Enzymatic oxidation of oleuropein and 3-hydroxytyrosol by laccase , peroxidase , and tyrosinase

Northumbria University has developed Northumbria Research Link (NRL) to enable users to access the University’s research output. Copyright © and moral rights for items on NRL are retained by the individual author(s) and/or other copyright owners. Single copies of full items can be reproduced, displayed or performed, and given to third parties in any format or medium for personal research or study, educational, or not-for-profit purposes without prior permission or charge, provided the authors, title and full bibliographic details are given, as well as a hyperlink and/or URL to the original metadata page. The content must not be changed in any way. Full items must not be sold commercially in any format or medium without formal permission of the copyright holder. The full policy is available online: http://nrl.northumbria.ac.uk/policies.html

The ability of these enzymes to attack phenols, diphenols, and so forth can be used for industrial applications such as wastewater treatment (Bucur et al., 2018;Durán et al., 2002;Janusz et al., 2020;Li et al., 2020).Its action also causes the polymerization of different compounds in a typical fruit of Mediterranean countries such as the olive.This fruit (Xie et al., 2021) has a bitter taste due to a substance called oleuropein (OL) (Ramírez et al., 2014); this compound has in its chemical structure 3-hydroxytyrosol (HT); in the olive processing, OL is transformed into a product that does not have a bitter taste (Ramírez et al., 2014).
On the other hand, one of the biggest environmental problems in the food industry comes from olive oil millwastewater (OMW) with a quantity of approximately 30 million tons per year worldwide (Xie et al., 2021).In addition, olive pomace, the solid residue obtained from the olive oil production (Xie et al., 2020), is rich in HT (Xie et al., 2021) and OL and the action of these oxidases can give rise to products in their reactions that can lead to polymerization, and this polymerization can have beneficial effects because it can increase the antioxidant capacity and therefore increase the health benefits with potential application on new functional foods or as feed ingredients (Xie et al., 2020).
When free radicals or o-quinones evolve towards polymerization (more than ten units), new advantages are obtained in these polymers (Xie et al., 2020).Furthermore, this polymerization is done under mild conditions.The polymerization process has been kinetically characterized, and changes and transformations in the structures of the enzymes involved have also been determined (Xie et al., 2021).
Among the compounds present in the olive oil extraction residues, HT and OL are abundant, they have a high capacity as antioxidants, but in the polymer this power becomes greater (Hachicha Hbaieb et al., 2015;Tikhonov et al., 2019).Yield can also be improved by treating oil mill wastewater with fungi such as Aspergillus niger, thus achieving a greater release of HT (Hamza et al., 2012), although the fruit ripening process is produced through the action of enzymes such as β-glucosidase, polyphenol oxidase, and peroxidase, increasing the release of OL and a series of enzymes that cause its transformation.
The kinetic characterization of the action of tyrosinase on HT has been carried out with the mushroom enzyme, using MBTH as a coupled reagent, obtaining a value of K M = 0.9 ± 0.07 mM (Espin-De Gea et al., 2002).The K M of grape tyrosinase has also been determined with a value of K M = 21.6 mM (García-García et al., 2013).
In this work, the action of these three enzymes: laccase, peroxidase, and tyrosinase on OL and HT substrates will be kinetically studied.This study will allow the kinetic characterization and thus obtain information regarding their mechanisms of action.In addition, the docking studies of these molecules in relation to the different enzymes will allow us to understand these catalytic processes.

