Purification and characterization of latent polyphenol oxidase from truffles (Terfezia arenaria)

Journal Pre-proofs Purification and characterization of latent polyphenol oxidase from Truffles (Terfezia arenaria) Farouk Benaceur, Rachid Chaibi, Fethi Berrabah, Aref Neifar, Hicham Gouzi, Kenza Benaceur, Wafa Nouioua, Asmaa Rezzoug, Horia Bouazarra, Ali Gargouri PII: DOI: Reference:

S0141-8130(19)36831-X https://doi.org/10.1016/j.ijbiomac.2019.09.126 BIOMAC 13371

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International Journal of Biological Macromolecules

Received Date: Revised Date: Accepted Date:

25 August 2019 26 September 2019 28 September 2019

Please cite this article as: F. Benaceur, R. Chaibi, F. Berrabah, A. Neifar, H. Gouzi, K. Benaceur, W. Nouioua, A. Rezzoug, H. Bouazarra, A. Gargouri, Purification and characterization of latent polyphenol oxidase from Truffles (Terfezia arenaria), International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/ j.ijbiomac.2019.09.126

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Purification and characterization of latent polyphenol oxidase from Truffles (Terfezia arenaria) Farouk Benaceur1*., Rachid Chaibi 1, Fethi Berrabah2,Aref Neifar3,4.,Hicham Gouzi1,Kenza Benaceur1,Wafa Nouioua5, Asmaa Rezzoug 1,Horia Bouazarra 1,Ali

1Laboratory

Gargouri3.

of Biological and Agricultural Sciences(LSBA), Amar Thelidji university,

Laghouat (UATL),03000, Algeria. 2Laboratory

of Exploration and Valorization of Steppic Ecosystems, Faculty of Nature and

Life Sciences, University of Ziane Achour, 17000 Djelfa, Algeria; 3Laboratory

of Molecular Biotechnology of Eukaryotes, Biotechnology Center of Sfax,

University of Sfax, Tunisia. 4Laboratory

of Aqua, National Institute of Sciences and Technologies of the Sea (INSTM),

Sfax, Tunisia. 5Department

of English, Faculty of Letters and Languages,University of Laghouat.

. 1- Polyphenol oxidase from truffles was purified and characterized. 2- PPO showed best affinity for catechol as substrate and stability under wide range of pH and temperature. 3- Cu+2,SDS , sarkosyl and Triton X100 activate latent form of enzyme. 4- AL+3,Kojic acid,L-cysteine,Metabisulfite sodium and sodium fluoride were excellent PPO inhibitors

Kojic acid amplify heat sensitivity of enzyme and reduce loss of antioxidant potential. *Author to whom correspondence should be addressed; E-Mail: [email protected]

Abstract The polyphenol oxidase was extracted and purified from truffles (Terfezia arenaria) and it exhibited a molecular weight of 67 kDa. The truffle PPO was able to oxidize monophenolic,odiphenolic and triphenolic substrates.Thus,the enzyme seems to be stable under wide range of pH and temperature. Best catalytic efficiency was observed for catechol as substrate (Kcat/km; 674.2S-1mM-1).The effect of detergents, chaotropic agents,metal ions and eleven different inhibitors on relative activity of Truffles PPO was also investigated. A latent form of enzyme was observed and its activity was stimulated using 4mM of SDS. Likewise, the type of inhibition and the values of KI and IC50 were reported for L-cysteine,Sodium fluoride, sodium metabisulfite and kojic acid. Besides, the effect of four concentrations of kojic acid(0.05.,0.1.,0.2 and 0.3mM) on thermal inactivation of PPO was performed in temperature range “ 60-75 ° C” .The use of Kojic acid increase the rate of inactivation process and disrupt enzymatic activity. Moreover, the combined effect of temperature and kojic acid prevent from enzymatic browning reaction and maintain high antioxidant activities including ABTS scavenging activity ,FRAP,and total phenolic contents. Keywords: Truffles; polyphenol oxidase; purification, characterization; ,kojic acid.enzymatic browning .

1.Introduction Truffles, known locally as '' Eterfes or Al-Kamaa “are ectomycorrhizal filamentous fungi that belong to Ascomycota Phylum and Terfeziaceae family[1].Those hypogeous fungi have no stalk,branch , or leaf and grow wild as polyphyletic group of soil [1].Moreover, truffles have number of distinctive characteristics including unique flavour and texture profiles.They showed a well equilibrate nutritional composition and they represent a good source of

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unsaturated fatty acid, vitamins,minerals, and protein[1].Likewise, the potential health benefits of truffles including anti-inflammatory, anti-bacterial, anti-oxidant,and anti-cancer properties were also reported [2]. Compared to other fruit and vegetables, truffles have a short shelf life.They lose their economical values and organoleptic qualities in a few days because of the senescence, the loss of water, the microbial attack and the enzymatic browning reaction[2].A positive correlation between the activity of oxidative enzymes in particular Polyphenol oxidase and the occurrence of this phenomena is currently well known[4].Polyphenol oxidase is a metalloenzyme that belong to oxidoreductase famil.It oxidize phenolic compounds into oquinone which polymerize to give rise to a brown black pigment [5]. The characterization of polyphenol oxidase in term of biochemical and kinetic properties is essential to understand its behavior and to control further its activity. Recently, polyphenol oxidases(PPOs) were purified and characterized from various sources such as Whangkeumbae pear [4],borage[5] and apricot [6].Moreover, the inhibition of its activity by inhibitors eg. organic acid [4,6],amino-acid[5,7],sulfites [5,8] and fluoride [9,10]was also suggested as effectives strategy to prevent its damage. Likewise ,the control of its activity by physical methods in particular heat treatment is also possible .However , its application must take into consideration the stability of enzyme and fruit quality [3]. Despites the commercial value of Truffles ,its browning reaction did not took an attention.Furthermore, the purification and characterization of its polyphenol oxidase was not reported before. Hence ,our objectives were to (a):Purify and characterize latent polyphenol oxidase from an original source “Terfezia arenaria )”(b) Determine the enzyme kinetics and its optimum conditions,(c) Investigate the effect of detergents ,chaotropic agents , and some inhibitors (d)Study the effect of kojic acid on thermal inactivation and thermodynamics parameters .Besides, the antioxidant properties are related to quality of fruit. Therefore (d)the

4

effect of the temperature and Kojic acid on total phenolic contents,ABTS Radical and FRAP was investigated to obtain information relevant to fruit quality loss.

2. Material and methods 2.1 Materials and chemical reagents Truffles was purchased from the local market of El-Bayadh (Algeria) on January 2018 .The truffles had a black brown color with a weight and average size equivalent to 55.61g and 5.6 cm, respectively (Fig.1).They were washed with distilled water, then stored at 4 ° C before their use.All substrates and chemical reagents used in this work were purchased from SigmaAldrich.

