Kinetics and molecular docking of purified polyphenol oxidase from rhizome of turmeric (Curcuma longa L.)

Kinetics and molecular docking of purified polyphenol oxidase from rhizome of turmeric (Curcuma longa L.) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Kinetics and molecular docking of purified polyphenol oxidase from rhizome of turmeric (Curcuma longa L.) Olutosin Samuel Ilesanmi, Victory Ayo Olagunju, Omowumi Funke Adedugbe, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4675546/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Polyphenol oxidase catalyzes oxidative conversion of polyphenols to their respective quinones. These have been exploited in various biotechnological processes. The kinetics and molecular docking interaction of turmeric PPO on some inhibitors are here described. The enzyme was purified using aqueous two-phase partitioning. The subunit and the native molecular masses of the purified turmeric PPO were 69 ± 2.0 kDa and 66.8 ± 3.5 kDa respectively, suggesting its monomeric nature. The K m and Vmax of the C. longa PPO for pyrogallol were 5.8 ± 0.6 mM and 722.9 ± 17.0 units/mg protein respectively leading to turnover number ( k cat) and first order rate constant ( k cat/ K m) of 831.6 ± 5.0 s -1 and 1.43 × 10 5 s -1 M -1 respectively. The purified enzyme was activated at the lowest concentration in KCl and CuSO 4, whereas was fairly stable in the presence of NaCl, ZnSO 4 and NH 4 Cl. The inhibition constant ( K i) obtained from Dixon plot for ascorbic acid, β-mercaptoethanol, citric acid, cysteine, EDTA, glutathione and kojic acid were 7.8, 1.7, 5.5, 2.0, 8.1, 3.3 and 6.4 mM respectively. In-depth analyses, revealed that cysteine was the most potent of all the inhibitors investigated. The binding interaction of the purified enzyme with inhibitors revealed that EDTA, Kojic acid and Cysteine have 2 hydrogen bonds formed while citric acid, ascorbic acid and glutathione had 4, 5 and 3 respectively. In conclusion, the kinetics and inhibition studies of the purified turmeric PPO could be deployed in the control of browning and several industrial and biotechnological applications. Biological sciences/Biochemistry Biological sciences/Biotechnology Biological sciences/Plant sciences Earth and environmental sciences/Environmental sciences Polyphenol oxidase Turmeric Kinetics Molecular Docking Biotechnological applications Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Polyphenol Oxidases (PPO) (E.C.1.10.3.1), also termed catechol oxidases, catecholases, diphenol oxidases, ortho-diphenolases, phenolases, and tyrosinases [ 1 ]. They are a group of copper-containing enzymes that catalyze the o -hydroxylation of monophenols to o -diphenols as well as the oxidation of o -diphenols to quinones in the presence of oxygen [ 2 ]. Polyphenol oxidase is broadly distributed among animals, fungi, and plants, although the studies are more extensive in plants [ 3 , 4 ]. The study of PPOs in plants has focused primarily on their role in the process of postharvest browning, whereby cut or damaged plant tissues turn brown due to the polymerization of PPO-generated quinones, generating phyto-melanins [ 5 ]. The enzyme can catalyze the oxidation of polyphenols and result in the browning of damaged or cut plant, which seriously affects quality and reduce the market value of the crop [ 2 , 6 ]. Polyphenol oxidase is widely present in plants, play an important role in the growth, development, and stress responses. Many studies have reported that PPO and peroxidases are induced in response to biotic and abiotic stress in plants, and it has been implicated in several functional processes such as participating in plant defense and the synthesis of plant-specific metabolites [ 7 , 8 ]. Plant PPO generally contain three conserved regions, N-terminal cTP, aCuA and CuB domain and a C-terminus [ 9 ], which are responsible for thylakoid lumen localization and enzyme activity. Polyphenol oxidase is found in many plant species such as banana [ 10 ], apple [ 11 ], potato [ 12 ], eggplant [ 13 ], strawberry [ 14 ], red cocoyam [ 15 ], bitter leaf [ 16 ] etc. The function and distribution of PPO differ in various plants [ 9 ]. Most PPO are transported to the thylakoid membrane in the chloroplast, or in cytosol and other organelles [ 17 ], whereas the phenolic compounds are localized to the vacuoles. Because of the different localization of the enzyme and its substrates, their interaction requires destruction of the cell and mechanical damage [ 18 ]. Plant PPOs have reported to possess various applications such as synthesis of drugs and other organic compounds [ 19 ]. Turmeric ( Curcuma longa ) is a perennial rhizomatous crop of the Zingiberaceae, a world-wide known spice whose medicinal properties has received interest from both the medical and scientific world as well as culinary enthusiasts, as it is the major source of the polyphenol curcumin [ 20 ]. Turmeric contains 3 to 6% polyphenolic compounds which is known as curcuminoids [ 21 ]. It aids in the management of oxidative and inflammatory conditions, metabolic syndrome, arthritis, anxiety, and hyperlipidemia. Most of these benefits can be attributed to its antioxidant and anti-inflammatory effects [ 20 ]. Like several plants, it suffers a reduction in its sensory qualities and health benefits as a result of spoilage which occurs due to browning. Recent research of novel anti-PPO systems is focused on mild alternatives to conventional treatments which could impair not only the sensory and nutritional properties of agro-food products but also the consumer health [ 22 ]. Milder processes of controlling enzymatic browning can be discovered as PPO studies are being carried out and this will help improve the shelf-life of turmeric and its products. Many researches have been conducted using turmeric rhizomes in many forms of investigation in the area of its characteristics, functionality, and applications [ 23 ]. Specifically, several studies have reported that turmeric possesses potent multiple properties such as anti-inflammatory, antioxidant, antitumor, antibacterial and anticoagulant, and antidiabetic based on its free-radical-scavenging activity expressed by the domicile bioactive compound. Among the bioactive components of turmeric, curcumin is the most frequently studied, it is fat soluble bioactive compound, whose characteristics and functionality is equable to its popularly reported medicinal, pharmacological (hypoglycemic, insulinotropic, and hypolipidemic) [ 24 ]. The potency of turmeric anti-oxidant and anti-inflammatory properties improves symptoms of depression, arthritis and Alzheimer’s disease. It contains natural antioxidants, polyphenols and phytochemicals which confer significant protection against free radicals’ damage [ 19 , 25 ]. Different sources of polyphenol oxidase have been reported including bacteria, fungi, animal and plants [ 26 ]. The PPO found in humans is responsible for skin pigmentation including development of freckles [ 27 ]. Plant PPO has an important role in plant stress resistance and physiological metabolism. Most of the PPO in plants are found in the chloroplasts of photosynthetic cells and the leucoplasts of storage cells [ 28 ]. Polyphenol oxidase is one of the most important industrial enzymes, considering its wide applications in several industrial and biotechnological application. There is need for continual searching for cheaper and readily available sources of the enzyme. Sufficient information on the relative occurrence and some physio-chemical properties of polyphenol oxidase from turmeric had been earlier reported [ 19 ]. However, in that work, the much-needed information on kinetics, inhibition studies and molecular docking of some important inhibitors and/or anti-browning agents of the purified turmeric PPO was lacking and greatly necessary. This was with a view to providing information on its mechanism of catalytic reactions, specificity towards substrates and binding interactions with inhibitors and anti-browning agents through molecular docking. Hence, the reason for this study. These could be deployed in several biotechnological applications and primarily for understanding the strategy in the control of browning in turmeric food products. 