Properties of Chlorophyllase from Capsicum annuum L. Fruits Dámaso Hornero-Méndez and Marí a Isabel Mínguez-Mosquera* Departamento de Biotecnologia de Alimentos, Instituto de la Grasa (CSIC), Av. Padre Garcia Tejero, 4, 41012-Sevilla, SPAIN. Fax: +34-954691262. E-mail: [email protected] * Author for correspondence and reprint requests Z. Naturforsch. 56c, 1015-1021 (2001); received June 27/August 6 , 2001 , Chlorophyllase, Capsicum annuum The in vitro properties of semi-purified chlorophyllase (chlorophyll-chlorophyllido hy­ drolase, EC 3.1.1.14) from Capsicum annuum fruits have been studied. The showed an optimum of activity at pH 8.5 and 50 °C. specificity was studied for chlorophyll (Chi) a, Chi b, pheophytin (Phe) a and Phe b, with K m values of 10.70, 4.04, 2.67 and 6.37 ^im respectively. Substrate inhibition was found for Phe b at concentrations higher than 5 ^m. Chlorophyllase action on Chi a ’ and Chi b' was also studied but no hydrolysis was observed, suggesting that the mechanism of action depends on the configuration at C-132 in the chloro­ phyll molecule, with the enzyme acting only on compounds with R132 stereochemistry. The effect of various metals (Mg2+, Hg2+, Cu2+, Zn2+, Co , Fe2+ and Fe3+) was also investigated, and a general inhibitory effect was found, this being more marked for Hg2+ and Fe2+. Func­ tional groups such as -SH and -S-S- seemed to participate in the formation of the enzyme- substrate complex. Chelating ion and the carbonyl group at C3 appeared to be important in substrate recognition by the enzyme. The method for measuring Chlase activity, including HPLC separation of substrate and , has been optimized.

Introduction 1996; Rüdiger et al., 1980). The recent cloning of genes for Chlase isozymes from different sources Chlorophyllase (Chlase) (Chlorophyll-chloro- (Chenopodium, Arabidopsis and Citrus) (Jacob- phyllide , EC 3.1.1.14), an intrinsic mem- Wilk et al., 1999; Tsuchiya et al., 1999) will help to brane-bound enzyme located in photosynthetic elucidate the physiological roles of Chlase and systems of higher plants and algae (Fernändez-Lö- other chlorophyll catabolic (Takamiya et pez et al., 1992; Shioi and Sasa, 1986; McFeeters al., 2000). Brown et al. (1991) have classified the et al., 1971), has been extensively studied since its reactions involved in Chi degradation into two discovery (Willstätter and Stoll, 1913). For long groups, type I and type II, with the reaction cata­ time there has been a controversy about the main lyzed by Chlase allocated to the first group. Type I function of the enzyme. Today it is widely ac­ reactions are those leading to loss of Mg or phytol, cepted that Chlase catalyses the in vivo hydrolysis oxidation, modification of peripheral substituents, of (Chls) to (Chlide) and opening of the isocyclic ring V, whereas type and phytol during degreening in leaf senescence II reactions are those leading to cleavage and and fruit ripening, and during normal turnover of opening of the porphyrin macrocycle. The order chlorophylls. Other studies have shown that in of the type I reactions is not clear - it is not well vivo Chlase also may add the alcohol phytol to the understood whether the excision of phytol is pre­ C-13 in Chlide at the final step of Chi biosynthesis ceded or not by Mg loss. Recent experiments using (Fiedor et al., 1992). However, an enzyme, named 14C-labeled Chls and a crude extract of Cheno­ Chi synthase, was reported to be responsible for podium album have suggested that phytol is split the final step of Chi synthesis in vivo (Lopez et al., before Mg loss (Shioi et al., 1991). Chlase exhibits a high specificity towards the substrate, and recently it has also been found to Abbreviations: ß-ME, ß-mercaptoethanol; Chi, chloro­ be stereospecific, the carboxymethyl in the C132 phyll; Chlide, chlorophyllide; DTT, dithiothreitol; IAE, chiral group playing a very important role in the iodoacetamide; MMTS, methylmethanethiosulfonate; NEM, N-ethylmaleimide; p-HMB, p-hydroxymercuri- interaction between substrate and enzyme (Fiedor benzoate; Phe, pheophytin; Pheide, pheophorbide. et al., 1992). Action of the enzyme on substrates