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2.2Purification and molecular weight of truffle PPO Trufffles (400 g) were washed and homogenized in 600 mL of 50 mM sodium phosphate buffer ( pH 7.0) containing 4% (w/v) of soluble polyvinyl pyrrolido pyrrolidone (PVP). The homogenate was laid overnight at 4 °C and then centrifuge at 10,000 × g for 20 min at 4 °C. The supernatant was collected for ammonium sulfate precipitation.Ammonium sulfate was added into the supernatant to achieve 80% saturation for precipitation of the enzyme, and then the precipitate was concentrated using Amicon ultra-15 centrifugal filter unit (10 kDa molecular weight cutoff). As a second step, an anion exchange chromatography “Q-Sepharose Big beads”was used. in order to separate protein according to their ionic charge. Equilibration and elution were performed first with 50 mM sodium phosphate buffer (pH 7) with linear salt gradient from 0.5M to 0 M NaCL ranging from 0.5M to 0M. The active fractions were collected and pooled in a single fraction . Amicon ultra-15 centrifugal filter unit (10 kDa molecular weight cutoff) was used another time to concentrate this fraction. Furthermore, Gel filtration HPLC (Zorbax C18 column, 3-300 kDa, Agilent 1260 Infinity) was then applied to separate proteins according to their molecular weight. The column was washed and equilibrated before use with phosphate buffer (0.5 mM and pH 7). During purification, the catechol (20 mM) was used to monitor the enzyme activity. Likewise, protein contents was estimated with the Bradford method using SAB as standard in order to determine the specific enzyme activity. Electrophoresis was then done to visualize enzymatic activity by immersing the gel on 80mL of catechol (40 mM) in 50mM phosphate buffer (pH 7). Similarly, to determine the molecular weight, denaturing electrophoresis SDS-PAGE with 5% stacking and 12% resolving polyacrylamide gel was used[11,12]. The denaturing conditions were ensured by adding β-mercaptoethanol and heating of fractions for 5 min at 100°C.The molecular weight

6

marker (MW) "PlusOneTM silver stain kit protein (17-1150-02 MW: 14-97 kDa) was used for molecular weight determination. 2.3.Enzme activity assay Truffles PPO activity was accessed in 1 mL of assay mixture using Shimadzu UV spectrophotometer (UV- 1800) .PPO activity was determined at optimal conditions of pH and temperature .The medium containing 0.1 ml of 20mM catechol solution in 50mM phosphate buffer (pH 7,40°C) and 10µl of the enzyme solution. The initial rate (v0) was estimated from the slope of absorbance versus time curve.A pre-incubation period of assay mixture for 5 min at 40°C was performed. Thus, one unit of PPO activity was defined as the amount of enzyme that caused an increase in absorbance of 0.001/min [4].All measurements of this work were carried out in triplicate and the averages of data were considered. The standard deviation was used to describe error bars (SD). 2.4Optimum pH and pH stability The activity of purified truffles PPO was studied in the pH range between 2 and 10 with three Buffer systems [acetate (50 mM, pH 2-5.5), phosphate (50 mM, pH 6-8) and Tris-HCl (50 mM, pH 8-10)].Likewise,for pH stability,the enzyme was incubated in McIIvaine buffer[6].The same range of pH (2-10) was used and the mixture was kept at 4°C for two days.Relative activity was estimated as percentage comparing to the optimum activity.

2.5Optimum Temperature and thermal stability The effect of temperature change on enzymatic activity was investigated in a temperature range between 10 and 90°C using catechol,4-methylcatechol ,pyrogallol and L-tyrosine as subtrates.Thus, thermal stability was also studied in the range of temperature 30-90°C using the four substrates above with pre-incubation of enzyme for 10 min at each temperature[3]. The enzymatic activity was expressed as percentage comparing to the maximum activity.

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2.6 Substrate specificity and Kinetic parameters The substrate specificity study was performed in presence of following substrates: catechol(410nm), pyrogallol(475nm), 4-methylcatechol(400nm), dopamine(385nm), chlorgenic acid(481nm),L-tyrosine(475nm),ferrulic acid(420nm), p-cresol(485nm),phenol (390nm) and tyramine(395nm) at a concentration of 10mM[3,4,5].Likewise, the kinetic param€eters (Vmax,Km ,Kcat and kcat/Km) were estimated in presence of only four highly oxidized substrates (Catechol,4-methylcatechol,pyrogalloll and L-tyrosine).A volume of 5μl of the purified enzyme was used for enzymatic activity measurement. 2.7Effect of detergents ,Chaotropic agents and Metals ions The effect of three different concentrations (0,1mM,2mM and 5mM) of ionic detergents (Sodium dodecyl sulfate, sarkosyl, sodium cholate, and sodium dodecyl cholate), nonionic detergents (TritonX-100, Tween 20, Tween 80, and NP-40), and chaotropic agents (urea and guanidine hydrochloride” GnHCl”) on purified truffles PPO was investigated . Moreover, the effect of pre-incubation of enzyme for 10min with two concentrations (2 and 5mM) of different metals ions (MgSO4.(5H20), CaCl2.(7H20), FeSO4.(7H20), CdSO4.(8H20) ,ZnSO4.(7H20), CuSO4.(5H20), MnCl2.(4H20), AlCl3(6H20)) was also reported.The catechol and L-Tyrosine were used as substrates to compare between odiphenolic and monophenolic sensitivity.respectively. 2.8Effect of inhibitors Several inhibitors different by their structure and effec were used in order to control the enzymatic browning reaction.The effect of sulfites (Sodium metabisulfite,Potassium metabisulfite ),of fluorides (Sodium fluorosilicate and Sodium fluoride ), of amino acids (Lcysteine,arginine,,Glutmaic acid ,Glycine ) and organic acids (Salicylic acid ,benzoic acid,kojic acid )were studied using three different concentrations(0,1Mm,2mM, and 5mM)for each reagent.Furthermore ,the values of IC50 and KI were estimated for potent inhibitor from

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each family (Sodium metabisulfite,L-cysteine ,Kojic acid & Sodium fluoride) . The inhibition type was deduced from Lineweaver-Burk curve using five concentrations of substrate. 2.9Effect of kojic acid on heat inactivation process The inactivation by temperature of purified truffles PPO was investigated in the range of temperature 60-75 °C at atmospheric pressure using catechol as substrate.Several tubes containing 2 ml of enzyme were incubated for different durations at the temperatures mentioned above. The inactivation reaction was stopped using ice bath. A volume of 15 μl of the purified enzyme was subsequently used to monitor enzymatic activity.Thus,the relative activity expressed on percentage was estimated in comparison with the activity of untreated enzyme. The first-order reaction model described well the inactivation process. The thermal inactivation parameters (K: the inactivation rate constant (min−1),D: Decimal reduction (min), t1/2: Half-life, Zt: temperature sensitivity parameter and the activation energy Ea(kj/min) were calculated using the following equation [13]; 𝐴

(1

ln 𝐴0 = ― 𝐾𝑡 𝐷 =

2,303

t1 = 2

Log

(2)

𝑘 Ln 2

(3

𝑘

( )=(

)