2. Materials and Methods 2.1 Materials The fresh turmeric ( Curcuma longa ) was obtained from farms in Owo environs, Southwestern Nigeria. The plant was authenticated at the Department of Plant Science and Biotechnology, Achiever University, Owo, Ondo State, Nigeria. 3,4-dihydroxyphenyl-L-alanine (L-DOPA), catechol, L-tyrosine, pyrogallol, blue dextran, acetic acid, citric acid, sodium citrate, bovine serum albumin (BSA), sodium phosphate dibasic (Na 2 HPO 4 ), anhydrous sodium phosphate monobasic (NaHPO 4 ), Coomassie brilliant blue R-250, glutathione was obtained from Sigma Chemical Company, St Louis, USA. Molecular weight standard for sodium dodecyl sulphate- polyacrylamide gel electrophoresis (SDS-PAGE) was obtained from Carl Roth GmbH, Karlsruhe, Germany. All other reagents were of analytical grade. 2.2 Methods 2.2.1 Extraction of PPO from turmeric (Curcuma longa) The fresh turmeric rhizome ( Curcuma longa ) rhizome was cut and peeled after which it was rinsed in distilled water and homogenized in 50mM potassium phosphate buffer, pH 6.5 on ice to obtain 30% homogenate [ 29 ]. The homogenate was centrifuged at 10,000× g for 30 min at 4 ˚C using cold centrifuge in order to obtain clear crude supernatants. The supernatants were assayed for PPO activity using catechol as substrate in a spectrophotometer at 410nm. The collected supernatant was stored in a freezer (at -20 ˚C). 2.2.2 Standard assay for polyphenol oxidase Polyphenol oxidase activity was determined using L-Tyrosine, L-DOPA, catechol and pyrogallol as substrates in the crude supernatants and during purification according to the method of Wititsuwannakul et al . [ 30 ] as modified by Ilesanmi et al . [ 31 ]. The reaction involved final concentration of 5 mM of the substrates, 50 mM phosphate buffer, pH 6.5 and appropriate volume of enzyme. Initial rate of product formation will be monitored spectrophotometrically. 2.2.3. Protein concentration determination The protein concentrations in the crude homogenates and purified PPO were determined as described by Bradford [ 32 ] using BSA as the standard protein. The mixture of Bradford working reagent, and the sample is run through the spectrometer at 595 nm, and then the absorbances is recorded for the standard and sample at 595 nm. 2.2.4. Purification of the crude polyphenol oxidase from turmeric Crude PPO from the rhizome of turmeric was purified as reported by Ilesanmi et al . [ 19 ]. The purification involved combination of aqueous two-phase partitioning and gel filtration chromatography. 2.2.5. Homogeneity test and determination of subunit molecular weight by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) Protein purity and the subunit molecular weight of the purified polyphenol oxidase were determined by SDS polyacrylamide gel electrophoresis using 12% (w/v) polyacrylamide (running gel) and 2.5% (w/v) stacking gel, according to the method of Laemmli [ 33 ] as modified by Weber and Osborn [ 34 ] using Tris-glycine buffer system at pH 8.3. the protein bands were obtained following staining and destaining procedures. 2.2.6. Characterization of the purified PPO from turmeric 2.2.6.1. Determination of kinetic parameters The effect of various concentrations of pyrogallol on the activity of purified PPO from Curcuma longa was determined. The kinetic parameters ( K m , V max , k cat and k cat/ K m) for pyrogallol were analyzed and estimated using non-regression software graph pad prism 8. 2.2.6.2. Effect of metals on the activity of purified PPO from turmeric This was done following the method used by Arabaci [ 35 ]. The effects of monovalent, divalent and trivalent metals like Na + (NaCl), K + (KCl), Cu 2+ (CuSO 4 ), Zn 2+ (ZnSO 4 ), Ba 2+ (BaCl 2 •H 2 O), and NH 4 + (NH 4 Cl), were determined at different concentrations of between 10 to 50 mM). 2.2.6.3. Effect of inhibitors and anti-browning agents on the purified PPO from turmeric The effects of anti-browning agents viz. L-cysteine, kojic acid, mercaptoethanol, glutathione (GSH), ethylenediamine tetra acetic acid (EDTA), L-ascorbic acid, and citric acid on the activity of the enzyme was determined as described by Liu [ 36 ]. This was carried out in varying concentrations of 1 to 10mM. 2.2.6.4. Inhibition constant (Ki) of some inhibitors The Inhibition constant ( K i) for Ascorbic Acid, L-cysteine, kojic acid, mercaptoethanol, glutathione (GSH), ethylenediamine tetra acetic acid (EDTA), and Citric acid were estimated using Dixon plot. 2.2.6.5. Molecular docking studies for binding interaction of inhibitors The X-ray crystallographic structure of polyphenol oxidase was retrieved from the RCSB protein database pdb id 4Z10. The protein preparation wizard of the Glide Schrödinger Suite 2017–1 was used to rectify specific errors in the protein during crystallographic structure and optimization was done. The structure-data file (SDF) structures of the seven (7) ligands were downloaded from the PubChem database, imported on the workspace of Maestro Schrödinger suite Interface. The molecular docking of the seven ligands was performed using extra precision (XP) and the co-crystallized ligand was re-docked into the catalytic site of 4Z10 to confirm the accuracy of the screening and docking scores. 3. Results 3.1. Extraction and purification The crude enzyme was homogenized and extracted in 50mM Phosphate Buffer to obtain 30% homogenate and after it was subjected to aqueous two-phase partitioning system (ATPS). The data in Table 1 shows the purification summary of purified C. longa PPO. The percentage yield obtained was 41% with a purification fold of 4.4. 3.2. Purity test and subunit molecular weight determination The purity test of the purified enzyme was determined on SDS polyacrylamide gel electrophoresis using 12% polyacrylamide (running gel) and 2.5% stacking gel using Tris-glycine buffer system at pH 8.3 which gave a single band protein estimated to be 69 ± 0.2 kDa. This is shown in the electrophoretogram (Figure 1a). Estimation of the subunit molecular weight of the purified PPO was carried by plotting the molecular weights of the standard protein against their relative mobility. 3.3. Kinetic parameters of purified PPO from turmeric The kinetic parameter of purified C. longa PPO in aqueous system was determined using non-linear regression plot of activity against concentration. This is shown in Table 2. The K m and Vmax of C. longa PPO for pyrogallol were 5.8 ± 0.6 mM and 722.9 ± 17.0 units/mg protein respectively. The k cat and k cat/ K m were 831.6 ± 5.0 s -1 and 1.43x10 5 s -1 M -1 respectively. 3.4. Effect of metals on the activity of purified PPO from turmeric The activity of PPO in the presence of varying concentrations of metals was determined. As shown in Figure 2, PPO was activated at the lowest concentration in KCl and CuSO 4 following a gradual decline while the enzyme remained fairly stable in the presence of NaCl, ZnSO 4 and NH 4 Cl. 3.5. Effects of inhibitors on PPO from turmeric The percentage residual activity of C. longa PPO in the presence of inhibitors from low to high concentrations were determined as shown in Figure 3. Cysteine was seen to show a good decline in activity compared to other inhibitors used on the enzyme. Furthermore, Dixon plot was used to determine the inhibition constant ( K i ) by plotting the inverse relative enzyme activity against the concentration of the inhibitor. This revealed Cysteine as the most potent inhibitor with the lowest K i of 2.0 mM. The inhibition constant ( K i ) summary for all the inhibitors is shown in Table 3. 