Journal of Colloid and Interface Science 646 (2023) 413–425 Contents lists available at ScienceDirect Journal of Colloid And Interface Science journal homepage: www.elsevier.com/locate/jcis Immobilization of Agaricus bisporus Polyphenol Oxidase 4 on mesoporous silica: Towards mimicking key enzymatic processes in peat soils Claudia Iriarte-Mesa a, b, Matthias Pretzler c, Cornelia von Baeckmann a, d, Hanspeter Kählig e, Regina Krachler f, Annette Rompel c, *, Freddy Kleitz a, * a Department of Inorganic Chemistry − Functional Materials, Faculty of Chemistry, University of Vienna, Währinger Str. 42, 1090 Vienna, Austria b Vienna Doctoral School in Chemistry (DoSChem), University of Vienna, Währinger Str. 42, 1090 Vienna, Austria c Universität Wien, Fakultät für Chemie, Institut für Biophysikalische Chemie, Josef-Holaubek-Platz 2, 1090 Vienna, Austria; www.bpc.univie.ac.at d Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC, and Barcelona Institute of Science and Technology, Campus UAB, Bellaterra, 08193 Barcelona, Spain e Department of Organic Chemistry, Faculty of Chemistry, University of Vienna, Währinger Str. 38, 1090 Vienna, Austria f Department of Inorganic Chemistry, Faculty of Chemistry, University of Vienna, Währinger Str. 42, 1090 Vienna, Austria G R A P H I C A L A B S T R A C T A R T I C L E I N F O A B S T R A C T Hypothesis: The use of immobilized enzyme-type biocatalysts to mimic specific processes in soil can be considered Keywords: Tyrosinase one of the most promising alternatives to overcome the difficulties behind the structural elucidation of riverine Mesoporous silica humic-derived iron-complexes. Herein, we propose that the immobilization of the functional mushroom Abbreviations: AbPPO4, Agaricus bisporus Polyphenol Oxidase 4; APTS, 3-aminopropyltriethoxysilane; ATR-FTIR, Attenuated total reflectance Fourier-transform infrared spectroscopy; BET, Brunauer-Emmet-Teller; CP, Cross-polarization; DSC, Differential scanning calorimetry; EA, Enzymatic activity; L-DOPA, 3,4-dihydroxy- L-phenylalanine; MAS NMR, Solid-state magic angle spinning nuclear magnetic resonance spectroscopy; MS, Mass spectrometry; NLDFT, Non-local density func­ tional theory; Pluronic P123, Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(-ethylene glycol); PPO, Polyphenol oxidase; PSD, Pore size distribution; SAHL, Small aquatic humic ligand; SBA-15, Santa Barbara Amorphous type material #15; SBA-15-NH2, APTS-modified SBA-15; SBA-15-NH2-Tyr, AbPPO4 loaded on APTS-functionalized silica; SBA-15-Tyr, AbPPO4 loaded on non-functionalized silica; SDS, Sodium n-dodecyl sulfate; TBC, 4-tert-butylcatechol; TEM, Trans­ mission electron microscopy; TEOS, Tetraethyl orthosilicate; TGA, Thermogravimetric analysis; UHPLC, Ultra-high-performance liquid chromatography. * Corresponding authors at: Department of Inorganic Chemistry − Functional Materials, Faculty of Chemistry, University of Vienna, Währinger Str. 42, 1090 Vienna, Austria. (F Kleitz). Universität Wien, Fakultät für Chemie, Institut für Biophysikalische Chemie, Josef-Holaubek-Platz 2, 1090 Vienna, Austria. (A Rompel). E-mail addresses: annette.rompel@univie.ac.at (A. Rompel), freddy.kleitz@univie.ac.at (F. Kleitz). https://doi.org/10.1016/j.jcis.2023.04.158 Received 7 March 2023; Received in revised form 24 April 2023; Accepted 28 April 2023 Available online 1 May 2023 0021-9797/© 2023 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). C. Iriarte-Mesa et al. Journal of Colloid And Interface Science 646 (2023) 413–425 Enzyme adsorption tyrosinase, Agaricus bisporus Polyphenol Oxidase 4 (AbPPO4) on mesoporous SBA-15-type silica could contribute Biocatalysis to the study of small aquatic humic ligands such as phenols. Phenol oxidation Experiments: The silica support was functionalized with amino-groups in order to investigate the impact of SBA-15 surface charge on the tyrosinase loading efficiency as well as on the catalytic performance of adsorbed AbPPO4. The oxidation of various phenols was catalyzed by the AbPPO4-loaded bioconjugates, yielding high levels of conversion and confirming the retention of enzyme activity after immobilization. The structures of the oxidized products were elucidated by integrating chromatographic and spectroscopic techniques. We also evaluated the stability of the immobilized enzyme over a wide range of pH values, temperatures, storage-times and sequential catalytic cycles. Findings: This is the first report where the latent AbPPO4 is confined within silica mesopores. The improved catalytic performance of the adsorbed AbPPO4 shows the potential use of these silica-based mesoporous bio­ catalysts for the preparation of a column-type bioreactor for in situ identification of soil samples. 