𝐷1

𝑇2 ― 𝑇2

𝐷2

𝑍𝑇

ln 𝑘 = ln (𝐴) ―

𝐸𝑎 𝑅𝑇

(4

(5

where A is the Arrhenius constant, R : is the gas constant (8.314 J / mole .K) and T is the absolute temperature (K). Thus,a complementary thermodynamic study was also performed. The values of variations in Gibbs free energy (ΔG), enthalpy (ΔH), and entropy (ΔS) were calculated using the following equation [3]:

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𝛥𝐺 = ―𝑅.𝑇.ln

𝐾 . ℎ𝑝 𝐾𝐵.𝑇

(6

𝛥𝐻 = 𝐸𝑎 ―𝑛𝑅𝑇

(7)

ΔH ― ΔG T

(8)

𝛥S =

where KB is the Boltzmann constant (1.3806 .10-23 m2 Kg / Ks) ,HP is the Planck constant (6.6262 .10-34 J s). In addition, the effect of kojic acid on those parameters was also studied using 4 different concentrations (0.05.,0.1.,0.2 and 0.3mM). 2.10Influence of combined effect of Kojic acid and temperature on crude PPO and on antioxidant activities of truffles The influence of Kojic acid and the temperature on crude truffles PPO as well as the composition and the antioxidant activities of truffles fruit was studied. The extracts were prepared as follows: First, 400g of the truffles were washed and cut in small fragments then solubilized in 600 ml of distilled water then centrifuged at 10000xg for 15 (4 ° C). The supernatant containing crude extract of the enzyme with other components was recovered and stored at 4 ° C for a day in the dark.The cooled solution was subsequently dispensed into small flasks and heated with stirring for 10 min at temperature between 60 ° C and 75 ° C. A thermostatic bath (Julabo) was used during the experiment. An immediate cooling in an ice bath of the heated flasks was applied to stop reaction. A second centrifugation took place at 1000 xg for 15 min at 4 ° C to obtain a clear and homogeneous solution. Likewise,the treated solution was dipped in different concentration of Kojic acid (0.1 to 0.3mM) .The supernatant activity was measured using 30 μl of the crude extract with catechol as substrate (20mM) .7 Moreover ,the effect of temperature and Kojic acid on the antioxidant properties of truffles extracts was also studied. Their influence on FRAP assay,ABTS radical and the total 10

phenolic contents were investigated .Herein,the FRAP and ABTS tests were performed, the absorbance at 700nm and 414 nm of the unheated extract were defined as 100% for each test ,respectively.[14,15].Likewise, for total phenolic contents of Truffles extract,the gallic acid was used as standard. The concentrations were expressed as mg of gallic acid equivalents per ml of truffles extract[16]. 2.11 Data Analysis SigmaPlot Version 12.0 (Copyright © 2011 Systat Software, Inc.) was used to fit linear and Nonlinear regressions.Thus,analysis of variance (ANOVA) was carried out to evaluate significant differences (P<0.005) between samples.

3. Results and discussion 3.1. Purification of truffles PPO The polyphenol oxidase of truffles “Terfezia arenaria” was purified 59.1-fold with recovery yield and specific activity equals to 34.2% and 1088.6 EU/mg ,respectively, using a process of three steps including precipitation by sulfate ammonium and chromatographic techniques (Table.1).In literature ,different purification protocols were reported.Overall,the purification-fold depends on methods used and localization of enzyme.The PPO from snake

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fruit was purified 10.5 fold involving Superdex 200 h 10/30 and FPLC method [17],while PPO from apricot was purified 3.9 fold using Q Sepharose FF anion exchange column and cation exchange chromatography [6]. The native-PAGE electrophoresis revealed a distinct enzymatic activity in comparison with the activity of the crude extract (Lane 5) whereas with SDS-PAGE ,a well separated band corresponding to purified enzyme fraction that exhibited a molecular weight of 67 kDa (Fig.2 ,lane 3).This value is similar to molecular weight of Apricot PPO[6], Plums PPO[18] and Fuji apple PPO [19] but different from that of Borage PPO (80.6kDa) [5] and wolf apple PPO(47.68kDa)[20]. 3.2Optimum pH and pH stability The influence of pH was studied using four different substrates in wide interval of pH (210) (Fig 3,A).The optimum pH values corresponding to the maximum activity were: pH 4 ;pH 6 ,pH 6,5 and pH 7 in presence of 4-methylcatechol, L-tyrosine ,pyrogallol and catechol as substrates, respectively. Catalytic efficiency of enzyme is related to its aptitude to oxidize substrate. The ionization state of the amino acids in the active site affects both the binding force and the affinity. The pH effect resides in the change of this state of ionization which consequently results in decrease of activity. Generally, the optimum pH of fruits and vegetables PPO varies depending on both the substrate used and source of enzyme [5,6,8]. For Borage PPO, optimum values of pH were pH 7,5,pH5,5 and pH 5 using catechol ,pyrogallol, and 4-methylcatechol as substrates,respectively [5],while a value of pH 5,5 for catechol and pH 4 for 4-methylcatechol were reported for Cape gooseberry PPO.Likewise, a lower activity was obtained at pH below than pH 2 or above to 8[8].Such decrease of activity was also reported for apricot PPO [6] and Whangkeumbae Pear PPO[4]. In addition,the range pH stability of truffles PPO was correlated to results obtained above.A ramrquable enzyme activity was recorded at pH range (3.5-4.5) for 4-

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methyclatechol, (5-6) for L-tyrosine , (6-6.5) for pyrogallol and (6.5-7.5 ) using catechol as substrate(Fig 3,B).The enzyme appears to be more stable in the neutral domain than the alkaline or acidic domain as we noticed that the decrease in enzymatic activity was accelerated beyond the value of pH 7,5. Our results are similar to those reported for pH stability of Plums PPO.At pH 7.5, and pH 8.0,plums PPO lost 61% and 73% of its o-diphenolic activity,respectively,whereas in acidic pH range (pH 4.0,4,5 and 5.0) enzyme lost just 32%,33% and 38%, respectively [18]. 3.3Optimum temperature and temperature stability The influence of temperature on purified enzyme was performed .Optimum temperatures values were 30°C ,35°C ,40°C and 45°C in presence of 4-methylcatechol,L-tyrosine, pyrogallol and catechol as susbtrates, respectively (Fig 3,C). At 50° C ,enzyme lost 59,%,49%,36% and 13% using,L-tyrosine , 4-methylcatechol ,pyrogallol and catechol ,respectively,while at 80°C,enzyme retained only 4,5%,12,38%, 16,5% and 22,7% of its activity for those four substrates ,respectively . Total inactivation occurred at a temperature up to 85 ° C. In general, the optimum temperature of polyphenol oxidase from fruit and vegetables were reported between 20°C to 60°C [5,8].Borage PPO showed following optimum temperature values: 5° C ,10°C,30°C using 4-methylcatechol,catechol and pyrogallol, respectively [5] ,while PPO from Cape gooseberry had optimum temperature values of 40 °C using catechol and 25°C for 4methylcatechol [8]. Furthermore, the thermal stability of purified truffles PPO was studied using the same four substrates in the range of temperature between 30-90°C under incubation time of 10 min .. The loss of activity was great and faster beyond 60 °C.Thus,up to75°C , the truffles PPO maintained 51%, 46% 38% and 29 % using 4-methylcatechol ,L-tyrosine, pyrogallol and catechol as substrates, respectively (Fig .3,D).