3.7. Binding interaction of purified PPO from turmeric with some selected inhibitors The X-ray crystallographic structure of polyphenol oxidase was retrieved from the protein databank with pdb id 4Z10 and the ligands were re-docked into the catalytic site of 4Z10. The binding energy was determined which was used to calculate the free binding energy. The docking score was also estimated. Figure 4 and 5 show a representation of the molecular docking score and binding energy for the ligands (inhibitors) while Table 4 gives a summary of the interaction of the docked inhibitors with C. longa PPO. 4. Discussion Polyphenol oxidase is one of the most important industrial enzymes with enormous applications in several technical and biotechnological processes [3,16]. The presence of polyphenol oxidase in the rhizome of turmeric ( Curcuma longa ) has been established in this study. The kinetics and interactions of the purified enzyme towards substrates, metals, inhibitors and/or anti-browning agents have also been documented. The extracted crude PPO from the turmeric was subjected to non-conventional purification-aqueous two-phase partitioning (ATPS) which resulted in a purified PPO [19]. The major advantages of the procedure used is that the method helps purify and also concentrate the enzyme in addition to requiring less purification time which makes it faster compared to other purification processes [37]. The purification scheme proved efficient enough as adjudged on the SDS-PAGE. The molecular weight of the purified PPO was confirmed by SDS-PAGE. The result on the slab gel showed a monomeric protein band with an estimated molecular weight of 69 ± 0.2 kDa. This confirmed the purity of the C. longa PPO and the effectiveness of the purification procedure. This aligns with similar findings of PPO obtained from Solanum lycocarpum of 68 kDa [38] . The kinetics of enzymes provide information on the mechanism of catalytic reactions and specificity of the enzyme towards substrates. The kinetic parameter of the purified turmeric PPO in aqueous solution was determined using non-linear regression plot of activity against the substrate concentration. The K m of C. longa PPO was 5.8 ± 0.6mM and Vmax of 722.9 ± 17.0 units/mg protein respectively, while the catalytic efficiency and specificity constant of the purified enzymes k cat/ K mwas 1.43x10 5 s -1 M -1 . Lower K m suggests higher affinity towards the substrate [39]. The kinetics revealed useful information about the catalytic mechanism, and mechanisms of inhibition of the purified turmeric PPO which would be exploited in several biotechnological processes. Metal ions play important roles in maintaining substrate binding in the active site of metalloenzymes and in controlling the redox activity of metalloenzymes in enzymatic reaction and metal ions can reversibly change its valence to regulate the enzymatic reaction [40]. In this work, NaCl, ZnSO 4 and NH 4 Cl showed a stable activity with average of 90 to 95% activity retained at concentrations between 10 to 50mM. The activity of PPO was observed to have been activated at concentration of 10mM which was followed by a gradual decline activity for KCl and CuSO 4 up to 40mM and another increased in at 50mM concentration. C. longa PPO activity with the introduction of BaCl 2 was observed to increase at 30mM and maintain 100% activity at 50mM concentration. The stability of PPO in the presence of Na + is similar to the results of Guo et al. [41] and Liu et al . [42]. The inhibition of enzymatic browning has been the subject of continuing concern among researchers and the food industry. Several compounds have been investigated for their effectiveness in preventing enzymatic browning in crude extracts of fruit and vegetables [43]. The residual activity of C. longa PPO plotted against different concentration of Citric acid, cysteine and EDTA. At the highest concentration in the presence of Citric acid, cysteine and EDTA, C. longa PPO had a residual activity of 41, 15 and 52% respectively. The inhibition constant K i was further used to ascertain the inhibitory prospect of the inhibitors using Dixon plot. Cysteine was the most potent inhibitor of C. longa PPO. This can be due to the presence of sulphur as sulphur containing compounds have been seen to have higher inhibitory effect on PPO in previous studies. Cysteine reacts with o -quinone and forms a stable colorless product which interferes with browning activities. The choice of cysteine as the most potent inhibitor is also supported by Liu et al. [41] who reported the inhibitory activity even at a concentration as low as 0.1mM. The inhibitory activity of EDTA is reported to not be obvious at low concentration but would increase as concentration increases and it works as a copper-binding ligand [44,45]. This information on the low values of K i for the cysteine of the enzyme may be deployed in food manufacturing industries to salvage increasing reports of food spoilage and more importantly to improve the qualities of turmeric The binding interaction showed that EDTA, Kojic acid and Cysteine have 2 hydrogen bonds formed while citric acid, ascorbic acid and glutathione had 4, 5 and 3 respectively. B -mercaptoethanol showed no hydrogen bonding interaction with the residues [46]. This infers these inhibitors inhibits the activity of PPO by preventing the substrate from binding with the key amino acid residues with which the hydrogen bonding interacts in the active site in the competitive process for its stronger affinity with the enzyme, which leads to its resultant of inhibition kinetics. β-mercaptoethanol and cysteine were observed to have the lowest docking score of -2.3060 and -2.6710. Both inhibitors also exhibited the higher binding free energy which further affirms the potency of cysteine. The inhibitory activity of Cysteine can also be due to the presence of sulphur which forms a stable complex making it a strong inhibitor of C. longa PPO. Conclusion The presence of PPO from turmeric ( Curcuma longa ) rhizome has been established and a shortened purification method (ATPS) was highly effective in removing unwanted proteins and contaminants from the desired enzyme. The kinetics and inhibition studies on the purified turmeric polyphenol oxidase could be deployed in several biotechnological applications and/or countering food spoilage considering how versatile and important turmeric is, in pharmaceutical and nutraceutical applications. Declarations Authorship Contribution Victory Ayo Olagunju - Performed the research, analyze data and wrote the paper Olutosin Samuel Ilesanmi – Conceived the research, analyze data and reviewed the paper Omowumi Funke Adedugbe – analyze data and proof-read the manuscript Adedeji Benedict Kayode – analyze data and proof-read the manuscript Data Availability Statement The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request. References Mayer, A.M. (2006). Polyphenol oxidases in plants and fungi: Going places? A review. Phytochemistry 67, 2318–2331. Qin, F., Hu, C., Dou, T., Sheng, O., Yang, Q., Deng, G., He, W., Gao, H., Li, C., Dong, T., Yi, G. and Bi, F. (2023). Genome-wide analysis of the polyphenol oxidase gene family reveals that MaPPO1 and MaPPO6 are the main contributors to fruit browning in Musa acuminata. Front. Plant Sci. 14:1125375. doi: 10.3389/fpls.2023.1125375. Moon, K. M., Kwon, E. B., Lee, B., and Kim, C. Y. 