Use of Immobilized Enzyme In Granular Activated Carbon And Chitosan Beads For Phenol Removal From E uents


 Tyrosinase enzyme present in a crude extract was immobilized in granular activated carbon (GAC) and activated chitosan beads (ACB). It was possible to immobilize up to 70.0 % of the enzymes in GAC in the conditions of 10.0 g of support, 15.7 rad/s of agitation and 90 minutes of contact time, and 100.0 % of enzymes in ACB when using 5 g of support, agitation of 15.7 rad/s and contact time of 120 minutes. In enzymatic oxidation tests, tyrosinase immobilized in GAC was able to achieve a final phenol concentration below the limit required by Brazilian law, 0.5 mg/L for phenol solutions with an initial concentration up to 20.0 mg/L while the enzyme immobilized in ACB was able to adapt solutions with initial concentrations of phenol up to 40.0 mg /L. It was possible to reuse the enzyme immobilized in GAC 2 times, maintaining the same phenol removal efficiency, while the enzyme immobilized in ACB maintained up to 98.0 % of its efficiency in 5 cycles of enzymatic oxidation of solutions with 10.0 mg/L of phenol initially. It was possible to maintain the same phenol removal efficiency as immobilized enzymes when stored for up to 2 weeks.


Introduction
One of the primary pollutants found in e uents is phenolic compounds commonly found in aqueous e uents from various industrial activities, such as the petrochemical, textile, plastics, resins, cellulose, and paper industries (Mohammadi et al. 2015). Based on the high toxicity of phenolic compounds, trace concentrations are already su cient to alter the organoleptic properties of water (Ilavský et  One way to reduce the cost of using enzymes in industrial processes, a signi cant limitation for their large-scale use, is to use enzymes derived from crude extracts, which are cheaper than commercial enzymes, and the use of the immobilized enzyme, which allows their reuse (Eş et al. 2015; Yamada et al. 2006).
The present work aims to study the use of granular activated carbon and activated chitosan beads as supports for the immobilization of the tyrosinase enzyme present in a crude enzyme extract, obtained from the mushroom Agaricus bisporus, optimizing the immobilization by studying the in uence of different parameters (mass support, agitation, contact time), for its future use in phenol removal from a synthetic e uent, in order to obtain an e cient alternative for phenol removal from industrial e uents.

Materials
The crude enzyme extract used containing the enzyme tyrosinase (EC 1.14.18.1) was produced from Agaricus bisporus macrofungus, purchased in lots at a local market.
For the phenol immobilization and enzymatic oxidation assays, two types of support were used, the commercial Carbotrat AP Granular Activated Carbon, supplied by Indústria Química Carbonífera Criciuma S.A, and chitosan beads, produced in the laboratory. All chemicals containing analytical purity and were purchased from Sigma-Aldrich.

Tyrosinase Enzyme Extraction Methodology
The enzymatic crude extract was obtained following the methodology developed by Kameda et al. (2006), in which the fruiting bodies of Agaricus bisporus are ground in 1:1 (g/mL) chilled acetone, with a maximum of 500.0 g at a time. The mixture was ltered under vacuum, and the residue was stored at 273K for 24 h. Then the mushroom paste was re-suspended with distilled water and stored again at 273K for 24 h to obtain the rst enzyme extract by centrifugation at 418.9 rad/s for 10 minutes. The process of re-suspension and centrifuging the slurry was performed again to obtain a second extract. The measurement of tyrosinase enzymatic activity was determined following the adapted procedure of Santos et al. (2013).

Supports
Carbotrat AP is a granular activated carbon produced in Brazil and supplied by Criciuma S.A Carboniferous Chemical Industry.
Chitosan beads were prepared based on the modi ed procedure of Zhou et al. (2013), where 2.0 g of medium molar weight chitosan was added in 100.0 mL of 5.0 % (v/v) acetic acid solution under stirring for 30 minutes. The solution was then dripped through an approximately 1mm plastic tip coupled to a peristaltic pump into 1000.0 mL of 2.0 mol/L sodium hydroxide solution and ethanol (4:1) for 24 hours. The formed beads were kept in contact with distilled water for 24 hours to remove traces of sodium acetate, sodium hydroxide, and acetic acid and were ltered and washed with distilled water until reaching a neutral pH value.

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The beads were kept in a watch glass for 24 hours to be dehydrated and, consequently, have their mechanical strength increased.
For more e cient immobilization, the chitosan beads were activated with glutaraldehyde (GA). Activation was performed by keeping the chitosan beads in contact with a solution containing up to 3.0 % GA (m/V) in gentle agitation for 90 minutes. After this time, beads were vacuum ltered and stored under refrigeration.