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Thermal stability was well studied for other PPOs from different sources. PPO is usually considered as thermostable enzyme with a stability range beyond to 80 °C . Plums PPO showed a higher stability in the range of temperature between 25 and 65 °C .Thus, at 70°C more than half activity was lost after heating time of 10 min while a loss of 100% of activity beyond 85°C value was reported[18]. Likewise ,a range of temperature stability between 20 and 80°C was also observed for mamey PPO with optimum stability in the range of 35-45°C [21]. 3.4 Substrate specificity and kinetic parameters The ability of truffles PPO to oxidize Ten different substrates including monphenolic, odiphenolic and triphenolic substrates was investigated .We noticed that the enzyme oxidized all substrates tested. A high activity was obtained using catechol (100%) followed by 4methylcatechol(91.1%) , pyrogallol (81.3%) ,L-tyrosine (73.9%) ,dopamine(60.7%), chlorogenic acid(57.6%), tyramine (45.8%) ,p-cresol(40.2%), ferrulic acid(35.9%) and phenol(30.4%). The ratio of monophenolase / o-diphenolase activities of the truffle PPO seems to be appreciable (0.73 compared to catechol and 0.8 compared to 4-methylcatechol). Based on the substrate specificity study, the values of kinetics parameters (Vmax,Km and Kcat) were estimated for catechol, 4-methylcatechol, pyrogallol and L-tyrosine using lineweaver-Burk plot.The Michaelis -Menten constant(Km) describe the affinity of enzyme towards its substrate.An enzyme with a high Km value has a low affinity for its substrate[6].Therefore,Among the four substrates used,the increasing order of affinity can be given as below: Catechol(Km: 1.5 mM) >L-tyrosine(Km:3.2mM) > 4-methylcatechol(Km: 6.1 mM ) >pyrogallol.(Km:8.7 mM). Furthermore,the value of Km seems to be correlated to substrate used.Thus,a value of Km equals to 3;68 for honeydew peach PPO [22] and 2,24mM for Borage PPO 5 were estimated

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in presence of catechol as substrate.Moreover, for Radish PPO ,the Km values were as follow ;Km equals 6,3mM for pyrogallol and 28,3mM or catechol,respectively. The values of Vmax (EU/mL) obtained were equal to 4331.4., 3458.3 .,2873.7 and 2247.2 while those of Kcat (S-1) were 1011.2 .,807.33.,670.68 and 524.6 using catechol,4methylcatechol , pyrogallol and L-tyrosine as susbtrates,respectively.,Hence,the catalytic efficiency (Kcat/km; S-1mM-1) followed the order below: Catechol (674.2)>L-tyrosine(163.9)> 4-méthylcatéchol (132.3) >pyrogallol (77.1). Several papers had reported the values of turnover number (s-1) and those of catalytic efficiency(s-1mM-1) of PPOs extracted from fruits and vegetables using different substrates. The values of (210

s-1,40s-1mM-1),(700 s-1 ,340 s-1mM-1),(590 s-1 ,50 s-1mM-1) using

catechol,4-methylcatechol and pyrogallol,respectively as substrates were reported for apricot PPO [6] .Thus,For parsley PPO ,values of (19.7 min-1,24937 min-1mM-1) ,(2.6 min-1 ,2680 min-1mM-1) ,(3.6 min-1 ,48.65 min-1mM-1) were estimated using catechol 4-methylcatechol, and pyrogallol ,respectively [24] but conversely high values were reported for borage PPO using catechol (2.2.104 s-1,9.8.109s-1mM-1),4-methylcatechol (10,7.104s-1,24.109s-1mM-1) and pyrogallol (1.15.104 s-1,4.9.109s-1mM-1) as substrates [5].

3.5 Effect of metal ions ,Detergents and Chaotropic agents The results of influence of metals ions and reagents on purified Truffles PPO are shown in table 2(A,B),respectively.For metals ions effect, both activation and inhibition behavior were noticed.The divalent ions (Cu+2,Mg+2 and Zn+2) reacted as activators while the effect of (Mn+2.,Fe+2,Ca+2,Cd+2) was negative on relative enzyme activity (Table.2,A).Besides ,up to 5mM , Cu+2 and Al+3 were the potent activator and inhibitor,respectively.Our results are

15

similar to those reported for Apricot PPO[6]and Tadela date fruit [13].In fact, the stimulation or inhibition of relative activity of PPO by metals ions might be due to the change of bivalent state of copper atom as prosthetic group in active site of enzyme. It was also reported that certain metal ions eg. Al+3

could disrupt enzymatic activity by modifying

stability of covalent bonds and interactions of amino acid chains in active site[6,13] In addition,we have noticed that chaotropic agents (urea & Guanidine hydrochloride)and nonionic detergents (Tween 20, Tween 80 & NP-40) inhibit the relative activity of enzyme . In contrast, Triton X-100 showed a positive effect at concentration of 5mM .However, Sodium cholate and Sodium dodecyl cholate inhibit enzyme at concentration of 5mM while no effect was remarked using Tween 20 and Tween 40. (Table.2.B).Overall,at high concentration,the o-diphenolase activity using catechol seems to be more stable compared to the monophenolic activity in the presence of L-tyrosine. The Loss of activity for monophenolic activity compared to o-diphenolic activity at a concentration of 5mM followed the order below : (88%, compared to 72%), (69% compared to 35%) ,(57% compared to 34),and (45% compared to 30%) in presence of Urea,GnHCl ,NP40 and Sodium cholate,respectively.

Moreover, a stimulation of activity was observed in presence of SDS and sarkosyl.Up to 2mM ,SDS and Sarkosyl increase activity by 289 % and 217% for tyrosinase activity,respectively .Such result was very significant and proves the presence of a latent forrm of PPO.Our results are similar to those reported for for apricot PPO [6] and mango PPO [25].Thus, for Dormant Saffron PPO.A positif effect on activity using SDS(+400%) and sarkosyl(+130%) was observed ,while a decrease of activity was found in presence of nonionic detergents and chaotropic agents [26].