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Journal of food science and technology , 55 (8), 3281-3291. Zhang, J., and Sun, X. (2021). Recent advances in polyphenol oxidase-mediated plant stress responses. Phytochemistry , 181 , 112588. Guo, L., Ma, Y., Shi, J., and Xue, S.J. (2009). The purification and characterization of polyphenol oxidase from green bean ( Phaseolus vulgaris L.). Food Chemistry, 117 , 143-151. Liu, W., Zou, L. Q., Liu, J. P., Zhang, Z. Q., Liu, C. M., and Liang, R. H. (2013). The effect of citric acid on the activity, thermodynamics and conformation of mushroom polyphenol oxidase. Food chemistry , 140 (1-2), 289-295. Lim, W. Y. and Wong, C. W. (2018). Inhibitory effect of chemical and natural anti-browning agents on polyphenol oxidase from ginger (Z ingiber officinale Roscoe). J Food Sci Technol. 55(8):3001-3007. doi: 10.1007/s13197-018-3218-7. Zhang, N., Huo, J., Yang, B., Ruan, X., Zhang, X., Bao, J., and He, G. (2018). Understanding of imidazolium group hydration and polymer structure for hydroxide anion conduction in hydrated imidazolium-g-PPO membrane by molecular dynamics simulations. Chemical Engineering Science , 192 , 1167-1176. Sikora, M., Świeca, M., Franczyk, M., Jakubczyk, A., Bochnak, J., and Złotek, U. (2019). Biochemical Properties of Polyphenol Oxidases from Ready-to-Eat Lentil ( Lens culinaris M .) Sprouts and Factors Affecting Their Activities: A Search for Potent Tools Limiting Enzymatic Browning. Foods (Basel, Switzerland), 8(5), 154. Nokthai, P., Lee, V. S., and Shank, L. (2010). Molecular modeling of peroxidase and polyphenol oxidase: substrate specificity and active site comparison. International Journal of Molecular Sciences , 11 (9), 3266–3276. Tables Tables 1 to 4 are available in the Supplementary Files section Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4675546","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":330398242,"identity":"49506c1b-e55e-4b81-b1c8-94662e2478f6","order_by":0,"name":"Olutosin Samuel Ilesanmi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBElEQVRIiWNgGAWjYDACdjApwcAGYTDw8DMwsOHXwszA2ADWwgzVItlAnBYwAwIMDhDQws/MY/7gwx+LPD5m5sMffu7YJmN8I/nZgw8VDPL8YgewapFs5jFsnNkmUczGzJYm2XvmNo/ZjTRzwxlnGAxnzk7AqsXgMI9hM2+DRGIbM48ZA28bSEuCmTRvG0OCwW08Wnj+gLTwf/74F6jFeEb6NyK0sIFtYZAG2WIgkYPfFslmtsKZQL8AtbCZScsCtUiceVMmOeOMBE6/8LM3b/jw4U9d4vz25scf37bdtudvT98m8aHCRp5fGrsWLEAArFKCWOVgiw+QonoUjIJRMApGAAAAp71VD1r/T+AAAAAASUVORK5CYII=","orcid":"","institution":"Achievers University","correspondingAuthor":true,"prefix":"","firstName":"Olutosin","middleName":"Samuel","lastName":"Ilesanmi","suffix":""},{"id":330398243,"identity":"5820c8a2-abd3-474b-bd25-a70018d1e390","order_by":1,"name":"Victory Ayo Olagunju","email":"","orcid":"","institution":"Achievers University","correspondingAuthor":false,"prefix":"","firstName":"Victory","middleName":"Ayo","lastName":"Olagunju","suffix":""},{"id":330398244,"identity":"ee43c608-c1ff-4d52-8f6b-208f5f0d6794","order_by":2,"name":"Omowumi Funke Adedugbe","email":"","orcid":"","institution":"Achievers University","correspondingAuthor":false,"prefix":"","firstName":"Omowumi","middleName":"Funke","lastName":"Adedugbe","suffix":""},{"id":330398245,"identity":"1f516dce-1847-4e6b-b836-1a0bf2b427d8","order_by":3,"name":"Adedeji Benedict Kayode","email":"","orcid":"","institution":"National Horticultural Research Institute (NIHORT)","correspondingAuthor":false,"prefix":"","firstName":"Adedeji","middleName":"Benedict","lastName":"Kayode","suffix":""}],"badges":[],"createdAt":"2024-07-02 16:17:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4675546/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4675546/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61295172,"identity":"6e6dd0aa-17ea-42e9-a7f5-175d8bf45d6d","added_by":"auto","created_at":"2024-07-29 08:01:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":70095,"visible":true,"origin":"","legend":"\u003cp\u003eElectrophoretogram of the Purified PPO from Turmeric. Lane (A) represent the purified enzyme and lane S represent the standard markers. The estimated subunit molecular weight was 69 ± 0.2 kDa.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4675546/v1/192ea2962cef77117b2efb2e.png"},{"id":61295171,"identity":"8b22a1e2-dedb-430e-b59a-0dd47ce8b11f","added_by":"auto","created_at":"2024-07-29 08:01:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":93030,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Metals on the Activity of Purified PPO from \u003cem\u003eC. longa\u003c/em\u003e. The relative activity of the PPO was plotted against different concentration of both monovalent and divalent metals. One hundred percent (100%) activity represents the activity of PPO in the absence of metal ions.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4675546/v1/dc57104fac12c470c58cd251.png"},{"id":61294247,"identity":"29663363-ad23-4c0d-9272-107a55c2b720","added_by":"auto","created_at":"2024-07-29 07:53:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":37874,"visible":true,"origin":"","legend":"\u003cp\u003eDixon Plot for estimation of Inhibition Constants (\u003cem\u003eK\u003c/em\u003ei). The Estimated \u003cem\u003eK\u003c/em\u003ei for (i) Citric acid (ii) Cysteine and (iii) EDTA were 5.5mM, 2.0mM and 8.1mM respectively.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4675546/v1/008be41107fbaf446d4c21bf.png"},{"id":61294245,"identity":"7c452ead-3ad8-4f01-819e-b5f486f15ce5","added_by":"auto","created_at":"2024-07-29 07:53:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":43148,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical Representation of the Molecular Docking Score and Prime/MM-GBSA binding energy (Δ\u003cem\u003eG\u003c/em\u003e\u003csup\u003ebind\u003c/sup\u003e) of Inhibitors viz 311-citric acid; 54670067-Ascorbic acid; 124886-Glutathione; 6049-EDTA; 3840-Kojic acid; 5862-Cysteine; 1567-β-mercaptoethanol\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4675546/v1/574d44751b20afa41cf61de8.png"},{"id":61294243,"identity":"1758b302-1096-4886-89df-915afa86ee72","added_by":"auto","created_at":"2024-07-29 07:53:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":163257,"visible":true,"origin":"","legend":"\u003cp\u003e2D Molecular Interaction of Inhibitors with Binding Pocket of \u003cem\u003eC. longa\u003c/em\u003e PPO viz 311-citric acid; 54670067-Ascorbic acid; 124886-Glutathione; 6049-EDTA; 3840-Kojic acid; 5862-Cysteine\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4675546/v1/821b7e88d3a5280f53087653.png"},{"id":82611014,"identity":"83db5d01-e900-4322-a0be-04c46bc94d84","added_by":"auto","created_at":"2025-05-13 10:47:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1197402,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4675546/v1/17718c05-79ba-480b-bfc6-b0215646b751.pdf"},{"id":61295170,"identity":"05399e73-5a89-4b5e-a262-e69337db5c38","added_by":"auto","created_at":"2024-07-29 08:01:02","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":37740,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-4675546/v1/70d2fa8c28edbad5786e7688.