Immobilization of Crude Polyphenol Oxidase Extracts from Apples on Polypyrrole as a Membrane for Phenol Removal

Received:1st November 2020 Revised: 8th March 2021 Accepted: 8th March 2021 Online: 15th March 2021 This research aims to make a polypyrrole (PPy) membrane and crude extract of polyphenol oxidase (PPO) as a membrane of mPPy/PPO apple extracts. The membrane of PPy/PPO-apple extract has been synthesized by the electrodeposition method. The electrolyte composition consists of a mixture of 0.10-0.20 M pyrrole (Py) and 50-100% PPO apple extract, which is stable using 50 mM of phosphate buffer solution at pH 6.80-7.00 and room temperature. The electrodeposition process is used 400 mesh steel gauze anode ST-304 and carbon plate cathode. Electrodeposition is carried out at potential = 5.00-6.00 V; current = 0.02-0.25 A; the distance from both electrodes = 1.00 2.00 cm for 300 500 seconds. The results from the deposition of PPy/PPO apple extract of the anode are a membrane of mPPy/PPO-apple extract, with total enzyme activity (U) = (957,1441, 2287 and 1754) using 2.00-5.00 mM phenol as a substrate which is measured based on the UV-visible spectrophotometric method. PPy and mPPy/PPO-apple extracts were characterized by SEM and SEM-EDS. The membrane of mPPy/PPO-apple extract can be used to remove phenol in industrial wastewater samples is 50-65% with a filtration capacity of 500 mL for 2 hours.


Introduction
Phenolic compounds and their derivatives are widely used as additives in several chemical and pharmaceutical industries. However, phenols are corrosive and toxic, which can pollute the environment. The increasing presence of phenols represents a significant environmental toxicity hazard; therefore, developing methods for removing phenols from industrial wastewater has generated significant interest [1]. The presence of phenols significantly reduces the biological degradation of other components [2].
According to Abd Garni [ 3 ], phenol ' s structure shows its reactivity, which leads to its properties like persistence in the environment, toxicity, and possible carcinogens against living organisms. The conventional methods of treating phenol waste are primarily chemical or physical, but this process creates a new secondary waste problem.
For example, phenolic compounds obtained from industrial wastewater can be eliminated using activated carbon. Meanwhile, activated carbon is removed by combustion. During the combustion process under normal operating conditions, several of new compounds can be produced. These compounds are usually dioxins and furans which have an impact on human health [ 4 ]. Polyphenol oxidase (PPO) is a group of enzymes that catalyze the oxidation of phenolic compounds to produce a brown color on cut the surface of fruit and vegetables [ 5 ]. PPO catalyzes the oxidation of polyphenols to quinones which react non-enzymatically to produce colored pigments [6].
In plant cells, phenolic compounds are located in vacuoles, whereas PPO is located in plastids. Damaged areas in cells allow contact between PPO and phenolic compounds [ 7 ]. PPOs usually cause browning after cell ℃ x.
damage due to aging, wounding, and the attack of pests and pathogens [8]. Apples that are mechanically damaged (peeled, bitten, and bumped) cause a browning reaction. As apples matured, there was an increase in damaged cells around the larger intercellular spaces [ 9 ].
The apple fraction ' s chemical composition is related to its enzymatic browning, which assesses apple pomace ' s suitability for extracting phenolic compounds [10]. PPO activity is crucial to control enzymatic browning. The factors that influence PPO activity include the type and amount of endogenous phenolic compounds, the presence of oxygen, and the targeted pH to prevent enzymatic browning [11].
In this research, PPOs extract can be isolated from apples in phosphate buffer 50 mM pH 6.80-7.00 and room temperature. PPO performance will be more effective through immobilized on the surface of the polypyrrole (PPy) and synthesized by electrodeposition at optimum conditions [12]. The electropolymerization of pyrrole (Py) has been synthesized for membrane fabrication using the electrolysis technique. Some previous researchers have synthesized PPy by chemical oxidative polymerization technique at room temperature using monomer 0.10 M Py [ 13 ], 0.025 , 0.05 , 0.1, 0.15 M Py [ 14 ]. Immobilized enzymes have many operational advantages over free enzymes, such as reusability, enhanced stability, continuous operational mode, rapid reaction termination, easy separation of biocatalyst from the product, and reduced operating costs [ 15 ]. PPO has immobilized on PPy film by electropolymerization using cyclic voltammetry method on a platinum electrode (0.1 M Py and 2 pg / mL PPO in a 50 mM phosphate buffer solution pH 6.5 at 25 ) [12]. PPO immobilization was carried out during the electrosynthesis of PPy films by adding 100 pL of the crude extract of avocado in the electropolymerization medium containing 0.1 M LiCl 04 and 0.07 M Py [16]. PPO was immobilized in PEO / PPy and CP / Ppy [ 17 ]. PPy is very promising for commercial applications because of its good environmental stability, smooth synthesis and higher conductivity than many other conducting polymers [ 13 ]

