Abdul-Hammed, M.

Ajanaku, K. O.

Bello, I. A.

Olajire, A. A.

English

Covenant University Repository

Kinetics Of Lycopene Biosynthesis In Tomato FruitsA.A.OlajireDepartment of  Pure and Applied Chemistry,Ladoke Akintola University of  Technology,Ogbomoso-(NIGERIA)E-mail: olajireaa@yahoo.comKEYWORDSKinetics;Lycopene;β-Carotene;Biosynthesis andtomato fruits.ABSTRACTThe rate at which tomato fruits ripe was investigated via the biosynthesisof lycopene in two tomato varieties under the conditions of field andambient temperature ripening. Results showed that the biosynthesis oflycopene follows a first-order kinetics in both varieties with calculated rateconstants of 1.2×10-2 and 1.4×10-2 d-1 for Ibadan local and Roma typesrespectively under the field ripening; and 2.1×10-2 and 5.3×10-2 d-1 forIbadan local and Roma types respectively at the ambient temperatureripening. The formation of  β-carotene follows zero-order kinetics in bothvarieties under the field ripening after 8 days (Roma type) and 10 days(Ibadan-local type), with respective calculated rate constants of 43.81and 54.7 ppm d-1. No simple order was obtained for the formation of  β-carotene at the ambient temperature ripening. The empirical rate law forthe lycopene biosynthesis under the field ripening suggests the expression:0tk1 ke]GGPP[kdt]Lycopene[d1−=− where GGPP is geranyl geranyldiphosphate. I.A.Bello, K.O.Ajanaku, M.Abdul-HammedDepartment of  Pure and Applied Chemistry, Ladoke Akintola Uni-versity of  Technology, Ogbomoso-(NIGERIA)Corresponding Author Co-AuthorsINTRODUCTIONTomato is any fruit of  the numerous cultivatedvarieties of Lycopersicon esculentum, a plant of thenightshade family(Solanaceae) and also the fruit ofLycopersicon pimpinelli, the tiny currant tomato[1].The crop has increased in popularity in the tropicsand subtropics until it is the second most importantvegetable in these regions since the end of the nine-teenth century[2]. The fruits are also grown in all partsof Nigeria.Lycopene, a member of  carotenoid family, whichaccumulates in human blood(~0.5µ mol/litreplasma) and the tissue(from 1nmol/g wet wt. in adi-2 Kinetics of lycopeneMevalonateslowGeranylgeranyldiphosphate(GGPP)CH2-O-P-PPhytoenePhytoflueneDesaturationNeurosporeneLYCOPENECyclization CyclizationCyclization CyclizationOxygenXanthophylls (Oxocarotenoids)DesaturationCyclizationDesaturationDesaturationBiosynthesis of Carotenoids showing the central position of lycopene-carotene-carotenezCyclization-carotene-carotene-zeacarotenegdbab-caroteneSCHEME 1pose tissue to up to 20nmol/g wet wt. in adrenalsand testes)[3], is an acyclic, strong unsaturated hy-drocarbon formed from isoprene units linked end toend[4]. Carotenoids are biosynthesized by a specialbranch of the terpenoid pathway(SCHEME 1)[5,6].Lycopene(11 conjugated and two non-conjugatedA.A.Olajire et al. 3double bonds) is one of the most potent antioxi-dants[7–11] with a singlet-oxygen quenching abilitytwice as high as that of β-carotene and 10 times higherthan that of α-tocopherol[10]. The mechanism of theenhanced reactivity of lycopene compared to otherC-40 carotenoids remains to be elucidated. It has beenspeculated that the increased reactivity of lycopeneis related to the presence of the two non-conjugateddouble bonds[10, 11].This study was undertaken to elucidate overallkinetics and mechanisms of lycopene biosynthesisby carrying out (i) the rate at which lycopene accumu-lates in tomato fruits under different stages of  ripen-ing, (ii) the rate of  formation of  β-carotene in tomatofruits, (iii) the dependence of  hydrogen ion concen-tration on the rate of  formation of  lycopene. To ourknowledge, it appears that no such kinetic study