| Periodate oxidation of oleuropein and 3-hydroxytyrosol
Periodate oxidation was performed to show the stability and λ max of quinones derivated from OL and HT and calculate its molar absorptivities in the different pH conditions under study.In this sense, the oxidation of target substrates by deficiency of sodium periodate (NaIO 4 ) (NaIO 4 <<< Substrate) (Muñoz et al., 2006)

| Spectrophotometricmethod
The products of the reaction of these three enzymes (laccase, peroxidase, and tyrosinase) when acting on OL and HT are semiquinones that evolve towards o-quinones, in the case of the first two (Manzano-Nicolas, Marin-Iniesta, et al., 2020;Manzano-Nicolas, Taboada-Rodriguez, et al., 2020;Rodríguez-López, Gilabert, et al., 2000) and directly o-quinones in the case of tyrosinase (Espín et al., 2001;Muñoz et al., 2006;Rodríguez-López, Fenoll, et al., 2000).It is known that o-quinones are unstable, especially at high pH values.Thus, oquinones absorb in the visible region of the spectrum and make it possible to measure the activity of these enzymes; however, their instability means that only laccase can be measured at pH = 4.0, at its optimal pH (Manzano-Nicolas, Marin-Iniesta, et al., 2020;Manzano-Nicolas, Taboada-Rodriguez, et al., 2020).The other two enzymes peroxidase (Rodríguez-López, Gilabert, et al., 2000) and tyrosinase (Rodríguez-López, Fenoll, et al., 2000), which have an optimal pH of 5.5 and 6.8, respectively, cannot be correctly measured, as shown below, in which case a spectrophotometric chronometric method is proposed, as it is described below.

| Chronometricspectrophotometricmethod
The enzymatic activity of peroxidase and tyrosinase on OL and HT was followed spectrophotometrically in the visible zone, measuring the formation of the corresponding products after the consumption of a determined amount of AH 2 (micromolar) by the reaction with the different quinones and semiquinones generated by the enzymes.Since in all cases the product absorbs in the visible area, the classic chronometric method is used (Muñoz et al., 2006), since the AH 2 spectrum does not influence the measurement (Manzano-Nicolas, Marin-Iniesta, et al., 2020;Manzano-Nicolas, Taboada-Rodriguez, et al., 2020;Rodríguez-López, Fenoll, et al., 2000;Rodríguez-López, Gilabert, et al., 2000).Except where otherwise indicated, the experimental conditions were as follows: pH 5.5 and 50-mM acetate buffer for peroxidase, while pH 6.8 and 50-mM phosphate buffer for tyrosinase; temperature was maintained at 25°C.Substrate and ascorbic acid concentrations are showed in Section 3.

| Antioxidantcapacityassays
Antioxidant capacity was obtained through the performing of enzymatic kinetic method (Munoz-Munoz et al., 2010).Experimental conditions of cuvette prepared for measuring antioxidant activity were 50-mM acetate buffer, pH = 5.5, ABTS 5 mM, H 2 O 2 100 µM, 0.66-nM peroxidase and 0-to 51-µM OL, and 0-to 56-µM HT.This method consists in the enzymatic production of ABTS radical with its subsequent increase of absorbance.In this way, the addition of different quantities of antioxidant ([A] 0 ) under study produces different lag periods without increase in their absorbance due to the ABTS radical consumption (similarly to previous chronometric method to analyse enzymatic activity).After that, the number of electrons (n) was obtained by calculating for linear regression of V 0 τ respect to [A] 0 .Finally, effective concentration (EC50 = 1/2n) and antioxidant capacity (ARP = 1/EC50) were obtained.