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Kinetics and molecular docking of purified polyphenol oxidase from rhizome of turmeric (Curcuma longa L.)","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePolyphenol Oxidases (PPO) (E.C.1.10.3.1), also termed catechol oxidases, catecholases, diphenol oxidases, ortho-diphenolases, phenolases, and tyrosinases [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. They are a group of copper-containing enzymes that catalyze the \u003cem\u003eo\u003c/em\u003e-hydroxylation of monophenols to \u003cem\u003eo\u003c/em\u003e-diphenols as well as the oxidation of \u003cem\u003eo\u003c/em\u003e-diphenols to quinones in the presence of oxygen [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Polyphenol oxidase is broadly distributed among animals, fungi, and plants, although the studies are more extensive in plants [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The study of PPOs in plants has focused primarily on their role in the process of postharvest browning, whereby cut or damaged plant tissues turn brown due to the polymerization of PPO-generated quinones, generating phyto-melanins [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The enzyme can catalyze the oxidation of polyphenols and result in the browning of damaged or cut plant, which seriously affects quality and reduce the market value of the crop [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Polyphenol oxidase is widely present in plants, play an important role in the growth, development, and stress responses. Many studies have reported that PPO and peroxidases are induced in response to biotic and abiotic stress in plants, and it has been implicated in several functional processes such as participating in plant defense and the synthesis of plant-specific metabolites [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePlant PPO generally contain three conserved regions, N-terminal cTP, aCuA and CuB domain and a C-terminus [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], which are responsible for thylakoid lumen localization and enzyme activity. Polyphenol oxidase is found in many plant species such as banana [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], apple [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], potato [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], eggplant [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], strawberry [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], red cocoyam [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], bitter leaf [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] etc. The function and distribution of PPO differ in various plants [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Most PPO are transported to the thylakoid membrane in the chloroplast, or in cytosol and other organelles [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], whereas the phenolic compounds are localized to the vacuoles. Because of the different localization of the enzyme and its substrates, their interaction requires destruction of the cell and mechanical damage [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Plant PPOs have reported to possess various applications such as synthesis of drugs and other organic compounds [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTurmeric (\u003cem\u003eCurcuma longa\u003c/em\u003e) is a perennial rhizomatous crop of the Zingiberaceae, a world-wide known spice whose medicinal properties has received interest from both the medical and scientific world as well as culinary enthusiasts, as it is the major source of the polyphenol curcumin [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Turmeric contains 3 to 6% polyphenolic compounds which is known as curcuminoids [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. It aids in the management of oxidative and inflammatory conditions, metabolic syndrome, arthritis, anxiety, and hyperlipidemia. Most of these benefits can be attributed to its antioxidant and anti-inflammatory effects [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Like several plants, it suffers a reduction in its sensory qualities and health benefits as a result of spoilage which occurs due to browning. Recent research of novel anti-PPO systems is focused on mild alternatives to conventional treatments which could impair not only the sensory and nutritional properties of agro-food products but also the consumer health [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Milder processes of controlling enzymatic browning can be discovered as PPO studies are being carried out and this will help improve the shelf-life of turmeric and its products. Many researches have been conducted using turmeric rhizomes in many forms of investigation in the area of its characteristics, functionality, and applications [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Specifically, several studies have reported that turmeric possesses potent multiple properties such as anti-inflammatory, antioxidant, antitumor, antibacterial and anticoagulant, and antidiabetic based on its free-radical-scavenging activity expressed by the domicile bioactive compound. Among the bioactive components of turmeric, curcumin is the most frequently studied, it is fat soluble bioactive compound, whose characteristics and functionality is equable to its popularly reported medicinal, pharmacological (hypoglycemic, insulinotropic, and hypolipidemic) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The potency of turmeric anti-oxidant and anti-inflammatory properties improves symptoms of depression, arthritis and Alzheimer\u0026rsquo;s disease. It contains natural antioxidants, polyphenols and phytochemicals which confer significant protection against free radicals\u0026rsquo; damage [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDifferent sources of polyphenol oxidase have been reported including bacteria, fungi, animal and plants [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The PPO found in humans is responsible for skin pigmentation including development of freckles [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Plant PPO has an important role in plant stress resistance and physiological metabolism. Most of the PPO in plants are found in the chloroplasts of photosynthetic cells and the leucoplasts of storage cells [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Polyphenol oxidase is one of the most important industrial enzymes, considering its wide applications in several industrial and biotechnological application. There is need for continual searching for cheaper and readily available sources of the enzyme. Sufficient information on the relative occurrence and some physio-chemical properties of polyphenol oxidase from turmeric had been earlier reported [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, in that work, the much-needed information on kinetics, inhibition studies and molecular docking of some important inhibitors and/or anti-browning agents of the purified turmeric PPO was lacking and greatly necessary. This was with a view to providing information on its mechanism of catalytic reactions, specificity towards substrates and binding interactions with inhibitors and anti-browning agents through molecular docking. Hence, the reason for this study. These could be deployed in several biotechnological applications and primarily for understanding the strategy in the control of browning in turmeric food products.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eThe fresh turmeric (\u003cem\u003eCurcuma longa\u003c/em\u003e) was obtained from farms in Owo environs, Southwestern Nigeria. The plant was authenticated at the Department of Plant Science and Biotechnology, Achiever University, Owo, Ondo State, Nigeria.\u003c/p\u003e \u003cp\u003e3,4-dihydroxyphenyl-L-alanine (L-DOPA), catechol, L-tyrosine, pyrogallol, blue dextran, acetic acid, citric acid, sodium citrate, bovine serum albumin (BSA), sodium phosphate dibasic (Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e), anhydrous sodium phosphate monobasic (NaHPO\u003csub\u003e4\u003c/sub\u003e), Coomassie brilliant blue R-250, glutathione was obtained from Sigma Chemical Company, St Louis, USA. Molecular weight standard for sodium dodecyl sulphate- polyacrylamide gel electrophoresis (SDS-PAGE) was obtained from Carl Roth GmbH, Karlsruhe, Germany. All other reagents were of analytical grade.