haspreviously been reported on lycopene biosynthesisin the literature.EXPERIMENTALSamplingFor field ripening, the tomato fruits were pluckedat random with respect to the ripening classes everymorning of  each period. The tomato fruits wereplucked at once in the mature green stage and wereleft to ‘self-ripe’ in a well-ventilated room of the re-search laboratory for ripening at the ambient tempera-ture. The description of the tomato varieties usedwith their ripening classes are given in TABLE 1 and2. About 3-5 whole fruits were chopped into smallerpieces in a mortar and samples were kept in the re-frigerator prior to analysis.ExtractionLycopene from tomato fruits was extracted withhexane, methanol, acetone(2:1:1), containing 2.5%BHT. The extract was treated with doubly distilledwater, methanol and 20% KOH/methanol(1:1:1) tosaponify any triglyceride present. The extract wasthen washed with doubly distilled water and re-dis-solved in hexane.Physical measurementsOptical density of the hexane extract was scannedspectrophotometrically in the wavelength range 400-TABLE 1: Description of  the tomato varieties usedType: Ibadan-local Roma Shape: Serrated Gourd Number of lobes: 8-10 2-4 Size: Small-big Small-big Color of unripe - fruits: Green Green Color of ripe - fruits: Red Red TABLE 2: Description of  the ripening classes of  thetomato fruitsRipening class Description# Time (days)## Breaker First appearance of external pink, red or tarnish yellow color not more than 10% 2, 3, 4 Turning Over 10% but not more than 30% red, pink or tarnish yellow 6, ( 7or 8)* Pink Over 30% but not more than 60% pinkish or red 10 Light-red Over 60% but not more than 90% red 12 Fully - red Over 90% red 13 #Percentage (%) refers to both color distribution and intensity;  ##daysto reach  the  ripening classes; * 7 days (Ibadan-local) and 8 days (Romatype)750nm against a hexane blank using UV-visible spec-trophotometer, Unicam Heλ10Sα V 2.05. Concen-trations of lycopene and β-carotene were calculatedat λmax. of  505 and 487nm respectively. The pH ofthe raw fruit juice at each ripening stage was mea-sured using pH meter (Mettler Toledo) MP 120.RESULTS AND DISCUSSIONThe description of  varieties of  tomato fruits usedand their ripening classes are given in TABLES 1 and2 respectively. TABLE 3 shows the concentrationsof  lycopene in tomato fruits. It could be observedthat the lycopene biosynthesis is slow in the first 7-8days of ripening but reached the maximal of 36.44and 43.22 ppm for Ibadan-local and Roma types re-spectively at the light-red stage on the parent plant(field ripening) and maximal of 24.07 and 21.04 ppmfor Ibadan-local and Roma types respectively at thepink stage in the detached tomato fruits ripened atthe ambient temperature. The highest concentrationpeaks of lycopene at these ripening stages may beassociated to the increasing concentration of geranylgeranyl diphosphate (GGPP) and/or the increasingactivation of the enzymes responsible for the conver-4 Kinetics of lycopenesion. The decrease in concentration of lycopene after12 days (field ripening) and 10 days(ambient tempera-ture ripening) may be due to conversion of lycopeneto β-carotene via cyclization reaction.The analytical data were subjected to kineticinvestigations, and the kinetic data of  tomato fruitslycopene at both field and ambient temperature rip-ening are given in TABLE 5. The variation of lyco-pene concentration with time is similar to a case ofconsecutive reaction and can be represented as:The rate of disappearance of GGPP is given as;x332013320 ]OPPHC[Kdt]OPPHC[dRate == (1)The rate of  formation of  lycopene and Beta-caro-tene are respectively given as;y56402x332015640 ]HC[k]OPPHC[Kdt]HC[dRate −== (2)y564025640 ]HC[kdt]HC[dRate =−β= (3)Where x and y are the order of reactions withTABLE 3: Concentration (ppm)# of  lycopene in tomato fruitsField ripening Ambient temperature ripening Ripening stage Ibadan-local Roma type Ibadan-local Roma type B2 0.94 ± 0.02 1.19 ± 0.03 0.97 ± 0.05 0.22 ± 0.02 B3 1.50 ± 0.10 1.56 ± 0.07 1.21 ± 0.04 1.75 ± 0.05 B4 1.85 ± 0.05 2.35 ±  0.10 2.09 ± 0.07 3.11 ± 0.10 T6 2.73 ± 0.10 3.48 ± 0.10 3.05 ± 0.04 4.88 ± 0.06 T8 3.15 ± 0.08* 4.49 ± 0.01 3.55 ± 0.07* 6.74 ± 0.08 P 9.00 ± 0.01 22.86 ± 0.02 24.07 ±  0.10 21.04 ± 0.10 LR 36.44 ±  0.01 43.22 ±  0.02 18.27 ±  0.05 16.60 ± 0.10 FR 18.59 ±  0.09 19.40 ± 0.06 13.73 ±  0.10 5.22 ± 0.05 #Values are means ± SEM, n = 5;  *Values at day 7 for Ibadan-local type; B2, B3 and B4 are breaker stage at days 2, 3 and 4 respectively; T6 and T8are turning stage at days 6 and 8 respectively; P is the pink stage; LR is light red stage; FR is the fully-red stageTABLE 4: Concentration (ppm)# of  β-carotene in tomato fruitsField ripening Ambient temperature ripening Ripening stage Ibadan-local Roma type Ibadan-local Roma type B2 29.98 ± 0.05 30.34 ± 0.10 5.92 ± 0.05 6.73 ± 0.15 B3 31.32 ± 0.20 31.18 ± 0.35 6.81 ± 0.02 7.34 ± 0.04 B4 30.62 ± 0.10 32.23 ±  0.05 5.96 ± 0.04 8.43 ± 0.05 T6 30.05 ± 0.10 30.88 ± 0.01 7.22 ± 0.05 7.92 ± 0.09 T8 29.84 ± 0.09* 37.64 ± 0.10 7.71 ± 0.02* 10.31 ± 0.10 P 48.35 ± 0.05 106.05 ± 0.20 17.79 ±  0.10 17.94 ± 0.05 LR 168.05 ±  0.50 209.21 ±  0.60 11.17 ±  0.05 9.18 ± 0.10 FR 210.04 ±  0.40 252.30 ± 0.92 14.59 ±  0.07 15.42 ± 0.08 *Values at day 7 for Ibadan-local typeTABLE 5: Kinetic data of  tomato fruits lycopeneField ripening Ripening at ambient temperature  t(days)  Ibadan local Roma type Ibadan local Roma type   In(C∞-Ct) In(C∞-Ct) In(C∞-Ct) In(C∞-Ct) 0 3.596 3.766 3.181 3.046 2 3.570 3.738 3.140 3.056 3 3.554 3.730 3.129 2.960 4 3.543 3.710 3.090 2.886 6 3.518 3.682 3.045 2.783 8 3.505* 3.657 3.021* 2.660 10 3.312 3.014 ∞ ∞ 12 ∞ ∞ - - 13 - - - - *values at day 7 for Ibadan Local typeC20H33OPP(GGPP)k1 C40H56(Lycopene)k2 β-C40H56(Beta -carotene)SCHEME 2A.A.Olajire et al. 5respect to GGPP and lycopene respectively.A plot of In(C∞-Ct ) against t gives a pseudo-firstorder kinetics (Figure 1), where C∞ is the maximumlycopene concentration in each case, Ct is the con-centration of lycopene at time t, and C∞-Ct is theconcentration of geranyl geranyl diphosphate, calcu-lable from lycopene concentration. It could be ob-served that the plots follow a first-order kinetics inboth varieties under the field and ambient tempera-ture ripening until after 7 days(Ibadan-local) and 8days(Roma type) when the plots deviated from thefirst-order kinetics; with respective calculated first-order rate constants of 1.2×10-2 and 1.4×10-2 d-1 un-der the field ripening and 2.1×10-2 and 5.3×10-2 d-1 atthe ambient temperature ripening. Since the forma-tion of lycopene follows first-order kinetics (Figure1), equation (1) can be integrated to give:tk0332033201e]OPPHC[]OPPHC[ −= (4)Where k1 is the first-order rate constant and[C20H33OPP]0 is the initial concentration of geranylgeranyl diphosphate. Combination of equations (2)and (4) gives equation (5) as follows:y56402tk033205640 ]HC[ke]OPPHC[dt]HC[d1−=− (5)The concentrations of β-carotene in tomatofruits are as shown in TABLE 4. The initial accu-mulation of β-carotene may be due to the conver-sion of neurosprene to b-zeacarotene, which in turnsforms γ-carotene that isomerizes to β-carotene andthe high accumulation of β-carotene after 10 daysin both varieties under the field ripening may be at-tributed to simultaneous conversion reactions of bothlycopene and ϒ-carotene (formed from β-zeacarotene)to β-carotene. Following