0939-5075/2001/1100-1015 $ 06.00 © 2001 Verlag der Zeitschrift für Naturforschung, Tübingen • www.znaturforsch.com • D 1016 D. Hornero-Mendez and M. I. Mmguez-Mosquera • Chlorophyllase from Capsicum annuum such as Chi a, Chi b , Phe a and Phe b have been -20 °C by using a homogenizer Ultraturrax T25 studied with enzyme preparations from different (Janke Kunkel IKa-Labortechnik), and left for sources (Schoch and Ihl, 1998; Fiedor et al., 1992; 15 min at -20 °C. Supernatant was collected by Tanaka et al., 1982). The enzyme shows no depen­ vacuum filtration through Whatman N°1 filter pa­ dence on the metal ion (Vezitskii and Sherbakov, per, and the filtrate extracted again with 100 ml of 1987), whereas Chi synthase exhibits a high depen­ acetone. This operation was repeated until no dence on the metal chelated by the macrocycle color was observed in the extract (5 times). Finally, (Helfrich and Rüdiger, 1992; Benz and Rüdiger, a white precipitate (acetone powder) was ob­ 1981). tained, dried at room temperature, and weighed. In the present study, Chlase from C. annuum Acetone powder (ca. 10 g) was extracted with fruits has been partially purified, and its proper­ 500 ml of 5 m M Na phosphate buffer (pH 7.0), con­ ties, including substrate specificity, have been taining 50 m M KC1 and 0.24% (w/v) Triton X-100. studied. C. annuum is a carotenogenic fruit, and The suspension was stirred for 1 h at room temper­ therefore during ripening a massive de novo syn­ ature and then filtered through four layers of thesis of carotenoid pigments takes place, which cheesecloth. The filtrate was centrifuged at is accompanied by a sharp decrease of Chls as a 15,000 x g for 15 min at 4 °C. The supernatant was consequence of the degeneration of chloroplasts semipurified by fractionation with (NH4)2S04 in chromoplasts. During this process the role of (30-60% w/v), and desalted through a PD-10 col­ Chlase seems to be important, and its activity is umn (Amersham Pharmacia Biotech, Uppsala, increased and/or activated by the ripening process, Sweden), previously equilibrated with extracting so that it is perhaps a factor modulating the de buffer. The resulting extract (35 ml) is the partially novo biosynthesis of carotenoid pigments. The purified extract used in this study. level of Chlase activity is affected by internal and external factors (Drazkiewicz, 1994). The increase Substrate and standards preparation in Chlase activity has been related with nutritional Chi a and Chi b were isolated from fresh spinach deficiencies, and physiological status of the plant leaves by means of acetone extraction and transfer such as senescent and maturation, when deorgani­ to diethyl ether. Both pigments were separated by zation of the chloroplast helps to put the latent TLC on silicagel GF2 54 using the developing mix­ enzyme and the substrates in contact (Terpstra and ture petroleum ether (65-95 °C)/acetone/diethyl- Lambers, 1983). Therefore a preliminary charac­ amine (10:4:1 v/v). Bands for Chi a and Chi b were terization of the enzyme, and optimization of the scraped off at Rf 0.51 and 0.45, respectively. Phe a activity assay, are needed before attempting to and Phe b were prepared from pure Chi solutions study the role of Chlase during ripening of Capsi­ in diethyl ether, by acidification with a few drops cum fruits. of HC1 10% (v/v). The resulting Phes were trans­ ferred to diethyl ether, evaporated and dissolved Materials and Methods in acetone. Purity of the products was checked by Plant material TLC under the same conditions used for Chls. Epi- merization of both Chi a and b was achieved as Green fresh fruits of peppers (C. annuum L.) cv. proposed by Fiedor et al. (1992). The pigment (0.5 Agridulce were used for the present study. Plants (imol) was dissolved in 25 ml of triethylamine and were cultivated in soil at “La Vera” (Caceres, stirred in the dark during 5 h at room temperature. Spain) by local growers, and harvested when the Chi a’ and Chi b ’ were separated and isolated from green fruits were fully developed. the reaction mixture by HPLC (Mmguez- Mosquera et al., 1991). Extraction and partial purification of the enzyme The method proposed by Terpstra and Lambers Activity assay (1983) was used with some modifications. One To characterize the enzyme, the following assay hundred grams of fruits, cleared of seeds, were procedure was optimized. The reaction mixture chopped and extracted with 100 ml of acetone at contained 0.1 ^imol of substrate (Chi a) dissolved D. Hornero-Mendez and M. I. Minguez-Mosquera • Chlorophyllase from Capsicum annuum 1017 in acetone (0.1 ml), 100 mM Tris (tris[hydroxy- Statistical analysis methyl]aminomethane)-HCl buffer at pH 8.5, and All experiments were carried out in triplicate. the purified chlorophyllase extract in a ratio 1:5:5 The data were subjected to analysis of variance (v/v/v; total volume 1.1 ml). The reaction was car­ (ANOVA) and Duncan’s method. Trends were ried out using Eppendorf® microfuge tubes in a considered significant when means of compared thermostatic bath at 50 °C in the dark during 1 h data differed at P<0.05. with continuous shaking. The reaction was stopped by freezing at -20 °C until analysis. For HPLC Results and Discussion analysis an aliquot of the sample (250 [il) was di­ luted with THF in a relation 1:1 and centrifuged Isolation and partial purification at 13,000 x g for 5 min, and 20 jil injected in the Chlorophyllase is associated with chloroplast chromatograph. HPLC analyses were carried out membranes which requires disruption and disin­ using a Waters 600E quaternary pump equipped tegration of the membrane structure to extract the with a photodiode array detector (PDA 996, enzyme. Our initial approach involved the direct Waters) and controlled with a Millennium data extraction of the enzyme from fresh fruit using Na acquisition station. Chromatographic separation phosphate buffer at pH 7.0 and containing 0.1% was carried out using the method of Mmguez- (w/v) Triton X-100. Chlorophyllase activity was Mosquera et al. (1991) but with a few modifica­ observed, however, the extract was greenish, indi­ tions in the gradient profile. This method allows cating the presence of chlorophyll which can dis­ the separation of both the remaining substrate and turb the results of measured activity. the reaction product Chlide. A reversed phase col­ Therefore protein precipitation with acetone umn (Spherisorb ODS2, 5 ^im, 25 cm x 0.46 cm), at -20 °C was chosen, five successive extractions and a binary solvent system consisted of A (water/ being necessary to get a white protein precipitate. tetrabutylammonium 0.5 m, ammonium acetate Approximately 10 grams of fresh fruit yielded 1 M/methanol, 1:1:8 v/v) and B (acetone/methanol, 1 gram of protein precipitate which was then ex­ 1:1 v/v) were used. The gradient profile was opti­ tracted with 5 m M Na phosphate buffer (pH 7.0), mized as follows: program started with a linear containing 50 m M KC1 and 0.24% (w/v) Triton X- gradient from 75% to 40% of A in 5 min, continu­ 100, in a ratio of 1:50 (w/v) for 1 hour at room ing by a convex gradient (curve 1 in the pump temperature and with continuous stirring. After Waters 600E) to 10% of A in 3 min, and followed filtration and centrifugation, a crude extract was by a linear step until 0% A (100% B) in 4 min, obtained. Enzymatic activity was maximal after and finally returned with a convex gradient to the 40 min, but 60 min was selected for the routine ex­ initial conditions in 3 min. In the case of using tractions. Phes as substrates, the gradient started at 90% of A partial purification was achieved by fraction­ A to allow the separation and detection of the ating the crude extract by precipitation with am­ Pheides polar products. Flow rate was 2.0 ml-min_1. monium sulfate. Chlorophyllase enzyme was pre­ Detection was performed using a detection wave­ sent in the protein fraction comprising 30 to 60% length of 432 nm for Chi a , 466 nm for Chi b, saturation. The precipitate was redissolved in a 410 nm for Phe a and 434 nm for Phe b. Quantifi­ minimum volume of extracting buffer and desalted cation was achieved by external calibration curves using a PD-10 column previously equilibrated with with pure standard solutions. extracting buffer; the resulting product was the Metal ions and functional group reagents were semi-purified extract. introduced in the Tris-HCl buffer at a concentra­ tion 10 [im and 1 mM respectively. Effect of pH and temperature Protein determination A broad peak of maximum chlorophyllase activ­ Protein content of different fractions was mea­ ity was found at pH 8.5. Decreases of 35 and 60% sured according to Bradford (1976). Bovine serum in activity were detected at pH 7.0 and 11.5 respec­ albumin (BSA) (Sigma Chemical Co., St Louis, tively. Similar results have been obtained by Tamai MO) was used as standard. et al. (1979) with semi-purified chlorophyllase 1018 D. Hornero-Mendez and M. I. Mfnguez-Mosquera • Chlorophyllase from Capsicum annuum from Chlorella, and by Khalyfa et al. (1993) with in agreement with previous studies on