Immobilization Of Enzyme Present In The Crude Extract
Adsorption immobilization on granular activated carbon The adsorption method used for immobilization on granular activated carbon as support was developed by Silva et al. (2005). For it to occur, a determined amount of support and 50.0 mL of enzymatic crude extract solution, which contained, on average, an enzymatic activity of 3000.0 U (in 0.1 mol/l phosphate buffer, pH 7.0), were placed in an erlenmeyer. It was in contact for 120 minutes, in constant agitation.
After that, the enzymes immobilized were vacuum ltered.
The enzyme immobilization rate in the activated carbon was made indirectly from data of the residual enzyme activity (U) of the enzymatic solution about to with concerning the initial enzymatic activity of the enzymatic crude extract solution. This method is used because it is not possible to determine the enzymatic activity of the enzyme immobilized on the support (

Covalent binding immobilization on activated chitosan beads
For covalent immobilization, 5.0 g of activated chitosan beads and 20.0 mL of enzymatic crude extract solution, which contained, on average, an enzymatic activity of 2000.0 U (in 0.1 mol/L phosphate buffer, pH 7.0), were placed in an erlenmeyer. Enzyme solution and beads were in contact for up to 120 minutes, with constant stirring and temperature, in 15.7 rad/s. After the contact period, vacuum ltration was performed to separate the residual enzyme solution from the support containing the adsorbed enzyme. The enzyme immobilization rate was indirectly determined, as described in previous item.
Enzymatic oxidation of phenol using immobilized enzyme Phenol removal was calculated considering the percentage of initial concentration and nal phenol concentration in the aqueous solution. The phenol concentration of the solutions was determined by the direct colorimetric method, described in Standard Methods (APHA 1995), applying 4-amino-antipyrine reagent as a complexing agent. This method allows the dosing of single phenol and substituted phenols, except in the para position.

Characterization
The characterization of Carbotrat AP granular activated carbon surface, before and after immobilization, and chitosan beads, before GA activation, after activation, and after enzyme immobilization, was performed by Scanning Electron Microscopy (SEM) analysis, providing the surface photographs and by analyzing the Fourier transform infrared (FT-IR) spectroscopy.  Some differences can be observed between the Carbotrat AP granular activated carbon FT-IR spectrum before and after immobilization of the enzyme. The spectrum of infrared support after immobilization presents peaks indicating the presence of histidine, which is part of the tyrosinase structure. It is also observed that the adsorption of the enzyme did not change the activated carbon structure. The appearance of two bands on 3700 -3600 cm −1 region in FT-IR spectrum of Carbotrat AP granular activated carbon infrared after immobilization (b), corresponding to the stretching frequency of the N-H binding, is due to the interaction of tyrosinase with the activated carbon matrix (Kennedy et al. 2007). In addition, the band that appears near 1400 cm −1 in the activated carbon spectrum after immobilization (b) is indicative of C-N bonding, which con rms the presence of tyrosinase immobilized on the matrix. It is noted that this band was absent in the activated carbon spectrum before immobilization (a).

Support Characterization
In immobilization using chitosan support, the enzyme tyrosinase was immobilized on the outside of the beads by covalent bonding with amino groups support followed by crosslinking with the bifunctional agent, GA. Beads had diameters between 3.31 mm and 4.15 mm. Fig. 3 (a) and 3 (b) show the scanning electron microscopy of the chitosan beads before and after activation with GA, and Fig. 3 (c) shows the scanning electron microscopy activated chitosan bead after immobilization with the tyrosinase enzyme contained in a crude enzyme extract.
The analysis of the images obtained by scanning electron microscopy provided the beads surface morphology. Chitosan beads before activation with GA had a rougher and non-uniform surface, whereas activation makes the bead surface more uniform and smooth. Figure 4 presents the comparison among FT-IR spectrum of a chitosan bead before activation (a), after activation (b), and after enzyme tyrosinase immobilization (c).
The infrared spectrum of chitosan beads is characterized by the presence of a large band between 3400 and 3200 cm −1 , which is attributed to the axial deformation of the O-H group associated with other polar groups via intra and intermolecular and axial deformation N-H, normally obscured by hydrogen bonding with OH groups. The activation with GA is con rmed with the appearance of a peak in the region around 2900 cm −1 and the region near 1600 cm −1 , as a result of the interaction of GA with chitosan functional groups. Just like observed the FT-IR spectrum of activated carbon after immobilization, the presence of tyrosinase is con rmed by the existence of the most pronounced peak in the region of 1100 cm −1 , indicative of the presence of histidine, the most signi cant component of the tyrosinase enzyme (Kennedy et al. 2007). Immobilization is also responsible for the disappearance of the peak in the region around 2900 cm −1 , indicating the interaction of tyrosinase with the modi ed chitosan surface.

