Kinetic Study of Peroxidase-Catalyzed Oxidation of 2-Hydroxyanthracene and 9-Phenanthrol in Presence of Biosurfactant Escin

: The kinetics of fungal peroxidase-catalyzed 2-hydroxyanthracene and 9-phenanthrol oxidation was investigated in presence of biosurfactant escin at pH 5.5 and 25 ◦ C. The kinetic measurements were performed using the ﬂuorimetric method and the critical micelle concentration (CMC) of escin was determined using the dynamic light scattering technique. Inactivation of peroxidase was observed in absence of biosurfactant escin. It was shown that escin, used in concentrations lower than CMC, decreases or completely stops the peroxidase inactivation and increases the conversion of 2-hydroxyanthracene as well as of 9-phenanthrol. The environmentally friendly method of peroxidase-catalyzed 2-hydroxyanthracene and 9-phenanthrol oxidation in presence of biosurfactant Escin has an advantage over traditional decontamination methods due to their less environmental impact.


Introduction
Aromatic hydroxyl derivatives (AHDs) are treated as pollutants that are produced and released into the environment by various industrial processes, and effective methods of their decontamination are very important [1,2]. Bio-utilization methods are usually not very expensive and environmentally friendly. Pollutants can be degraded with the help of enzymes, synthesized by bacteria, fungi or plants [3][4][5]. Peroxidases and other phenol-oxidizing enzymes have great potential to decrease environmental pollution by bioremediation of waste water containing a broad spectrum of aromatic hydroxyl derivatives [5]. Progress in genetic engineering also provides possibilities for application of recombinant peroxidases that demonstrate an elevated stability compared with native peroxidases [6].
Peroxidases undergoes a cyclic reaction when reacting with AHDs. This sequence is summarized in the following reactions [7]: cpd I + AHD k 2 → cpd II + AHD• (2) cpd II + AHD Native peroxidase (E) is oxidized by hydrogen peroxide (H 2 O 2 ) with compound I (cpd I) formation. The cpd I in its turn oxidizes AHDs (AHD) via the formation of radical (AHD • ) and compound II (cpd II) (reactions 1-3). The resulting radicals can react chemically, forming oligomers (Olig, reaction 4) that are subject to further oxidation by peroxidase to constitute higher polymers until the solubility limit is reached. Unfortunately, biocatalytic oxidation of AHDs has some limitation, i.e., inactivation of enzymes during the reaction. It was proved that peroxidases and other phenol-oxidizing enzymes are inactivated by the polymer product formed during the reaction due to formation of polymer-peroxidase conjugates [8][9][10]. In an attempt to decrease the inhibition rate, some additives were used, i.e., non-ionic polymeric compounds and surfactants [8,11,12]. Synthetic surfactants such as SDS, CTAB and Triton X-100 are very poisonous to aquatic organisms, and they can influence the aquatic environment by causing long-term adverse effects [13,14]. Recently, biosurfactants have received a lot of attention due to biodegradability, low toxity and safety [15].
The task of this investigation was to explore recombinant Coprinus cinereus peroxidase-catalyzed oxidation of 2-hydroxyanthracene and 9-phenanthrol in the presence of environmentally friendly biosurfactant escin (Figure 1).
Native peroxidase (E) is oxidized by hydrogen peroxide (H2О2) with compound I (cpd I) formation. The cpd I in its turn oxidizes AHDs (AHD) via the formation of radical (AHD • ) and compound II (cpd II) (reactions 1-3). The resulting radicals can react chemically, forming oligomers (Olig, reaction 4) that are subject to further oxidation by peroxidase to constitute higher polymers until the solubility limit is reached. Unfortunately, biocatalytic oxidation of AHDs has some limitation, i.e., inactivation of enzymes during the reaction. It was proved that peroxidases and other phenol-oxidizing enzymes are inactivated by the polymer product formed during the reaction due to formation of polymer-peroxidase conjugates [8][9][10]. In an attempt to decrease the inhibition rate, some additives were used, i.e., non-ionic polymeric compounds and surfactants [8,11,12]. Synthetic surfactants such as SDS, CTAB and Triton X-100 are very poisonous to aquatic organisms, and they can influence the aquatic environment by causing long-term adverse effects [13,14]. Recently, biosurfactants have received a lot of attention due to biodegradability, low toxity and safety [15].
The task of this investigation was to explore recombinant Coprinus cinereus peroxidase-catalyzed oxidation of 2-hydroxyanthracene and 9-phenanthrol in the presence of environmentally friendly biosurfactant escin (Figure 1). 2-hydroxyanthracene and 9-phenanthrol (also called 9-hydroxyphenanthrene) are tricyclic aromatic hydrocarbons with hydroxyl group compounds, that being considered persistent, bioaccumulative and toxic for freshwater and marine ecosystems [17,18]. Escin is the main natural constituent of the saponin extract from the seeds of Aesculus hippocastanum (known also as the horse chestnut tree) [19]. Horse chestnut seed extract, containing around 20 wt % escin, is widely used in medicine for its venotonic effect, vascular protection and free radical scavenging properties [20]. The experiments of other scientists show that escin is obtained from crude extracts of aesculus plants with high efficiency and it is not relatively expensive [21]; it is also sold as a market product. 2-hydroxyanthracene and 9-phenanthrol (also called 9-hydroxyphenanthrene) are tricyclic aromatic hydrocarbons with hydroxyl group compounds, that being considered persistent, bioaccumulative and toxic for freshwater and marine ecosystems [17,18]. Escin is the main natural constituent of the saponin extract from the seeds of Aesculus hippocastanum (known also as the horse chestnut tree) [19]. Horse chestnut seed extract, containing around 20 wt % escin, is widely used in medicine for its venotonic effect, vascular protection and free radical scavenging properties [20]. The experiments of other scientists show that escin is obtained from crude extracts of aesculus plants with high efficiency and it is not relatively expensive [21]; it is also sold as a market product.