| Computational docking
The chemical structures information for all ligands are available in the PubChem Substance and Compound database (Kim et al., 2016) through the unique chemical structure identifier CID 82755 for 3-hydroxytyrosol and 5281544 for oleuropein.The molecular structure of the enzymes was obtained from the Protein Databank: laccase from the Fungus Trametes versicolor (PDB ID:1GYC) (Piontek et al., 2002), peroxidase from horseradish (Armoracia rusticana, PDB ID:1HCH) (Berglund et al., 2002), and the deoxy-form of tyrosinase from Agaricus bisporus (PDB ID:2Y9W, Chain A) (Ismaya et al., 2011).
Input protein structures were prepared by adding hydrogen atoms and removing nonfunctional water molecules.Rotatable bonds in the ligands and Gasteiger's partial charges were assigned by AutoDockTools4 software (Morris et al., 2009;Sanner, 1999).The met and oxy forms of tyrosinase were built by a slight modification of the binuclear copper-binding site as previously described (Maria-Solano et al., 2016).AutoDock 4.2.6 (Morris et al., 2009) package was employed for docking.Lamarkian Genetic Algorithm was chosen to search for the best conformers.The maximum number of energy evaluations was set to 2,500,000, the number of independent docking to 200 and the population size to 150.Grid parameter files were built using AutoGrid 4.2.6 (Huey et al., 2007).The grid box was centered close to T1 copper for laccase, the copper ions for tyrosinase, and the Fe atom of the heme group.Other AutoDock parameters were used with default values.PyMOL 2.3.0 (Schrödinger, n.d.) and AutoDockTools4 (Morris et al., 2009) were employed to edit and inspect the docked conformations.LigPLot software was used for two-dimensional representations (Wallace et al., 1995).Docking conformations were selected according to the minimum free energy criteria after a cluster analysis in the binding region.

| Kinetic analysis
To quantitatively measure the rate of action of peroxidase and tyrosinase, it is necessary to obtain an analytical expression for the rate as a function of the parameters obtained experimentally.
Similarly, it is necessary, to obtain the antioxidant capacity of a compound by the enzymatic method, to describe an analytical expression that defines it.
In concentration, the quantity of radical formed over time is as follows: In presence of AH 2, it happens that: In the time, t = , results: Therefore For a given concentration of AH 2 , a lag period is obtained τ, which allows the calculation of V 0 , which will be designated as V Lag .
Therefore, the stoichiometry is 1 mole of O 2 /2 moles of D/2 moles of AH 2 .
The quantity of quinone accumulated over time is: In presence of AH 2 , the following correlation is achieved: In the time, t = results: Therefore,

| Antioxidant capacity determination
Olive oil is rich in antioxidants, such as HT, OL, and oleacin (Czerwińska et al., 2012).It has been shown that tyrosol (T), "the largest constituent in olive oil", restores antioxidant defences despite its weak efficiency as antioxidant (Di Benedetto et al., 2007), probably because of its intracellular accumulation.It has been studied the addition of extra-virgin olive oil on animals diets, reducing the lipid peroxidation by increasing antioxidant defence system (1) (2) ( Tufarelli et al., 2016).In general, extra-virgin olive oil enriched diet increase the antioxidant status (Oliveras-López et al., 2013, 2014).
In olive browning reactions, the enzymes tyrosinase and peroxidase intervene, acting mainly on HT (Segovia-Bravo et al., 2009).
Given the importance of HT, different methods of obtaining it have been published and even patented (Bernini et al., 2012;Britton et al., 2019;Espin-De Gea et al., 2002).
For the determination of the antioxidant power of OL and HT, the enzymatic kinetic method was used (Munoz-Munoz et al., 2010).
The method uses the peroxidase system (POD/ABTS/H 2 O 2 ) to generate the free radical ABTS •+ , in the presence of an antioxidant, A, the following material balance is met: where V 0 is the initial rate of action of the enzyme, t, time and n is the number of radical molecules that an antioxidant molecule captures, also called stoichiometric factor.A representation of V 0 τ versus [A] 0 , according to Equation 9, where τ is the delay period at a given antioxidant concentration, allows to obtain the value of n "stoichiometric factor".From this value, the antioxidant power or capacity (ARP) can be determined.
The effective concentration can also be obtained: This parameter is defined as the ratio of the antioxidant concentration necessary to decrease the initial concentration of radical by 50%.
Therefore, the characterization of the primary antioxidant capacity of a compound is defined by n, EC50 and ARP.

| RE SULTS AND D ISCUSS I ON
The chemical structures of the compounds studied in this work, OL and HT, are shown in Figure 1; note that HT is included in the OL structure.

