2.1. Eggplant Extract Polyphenol Oxidase Activity and Substrate Specificity
Previously, eggplant has been reported to have polyphenol oxidase activity [
12,
17,
18]. Purified eggplant PPO was characterized by much lower cresolase activity than catecholase activity [
12], in another study purified eggplant PPO lacked any cresolase activity [
18]. The substrate specificity of purified eggplant PPO showed that it belonged to the catechol oxidase family (EC 1.10.3.1) as the maximum activity was observed with catechol or its derivative substrates [
18].
Eggplant crude extract [
17] and tissues [
19,
20] also demonstrate PPO activity. Catechol, 4-methylcatechol, caffeic, chlorogenic, 3,4-dihydroxyhydrocinnamic acids, and paracetamol were shown to be substrates of eggplant PPO. The eggplant tissue [
19] showed activity towards monophenol substrate (paracetamol) on the contrary to purified eggplant PPO, which may be explained by the loss of some enzyme activity during purification procedure. Using a native enzyme preparation, such as crude extract, seems to be a perspective way to keep all the benefits of enzymatic analytical procedures and broaden the number of possible analytes. In the present work, we aimed to study EE activity towards broad spectrum of different poly- and monophenolic compounds oxidation to determine the possible analytes and propose analytical procedures. The following phenolic compounds were used: catechol, gallic, caffeic, ferulic, and chlorogenic acids, quercetin, rutin, dihydroquercetin,
l-DOPA,
l-tyrosine, paracetamol, phenol and
p-nitrophenol.
The polyphenol oxidase activity of crude eggplant extract (EE), prepared in this study, was determined by comparing the reaction speed of catechol oxidation in the presence of EE and purified mushroom tyrosinase. EE was characterized by 160 ± 6 U/mL (n = 3,
P = 0.95) polyphenol oxidase activity which is 2.5 times higher than for banana extract that we studied before [
4]. Total protein content of EE was also determined (1.1 ± 0.3 mg/mL,
n = 3,
P = 0.95). When stored at −20 °C EE retained 75% of activity after 6 months, when stored at +4 °C EE retained 75% of activity after 3 days and 40% of activity after 2 months.
We investigated EE activity towards various phenolic compounds in order to develop analytical procedures for their fast determination. Catechol, caffeic and chlorogenic acids, and
l-DOPA were rapidly oxidized in presence of EE with the formation of colored products. Their oxidation products had the following absorbance maxima: 410 nm for catechol, 420 nm for caffeic and chlorogenic acids, 480 nm for
l-DOPA. Similarly to [
18] in our work EE activity towards
l-DOPA oxidation was lower than for other catechol derivatives. For other studied phenolic compounds (gallic acid, ferulic acid, quercetin, rutin, dihydroquercetin,
l-tyrosine, paracetamol, phenol,
p-nitrophenol) no colored oxidation products were observed in the presence of EE during the observation time (4 min).
Monophenolic compounds, such as paracetamol, were previously shown to interact with eggplant PPO [
19]. We suggested that the oxidation reaction was much slower for monophenolic compounds because of cresolase reaction step, and indeed, some colored products were observed for paracetamol and
l-tyrosine if the reaction mixture was left overnight. Such slow reaction speed was not acceptable for our analytical application, so we did not study monophenol substrates further.
Gallic acid was not oxidized in the presence of EE similarly to purified eggplant PPO [
18]. Ferulic acid, quercetin, rutin, and dihydroquercetin interaction with eggplant PPO was studied for the first time. These compounds did not serve as substrates for EE and furthermore, these compounds were found to inhibit catechol oxidation by EE. Ferulic acid is a known PPO inhibitor [
23] and quercetin is known to inhibit tyrosinase [
24].
The substrate specificity study allowed us to select four phenolic compounds (catechol, caffeic acid, chlorogenic acid, and l-DOPA) in order to develop fast analytical procedures for their determination.
2.2. Eggplant Extract Polyphenol Oxidase Interaction with Catechol, Caffeic Acid, Chlorogenic Acid, and l-DOPA
In order to choose the reaction conditions we investigated the influence of pH and temperature on oxidation of catechol, caffeic acid, chlorogenic acid, and l-DOPA. The reaction speed was calculated as a difference between absorbance at 30 s and at 150 s after the reaction start (ΔA).
The influence of pH on the reaction speed was studied in the range 2.0–9.0 (
Figure 1), for all studied substrates the reaction speed reached maximal values in the 5.0–7.0 pH range. All the further activity measurements were conducted at pH 6.0. We studied the influence of temperature (22–45 °C): no increase of the reaction speed was detected and at temperatures higher than 35 °C reaction became significantly slower (
Figure 2). We conducted all the further experiments at room temperature (22 °C). The influence of enzyme amount (EE volume) was also investigated and the 1.0 mL volume was chosen for further experiments (
Figure 3).
3-Methyl-2-benzothiazolinone hydrazone (MBTH) is known to couple with the product of phenolic compounds oxidation (both enzymatic and non-enzymatic) and accelerate the reaction speed [
4,
25,
26,
27]. MBTH is widely used in optical biosensors based on laccase and tyrosinase enzymes [
28,
29,
30,
31,
32]. The interaction of MBTH with the enzymatically produced quinones leads to optical signal amplification because the reaction equilibrium is shifted towards the product and the azo dye product is intensely colored.
The influence of MBTH on eggplant PPO catalyzed oxidation was studied for the first time. In presence of MBTH the spectra of the products of enzymatic oxidation by EE demonstrated the following absorbance maxima: 460 nm for catechol, 520 nm for
l-DOPA and caffeic and chlorogenic acids. The reaction speed increased with the increase of MBTH concentration (
Figure 4) and 2.0 × 10
−3 M concentration of MBTH was chosen for further experiments.
The enzymatic activity of EE towards catechol, caffeic acid, chlorogenic acid, and
l-DOPA was studied in the broad range of substrate concentrations. The enzymatic oxidation reaction Michaelis constants (K
m) and maximum rates (V
max) were calculated using Lineweaver–Burk plots (
Table 1). For catechol, caffeic and chlorogenic acids Michaelis constants had similar values, because the studied substrates have similar structure. The maximum value of V
max was observed for chlorogenic acid.
Kinetic parameters of all the studied phenolic compounds oxidation in presence of MBTH were determined using Lineweaver–Burk plots (
Table 1). K
m values decreased by a factor of 2 to 3 for the studied substrates. V
max increased significantly only for catechol, which can be explained by its simple structure: V
max increases because of the azo dye formation which increases absorbance of the mixture, and the simpler is the structure of the quinone the easier it is to form the azo dye.
The effect of MBTH on the EE enzymatic oxidation (decrease of K
m values and increase of V
max values) is similar to that observed for other crude plant extracts [
4]. K
m values decreased by a factor of 2 to 3 on the average for EE, which is a less pronounced effect than for banana extract (a factor of 12 on the average). However, this decrease was similar for all the studied substrates for both PPO extracts (eggplant and banana) compared to an uneven effect for peroxidase containing green bean extract (from 2 to 20 times K
m values decrease) [
4]. This characteristic of PPO extracts makes them preferable for total phenol content determination procedures.
The proposed system (EE+MBTH) was used for the development of spectrophotometric procedures of phenolic compounds determination.