Immobilization on Granular Activated Carbon
The immobilization in granular activated carbon occurs by physical adsorption, so enzyme desorption is easier to take place in case of operational changes. Immobilization assays were performed, using as support the Carbotrat AP Granular Activated Carbon, in the amount of 10.0 g and an enzyme solution with 3000.0 U of activity, up until 120 minutes of contact between the support and enzymatic crude extract. Some authors have observed that during immobilization of enzymes in GAC, the percentage of immobilized enzymes stabilized in less than 120 minutes of contact time between enzyme and support   Table 1 presents the immobilization rates found in different stirring speeds after the contact time between the enzymatic solution and support.

Immobilization on Activated Chitosan Beads
The immobilization using activated chitosan bead support occurs by covalent bonding. Immobilization Assays were performed using 5.0 g of chitosan beads activated with 1.  From these results, the best contact time was 120 minutes and using chitosan beads activated with 3.0 % of GA as support, resulting in immobilization of approximately 100.0 %, higher than that obtained with immobilization by adsorption on granular activated carbon and similar to that found by Chavita (2010) and Miyaguti (2011), but higher than that found by Santos et al. (2013).

Enzymatic Oxidation Assays
In uence of Initial Phenol Concentration  Table 2.

Reuse of Immobilized Enzyme
Immobilized enzymes with the initial enzymatic activity of 1500.0 U on granular activated carbon support and initial enzymatic activity of 2000.0 U on activated chitosan beads were used for the assays. After each assay, the immobilized enzyme was washed with distilled water and phosphate buffer pH 7.0 and subjected to vacuum ltration. Assays were performed using the immobilized enzyme for phenol removal from phenol-containing solutions at concentrations of 10.0 and 60.0 mg/L.
For phenol removal on 10.0 mg phenol/L solutions using the immobilized enzyme in activated carbon support Carbotrat AP, it was observed that, in the second cycle of use, there was a 4.5 % drop in yield over the 100.0 % removal yield of the rst cycle, and yield drop greater than 50.0 % in its third use ( Fig. 7 (a)).
Removal using the immobilized enzyme on activated chitosan bead was able to remove up to 100.0 % of phenol in the rst three uses. From the fourth use onwards, there was a 15.0 % drop in yield. The enzyme immobilized on activated S24 beads showed lower reductions than the enzyme immobilized on activated carbon, maintaining the phenol removal rate close to 70.0 % until the sixth use ( Fig. 7 (b)).
Considering the removal of phenol in 60.0 mg/L solutions, it could be seen again that the use of the immobilized enzyme in Carbotrat AP activated carbon made a yield drop from the second cycle onwards, with an 18.9 % decrease compared to 66.2 % during the rst cycle, and at the third use, the removal yield drop was 63.8 % (Fig. 8 (a)). Immobilized enzymes on activated chitosan beads were able to remove 9 8.0 % of phenol in its rst use, and from the second use, there was a yield drop of 1.2 % and, differently from removal using GAC as support, only in the fourth cycle of use phenol removal yield drop was greater than 50.0 % (Fig. 8 (b)).
The drop in enzymatic oxidation yield may cause prolonged exposure of the enzyme to the phenol degradation product (which explains a higher yield drop in more concentrated phenol solutions), the adsorption of this degradation product to the support (which causes inactivation of the tyrosinase enzyme) and also by the loss of support that occurs between uses either during enzymatic oxidation or during the washing and ltration process after testing. In addition, the sharpest drop in yield during reuse of the immobilized enzyme in GAC is caused by the enzyme desorption due to the stirring of the system during enzymatic oxidation.