Kinetics of Peroxidase-Catalyzed 2-Hydroxyanthracene and 9-Phenanthrol Oxidation in the Absence and Presence of Biosurfactant Escin
The oxidations of 2-hydroxyanthracene and 9-phenanthrol were not complete in the absence of biosurfactants (Figure 2a,b curves 1). During oxidation of these compounds, kinetic curves have saturated. The fluorescence is not noticeable in enzymes, hydrogen peroxide and the products of the reaction. So fluorescence intensity is minimized only due to 2-hydroxyanthracene or 9-phenanthrol concentration decrease. The addition of a new portion of hydrogen peroxide did not affect the reaction.
On the other hand, the addition of a new portion of enzyme has caused further 2-hydroxyanthracene and 9-phenanthrol oxidation. This proves the peroxidase inactivation during the reaction. Other curves on Figure 2a,b (curves 2-6) show the kinetics of peroxidase-catalyzed 2-hydroxyanthracene (a) and 9-phenanthrol (b) oxidation in presence of biosurfactant escin. With small quantities (up to 1 µmol L −1 ) of biosurfactant, the conversion of both pollutants has risen up gradually. In presence of 2 µmol L −1 -0.2 mmol L −1 concentration of escin the conversion of both substrates was fully completed, so the enzyme inhibition was stopped. Also, escin (up to 0.2 mmol L −1 ) did not impact the initial oxidation rate of both pollutants. When the concentration of escin exceeded 0.2 mmol L −1 , the initial rate of 2-hydroxyanthracene and 9-phenanthrol oxidation gradually decreased as well as the degree of both AHDs' conversion.
Catalysts 2019, 9, x FOR PEER REVIEW 3 of 8 The oxidations of 2-hydroxyanthracene and 9-phenanthrol were not complete in the absence of biosurfactants (Figure 2a,b curves 1). During oxidation of these compounds, kinetic curves have saturated. The fluorescence is not noticeable in enzymes, hydrogen peroxide and the products of the reaction. So fluorescence intensity is minimized only due to 2-hydroxyanthracene or 9-phenanthrol concentration decrease. The addition of a new portion of hydrogen peroxide did not affect the reaction. On the other hand, the addition of a new portion of enzyme has caused further 2hydroxyanthracene and 9-phenanthrol oxidation. This proves the peroxidase inactivation during the reaction. Other curves on Figure 2a,b (curves 2-6) show the kinetics of peroxidase-catalyzed 2hydroxyanthracene (a) and 9-phenanthrol (b) oxidation in presence of biosurfactant escin. With small quantities (up to 1 μmol L −1 ) of biosurfactant, the conversion of both pollutants has risen up gradually. In presence of 2 μmol L −1 -0.2 mmol L −1 concentration of escin the conversion of both substrates was fully completed, so the enzyme inhibition was stopped. Also, escin (up to 0.2 mmol L −1 ) did not impact the initial oxidation rate of both pollutants. When the concentration of escin exceeded 0.2 mmol L −1 , the initial rate of 2-hydroxyanthracene and 9-phenanthrol oxidation gradually decreased as well as the degree of both AHDs' conversion.  6)).