olive ( Olea chlorophyllase from Phaeodactylum tricornutum. europaea) (Mfnguez-Mosquera et al., 1994) and During ripening chloroplasts are transformed into ‘satsuma’ mandarin (Shimokawa, 1979). Neverthe­ chromoplasts, which leads to deorganization of the less, the enzyme showed its strongest affinity for thylakoid membranes, and may provide enzyme Phe a ( Km= 2.67 |i m ) , although with a Vmax lower access to Chi molecules. Moreover, the fact that than for Chi a. These results suggest the impor­ the intra-thylakoid space (as a consequence of the tance of some structural features for the recogni­ photosynthetic activity) has basic pH conditions, tion of the substrate by the enzyme. A higher K m close to the optimum chlorophyllase pH, maybe value for Mg-free substrates (i.e. pheophytins) important for the in vivo action of the enzyme. suggested an important role of the chelated ion, Activity was measured in the temperature range which is less specific and therefore yields a lower 30-70 °C, and exhibited a maximum at 50 °C. Vmax- Structural differences between a and b chlo­ These results were in agreement with those found rophylls at the C3 carbon (-CH3 and -CHO, in Olea europaea (Mfnguez-Mosquera et al., 1994) respectively) may also explain the higher apparent and Canola (Levadoux et al ., 1987). Denaturation affinity for Chi b, which indicates an involvement kinetics were determined at four temperatures of the carbonyl group in substrate recognition. The (40, 50, 60 and 70 °C). The enzyme remained sta­ substrate inhibition shown by the enzyme for ble at 40 °C for 1 hour, whereas at 50 °C it was pheophytin b might also corroborates this point, slightly degraded after 40 min, and more rapidly since this pigment has both structural characteris­ at higher temperatures. Storage temperature con­ tics (Mg-free and carbonyl at C3) which makes this ditions at -20, 4 and 25 °C were also assayed. At substrate less specific. 25 °C, the enzyme lost 50% of activity in 6 days, During the course of the present study, an inter­ whereas at 4 °C it took 18 days to reach the same esting phenomenon was observed: the standard value. At -20 °C, its activity remained unchanged solutions of Chi a and b always contained small for at least one year. amounts of their C132 epimers, Chi a ’ and 6’ respectively. When these solutions were used for the chlorophyllase assay, a preferential action on Substrate specificity the non-epimeric forms was found. To investigate Substrate specificity has been studied for Chi a this observation standard solutions of Chi a’ and and b, as well as for Phe a and b, measuring in b ’ were prepared and incubated with chloro­ each case the amount of product formed, Chlides phyllase at various concentrations. No de-esterifi- and Pheides respectively. Incubations were carried cation was observed with the C132 epimeric forms, out with increasing concentrations of substrate in which is in accordance with previous reports (Nis­ the range 2 to 60 |i m . Fig. 1 shows the kinetic hiyama et al., 1994; Fiedor et al., 1992). They con­ curves obtained, in each case, all four substrates, cluded that chlorophyllase recognizes the C132 exhibited a typical Michaelis-Menten kinetic stereochemistry, hydrolyzing only the a and b curve. However, substrate inhibition was observed forms whose stereochemistry is 132R, that is, ori­ for Phe b at concentration higher than 5 |i m . Li- ented in opposite direction to the propionic acid neweaver-Burk plots (Fig. 1) were used to calcu­ residue on C17. Fiedor et al. (1992) suggested that late kinetic parameters, K m and Vmax. The ob­ C132 could be involved in the formation of the tained values were similar to those found by other enzyme-substrate complex with an enzyme similar authors using other plants and algae; for instance, to the normal chlorophyllase being necessary for K m is 10 [.im for Chi a in tea leaves (Kuroki et al., the C132 epimeric forms. Such enzyme might be a 1981), 12 |.im in rye seedlings (Tanaka et al., 1982), modified chlorophyllase. and 7.0 |i m in Chlorella regularis (Nishiyama et al., 1994). Effects of metal ions The K m value indicates that the enzyme has a higher affinity for Chi b than for Chi a; however, Extracts were incubated for 24 h at 4 °C in the Vmax was higher for Chi a, indicating a faster trans­ presence of various metal ions (Mg2+, Mn2+, Hg2+, formation of this substrate by the enzyme, which is Cu2+, Zn2+, Co2+, Fe2+ and Fe3+) at 10 [im of con- D. Hornero-Mendez and M. I. Mmguez-Mosquera • Chlorophyllase from Capsicum annuum 1019