| Stability of oleuropein and 3-hydroxytyrosol oxidation products
The oxidation of these compounds by NaIO 4 gives the same products as the enzymatic oxidation; therefore, this reagent was used to study the stability at the different optimal pH of the enzymes (laccase pH = 4, peroxidase pH = 5.5 and tyrosinase pH = 6.8) (Muñoz et al., 2006).
In the spectrophotometric records shown in Figure 4a, a spectrum of OL is shown at pH = 4 (a) and its oxidation product by (9) In Figure 4b, the oxidation of OL with NaIO 4 at pH = 5.5 is shown.In Figure 4b (insert) the instability of o-quinone can be seen and this instability becomes greater in Figure 4c and Figure 4c (insert) at pH = 6.8, these processes must be taken into account for the correct determination of the enzymatic activity.

| Oxidation of 3-hydroxytyrosol by sodium periodate
HT was oxidized by NaIO 4 in a 5:1 ratio, with different pH values.
Figure S1, HT oxidation at pH = 4.0 is shown giving a stable o-quinone.
Figure S1b,c shows the spectrophotometric recordings at pH = 5.5 and 6.8 where the instability of the o-quinone is demonstrated.

| Enzymatic oxidation of oleuropein
Figure 5a shows the oxidation of OL with laccase.The product absorbs in the visible zone as in Figure 4a. Figure 5b,c shows the oxidation by peroxidase and tyrosinase, respectively; the instability of o-quinone can be appreciated, as occurred in Figure 4b,c.however, with peroxidase and tyrosine, as the measurement pH is higher, o-quinone is more unstable (Figure S2b,c) and the spectrophotometric chronometric method described in Materials and Methods section should be used.

Oxidation of oleuropein by laccase
Figure 6 shows the hyperbolic dependence of the steady-state velocity, V SS , with respect to the OL concentration; the analysis by nonlinear regression to the Michaelis equation allows obtaining V L , OL max and K L , OL M (L = laccase).Table 1 shows the kinetic parameters of the laccase reaction on HT and OL.Furthermore, they are compared to other o-diphenols.From these data, it appears that laccase has the highest affinity for these substrates (see below).The speed of catalysis is related to the values of the chemical shifts of the carbons that support the phenolic hydroxyl group.Furthermore, these data show a greater speed with positively charged substrates in the side chain.
Note that when measuring the activity of the enzyme at pH = 4.0, the formation of a fairly stable o-quinone is achieved, with which the direct spectrophotometric measurement of o-quinone formation is sufficient.

Oxidation of oleuropein by peroxidase
Figure 7 shows the oxidation of OL with peroxidase, in this case, because the optimal pH is 5.5, and according to Figures 4 and 5, b Limiroli et al. (1995).
F I G U R E 7 Action of peroxidase on oleuropein.Representation of the steady-state rate values obtained with the chronometric method, V Lag , at λ = 400 nm.Experimental conditions were as follows: 50-mM sodium acetate buffer, pH = 5.5, and E 0 = 0.66 nM; ascorbic acid 37 μM and oleuropein concentration were varied from 0.056 to 4.44 mM.Insert: spectrophotometric recordings obtained by applying the chronometric method to the action of peroxidase on oleuropein (a-l) (insert), starting from the lag period and according to Equation 4the value of V 0 = V Lag is obtained (for peroxidase acting on oleuropein).From the adjustment of the values of V Lag versus OL 0 , the kinetic parameters V POD, OL max and K POD, OL M are obtained (Table 2).Note that the values of the chemical displacements are practically the same, and therefore, the k cat values are in the same order; however, the K M values are lower than the substrates that carry a positive charge in the side chain (dopamine and L-noradrenaline).3. The values of the kinetics parameters show that the charged substrates have higher

Michaelis constants (K S M
).The catalytic constants are related to the values of the chemical displacement.In the case of HT, the catalytic constant is higher, because the nucleophilic attack of the C-4 hydroxyl oxygen is more powerful because the value of chemical displacement is lower and therefore the electronic density is higher.