Methodology
The chemicals, laboratory apparatus and instrumentations, and experiment procedure are explained in the sub-section below.

. The Applications of mPPy / PPO apple extract
The application of mPPy / PPO-apple extract used the industrial sample containing phenol. The filtration unit was feed phase, membrane, permeate container, a pump, and manometer). A filtration set was combined with the mPPy / PPO-apple extract ' s housing membrane with a dead-end flow system and pressure (0. 10 [21]. The resulting permeate solution was collected in a permeate container.

Membrane Filtration Method
A microfiltration set consists of the feed phase, membrane housing, permeate container, manometer, and a pump.

. The membrane of PPy / PPO-apple extract (mPPy / PPO-apple extract)
This research describes an effective process of synthesizing the mPPy / PPO-apple extracts using the electrodeposition method. The electrodeposition uses the steel gauze ( 1.50 x 1.50 cm) as an anode and a carbon plate as the cathode. The electrolysis was controlled by electrolytic optimum (the distance between the two electrodes = 1.50 cm and electrolysis time = 500 seconds).
The composition of mPPy / PPO-apple extract has achieved 0.15 M Py and 75 % (v / v) PPO-apple extract.
Physically, mPPy / PPO-apple extract can be seen in However, the observations for these membrane images are not visible, and this will be discussed further in the SEM images for membranes.
consisting of Py and PPO in phosphate buffer pH 6.8-7.0 .
The amount of charge transported is also proportional to the electron transfer of the redox reaction between Py + and Afrom the 0.15 M PPy deposition process and the 75 % (v / v) PPO extract-apple attached on the steel gauze anode. The other membrane parameters that can be studied in Table 1 are the observation digital multimeter measurement results. The thickness of mPPy / PPO-apple extract (mm) was 0.122-0.180; apple-extract PPy / PPO layer thickness (mm) was 0.038 -0.092; so the predicted thickness of steel gauze (mm) was 0.084 -0.088.   Mn, Si, and Cu). The conditions for electrolysis also have an impact on the weight and thickness of the membrane. The PPy with Py oxidation occurring at the steel gauze anode and A as dopant anions [22], and the possible redox reactions that occur at steel gauze anodes and carbon plate cathodes are:  Table 1 shows the influence of potential ( 3 ,00-6.00 V) on membrane characteristics of mPPy / PPO-apple extract (M-l to M-4 ), which were synthesized based on electrodeposition at optimum conditions (current, weight, and thickness membrane). The current response ( 0.04 -0.22 A) shows the impact of the electrodeposition process. It is easy regulating during the PPO-apple extract is immobilization on PPy film growth at 3.00 -6.00 V, so that it is as the difference in the shape the physically of the M-l to M-4 of mPPy / PPO-apple extract (Figure-i). The potential increase in proportion to the increase in current. As explained by Wang [22], there is a relationship between current flow and mobility of PPy + and Aions (counterion), in which dopant anion concentration correlates with electrolyte solution (M-4 ) ( 0 cathode: (ii) overall: (iii) The quality of the film produced on electropolymerization depends on the interaction between polymer and dopant, which will direct the growth of polymer chains on the electrode ' s surface [26]. PPy can be used as a supporting material for PPO immobilization in apple extracts synthesized by electrodeposition technique. The electrochemical method involves the entrapment of biomolecules in organic polymers during the electro-generation of their electrode surfaces. The formation of polymers using electrolysis at a controlled potential of aqueous solutions containing monomers and biomolecules [ 27 ]. The stability of PPO (from apple extract) in a 50 mM phosphate buffer solution can affect the degradation process, so it is necessary to prepare it at optimum pH and room temperature and achieve the mPPy / PPO electrodeposition process. In Table 1, the achievement of PPO activity on the mPPy / PPO-apple extract membrane was 957 -2287 U PPO activity before the electrodeposition process. In this process, PPO-apple extract has the highest activity. Before the electrodeposition process, apple extract characteristics have been studied based on the PPO activity, the protein content, and the specific activity at the pH variation (6.00; 6.20; 6.40 ; 6.60; 6.80; and 7.00 ) are shown in Fig  2 and 3 . In Fig 2, the highest PPO activity and total PPO activity is shown (U / mL) = 76.23 or (U) = 7623 in 100 mg apple extract; 2.5 mM phenol; pH 7 in a 50 mM phosphate buffer solution and room temperature).