the biosynthesis of  β-caro-tene (SCHEME 1), there is non-existence of ‘induc-tion period’ (the period during which no β-carotene isobtained). The concentration of β-carotene is ex-pected to be low at initial stage with high concentra-tion of lycopene if β-carotene is biosynthesizedmainly from lycopene; but this is contrary to our ob-servation (TABLES 3 & 4). For example, at the ini-tial stage (i.e. B2 stage), lycopene concentration was0.94 ppm (TABLE 3) while β-carotene concentra-tion was 29.98 ppm (TABLE 4). This is an indica-tion that β-carotene is also formed from other inter-mediate compounds, such as β-zeacarotene, apartfrom lycopene (SCHEME 1 refers). Our observationsuggests that the rate of  β-carotene biosynthesis isindependent of lycopene concentration at a certainperiod of  ripening.The kinetic plot of β-carotene concentrationagainst the period(days) of  ripening of  tomato fruitsshowed zero-order kinetics after 8 days (Roma type)and10 days(Ibadan-local type) (Figure 2), with re-spective zero-order rate constants of 43.81 and54.7ppm d-1. Hence, equation(5) can then be re-writ-ten as:0tk033205640 ke]OPPHC[dt]HC[d1−=− (6)Where k0 is the zero-order rate constant. No simpleorder was obtained for the β-carotene formation inFigure 1: Plot of ln(C∞-Ct) against time under thefield and ambient temperature ripening3.453.53.553.63.653.73.753.80 2 4 6 8 10Time (days)ln(C∞  -  Ct)Ibadan - local type Roma typeField ripening2.62.72.82.933.13.23.30 2 4 6 8 10Time ( days)ln(C∞  - Ct)Ibadan - local type Roma typeAmbient temperature ripening6 Kinetics of lycopeneFigure 2: Plot of β-carotene concentration againsttime under the field and ambient temperatureripening0501001502002503000 2 4 6 8 10 12 14Time ( days)[ - carotene ];  ppmIbadan - local type Roma typeField ripening024681012141618200 2 4 6 8 10 12 14Time (days)[-carotene] ; ppmIbadan - local type Roma typeAmbient temperature ripeningTABLE 6: Hydrogen ion concentration* of  the tomato fruitsField ripening Ripening at ambient temperature t (days) Ibadan local Roma type Ibadan local Roma type 2 2.300 ± 0.071# 3.398 ± 0.096 4.211 ± 0.100 3.902 ± 0.212 3 2.319 ± 0.571 3.708 ± 1.080 4.169 ± 0.095 3.719 ± 0.170 4 3.412 ± 0.619 4.216 ± 0.111 4.192 ± 0.010 3.811 ± 0.067 6 5.188 ± 0.204 5.206 ± 0.166 3.986 ± 0.246 3.912 ± 1.124 8 6.026 ± 0.510 5.888 ± 0.200 4.075 ± 0.250 4.169 ± 0.135 10 5.756 ± 0.133 5.188 ± 0.012 6.918 ± 0.400 6.614 ± 0.304 12 6.719 ± 0.690 6.717 ± 0.657 7.099 ± 0.490 7.765 ± 0.179 13 8.428 ± 0.485 13.581 ±1.560 6.918 ± 0.280 6.317 ± 0.290 * [H+]( 10-5 mol dm-3); #values are means ± SEM, n = 5both tomato varieties under ambient temperature rip-ening (Figure 3). This may be as a result of inhibitionof enzymes, resulting from unfavorable condition forthe enzymes, thereby suggesting the biosynthetic re-action pathways to be enzymic in nature.In order to confirm whether the lycopene bio-synthesis is acid-catalyzed or not, the pH at eachripening stage was measured under the conditionsstudied and the result is given in TABLE 6. For acid-catalyzed enzymic reaction:]Substrate][H[kRate * += (7)Where lycopene is the substrate and k* is the acid-catalyzed rate constant. Based on equation (7), weinvestigated the dependence of [H+] concentrationon the rate of  formation of  lycopene at both fieldand ambient temperature ripening as a means of con-firming whether the enzymic reaction for lycopenebiosynthesis is acid-catalyzed or not. The calculatedrates of  formation of  lycopene were plotted againstthe product of concentrations of hydrogen ion andlycopene. The rate of  formation of  lycopene is lin-early correlated with [H+][Lycopene] (Figure 3), thelinear least squared fit to the plotted data