| Oxidationof3-hydroxytyrosolbylaccase, peroxidase and tyrosinase
We followed the same methodology as with OL as shown in Figure S2.For the kinetic characterization of laccase, the spectrophotometric method was used (Figure S3) and with peroxidase to the stoichiometric factor between the free radical and the antioxidant.From the value of "n", the EC50 parameters (Equation 11) and the antioxidant capacity ARP (Equation 10) can be determined (Table 4).Similar results of the ARP value were obtained with other molecules as has been reported by Muñoz-Muñoz et al., (Munoz-Munoz et al., 2010).
Similar results are obtained with HT (see Table 4).The polymerization of the radicals generated can occur over a long time, increasing the antioxidant capacity (Xie et al., 2020(Xie et al., , 2021)).
Table 4 shows that the antioxidant capacity of HT and OL are in the same magnitude order than ascorbic acid and Trolox (Munoz-Munoz et al., 2010).

| Molecular docking study
We have used molecular docking to study binding of HT and OL to laccase, tyrosinase, and peroxidase to identify the interactions of these ligands in the catalytic centre of the enzymes where electron  with laccase is greater than in the case of 3-hydroxytyrosol yielding a lower K d for oleuropein (Table 5).A 2D view of these results is shown in Figures S6 and S7.Our docking results with laccase are in good agreement with previously reported works, where participation of His458 and Asp206 in hydrogen bonds formation with substrates of laccase (Madzak et al., 2006;Manzano-Nicolas, Taboada-Rodriguez, et al., 2020;Piontek et al., 2002;Polyakov et al., 2019), and Π-interactions of substrates with Phe265 have been suggested (Manzano-Nicolas, Taboada-Rodriguez, et al., 2020).
Tyrosinase carries out two consecutive reactions in the pres- HT and OL binding to the met-form of tyrosinase from Agaricus bisporus shows a full overlap of the diphenol group of both ligands (Figure 11).The main interactions of tyrosinase with the diphenol groups are from copper ions, the hydroxyl group, and Phe264 residue by hydrogen bonds, from His263 residue by Π-interactions with the aromatic rings and from Val283 residue by hydrophobic interactions (Figure S8).Besides, OL presents additional hydrogen bonds to Asp191 and Glu189 residues (Figure S9).Again, the number of interactions in OL is greater than in HT yielding a lower K d for OL (Table 5).A 2D view of these results is shown in Figures S8   and S9.Similar results for binding of other ligands to tyrosinase have been reported from docking studies (Garcia-Jimenez et al., 2016;Nokinsee et al., 2015).
Ligands binding to the oxy-form of tyrosinase are shown in Figure S10 where a good overlap of the diphenols groups is observed.
Hydrogen bonds are formed from the molecular oxygen to the diphe- was scan recorded at 250-550 nm at 10 min, allowing to (a) select the measurement wavelength in enzymatic assays (λ max or λ i ); (b) calculate molar absorptivity, taking into account the amount of [NaIO 4 ] 0 and the stoichiometric relation of reaction studied; and (c) test the stability of the o-quinones produced with the presence or not of decay of absorbance over the time.
Action of peroxidase (HRP) on ABTS in presence of AH 2 (Rodríguez-López, Gilabert, et al., 2000)F I G U R E 3 Action of tyrosinase (PPO) on o-diphenols in presence of AH 2(Rodríguez-López, Fenoll, et al., 2000) Steady-state rates (V 0 or V Lag ) values are determined from the spectrophotometric recordings, and these values are adjusted to the Michaelis-Menten equation through the Sigma Plot program for Windows (Jandel-Scientific, 2016), providing the values of V max and K M .Data were recorded as mean ± standard deviation of at least triplicate determinations.
Spectrophotometric recordings of oleuropein oxidation by deficiency of NaIO 4 at different pH.(a) Oxidation at pH = 4.0.Oleuropein 0.44, 50 mM sodium acetate buffer (recording a) was oxidized with NaIO 4 , 0.11 mM (recording b).(b) Oxidation at pH = 5.5.Oxidation was done as in (a) but at pH = 5.5.Insert.Variation of absorbance at this pH, the recordings were made every minute (b-j).(c) Oxidation at pH = 6.8.Oxidation was done as in (a) but at pH = 6.8 in 50 mM sodium phosphate buffer.Insert.Variation of absorbance at this pH, the recordings were made every minute (b-j) deficiency of NaIO 4 , Figure 4a(Durán et al., 2002).Note the stability of the o-quinone at this pH.

