementary precision for the analysis of the volatile compounds appeared during a reaction. Instability of 3(Z)-aldehydes after hydroperoxide cleavage had been discussed in the literature (Hatanaka, 1996; Gargouri, 2001). The isomerization is due to the action of 3(Z)-2(E)-enal-isomerase and mainly to nonenzymatic rearrangement. We obtained similar results of the production of the two isomers of hexenal when using mint extracts from different origins. In both cases, the aldehyde production increased to more than 0.65 mol when using 1–5 mM of substrate and stopped increasing above. This result implies that the aldehydes appearing in the medium are inhibitory for the HPLS reaction (Whitehead et al., 1995). In perspective to this work we suggest to perform in situ a reduction of the aldehydes to their
conversion yield (%)
hexanal (micromole)
was as found in tea chloroplasts (Hatanaka et al., 1982a). At pH lower than 5 and higher than 9, small amounts of aldehyde accumulated. Optimum pH for HPLS activity found in other higher plants seems to be spread on a wide range of pH between 5.5 and 9 (Gardner, 1991; Ol´ıas et al., 1990; Matsui et al., 1989; Kajiwara et al., 1982). The production of hexanal was studied as function of the substrate 13(S)-hydroperoxy-linoleic acid concentration. The reaction was achieved at 15 ◦ C in the presence of aqueous extract of two different varieties of mint in a sealed flask, followed by the analysis of the produced hexanal (Fig. 1). The same figure presents the evolution of the conversion yield. Considerable quantities of hexanal were produced by cleavage of 13(S)-hydroperoxy-linoleic acid especially in presence of extract of M. veridis (Fig. 1). Aldehyde production increased when increasing the substrate concentration till 10 mM. When using higher concentration of substrate, the aldehyde production decreased in the medium. The conversion yield decreases as function of initial substrate concentration. The reaction in presence of both the mint extracts produced very low amounts of hexanol (around 10 nmol) produced from hexanal by the alcohol-dehydrogenase activity present in the preparation. Only 1/10 of this amount was detected in the extract before the reaction. It was due to the endogenous activity. The analysis was achieved using dynamic headspace, in this section and the following section. The 13(S)-hydroperoxy-linolenic acid lead to a molecule of oxo-acid and an unsaturated aldehyde, the
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5
10
15
20
13S-hydroperoxy-linoleic acid(mM)
Fig. 1. Hexanal (mol) appeared and conversion yield after 15 min of cleavage of 13(S)-hydroperoxy-linoleic acid by HPLS in M. veridis (A) and M. pulegium (B) extracts at standard conditions. The conversion yield = 100% corresponds to the total conversion of hydroperoxide to hexanal.
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(A)
Identification of the products: hexanal, 3(Z)-hexenal and 2(E)-hexenal obtained after respective incubation of 13(S)-hydroperoxy-linoleic acid or 13(S)hydroperoxy-linolenic acid with M. veridis extract was carried out by GC–MS analysis. High HPLS activity was found in mint leaves from two different varieties. Hexanal and hexenals were produced in high concentrations and conversion yields from the corresponding hydroperoxy-fatty acids. The activity found in this source is interesting when compared to those described by Hatanaka (1993) and Whitehead et al. (1995). The starting materials used in this work are of natural biological origin. This gives to the produced green flavors a considerable importance in industry.
Acknowledgements
(B)
The authors thank Pr. Mohamed Hammami from the Medicine Faculty in Monastir for help in GC–MS analysis, and Pr. Olle Holst from Lund University for valuable comments. This research was supported in part by the International Foundation for Science, Stockholm, Sweden, and the Organization for the Prohibition of Chemical Weapons the Hague, The Netherlands through a grant No. E/3346-1.
References
(C) Fig. 2. 3(Z)-Hexenal and 2(E)-hexenal production (mol) after 15 min of cleavage reaction of 13(S)-hydroperoxy-linolenic acid in the presence of Mentha viridis (A) and M. pulegium (B) extracts at standard conditions. (C) presents the production results in presence of fresh extract of M. viridis (1 h after gathering). The conversion yield = 100% corresponds to the total conversion of hydroperoxide to hexenals.
corresponding alcohols in presence of a source of alcohol-dehydrogenase, in order to avoid the inhibition of the HPLS by its products. As observed in the previous case, this reaction produces a small amount of 3(Z)-hexenol (around 10 nmol).
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