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3.6Effect of inhibitors Eleven different inhibitors often used in food industry as food additives and known by their effect on the PPO were tested on the enzymatic activity of Truffles PPO using catechol and Ltyrosine as substrates.The results obtained are grouped in Table (3).The monophneolic activity seem to be more sensitive than o-diphenolic activity. A total inhibition was noted above a concentration of 2 mM using sodium metabisulfite and sodium fluoride.Such a potent inhibitory effect was also reported for borage PPO[5] and eggplant PPO[23].In addition,the effect of different amino-acids (Non-polar,aliphatic residues: Glysine.,Polar,non-charged residues :L-Cysteine,Positively charged residues:Lysine and Negatively charged residues:Glutamic acid) was also studied.Among this list, up to 2mM, a decrease of monophenolic activity by 89% , 70%,65% and 17% was found in presence of Lcysteine,Glutamic acid ,Glycine and Arginine.In litterature,the efficiency of L-cysteine was also reported for Whangkeumbae pear PPO [4 ] and European pear

PPO[27] while the

inhibitory of Glutamic acid has been remarked for mushroom and Ocimum basilicum L.PPOs [28] . Furthermore ,the study of the effect of organic acids(salicylic acid,benzoic acid and kojic acid) showed a significant inhibition of tyrosinase activity at concentration of 2mM as follow: kojic acid (90%) followed by benzoic acid (80%) and salicylic acid (64%).Likewise,the inhibition of PPO activity was reported for Fresh-cut Chinese chestnutusing salicylic acid[7],for Ataulfo mango [25] and Whangkeumbae pear PPO [4] in presence of kojic acid as inhibitor, while a reliable inhibition by benzoic acid was observed for plums PPO[18] and blueberry PPO[29]. Furthermore,a description of inhibition type and kinetic parameters (IC50 ,KI) values are given in Table.4.The best inhibitor of each category was selected(Sodium metabisulfite, sodium fluoride, L-cysteine and Kojic acid). The IC50 value reflects the lowest concentration

17

that can decrease 50% of the enzyme activity..Therefore,we can classify the four inhibitors according to their inhibition potency as follows: Metabisulfite sodium> sodium fluoride >Kojic Acid >L-cysteine . Thus, different inhibition types were found for each inhibitor and it depends of substrate used .In literature ,a noncompetitive inhibition for sodium metabisulfite and uncompetitive inhibition for L-cysteine using catechol as susbtrate were found while a mixed inhibition and noncompetitive were observed for L-cysteine and sodium metabisulfite using 4methylcatechol as susbtrate for ginger PPO [30] .Besides,L-cysteine and sodium metabisulfite reacted as noncompetitive inhibitors of Cape gooseberry PPO using 4-methylcatechol as substrate and as competitive inhibitors using chlorogenic acid as substrate [8 ]. For Lcysteine, three mechanisms have been cited either by direct action on the enzyme or by forming a colored cys-quinone complex or by reducing o-quionone to o-dihydroxyphenol [31 ]. Thus,The mechanism of inhibition by sodium fluoride has not been clarified yet .We suggested that their effect is related to its ability to chelate copper which destabilizes the active site of the enzyme. Despites that sulphites showed high inhibition efficiency on PPO due to their combination with the product of the reaction (O-quinone)which prevent the formation of melanin pigment, their use with also dluorides is not very recommended due to their side effects [8]. In addition,for kojic acid, a competitive inhibition was observed for eggplant PPO[23].The use of this organic acid as a promising inhibitor to prevent enzymatic browning was also reported for Ataulfo mango PPO [ 26].In fact,the effect of this fungal secondary metabolite was accredited to its aptitude to chelate copper atom from active site [23]. 3.7Combined effect of kojic acid and heat treatement on Thermal inactivation process The combined effect of kojic acid and heat treatment on the enzymatic activity of enzyme was studied in the temperature range between 60 ° C and 75 ° C (increments in 5°C)

18

using catechol as substrate. The values of the kinetic thermal inactivation and thermodynamics parameters are shown in Table 5. The thermal effect disrupts the enzyme stability and causes conformational change making the enzyme inactive .Such an effect is all the more rapid and irreversible as the exposure time is long at a high temperature.Besides,the heat sensivity of enzyme was predicted by zt and Ea values[11].When the value of the activation energy «Ea» is high, this implies that the reaction requires to bring more energy to maintain the intermediate state which decreases the stability of the enzyme by increasing its sensitivity to the change of temperature. The low value of zt indicates that the required change of temperature to modify the value of D by a factor of 10 is faster with an increase of sensitivity [32]. Comparing the zt(°C) and Ea(Kj/mol) values of Truffles PPO with other PPOs using catechol as susbtrate,we found that Truffles PPO is more thermostable than

plums PPO (

15,15°C and 150,46 Kj/mol) [ 18],strawberry PPO ( 14°C and 147 Kj/mol)and apple PPO ( 17°C and 78 Kj/mol)[33] but less thermostable than edible Yam PPO

(29.4°C and 67.6

Kj/mol )[33] and pineapple PPO(104.2°C and 23.7 Kj/mol )[35]. Moreover, the thermodynamic study was carried out and their parameters(the enthalpy (ΔH), the entropy (ΔS) and the free energy (ΔG)) were estimated (Table 5). Enthalpy is commonly used in estimate the energy disruption of system in chemical, biological or physical processes. The value of enthalpy is proportional to the number of broken bonds during the process.The higher the value, the higher is the rate of deformation, and greater is the instability[11]. Likewise, the variation of free enthalpy or free energy of Gibbs is studied to predict the evolution of the transformation and to have an idea about the spontaneity of the reaction.Their positive values is correlated to the increase of inactivation constant value (K). Besides, the entropy“ΔS” measures the reversibility of a transformation, and characterizes the state of

19

disorder and /or the degree of homogeneity of the system[11]. The value of entropy was positive involving a reversible reaction and a phenomenon of aggregation associated to a partial disorder [3,11]. In addition, the effect of different concentrations of kojic acid on those parameters were investigated using four different concentrations .We noticed an acceleration in the process of thermal inactivation. The value of constant of inactivation “K” was amplified by 1.18 .,1.63.,2.67 and 4.77 at concentration of 0.05.,0.1.,0.2 and 0.3mM,respectively. We suggest that kojic acid increase instability of enzyme during heat treatment and accelerates further its inactivation. Such stimulation and loss of stability is also correlated to the increase of entropy value”ΔS”.To our knowledge, the effect of kojic acid on thermal inactivation process has not been reported before,whereas ,the effect of other organic acids eg. citric acid [36 ] and ascorbic acid[ 37] on thermal inactivation process of Agaricus bisporus was reported .Both citric acid and ascorbic acid increased sensitivity of PPO towards heat treatment .Thus,they cause unfolding of PPO conformation by change of its secondary structure which led to its denaturation [36,37].

3.8Influence of combined effect of kojic acid and heat treatment on crude PPO and on antioxidant activities of truffles The combined effect of heat treatment and kojic acid on crude PPO and on antioxidant potential of truffles was investigated. The heat treatment effect during 15 min was studied in the temperature range between 60 and 75 (Increments in 5 ° C) using the catechol as substrate in presence of four concentrations of Kojic acid (0.05.,0.1.,0.2 and 0.3mM). (Fig.4). We observed a loss on activity as the temperature and concentration of inhibitor increase. Up to 3mM of kojic acid,the enzyme maintains 41%, 31% ,20% and 11% of its activity at 60 ° C, 65 ° C, 70 ° C and 75 ° C, respectively (Fig.4,A).Moreover, the inhibition of enzymatic