Enhancing Effect of Escin Monomers on Biocatalytic Oxidation of 2-Hydroxyanthracene and 9-Phenanthrol
Escin is the amphiphilic molecule. At low concentration, it forms as monomers. At high concentration, it may form a micelle. The concentration of the biosurfactant above which micelles are formed is described as the critical micelle concentration (CMC, Figure 3).

Enhancing Effect of Escin Monomers on Biocatalytic Oxidation of 2-Hydroxyanthracene and 9-Phenanthrol
Escin is the amphiphilic molecule. At low concentration, it forms as monomers. At high concentration, it may form a micelle. The concentration of the biosurfactant above which micelles are formed is described as the critical micelle concentration (CMC, Figure 3).
The CMC of escin at 25 • C in 50 mmol L −1 sodium acetate buffer (pH 5.5) employing the dynamic light scattering method was determined. Figure 3 presents the variation of the hydrodynamic diameter with the escin concentration at 25 • C. It shows that the CMC of escin was 0.2 mmol L −1 in the sodium acetate buffer. As shown in Figure 2, the conversion of 2-hydroxyanthracene and 9-phenanthrol was high in the escin concentration range 2 µmol L −1 -0.2 mmol L −1 . Within this concentration range, escin exists as a monomer. This shows that the monomer receives a better augmenting effect on the conversion of 2-hydroxyanthracene and 9-phenanthrol than micelle. The CMC of escin at 25 °C in 50 mmol L −1 sodium acetate buffer (pH 5.5) employing the dynamic light scattering method was determined. Figure 3 presents the variation of the hydrodynamic diameter with the escin concentration at 25 °C . It shows that the CMC of escin was 0.2 mmol L −1 in the sodium acetate buffer. As shown in Figure 2, the conversion of 2-hydroxyanthracene and 9phenanthrol was high in the escin concentration range 2 μmol L −1 -0.2 mmol L −1 . Within this concentration range, escin exists as a monomer. This shows that the monomer receives a better augmenting effect on the conversion of 2-hydroxyanthracene and 9-phenanthrol than micelle.

Mechanism of Escin Action
The saturation reaction rate during 2-hydroxyanthracene and 9-phenanthrol oxidation indicates that enzyme is inhibited during the reaction (Figure 2a,b curves 1). The prevention of inhibition by addition of biosurfactant escin indicates that the inhibition proceeds by enzyme interaction with oxidation products. The results are accounted for by a scheme, which contains a stage of enzyme inhibition by oligomers (Reactions (1)-(6)), using KinFitSim program. The second order constant of hydrogen peroxide reaction with the native enzyme (Reaction (1)) was taken as k1 = 7.1 × 10 6 mol −1 L s −1 at 25 °C [22]. The fitting of data gave constants of 2-hydroxyanthracene or 9-phenanthrol reactivity with the ferryl compounds of rCiP (with compound I (k2) and with compound II (k3)), which were similar at different biosurfactant concentrations (Table 1).

Mechanism of Escin Action
The saturation reaction rate during 2-hydroxyanthracene and 9-phenanthrol oxidation indicates that enzyme is inhibited during the reaction (Figure 2a,b curves 1). The prevention of inhibition by addition of biosurfactant escin indicates that the inhibition proceeds by enzyme interaction with oxidation products. The results are accounted for by a scheme, which contains a stage of enzyme inhibition by oligomers (Reactions (1)-(6)), using KinFitSim program. The second order constant of hydrogen peroxide reaction with the native enzyme (Reaction (1)) was taken as k 1 = 7.1 × 10 6 mol −1 L s −1 at 25 • C [22]. The fitting of data gave constants of 2-hydroxyanthracene or 9-phenanthrol reactivity with the ferryl compounds of rCiP (with compound I (k 2 ) and with compound II (k 3 )), which were similar at different biosurfactant concentrations (Table 1). Table 1. Kinetic parameters of biocatalytic oxidation of 2-hydroxyanthracene and 9-phenanthrol in presence of escin, when concentrations of biosurfactant are less than CMC, in 50 mmol L −1 acetate buffer pH 5.5, 25 • C.