had an R2values of 0.9899 and 0.8266 for Ibadan-local andRoma types respectively under the field ripening (Fig-ure 3a); and 0.9922 and 0.9453 for Ibadan-local andRoma types respectively at the ambient temperatureripening (Figure 3b). Thus, the rate of  formation oflycopene is directly proportional to the hydrogen ionconcentration for both conditions studied; and thelinear least squares fit equation can be used as a pre-dictive equation for the rate of lycopene biosynthe-sis, by measuring the lycopene content and pH ofthe tomato fruit juice, and inserting each value intothe linear equation.A.A.Olajire et al. 7Figure 3b: Correlation of  rate of  formation of  ly-copene with the product of concentrations of hy-drogen ion and lycopene at the ambient tempera-ture ripening. The R2 value and the linear leastsquare fit equations are given in the figuresy = 0.04x + 0.12R2 = 0.990123456780 50 100 150 200([H+][L] x 10-2, (ppm)2Rate (ppm d-1)y = 0.04x + 0.53R2 = 0.940123456780 50 100 150([H+][L]) x 10-2   (ppm)2Rate  (ppm d-1)TABLE 7: Dependence of  rate of  lycopene biosynthesis on the hydrogen ion concentrationField ripening Ripening at ambient temperature  t(days)  Ibadan local Roma type Ibadan local Roma type   Rate (ppm/day) *[H+] [L]× 10-2 (ppm)2 Rate (ppm/day)  *[H+][L] × 102 (ppm)2 Rate (ppm/day) *[H+][L] × 10-2 (ppm)2 Rate (ppm/day) *[H+][L] × 10-2 (ppm)2 2 0.470 2.162 0.595 4.044 0.485 4.085 0.110 0.858 3 0.560 3.479 0.370 5.784 0.240 5.044 1.530 6.508 4 0.350 6.312 0.790 9.908 0.880 8.761 1.360 11.852 6 0.440 14.163 0.565 18.117 0.480 12.157 1.770 19.091 8 0.420 18.982 0.505 26.437 0.500 14.467 0.930 28.099 10 1.950 51.804 9.185 118.598 6.840 166.516 7.150 139.159 12 13.72 244.840 10.180 290.031 -2.900 129.699 -4.440 128.899 13 -17.85 156.677 -23.820 263.471 -4.54 94.984 -11.380 32.975 *[H+], mol dm-3 is equivalent to 1000 ppm; [L] = [Lycopene] in ppmFigure 3a:  Correlation of  rate of  formation of  ly-copene with the product of concentrations of hy-drogen ion and lycopene under  the field ripening.The R2 value and the linear least square fit equa-tions are given in the figuresy = 0.06x - 0.17R2 = 0.9902468101214160 50 100 150 200 250 300([H+][L]) x 10-2  (ppm)2Rate (ppm d-1)y = 0.04x + 0.59R2 = 0.83024681012140 100 200 300 400([H+][L])  x 10-2   (ppm)2Rate  (ppm d-1)8 Kinetics of lycopeneREFERENCES[1] ‘The New Encyclopedia Britannica’, EncyclopediaBritannica Inc; Chicago, 15th Ed., 11, 834-836 (1990).[2] ‘The Encyclopedia Americana’, Grolier Incorporated,USA, International Ed., 26, 832-833 (1990).[3] W.Stahl, H.Sies; Lycopene: Archives of  Biochemis-try and Biophysics, 336(1), 1-9 (1996).[4] C.Alais, G.Linden; Food Biochemistry, Aspen Pub-lishers Inc., Gaithersburg, Maryland, 65 (1999).[5] J.G.Atherton, J.Rudich; ‘The Tomato Crop’, Chapmanand Hall, New York, 84 (1986).[6] A.Young, G.Britton; ‘Carotenoids in Photosynthesis’Chapman and Hall, London, (1993).[7] N.J.Miller, J.Sampson, L.P.Candeias, P.M.Bramley,C.A.Rice-Evans; Antioxidant activities of carotenesand xanthophylls. FEBS Lett., 384, 240 -246 (1996).[8] A.Mortensen, L.H.Skibsted; FEBS Lett., 417, 261-266 (1997).[9] A.A.Woodall; S.W.M.Lee, R.J.Weesie, M.J.Jackson,G.Britton; Biochim. Biophys.Acta, 1336, 33-42 (1997).[10] P.Di Mascio, S.Kaiser, H.Sies; Lycopene as the mosteffective biological carotenoid singlet oxygen quencherArchives of Biochemistry and. Biophysics, 274, 532-538 (1989).[11] P.F.Conn, W.Schalch, T.G.Truscott; J.Photochem.Photobiol. B. Biol; 11, 41-47 (1991).

Kinetics Of Lycopene Biosynthesis In Tomato Fruits

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Kinetics Of Lycopene Biosynthesis In Tomato Fruits

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