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Methods\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Extraction of PPO from turmeric (Curcuma longa)\u003c/h2\u003e \u003cp\u003eThe fresh turmeric rhizome (\u003cem\u003eCurcuma longa\u003c/em\u003e) rhizome was cut and peeled after which it was rinsed in distilled water and homogenized in 50mM potassium phosphate buffer, pH 6.5 on ice to obtain 30% homogenate [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The homogenate was centrifuged at 10,000\u0026times;\u003cem\u003eg\u003c/em\u003e for 30 min at 4 ˚C using cold centrifuge in order to obtain clear crude supernatants. The supernatants were assayed for PPO activity using catechol as substrate in a spectrophotometer at 410nm. The collected supernatant was stored in a freezer (at -20 ˚C).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 Standard assay for polyphenol oxidase\u003c/h2\u003e \u003cp\u003ePolyphenol oxidase activity was determined using L-Tyrosine, L-DOPA, catechol and pyrogallol as substrates in the crude supernatants and during purification according to the method of Wititsuwannakul \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] as modified by Ilesanmi \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The reaction involved final concentration of 5 mM of the substrates, 50 mM phosphate buffer, pH 6.5 and appropriate volume of enzyme. Initial rate of product formation will be monitored spectrophotometrically.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3. Protein concentration determination\u003c/h2\u003e \u003cp\u003eThe protein concentrations in the crude homogenates and purified PPO were determined as described by Bradford [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] using BSA as the standard protein.\u003c/p\u003e \u003cp\u003eThe mixture of Bradford working reagent, and the sample is run through the spectrometer at 595 nm, and then the absorbances is recorded for the standard and sample at 595 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.2.4. Purification of the crude polyphenol oxidase from turmeric\u003c/h2\u003e \u003cp\u003eCrude PPO from the rhizome of turmeric was purified as reported by Ilesanmi \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The purification involved combination of aqueous two-phase partitioning and gel filtration chromatography.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.2.5. Homogeneity test and determination of subunit molecular weight by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)\u003c/h2\u003e \u003cp\u003eProtein purity and the subunit molecular weight of the purified polyphenol oxidase were determined by SDS polyacrylamide gel electrophoresis using 12% (w/v) polyacrylamide (running gel) and 2.5% (w/v) stacking gel, according to the method of Laemmli [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] as modified by Weber and Osborn [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] using Tris-glycine buffer system at pH 8.3. the protein bands were obtained following staining and destaining procedures.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.2.6. Characterization of the purified PPO from turmeric\u003c/h2\u003e \u003cdiv id=\"Sec11\" class=\"Section4\"\u003e \u003ch2\u003e2.2.6.1. Determination of kinetic parameters\u003c/h2\u003e \u003cp\u003eThe effect of various concentrations of pyrogallol on the activity of purified PPO from \u003cem\u003eCurcuma longa\u003c/em\u003e was determined. The kinetic parameters (\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e, V\u003csub\u003emax\u003c/sub\u003e, \u003cem\u003ek\u003c/em\u003ecat and \u003cem\u003ek\u003c/em\u003ecat/\u003cem\u003eK\u003c/em\u003em) for pyrogallol were analyzed and estimated using non-regression software graph pad prism 8.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section4\"\u003e \u003ch2\u003e2.2.6.2. Effect of metals on the activity of purified PPO from turmeric\u003c/h2\u003e \u003cp\u003eThis was done following the method used by Arabaci [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The effects of monovalent, divalent and trivalent metals like Na\u003csup\u003e+\u003c/sup\u003e (NaCl), K\u003csup\u003e+\u003c/sup\u003e (KCl), Cu\u003csup\u003e2+\u003c/sup\u003e (CuSO\u003csub\u003e4\u003c/sub\u003e), Zn\u003csup\u003e2+\u003c/sup\u003e (ZnSO\u003csub\u003e4\u003c/sub\u003e), Ba \u003csup\u003e2+\u003c/sup\u003e (BaCl\u003csub\u003e2\u003c/sub\u003e\u0026bull;H\u003csub\u003e2\u003c/sub\u003eO), and NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e (NH\u003csub\u003e4\u003c/sub\u003eCl), were determined at different concentrations of between 10 to 50 mM).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section4\"\u003e \u003ch2\u003e2.2.6.3. Effect of inhibitors and anti-browning agents on the purified PPO from turmeric\u003c/h2\u003e \u003cp\u003eThe effects of anti-browning agents viz. L-cysteine, kojic acid, mercaptoethanol, glutathione (GSH), ethylenediamine tetra acetic acid (EDTA), L-ascorbic acid, and citric acid on the activity of the enzyme was determined as described by Liu [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. This was carried out in varying concentrations of 1 to 10mM.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section4\"\u003e \u003ch2\u003e2.2.6.4. Inhibition constant (Ki) of some inhibitors\u003c/h2\u003e \u003cp\u003eThe Inhibition constant (\u003cem\u003eK\u003c/em\u003ei) for Ascorbic Acid, L-cysteine, kojic acid, mercaptoethanol, glutathione (GSH), ethylenediamine tetra acetic acid (EDTA), and Citric acid were estimated using Dixon plot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section4\"\u003e \u003ch2\u003e2.2.6.5. Molecular docking studies for binding interaction of inhibitors\u003c/h2\u003e \u003cp\u003eThe X-ray crystallographic structure of polyphenol oxidase was retrieved from the RCSB protein database pdb id 4Z10. The protein preparation wizard of the Glide Schr\u0026ouml;dinger Suite 2017\u0026ndash;1 was used to rectify specific errors in the protein during crystallographic structure and optimization was done. The structure-data file (SDF) structures of the seven (7) ligands were downloaded from the PubChem database, imported on the workspace of Maestro Schr\u0026ouml;dinger suite Interface. The molecular docking of the seven ligands was performed using extra precision (XP) and the co-crystallized ligand was re-docked into the catalytic site of 4Z10 to confirm the accuracy of the screening and docking scores.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cem\u003e3.1. Extraction and purification \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe crude enzyme was homogenized and extracted in 50mM Phosphate Buffer to obtain 30% homogenate and after it was subjected to aqueous two-phase partitioning system (ATPS). The data in Table 1 shows the purification summary of purified \u003cem\u003eC. longa\u003c/em\u003e PPO. The percentage yield obtained was 41% with a purification fold of 4.4.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.2. \u003c/em\u003e\u003cem\u003ePurity test and subunit molecular weight determination \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe purity test of the purified enzyme was determined on SDS polyacrylamide gel electrophoresis using 12% polyacrylamide (running gel) and 2.5% stacking gel using Tris-glycine buffer system at pH 8.3 which gave a single band protein estimated to be 69 ± 0.2 kDa. This is shown in the electrophoretogram (Figure 1a). Estimation of the subunit molecular weight of the purified PPO was carried by plotting the molecular weights of the standard protein against their relative mobility.