AHDs
Escin On the terms of 2-hydroxyanthracene, a mean value of k 2 was (9.7 ± 0.4) × 10 7 mol −1 L s −1 and k 3 was (9.8 ± 0.3) × 10 7 mol −1 L s −1 if escin concentrations had varied between 0 and 0.2 mmol L −1 ( Table 1). In the event of 9-phenanthrol, a mean value of k 2 as well as k 3 was (3.1 ± 0.1) × 10 7 mol −1 L s −1  Table 1). The results show that k 2 and k 3 values did not change at different biosurfactant concentrations for both derivatives. This means that biosurfactant escin does not interact with peroxidase. The calculated inhibition constant k in value has decreased for 2-hydroxyanthracene and 9-phenanthrol if biosurfactant escin concentration was increased ( Table 1). The decline of k in when biosurfactant concentration was increased could be illustrated by reversible oligomeric aromatic hydroxyl derivatives oxidation products interaction with biosurfactant escin.
Demonstration of the mechanism of peroxidase inactivation by the molecular clothing of active center by oligomeric derivatives has been submitted previously [10]. Docking and molecular dynamics computations have indicated that oligomeric AHDs have interacted with the enzyme better than the substrates. Opposed to the substrate, the binding of oligomeric derivatives did not form effective complexes, and closed the active center. With small quantities of biosurfactant (less than critical micelle concentrations), the enzyme inhibition was prevented. This may be defined by the fact that monomers of biosurfactant connect with oligomeric compounds, which show strict hydrophobicity [12] and interact with biosurfactant's monomers.

CMC Determination
The CMC of biosurfactants in 50 mmol L −1 sodium acetate buffer at pH 5.5 and 25 ± 0.1 • C was determined by using a Zetasizer Nano-ZS analyzer (Malvern Instruments, UK). This gadget contains a 4 mW He-Ne laser operating at a wavelength of 633 nm and an avalanche photodiode detector. The scattered light is analyzed at an angle of 173 • and optics arrangement maximizes the detection of scattered light while maintaining signal quality. For dynamic light scattering measurements, buffer solution was filtered (pore size = 200 nm). The escin concentration in the solutions was ranged to 0.2 mmol L −1 with an step of 0.02 mmol L −1 and up to 1 mmol L −1 with an step of 0.1 mmol L −1 . All prepared solutions were left to equilibrate for 15 to 20 min then they were analyzed.

Kinetic Analysis
The kinetic analysis was done by using a Aminco Bowman luminescence spectrometer (Thermo Electron Corporation, USA). The fluorescence of 2-hydroxyanthracene was determined at 526 nm and at excitation 333 nm. The fluorescence of 9-phenanthrol was determined at 470 nm and at excitation 340 nm. The kinetic analysis was done in 50 mmol L −1 sodium acetate buffer solution at pH 5.5 and 25 • C and the reaction mixture contained 25 µmol L −1 2-hydroxyanthracene or 9-phenanthrol, 0.3-1 nmol L −1 rCiP, 100 µmol L −1 H 2 O 2 , 0-1 mmol L −1 of escin. The reactions began when adding the Catalysts 2019, 9, 591 6 of 7 enzyme solution. All the experiments were carried out 3 times. Out of 3 kinetic curves the best curve was chosen and it was marked using signs in Figure 2.

Mathematical Calculations
The fluorescence 2-hydroxyanthracene and 9-phenanthrol was normalized to the initial concentrations. Computer programs Grafit 7 and Mathcad 15 were employed for data processing. The kinetics of the process were interpreted according to the Dunford scheme (reactions 1-4), adding the steps 5 and 6 corresponding to the enzyme inhibition by oligomers [12]: cpd I + Olig k 6 → E in (6) According to the scheme (reactions 1-6), all the best kinetic curves ( Figure 2) were fitted 3 times and kinetic parameters (k 2 , k 3 , k in ) were calculated 3 times also using the KinFitSim 2.1 program [24]. The fitting of data gave chi-square value (χ2). According to the best χ2 value, the solid lines were marked in the Figure 2. The mean values of kinetic parameters and standard errors were marked in Table 1.

Conclusions
The performed investigations of biocatalytic oxidation of 2-hydroxyanthracene and 9-phenanthrol in presence of escin can be used for further intensification of peroxidase-catalysed biotechnological processes that can be applied while developing environmentally friendly decontamination methods. It was shown that the biosurfactant escin, used with concentrations lower than the CMC, decreased or completely stopped the peroxidase inactivation and increased the conversion of 2-hydroxyanthracene as well as of 9-phenanthrol. Environmentally friendly methods has an advantage over traditional decontamination methods due to the lesser environmental impact.
Funding: This research received no external funding.