Chlorophyll a 2.5

2.0 ------• -

1.5

1.0

0.5

1/[S] 0.0 0 20 40 60 [Substrate] (^M) [Substrate] (^M)

Pheophytin a Pheophytin b

3.0

2.5 - o ^ 2 0 cn 0 1 15 H > 1.0 H

0.5

1/[S] 0.0 20 40 60 [Substrate] (nM) [Substrate] (nM)

Fig. 1. Enzyme kinetic curves and Lineweaver-Burk transformation plots for chlorophyllase activity with Chi a, Chi b, Phe a and Phe b. Km (um) and Vmax (x 106//mol s_1) were 10.70 ± 0.53 and 9.33 ± 0.38 for Chi a, 4.04 ± 0.17 and 4.48 ± 0.21for Chi b, 2.67 ± 0.06 and 5.01 ± 0.18 for Phe a, and 6.37 ± 0.25 and 7.49 ± 0.27 for Phe b, respectively. centration. In parallel, an enzyme control solution chlorophyllase. Metals such as Cu, Zn, and Co without any metal ion was incubated under the may react with sulfhydryl groups of the amino acid same conditions, and the activity was assayed as residue cysteine, indicating the need for such described before. The results are documented in groups in the action mechanism of the enzyme. Table I. All ions inhibited, although Mg and This was observed again with Hg and Fe(II), which Fe(III) only slightly, which are both closely related are highly reactive with these functional groups, to the Chi biosynthetic pathway, and therefore leading to a high degree of inhibition. they may be present in the in vivo environment of 1020 D. Hornero-Mendez and M. I. Mfnguez-Mosquera • Chlorophyllase from Capsicum annuum

Table I. Metal effect on chlorophyllase activity. Metal Table II. Involvement of functional groups in chloro­ concentration was 10 |!M in 100 m M Tris-HCl pH 8.5. In­ phyllase activity. Reagent concentration was 1 mM in cubation time was 24 h prior to enzyme activity assay. 100 mM Tris-HCl, pH 8.5. The enzyme was incubated in presence of the reagent for 24 h at 4 °C. Metal ion Inactivation (%) Reagent Activity (%) Control 0 Mg2+ 12 ± 2 a Control 100 Mn2+ 67 ± 4 IAE 93 ± 3a Hg2+ 85 ± 5 NEM 87 ± 4 Cu2+ 55 ± 4 p-HMB 17 ± 2 Zn2+ 60 ± 4 MMTS 97 ± 2 Co2+ 56 ± 3 DTT 113 ± 8 Fe2+ 89 ± 5 ß-ME 115 ± 6 Fe3+ 13 ± 2 a Mean and standard deviation of three replicates. a Mean and standard deviation of three replicates. these functional groups in chlorophyllase from A i- lanthus altissima. Functional groups involved The involvement of -S-S- bonds was investigated Little effect of iodoacetamide (IAE), N-ethyl- using dithiothreitol (DTT) and ß-mercaptoethanol maleimide (NEM), and methyl methanethiosulfo- (ß-ME) as reducing agents. The results obtained nate (MMTS) was found, but /7-hydroxymercuri- (Table II) indicate an activating effect of the en­ benzoate (p-HMB) almost totally inhibited zyme when the -S-S- groups are reduced to -SH. activity (Table II). These results not only indicate As mentioned before the blocking of -SH groups the existence of sulfhydryl groups in the enzyme, leads to an inhibitory effect. In this case, reduction but also suggest the involvement of some of them of -S-S- bonds may modify the active conforma­ at the active sites, which are not easily available to tion, increasing the accessibility of the substrate to weak reagents. We can deduce that the the active site. may contain at least one sulfhydryl group partici­ pating in the formation of the substrate-enzyme A cknowledgemen ts complex, which was inhibited by p-HMB. The re­ Authors express their sincere gratitude to CI- sults agree with those of Kuroki et al. (1981) for CYT (Comisiön Interministerial de Ciencia y Tec- chlorophyllase from tea leaves, although Mc- nologla, Spanish Government) for supporting this Feeters et al. (1971) concluded the absence of research project, (ALI94-0777 and ALI97-0352). D. Hornero-Mendez and M. I. Mmguez-Mosquera • Chlorophyllase from Capsicum annuum 1021

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