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.3. Kinetic parameters of purified PPO from turmeric\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe kinetic parameter of purified \u003cem\u003eC. longa\u003c/em\u003e PPO in aqueous system was determined using non-linear regression plot of activity against concentration. This is shown in Table 2. The \u003cem\u003eK\u003c/em\u003em and Vmax of C. longa PPO for pyrogallol were 5.8 ± 0.6 mM and 722.9 ± 17.0 units/mg protein respectively. The \u003cem\u003ek\u003c/em\u003ecat and \u003cem\u003ek\u003c/em\u003ecat/\u003cem\u003eK\u003c/em\u003em were 831.6 ± 5.0 s \u003csup\u003e-1\u003c/sup\u003e and 1.43x10\u003csup\u003e5\u003c/sup\u003e s \u003csup\u003e-1\u003c/sup\u003eM\u003csup\u003e-1\u003c/sup\u003e respectively.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.4. Effect of metals on the activity of purified PPO from turmeric\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe activity of PPO in the presence of varying concentrations of metals was determined. As shown in Figure 2, PPO was activated at the lowest concentration in KCl and CuSO\u003csub\u003e4\u003c/sub\u003e following a gradual decline while the enzyme remained fairly stable in the presence of NaCl, ZnSO\u003csub\u003e4\u003c/sub\u003e and NH\u003csub\u003e4\u003c/sub\u003eCl.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.5. Effects of inhibitors on PPO from turmeric\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe percentage residual activity of \u003cem\u003eC. longa \u003c/em\u003ePPO in the presence of inhibitors from low to high concentrations were determined as shown in Figure 3. Cysteine was seen to show a good decline in activity compared to other inhibitors used on the enzyme. Furthermore, Dixon plot was used to determine the inhibition constant (\u003cem\u003eK\u003c/em\u003e\u003csub\u003ei\u003c/sub\u003e) by plotting the inverse relative enzyme activity against the concentration of the inhibitor. This revealed Cysteine as the most potent inhibitor with the lowest \u003cem\u003eK\u003c/em\u003e\u003csub\u003ei \u003c/sub\u003eof 2.0 mM. The inhibition constant (\u003cem\u003eK\u003c/em\u003e\u003csub\u003ei\u003c/sub\u003e) summary for all the inhibitors is shown in Table 3.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.7. Binding interaction of purified PPO from turmeric with some selected inhibitors\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe X-ray crystallographic structure of polyphenol oxidase was retrieved from the protein databank with pdb id 4Z10 and the ligands were re-docked into the catalytic site of 4Z10. The binding energy was determined which was used to calculate the free binding energy. The docking score was also estimated. Figure 4 and 5 show a representation of the molecular docking score and binding energy for the ligands (inhibitors) while Table 4 gives a summary of the interaction of the docked inhibitors with \u003cem\u003eC. longa\u003c/em\u003e PPO.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003ePolyphenol oxidase is one of the most important industrial enzymes with enormous applications in several technical and biotechnological processes [3,16]. The presence of polyphenol oxidase in the rhizome of turmeric (\u003cem\u003eCurcuma longa\u003c/em\u003e) has been established in this study. The kinetics and interactions of the purified enzyme towards substrates, metals, inhibitors and/or anti-browning agents have also been documented. The extracted crude PPO from the turmeric was subjected to non-conventional purification-aqueous two-phase partitioning (ATPS) which resulted in a purified PPO [19]. The major advantages of the procedure used is that the method helps purify and also concentrate the enzyme in addition to requiring less purification time which makes it faster compared to other purification processes [37]. The purification scheme proved efficient enough as adjudged on the SDS-PAGE. The molecular weight of the purified PPO was confirmed by SDS-PAGE. The result on the slab gel showed a monomeric protein band with an estimated molecular weight of 69 ± 0.2 kDa. This confirmed the purity of the \u003cem\u003eC. longa\u003c/em\u003e PPO and the effectiveness of the purification procedure. This aligns with similar findings of PPO obtained from \u003cem\u003eSolanum lycocarpum\u003c/em\u003e of 68 kDa [38]\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe kinetics of enzymes provide information on the mechanism of catalytic reactions and specificity of the enzyme towards substrates. The kinetic parameter of the purified turmeric PPO in aqueous solution was determined using non-linear regression plot of activity against the substrate concentration. The \u003cem\u003eK\u003c/em\u003em of \u003cem\u003eC. longa\u003c/em\u003e PPO was 5.8 ± 0.6mM and Vmax of 722.9 ± 17.0 units/mg protein respectively, while the catalytic efficiency and specificity constant of the purified enzymes \u003cem\u003ek\u003c/em\u003ecat/\u003cem\u003eK\u003c/em\u003emwas 1.43x10\u003csup\u003e5\u0026nbsp;\u003c/sup\u003es\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eM\u003csup\u003e-1\u003c/sup\u003e. Lower \u003cem\u003eK\u003c/em\u003em suggests higher affinity towards the substrate [39]. The kinetics revealed useful information about the catalytic mechanism, and mechanisms of inhibition of the purified turmeric PPO which would be exploited in several biotechnological processes.\u003c/p\u003e\n\u003cp\u003eMetal ions play important roles in maintaining substrate binding in the active site of metalloenzymes and in controlling the redox activity of metalloenzymes in enzymatic reaction and metal ions can reversibly change its valence to regulate the enzymatic reaction [40]. In this work, NaCl, ZnSO\u003csub\u003e4\u003c/sub\u003e and NH\u003csub\u003e4\u003c/sub\u003eCl showed a stable activity with average of 90 to 95% activity retained at concentrations between 10 to 50mM. The activity of PPO was observed to have been activated at concentration of 10mM which was followed by a gradual decline activity for KCl and CuSO\u003csub\u003e4\u0026nbsp;\u003c/sub\u003eup to 40mM and another increased in at 50mM concentration. \u003cem\u003eC. longa\u003c/em\u003e PPO activity with the introduction of BaCl\u003csub\u003e2\u0026nbsp;\u003c/sub\u003ewas observed to increase at 30mM and maintain 100% activity at 50mM concentration. The stability of PPO in the presence of Na\u003csup\u003e+\u003c/sup\u003e is similar to the results of Guo \u003cem\u003eet al.\u003c/em\u003e [41] and Liu \u003cem\u003eet al\u003c/em\u003e. [42].\u003c/p\u003e\n\u003cp\u003eThe inhibition of enzymatic browning has been the subject of continuing concern among researchers and the food industry. Several compounds have been investigated for their effectiveness in preventing enzymatic browning in crude extracts of fruit and vegetables [43]. The residual activity of \u003cem\u003eC. longa\u003c/em\u003e PPO plotted against different concentration of Citric acid, cysteine and EDTA. At the highest concentration in the presence of Citric acid, cysteine and EDTA, \u003cem\u003eC. longa\u003c/em\u003e PPO had a residual activity of 41, 15 and 52% respectively. The inhibition constant \u003cem\u003eK\u003c/em\u003e\u003csub\u003ei\u003c/sub\u003e was further used to ascertain the inhibitory prospect of the inhibitors using Dixon plot. Cysteine was the most potent inhibitor of \u003cem\u003eC. longa\u003c/em\u003e PPO. This can be due to the presence of sulphur as sulphur containing compounds have been seen to have higher inhibitory effect on PPO in previous studies. Cysteine reacts with \u003cem\u003eo\u003c/em\u003e-quinone and forms a stable colorless product which interferes with browning activities. The choice of cysteine as the most potent inhibitor is also supported by Liu \u003cem\u003eet al.