Figure
Figure S2 shows the spectrophotometric recordings of the oxidation of HT by laccase, peroxidase and tyrosinase (Figure S2a-c respectively); the spectra are similar to those obtained in Figure S1.It is shown that measurements with laccase can be made at pH = 4.0, measuring the formation of o-quinone due to its great stability; -quinone is more unstable than at pH = 4.0; the spectrophotometric chronometric method is used.The experimental recordings necessary to obtain the initial rate are shown in Figure 7 F I G U R E 5 Action of laccase, peroxidase and tyrosinase on oleuropein.(a) Action of laccase (17 μg/ml) on oleuropein 0.55 mM in 50-mM sodium acetate buffer, pH = 4; scans were made every minute (a-j).(b) Action of peroxidase (0.65 nM) on oleuropein 0.55 mM at pH = 5.5, scans were made every minute (a-j).(c) Action of tyrosinase (23 nM) on oleuropein 0.55 mM in 50-mM sodium phosphate buffer, pH = 6.8, in recordings a-b, absorbance increases while in recordings c-j, this decreases F I G U R E 6 Action of laccase on oleuropein.Representation of the steady-state rate values, V 0 , obtained by measuring the increase in absorbance over time at λ = 400 nm.The experimental conditions were as follows: 50-mM sodium acetate buffer, and pH = 4; laccase and oleuropein concentrations were 16.7 μg/ml and (0.056-10 mM), respectively.Insert, Recordings of the increase in absorbance over time.The oleuropein concentrations were (mM): 0.056 (a), 0.11 (b), 0.28 (c), 0.56 (d), 0.83 (e), 1.11 (f), 1.67 (g), 3.33 (h), 5 (i), 6.67 (j), and 10 (k) TA B L E 1 Parameters and kinetic constants, which characterize the action of laccase on different compounds Substrate

Figure
Figure 8a represents the values of V 0 , obtained from the increase in absorbance with time, with respect to the concentration of OL, and Figure 8b (insert) shows the spectrophotometric recordings of the absorbance measurement with time, according to the chronometric method.From Equation 8, the values of the initial rate (V Lag ) are obtained.From the nonlinear regression adjustment of V Lag versus [OL] 0 , V PPO,OL max and K PPO, OL M

TA B L E 2 Figure 9
Figure S5 shows the values (V Lag ) obtained for tyrosinase according to Equation 8. Nonlinear regression analysis to the Michaelis equation of the data in Figures S3-S5 allowed to obtain the kinetic parameters as shown in Tables 1-3.

b
Figure 10.The main amino acid residues involved in the interactions ence of molecular oxygen: hydroxylation of monophenols to form odiphenols by the oxy-form of tyrosinase and oxidation of o-diphenols to o-quinones by the met-form of tyrosinase that the oxy-form of tyrosinase can also oxidize o-diphenols to o-quinones(Zolghadri et al., 2019).Therefore, HT and OL docking have been done to both the met-form and the oxy-form of tyrosinase.
nol groups.The aromatic ring position of the diphenol groups is stabilized by Π-interactions from His263 and by hydrophobic interactions with Val283.The OL tail is further stabilized by hydrogen bonds interactions with Asn260, Thr261, and Arg268 and by hydrophobic interactions with Val248 (Figure S10).It is to note that in the case of oxy-form, there are no interactions from the ligands to the copper F I G U R E 11 Docked conformations of 3-hydroxytyrosol and oleuropein in the met-form of Agaricus bisporus tyrosinase.Color scheme as in Figure 10 Parameters and kinetic constants, which characterize the action of tyrosinase on different compounds Kalampaliki et al. (2019)).