(100% of relative activity), followed by caffeic acid ( 18.5 %), catechin ( 17.5 %), and gallic acid ( 4.2 %) PPO activity was measured based on the spectrophotometric method and the initial rate of decrease in absorbance (phenol concentration) and the reaction time of quinone formation (curve at phenol = 282 nm; y = 1.2929 + 0.0041 ; R = 0.9970 ). Meanwhile, the protein content was (mg / mL) = 0.39 ; total protein (mg) = 39 or 0.4 % in 100 g crude apple extract, which was measured based on the Biuret method (standard curve BSA max = 278 nm; y = o.ooilx + 0.04 ; R = 0.9966 ). In Fig. 3 , showing of the specific activity (U / mg) = 195.46 and total specific activity (U) = 19.5 . PPO activity as a biocatalyst in a mPPy / PPO-apple extract can affect the function of phenol removal in industrial wastewater contaminated with phenol. Therefore, it needs to have a useful technique in making mPPy / PPO-apple extract during the electrodeposition process. It is necessary to ideal design between the filtration device unit and mPPy / PPO-apple extract for good performs smoothly in phenol ' s degradation process to a quinone. The PPO in a solution is deactivated by the presence of the reaction product. But, if it is immobilized, the separation of the product from this enzyme will reduce this inhibition. Immobilization can also increase stability and allow for continuous use of the enzyme [  pore size (about 10-20 pm) and a pressure of 0.5 Bar, and this is still close to the standard technique for the membrane microfiltration group (dead-end system) according to Mulder [21]. The thin film of PPy has uniform granular morphology and average grain size of 0.7 pm [ 13 ]. The research results from Yussuf et al. [ 14 ] have studied the SEM photograph of PPy films has been made of Py ( 0.05 M) / FeCl 3 (0.1 M), which shows the fibrillar morphology. While the SEM image of PPy films that Py has made ( 0.05 M) / N2HsS208 (0.1 M) showed spherical morphology. Accordingly, changes in the morphology of PPy are very dependent on the oxidant type used during the chemical polymerization. PPy morphology structure significantly effects its conductivity. It was found that the fibrillar morphology sample have higher conductivity than the sample with a spherical morphology. Based on these assumptions, if we observe the SEM images in Fig. 4 a and

as a substrate
A previous research [28] studied mPPO on Fuji apples purified by a combined protocol involving temperature induced phase partitioning ( 1.5 % Triton X-100; 150 mL; total activity = 1, 071 ,000 U; total protein = 157.50 mg, and specific activity Meanwhile, the research results of Queiroz et al [ 6] have studied that PPO in cashew apple shows maximum activity (pH 6.5 ) and remains in the range of 63 % (pH 7.0 ). PPO activity is not visible (pH above 7.0 ) because of the substrate oxidation. Cashew apple PPO showed the highest activity with 0-dihydroxy-phenol catechol  27.44 to 28.67 %. This is possible because the PPy will be more dominant with the polymer ' s growth covering the steel gauze's surface as mPPy / PPO-apple extract. For this reason, the SEM-EDS profile of PPy to mPPy / PPOapple extract also has an impact on steel gauze as the main support for the membrane. Fe is the main component in steel alloys, so that the mass scale decreases, Fe (6,

. The Applications of mPPy / PPO apple extract
The mPPy / PPO-apple extract (M-i M-4 ) has been applied to an industrial sample ' s initial solution containing 1.00 mM of phenol. The results of a permeate solution containing phenol remeasured phenol concentration in the final solution (Fig. 5 a and