20

browning reaction must take into consideration composition and antioxidant properties .Herein,its influence on FRAP, ABTS radical and total phenolic contents are shown in the figure ( 4,B,C & D ) respectively. According to figure (4,B) ,we noticed a slow decrease of ferric reducing power (FRAP) with the increase of both temperature and kojic acid concentration. Up to 0.2mM, the FRAP rates were 87%, 80.5%,78.5% and 72 % at 60 ° C, 75 ° C, 65 ° C and 70 ° C, respectively. Thus,an interesting increase was observed at 75 °C value.The scanvenger activity in turn was affected by heat treatment and kojic acid .At concentration of 0.2mM ,the ABTS values were 92.5%,90.5% 89.5% and 85.5% under treatement at 60°C,65°C,70°C,75°C.respectively while the same increase as FRAP was detected for ABTS activity at 75°C. In fact,such decrease of antioxidant properties under heat treatment was also reported for six onion varieties [38],for white tea [39] and for jackfruit extracts [40].The decrease of the antioxidant activities might be ascribed to the release of some compounds playing an opposite action by their pro-oxidant effect, while the increase of this activity at high temperature value can be explained by the thermal activating effect which accelerates the reaction by allowing the appearance of new compounds having a high antioxidant potential [40]. Moreover, the combined effect of temeperature and kojic acid on total phenolic content was also investigated ( Fig 4, D).We observed a low decrease in total phenolic contents with increase of kojic acid concentration.The total phenolic contents was correlated to ABTS activity and FRAP change.Such results might be accredited to destabilization of plant wall, which allows the release of phenolic compounds into the medium[39].Likewise,a positive effect of prestorage of kojic acid on antioxidant activities and sensory properties was recently reported for litchi fruit[41]. As far as our literature survey could as certain, no report is available in the literature for the combined effect of kojic acid and temperature on those parameters.Finally,the combined

21

effect of Kojic acid at concentration of 0.2mM with heat treatment at 75 for 15 min ° C could be used to disrupt PPO activity and to maintain high antioxidant properties of fruits and vegetables. 4.Conclusion The detailed characterization and purification of truffles PPO was reported for the first time.The enzyme exhibited a molecular weight of 67kDa and showed variable affinity towards substrates tested.Best catalytic efficiency was observed for catechol as substrate.Latent form of PPO was stimulated by SDS,sarkosyle and some divalent ions (Cu+2,Mg+2 and Zn+2) while chaotropic agents and nonionic detergents reacted as inhibitors. Likewise,sodium metabisulfite ,sodium, fluoride,L-cysteine and kojic acid were the potent inhibitors of truffles PPO.The mechanism and inhibition efficiency depended on both inhibitor and substrate used.The thermal inactivation followed a first order model. The rate of thermal inactivation was amplified five times under treatment by Kojic acid at 0.3mM .The combined effect of kojic acid (0.2 mM) and heat Treatment (75° C ,15 minutes) is suggested as a suitable strategy to control enzymatic browning and to maintain antioxidant potential of fruits. Finally, the high ratio of monophenolase / o-diphenolase activities of this enzyme let to suggest its use as alternative to Agaricus bisporus PPO especially in pharmaceutical industry.

22

References [1].Khalifa,A.M.S.,Farag.M.A.,Yosri.N.,Sabirf.J.S.M.,Saeed.A.,Al-Mousawi,S.M., Taha,W.,Musharraf,S.G.,Patel,S.,El-Seedi.H.R.Truffles: From Islamic culture to chemistry, pharmacology, and food trends in recent times.Trends in Food Science & Technology 91 (2019) 193–218. [2].Dahham,S.S.,Al-Rawi.S.S., Ibrahim,A.H.,Majid.A.S.A., Majid.S.A.AM .AAntioxidant, anticancer, apoptosis propertiesand chemical composition of black truffle Terfezia claveryi.Saudi Journal of Biological Sciences (2018) 25, 1524–1534 [3]. Gouzi, H., Depagne, C.,Coradin, T. Kinetics and thermodynamics of the thermal

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inactivation of polyphenol oxidase in an aqueous extract from Agaricus bisporus. J of Agric Food Chemy. 60(2012) 500–506 . [4]. Zhou, X.,Xiao, Y., Meng,X.,& Bingjie,L. Full inhibition of Whangkeumbae pear polyphenol oxidase enzymatic browning reaction by L-cysteine. Food Chem,266(2018)1-8. [5]. Alici,E.H.,&Arabaci.,G.Purification of polyphenol oxidase from borage (Trachystemo norientalis L.) by using three-phase partitioning and investigation of kinetic properties. Int BiolMacromol.93(2016)1051-1056. [6].Derardja,M. Pretzler, I.Kampatsikas,Barkat,M., &Rompel,A.Purification and Characterization of Latent Polyphenol Oxidase from Apricot (Prunus armeniaca L.). J Agric Food Chem.65(2017)8203–8212. [7].Zhou,D., Li,L.,Wu,Y., Fan,J., & Ouyang,J.Salicylic acid inhibits enzymatic browning of fresh-cut Chinese chestnut (Castanea mollissima) by competitively inhibiting polyphenol oxidase. Food Chem. 171(2015)19-25. [8].Bravo.K.,Osorio.E.Characterization of polyphenol oxidase from Cape gooseberry (Physalis peruviana L.) fruit. Food Chem.197(2016)185-190.

[9].Gouzi.H., Coradin.T.,Delicado.E.N.,ÜmitÜnal.M.,&Benmansour.A.Inhibition Kinetics of Agaricus bisporus (J.E. Lange) Imbach Polyphenol.The Open Enzyme Inhibition Journal.3(2010)1-7. [10].Rescigno,A.,Sollai,F.,Pisu,B.,Rinaldi.A.,& Sanjust.E. Tyrosinase inhibition: general and applied aspects. J Enzyme Inhib Med Chem.17(2002)207-18. [11].Davis,B.J.Disc electrophoresis. II. Method and application to human serum proteins.Ann N Y Acad Sci.121(1964)404-27. [12].Laemmli,U. K.Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4.Nature.227(1970)680–685 24

[13].Benaceur.F., Gouzi .H.,Meddah .B., Neifar .A., Guergouri .A.Purification and characterization of catechol oxidase from Tadela (Phoenix dactylifera L.) date fruit.Int J Biol Macromol.125( 2019 ) 1248-1256. [14].Benzie.I.F.,&Strain.J.J.The ferric reducing ability of plasma (FRAP) as a measure of "antioxidant power": the FRAP assay.Anal Biochem.239(1996)70-6. [15].Choi.Y.,Lee.S.M.,Chu.J.,Lee,H.B., &Lee,J.Influence of heat treatment on the antioxidant activities and polyphenolic com-pounds of Shiitake (Lentinu sedodes) mushroom.Food Chem.99(2006) 381 – 38. [16].Blainski.A.,Lopes.G.C.,& Palazzo de Mello,J.C .Application and Analysis of the Folin Ciocalteu Method for the Determination of the Total Phenolic Content from Limonium Brasiliense L.Molecules.18(2013)6852-6865. [17].Zaini.N.A.M.,Osman.A.,Hamid.A.A.,Ebrahimpour,A.,Saari.N.Purification and characterization of membrane-bound polyphenol oxidase (mPPO) from Snake fruit [Salacca zalacca (Gaertn.) Voss]. Food Chem.136(2013) 407–414. [18].Ionita,E.,Gurgu,L.,Aprodu,J.,Stanciuc,N., Dalmadi,I.,Bahrim,G., & Rapeanu,G. Characterization, purification, and temperature/pressure stability of polyphenol oxidase extracted from plums (Prunus domestica).Process Biochem.56(2018)177-185. [19].Liu, F., Zhao, J., Wen, X., & Ni, Y.Purification and structural analysis of membranebound polyphenol oxidase from Fuji apple. Food Chem. 183(2015)72–77. [20].Batista.K.A,.Batista.G.L.A., Alves.G.L.,Fernandes.K.F.Extraction,partial purification and characterization of polyphenol oxidase from Solanum lycocarpum fruits. J. Mol. Catal. B Enzym.102(2014)211–217. [21].Palma-Orozco,G.,Ortiz-Moreno,A.,Dorantes-Alvarez,L.,Sampedro,J.G., Nájera,H. Purification and partial biochemical characterization of polyphenol oxidase from mamey (Pouteria sapota).Phytochemistry.72(2011)82-8.