Support Reuse
The reuse of the support in future immobilizations is very interesting from the economic point of view, but it was only possible to reuse the support for immobilizations in GAC, due to the type of immobilizationphysical adsorption -in which it is possible to remove the immobilized enzyme after its loss of activity. Table 3 shows the reuse of the support as a function of the immobilized enzyme rate, when the initial enzymatic activity refers to the enzymatic solution and the nal enzymatic activity refers to the estimated activity of the immobilized enzyme on the support. The rate of immobilization declined with each use of the same support, but this decrease was less than 10.0 %, which can be considered a very positive result, as the reuse of the support would help lower the cost of using the immobilized enzyme, giving higher economic viability for it.
Enzyme oxidation assays were performed using the immobilized enzyme on reused support (second immobilization with the support), also reusing the immobilized enzyme on this support to remove phenol from an aqueous solution containing 10 mg/L of phenol.
The results of enzymatic oxidation as a function of phenol removal rate are expressed in Fig. 9.
It was possible to reach the limit established by CONAMA 430 (MMA 2011) for phenol-containing e uent disposal in the rst two uses of the immobilized enzyme. There was also a sharp drop in phenol removal from the rst use to the third. Just the same as with the immobilized enzymes in new support, it was also veri ed, but in a smaller proportion. The drop in removal can still be explained by the loss of the enzyme during the test and its inactivation in the activated carbon.
Since it is not possible to reuse the activated chitosan beads support because adsorption occurs by covalent bonding (chemically joining the enzyme to the support surface), it has been studied a way of decreasing the yield drop of the immobilized enzyme in this support type. As a cause for the drop in phenol removal yield is the adsorption of o-quinone on the bead surface, inactivating the tyrosinase enzyme, successive washing steps with pH 6.7 and 8.0 buffers, and distilled water after each cycle were added to the enzymatic oxidation test. To verify the e ciency of the use of these new steps, enzymatic oxidation assays were carried out using the enzyme-support system for 5 cycles in 10.0 and 100.0 mg phenol/L solutions. In the 10.0 mg phenol/L removal, the new cleaning methodology after each assay allowed the phenol removal yield to fall only 0.9 % after 5 cycles, unlike the enzymatic oxidation cycles using the old method. From the fourth cycle onwards, it dropped by 15.0 %, reaching 28.8 % on the fth cycle. Yield to enzymatic oxidation to phenol is shown in Fig. 10.
For the removal of 100.0 mg/L (Fig. 11), although the use of successive washing steps after each cycle was not su cient for the maintenance of enzymatic activity, its application still caused the decrease of phenol removal yield in the initial cycles, allowing the immobilized enzyme to be used ve times with a phenol removal above 50.0 %. There has been a 33.7 % yield drop after 5 uses of the immobilized enzyme, whereas, with the old cleaning methodology, this drop was almost 50.0 %.

Immobilized enzyme storage
One of the most important parameters to consider in enzymatic immobilization is storage stability. In general, storing enzymes in solution makes them unstable and leads to a decline on activity.
In order to verify the behavior of the enzymatic activity concerning the storage time, 5 immobilizations were performed under the same conditions for each support used, using immobilized enzymes with activities of approximately 1500.0 U for immobilization in GAC and 2000.0 U for immobilization in beads. Activated chitosan was stored under refrigeration for later use in enzymatic oxidation assays at different times: 1 day, 1 week, 2 weeks, 3 weeks, and 4 weeks after immobilization. Enzymatic oxidation using immobilized tyrosinase 1 day after immobilization reached 100.0 % of phenol removal within 60 minutes for immobilization on both support types and similar to that found by some authors such as Chavita (2010) and Pigatto (2013). Analyzing the graphs, it is shown that phenol removal slows down as the time between immobilization and enzymatic oxidation increases and that after the fourth week of storage, the drop in activity is more pronounced in both cases.
The graph in Fig. 14  The stability of the immobilized enzyme in GAC and ACB concerning the storage time between its rst and second use in enzymatic oxidation assays was also investigated. Again, 5 immobilizations were performed for each support type, under the same operating conditions, generating 5 support enzyme systems containing an initial enzyme activity of 1500.0 U and 2000.0 U, respectively. Each enzyme support system was then used in enzymatic oxidation of an aqueous 10.0 mg phenol/L solution and stored under refrigeration after the assay. Once reserved, each enzyme support system was reused with time between two different uses: 1 day, 1 week, 2 weeks, 3 weeks, and 4 weeks after their rst use.
The graphs show in Fig. 15 and 16 illustrate the phenol concentration decay concerning the contact time of the aqueous phenol solution, with the initial concentration of 10.0 mg/L, with the immobilized enzyme on GAC (Fig. 15) and ACB (Fig. 16)   Phenol removal yield remains unchanged using immobilized enzymes on GAC and ACB for up to 2 weeks. After 4 weeks, a 15.0 % yield drop occurs using GAC immobilized enzymes and 12.6 % using ACB immobilized enzymes. After its rst use, the immobilized enzyme on ACB is more stable, with only a 4.4 % drop in phenol removal yield after 4 weeks.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Availability of data and materials
The raw data of experimental results reported, as well as the materials used in this work, are available upon reasonable request.

Competing interests
The authors declare that they have no competing interests.  Enzymatic oxidation using (a) immobilized tyrosinase on GAC 1 day before of its use, (b) immobilized tyrosinase 1 week before use, (c) immobilized tyrosinase 2 weeks before use, (d) immobilized tyrosinase 3 weeks before use, and (e) immobilized tyrosinase 4 weeks before use. Experimental conditions: initial activity of 1500.0 U, constant stirring of 15.7 rad/s, 298 K, and 120 minutes contact time