\u003c/em\u003e [41] who reported the inhibitory activity even at a concentration as low as 0.1mM. The inhibitory activity of EDTA is reported to not be obvious at low concentration but would increase as concentration increases and it works as a copper-binding ligand [44,45]. This information on the low values of \u003cem\u003eK\u003c/em\u003e\u003csub\u003ei\u0026nbsp;\u003c/sub\u003efor the cysteine of the enzyme may be deployed in food manufacturing industries to salvage increasing reports of food spoilage and more importantly to improve the qualities of turmeric\u003c/p\u003e\n\u003cp\u003eThe binding interaction showed that EDTA, Kojic acid and Cysteine have 2 hydrogen bonds formed while citric acid, ascorbic acid and glutathione had 4, 5 and 3 respectively. \u003cem\u003eB\u003c/em\u003e-mercaptoethanol showed no hydrogen bonding interaction with the residues [46]. This infers these inhibitors inhibits the activity of PPO by preventing the substrate from binding with the key amino acid residues with which the hydrogen bonding interacts in the active site in the competitive process for its stronger affinity with the enzyme, which leads to its resultant of inhibition kinetics.\u0026nbsp;β-mercaptoethanol and cysteine were observed to have the lowest docking score of -2.3060 and -2.6710. Both inhibitors also exhibited the higher binding free energy which further affirms the potency of cysteine. The inhibitory activity of Cysteine can also be due to the presence of sulphur which forms a stable complex making it a strong inhibitor of \u003cem\u003eC. longa\u003c/em\u003e PPO.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe presence of PPO from turmeric (\u003cem\u003eCurcuma longa\u003c/em\u003e) rhizome has been established and a shortened purification method (ATPS) was highly effective in removing unwanted proteins and contaminants from the desired enzyme. The kinetics and inhibition studies on the purified turmeric polyphenol oxidase could be deployed in several biotechnological applications and/or countering food spoilage considering how versatile and important turmeric is, in pharmaceutical and nutraceutical applications.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthorship Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVictory Ayo Olagunju - Performed the research, analyze data and wrote the paper\u003c/p\u003e\n\u003cp\u003eOlutosin Samuel Ilesanmi – Conceived the research, analyze data and reviewed the paper\u003c/p\u003e\n\u003cp\u003eOmowumi Funke Adedugbe – analyze data and proof-read the manuscript\u003c/p\u003e\n\u003cp\u003eAdedeji Benedict Kayode – analyze data and proof-read the manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eMayer, A.M. (2006). Polyphenol oxidases in plants and fungi: Going places? A review. Phytochemistry 67, 2318\u0026ndash;2331.\u003c/li\u003e\n \u003cli\u003eQin, F., Hu, C., Dou, T., Sheng, O., Yang, Q., Deng, G., He, W., Gao, H., Li, C., Dong, T., Yi, G. and Bi, F. (2023). Genome-wide analysis of the polyphenol oxidase gene family reveals that MaPPO1 and MaPPO6 are the main contributors to fruit browning in \u003cem\u003eMusa acuminata.\u0026nbsp;\u003c/em\u003e\u003cem\u003eFront. Plant Sci.\u003c/em\u003e 14:1125375. doi: 10.3389/fpls.2023.1125375.\u003c/li\u003e\n \u003cli\u003eMoon, K. M., Kwon, E. B., Lee, B., and Kim, C. Y. (2020). Recent trends in controlling the enzymatic browning of fruit and vegetable products. \u003cem\u003eMolecules\u003c/em\u003e, \u003cem\u003e25\u003c/em\u003e(12), 2754.\u003c/li\u003e\n \u003cli\u003eIlesanmi, O. S., Adedugbe, O. F. and Oyegoke, D. A. (2022). 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Molecular modeling of peroxidase and polyphenol oxidase: substrate specificity and active site comparison. \u003cem\u003eInternational Journal of Molecular Sciences\u003c/em\u003e, \u003cem\u003e11\u003c/em\u003e(9), 3266\u0026ndash;3276.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 4 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Polyphenol oxidase, Turmeric, Kinetics, Molecular Docking, Biotechnological applications","lastPublishedDoi":"10.21203/rs.3.rs-4675546/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4675546/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePolyphenol oxidase catalyzes oxidative conversion of polyphenols to their respective quinones. These have been exploited in various biotechnological processes. The kinetics and molecular docking interaction of turmeric PPO on some inhibitors are here described. The enzyme was purified using aqueous two-phase partitioning. The subunit and the native molecular masses of the purified turmeric\u003cem\u003e \u003c/em\u003ePPO were 69 ± 2.0 kDa and 66.8 ± 3.5 kDa respectively, suggesting its monomeric nature. The \u003cem\u003eK\u003c/em\u003em and Vmax of the \u003cem\u003eC. longa\u003c/em\u003e PPO for pyrogallol were 5.8 ± 0.6 mM and 722.9 ± 17.0 units/mg protein respectively leading to turnover number (\u003cem\u003ek\u003c/em\u003ecat) and first order rate constant (\u003cem\u003ek\u003c/em\u003ecat/\u003cem\u003eK\u003c/em\u003em) of 831.6 ± 5.0 s\u003csup\u003e-1 \u003c/sup\u003eand 1.43 × 10\u003csup\u003e5\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e M\u003csup\u003e-1 \u003c/sup\u003erespectively. The purified enzyme was activated at the lowest concentration in KCl and CuSO\u003csub\u003e4,\u003c/sub\u003e whereas was fairly stable in the presence of NaCl, ZnSO\u003csub\u003e4\u003c/sub\u003e and NH\u003csub\u003e4\u003c/sub\u003eCl. The inhibition constant (\u003cem\u003eK\u003c/em\u003ei) obtained from Dixon plot for ascorbic acid, β-mercaptoethanol, citric acid, cysteine, EDTA, glutathione and kojic acid were 7.8, 1.7, 5.5, 2.0, 8.1, 3.3 and 6.4 mM respectively. In-depth analyses, revealed that cysteine was the most potent of all the inhibitors investigated. The binding interaction of the purified enzyme with inhibitors revealed that EDTA, Kojic acid and Cysteine have 2 hydrogen bonds formed while citric acid, ascorbic acid and glutathione had 4, 5 and 3 respectively. \u0026nbsp;In conclusion, the kinetics and inhibition studies of the purified turmeric PPO could be deployed in the control of browning and several industrial and biotechnological applications.\u003c/p\u003e","manuscriptTitle":"Kinetics and molecular docking of purified polyphenol oxidase from rhizome of turmeric (Curcuma longa L.)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-29 07:52:58","doi":"10.21203/rs.3.rs-4675546/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8d7c4c1b-d728-4295-bed3-a2a775715cac","owner":[],"postedDate":"July 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":34975813,"name":"Biological sciences/Biochemistry"},{"id":34975814,"name":"Biological sciences/Biotechnology"},{"id":34975815,"name":"Biological sciences/Plant sciences"},{"id":34975816,"name":"Earth and environmental sciences/Environmental sciences"}],"tags":[],"updatedAt":"2025-05-13T10:38:58+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-29 07:52:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4675546","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4675546","identity":"rs-4675546","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}