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[22].Liu, L., Cao, S., Yang, H.,& Qi, X. Pectin plays an important role on the kinetics properties of polyphenol oxidase from honeydew peach. Food Chem. 168 (2015)14– 20. [23].Mishra, B.B.,Gautam,S.,& Sharma,A.Purification and characterization of polyphenol oxidase (PPO) from eggplant (Solanum melongena).Food Chem.134 (2012)1855–1861. [24].Doğru, YZ., Erat, M.. Investigation of some kinetic properties of polyphenol oxidase from parsley (Petroselinum crispum, Apiaceae).Food Research International 495(2012)411-415. [25].Cheema,S. ,&Sommerhalter,M. Characterization of polyphenol oxidase activity in Ataulfo mango. Food Chem. 171(2015) 382–387 [26].Saeidian,S., Keyhani,E., Keyhani ,J.(2007). Effect of ionic detergents, nonionic detergents, and chaotropic agents on polyphenol oxidase activity from dormant saffron (Crocus sativus L.) corms. J Agric Food Chem. 55,3713-9. [27].Saeidian,S.Inhibitory Effect of Cysteine and Glycine Upon Partial Purified Polyphenol Oxidase of Pyruscommunis.Advances in Biological Research.6(2013)216-222. [28].Dogan ,S., Turan,P.,Dogan,M.,Arslan,O., &Alkan M.Purification and characterization of Ocimum basilicum L. polyphenol oxidase.J Agric Food Chem. 26(2005)10224-30. [29].Siddiq,M.,&Dolan K.D.Characterization of polyphenol oxidase from blueberry (Vaccinium corymbosum L.).Food Chem,218 (2017)216-220. [30]. Lim,W.Y., &Wong,C.W. Inhibitory effect of chemical and natural anti-browning agents on polyphenol oxidase from ginger (Zingiber officinale Roscoe).J Food Sci Technol. 55(2018)3001-3007. [31].Hussein,M. A. M. El-Gizawy, A.,. El-Bassiouny,R.E.I.,&. Saleh,M.A.Browning inhibition mechanisms by cysteine, ascorbic acid and citric acid, and identifying PPOcatechol-cysteine reaction products. J Food Sci Technol.52(2015) 3651-3659. 26

[32].Goyeneche,R.,DiScala,K.,& Roura,S.Biochemical characterization and thermal inactivation of polyphenol oxidase from radish (Raphanussativus var. sativus).LWTFood Sci Technol.54(2013)57-62. [33].Sulaiman,A.,Soo,M.J.,Farid,M.,& Silva, F.V.M.Thermosonication for polyphenol oxidase inactivationin fruits: modeling the ultrasound and thermal kinetics in pear,apple and strawberry purees at different temperatures. J Food Eng.16(2015)133-140. [34].Gnangui,S,N.,Dué, E. A.,N’guessan Kouadio, J-P. E.,Kouamé, L. P. Effect of heat treatment on edible yam polyphenol oxidase activity: kinetic and thermodynamic analysis. Journal of animal & plant sciences. 2,(2009)128 – 137. [35].Chutintrasri, A.,& Noomhorm,Thermal inactivation of polyphenoloxidase in pineapple puree. LWT-FOOD SCI TECHNOL.39(2006)492-495 [36]. Wei ,L , Li-Qiang Z, Jun-Ping L, Zhao-Qin Z, Cheng-Mei L, Rui-Hong L.The effect of citric acid on the activity, thermodynamics and conformation. Food Chemistry 140(2013)289–295.

[37].Aka,J,P.,Courtois,F.,Louarme,L.,Nicolas.,J,Billaud,C.Modelling the interactions between free phenols, L-ascorbic acid, apple polyphenoloxidase and oxygen during a thermal treatment. Food Chemistry 138(2013)1289–1297. [38].Sharma,K.,Ko,E.Y.,Assefa,A.D.,Ha,S.,Nile,S.H.,Lee,E.T.,& Park,S.W.Temperaturedependent studies on the total phenolics, flavonoids, antioxidant activities, and sugar content in six onion varieties. J Food Drug Anal.23(2015)243-252. [39].Pérez-Burillo,S.,Giménez,R.,Rufián-Henares,J., Pastoriza,S.Effect of brewing time and temperature on antioxidant capacity and phenols of white tea: Relationship with sensory properties. Food Chem.248 (2018)111-118. [40].Tao,Y.M.,Yao,L.Y.,Qin,Q.Y.,& Shen,W..Purification and characterization of polyphenol

27

oxidase from jackfruit (Artocarpus heterophyllus) bulbs. J Agric Food Chem.61(2013) 2662-12669. [41]. Shah.H.M.S. , Khan,A.S.,Sajid,A.Pre-storage kojic acid application delays pericarp browning and maintain antioxidant activities of litchi fruit.Postharvest Biology and Technology 132 (2017)154–161.

Fig. 1 Truffles “Terfezia arenaria” was purchased from the local market of El-Bayadh on January 2018 ,located southwest of Saharan Atlas (Amour range) about 520 miles from the capital (Algiers). Fig.2:Denaturing SDS- PAGE ;Lane 1: molecular weight marker,Lanes 2: crude extract and Lane 3: Purified truffles PPO stained with Coomassie brilliant blue R-250. (D)Native-PAGE : Lanes 4 and 5 for

crude and purified PPO,respectively. Truffles PPO exhibited a

molecular weight of 67kDa . Fig. 3 :Influence of pH and temperature on the activity of purified Truffles PPO. A) optima pH .B)pH stability.C) Optima temperature and D) Temperature stability. Each data point is the mean of three experiments .Vertical bars represent standard deviations. Truffles PPO seems to be highly active under wide range of pH and temperature. Optimum conditions of PPO depend on substrate used. Fig.4.Effect of temperature and kojic acid on PPO in Truffles extracts and their antioxidant activities (A)Crude PPO inactivation (B) FRAP assay (C) ABTS radical, D)Total phenolic contents.Treatement of truffles extracts by Kojic acid increase inactivation process of PPO and maintain their antioxidant properties.

28

Tables caption: Table 1.Purification steps of polyphenol oxidase from truffle”Terfezia arenaria”. PPO wad purified 59 fold and showed specific activity equal to 1088.6 EU/mg. Table 2.Effect of reagents on purified Tanbouchet PPO .A) Effect of metals ions ,B)Effect of detergents and chaotropic agents .SDS, sarkosyl and triton x100 react as activators of latent form of PPO. Table 3. Effect of different inhibitors on truffles PPO activity.The potency of inhibitor depend of substrate used and concentration applied. Table 4.Kinetic parameters of inhibition (KI ,IC50) and type. Kojic acid showed independence of inhibition type towards substrates used . Table 5.Effect of kojic acid on Thermal inactivation and thermodynamic parameters of heat inactivation of purified truffles PPO.Kojic acid react as accelerators of heat inactivation.by reducing stability of enzyme.conformation .

29

Table 1.

Total volume (ml)

Total activity (EU)

Total protein (mg)

250

3511.6

190.7

Specific activity (EU/mg) 18.4

Ammonium sulfate precipitation (80%)

60

2759.3

110.2

Q-Sepharose “Big beads”

7

1815.4

5

1197.5

Purification steps Crude extract

HPLC Zorbax

30

Yield (%)

Purification (n-fold)

100

1

25.1

78.8

1.3

13.5

134.4

51.6

7.3

1.1

1088.6

34.2

59.1

Table2 (A)

Metal ions

PPO relative activité (%) Pyrocatechol

L-tyrosine

2mM

5mM

2mM

5mM

MgSO4.(5H20)

101

105

103

111

FeSO4.(7H20)

70

50

60

37

CaCl2.(7H20)

85

47

79

33

CdSO4.(8H20)

61

45

53

28

ZnSO4.(7H20)

102

105

103

108

CuSO4.(5H20)

104

123

106

140

MnCl2.(4H20)

95

81

88

64

AlCl3(6H20)

45

22

39

7

31

(B)

PPO relative activité (%)

Reagents

Catechol

L-tyrosine

a-Ionic Detergents

0 .1 mM

2mM

5mM

0.1mM

2mM

5mM

Sodium dodecyl sulfate

+15.1

+253.3

+260.6

+25.2

+185.3

+189.1

Sarkosyl

+7.2

+170.4

+180.9

+17.6

+147.9

+150.2

Sodium cholate

NE

-15.1

-30.3

NE

-28.3

-45.7

Sodium dodecyl cholate

NE

NE

-20.1

NE

-9.3

-35.2

Triton X-100

NE

NE

+28.1

NE

+2.3

+35.7

Tween 20

NE

NE

NE

NE

NE

NE

Tween 80

NE

NE

NE

NE

NE

NE

NP-40

-9.7

-22.4

-34.9

-20.3

-37.6

-57.8

urea

-42.1

-63.2

-72.4

-48.7

-70.3

-88.4

Guanidine hydrochloride

-30.4

- 41.8

-53.6

-35.9

-47.6

-69.3

b-Non ionic Detergents

c-Chaotropic agents

NE :No effect (-) & (+) mean Inhibition.and activation. (a )Mean (± SD) for triplicate experiments

32

Table 3 Inhibition of relative activity (%)

Inhibitor

Pyrocatechol

L-Tyrosine

a-Sulfites

0 .1 mM

2mM

5mM

0.1mM

2mM

5mM

Potassium metabisulfite

48.3

79.6

100

53.2

86.7

100

Sodium metabisulfute

62.9

95.3

100

75.2

100

100

Sodium fluoride

54.2±1.4

76.1

95.6

60.2

82.6

100

Sodium fluorosilicate

39.5 ±1.3

70.3

85.2

43.4

85.6±1

100

Arginine

NE

NE

34.5

NE

17.2

42.3

L-cysteine

47.2

83.1

100

71.3

89.5

100

Glycine

34.8

59.3

75.6

44.2

65.4

86.1

Glutamic acid

48.3

62.7

81.9

53.7

70.1

84.6

Kojic acid

54.3

85.2

100

68.3

90.3

100

Salicylic acid

29.6

60.3

85.1

35.5

64.7

88.9

Benzoic acid

51.3

72.1

97.6

55.2

80.4

100

b-Fluorides

c-Amino acids

d-Organi cacids

Table 4. Inhibitor

Catechol

L-Tyrosine

Substrate

Sodium metabisulfute

Sodium fluoride

L-cysteine

Kojic Acid

IC50(mM)

0.09

0.19

1.17

0.91

KI(mM)

0.07

0.22

1.03

0.87

R2

0.998

0.997

0.998

0.999

Inhibition type

Uncompetitive

Noncompetitive

Competitive

Mixed type 1

IC50 (mM)

0.18

0.58

1.49

1.21

KI (mM) R2

0.23 0.997

0.51 0.997

1.38 0.999

1.12 0.998

Inhibition type

Noncompetitive

Uncompetitive

Competitive

Mixed type 1

33

Table 5.

T(°C) and kojic acid concentration

Catechol K (min-1)

D (min)

t½ (min)

60°C 65°C

0,00609 0,01107

378,15 207.88

113.81 62.56

70°C

0,1856

124.06

37.33

75°C

0,0270

84.99

60°C

0.0042

65°C

Zt (°C)

Ea (kJ/mol)

∆H (kJ/mol)

∆G (kJ/mol)

∆S (J/mol K-1)

115.10 115.051±1.0 115.19

480.72 176.741±2.0 473.21

[I]=0 mM 275.18 275.13 23.05

277.95

275.09

115.47

465.38

25.58

275.05

116.11

456.76

537.01

161.62

308.19

0.0072

315.54

94.97

308.14

116.07 114.693±3.0 116.36

70°C

0.0139

165.57

49.83

308.10

116.29

559.22

75°C

0.022

101.77

30.63

308.06

116.62

550.12

60°C

0.0024

927.00

279.00

359.49

65°C

0.0054

421.93

126.99

359.44

117.59 113.339±2.0 117.18

70°C

0.0091

252.25

75.92

359.40

117.49

705.28

75°C

0.0180

127.81

38.47

359.36

117.28

695.64

60°C

0.0009

2345.22

705.85

449.33

120.16

988.50

65°C

0.00232

990.32

298.06

449.28

119.58 119.51

975.46 961.30

[I]=0.05

20.43

310.96

576.91 175.063±2.0 567.39

[I]=0.1 mM

17.82

362.26

726.42 176.152±2.0 716.75

[I]=0.2mM 14.23

452.10

70°C

0.0044

512.99

154.39

449.24

75°C

0.0116

197.07

59.31

449.20

111.235±4.0 118.53

60°C

0.0003

6363.43

1970.11

568.62

122.91

1338.46

65°C

0.0008

2830.74

851.98

568.57

122.53

1319.65

120.87

1305.14

119.73

1289.54

177.067±3.0 950.20

[I]=0.3mM

70°C

0.0027

824.78

248.24

75°C

0.0077

298.33

89.79

11.06

571.39

568.53 568.49

34

35