1. Introduction the production of 6-aminopenicillanic acid from native penicillins and the production of fructose from starch.[23,24] Iron plays a crucial role in the maintenance of marine ecosystems as The immobilization of enzymes in ordered mesoporous materials has it is involved in regulating ocean carbon equilibria and biogeochemical been studied for almost two decades and these have proven to be cycles.[1,2] Complexation with biogenic ligands present in seawater excellent supports to host enzymes.[24–27] Mesoporous materials increases iron solubility and ensures a life-sustaining concentration. possess suitable porosity and have been used successfully to encapsulate [3–5] Small aquatic humic ligands (SAHLs) are inherently iron- a variety of enzymes and other biomolecules.[28] In particular, robust containing compounds that play a key role in reducing the iron limita­ mesoporous silica (pore size between 2 and 50 nm) can be easily pre­ tion of phytoplankton growth in the marine environment, thereby pared by a combination of templating (using surfactants) and inorganic enhancing the marine biological carbon pump, a key process for sol–gel processes, generally under aqueous conditions.[29] The pres­ capturing CO2 on a global scale.[6] SAHLs have recently been shown to ence of surface silanol groups (Si-OH) allows interactions with enzymes be the most important group of iron-bearing humic substances in peat- and the grafting of a wide variety of functionalities through coupling affected rivers.[7] However, up to now, little is known about the mo­ reactions with organosilanes.[30–32] Ordered mesoporous silica of the lecular structures of these iron-chelating ligands, due to their hetero­ SBA-15-type family represents an excellent starting point for the geneity, low overall concentrations and various limitations associated adsorption and immobilization of polyphenol oxidases.[33–36] These with the structural analysis of humic substances.[8,9] silica materials usually have a high surface area (≥800-1000 m2⋅g− 1) Low-molecular-weight phenolic compounds are exceptionally providing many adsorption sites, a high pore volume (>1 cm3⋅g− 1) and abundant in peat soils and can be converted into multidentate Fe- large pore diameter (6–12 nm) for the enzyme to penetrate.[35] The binding SAHLs due to the activity of polyphenol oxidases (PPOs) pro­ morphology and dimensions of the particles can be tuned [37,38] and duced by soil bacteria.[10,11] PPOs are type 3 copper-containing en­ the materials can be fabricated into monolithic objects if required. zymes requiring O2 for their catalytic transformation of phenolic [39,40]. substances.[12,13] PPOs catalyze two different enzymatic reactions To develop an accurate model for the formation of iron-chelating (Scheme 1), the ortho-hydroxylation and subsequent oxidation of ligands, we focused on the immobilization of a functional mushroom monophenols to o-quinones (monophenolase, cresolase activity, EC tyrosinase, Agaricus bisporus Polyphenol Oxidase 4 (AbPPO4, 65 kDa) on 1.14.18.1) and the oxidation of catechols to o-quinones (diphenolase, mesoporous SBA-15. AbPPO4 was heterologously expressed by a catecholase activity, EC 1.10.3.1).[14,15] The reaction products un­ reproducible protocol that provides large quantities of the tyrosinase, dergo many secondary reactions, both with each other and with other free of isoenzyme admixtures and side-activities commonly present in substances (Scheme S2).[16,17] Hence, it is an important task to iden­ commercial preparations isolated from mushrooms.[14] AbPPO4 was tify the products collected from soils in situ. applied as the latent form of the enzyme containing the tyrosinase To overcome the difficulties behind the structural elucidation of iron domain responsible for the catalytic activity as well as the C-terminal complexes from river humus, attempts to immobilize peat-soil derived domain that blocks substrate access to the active site. Activation was PPOs have gained increasing interest.[18–22] Studies on enzyme effected by addition of the anionic detergent sodium n-dodecyl sulfate immobilization on different supports via a variety of routes have (SDS).[12] AbPPO4 was incorporated into the silica mesopores via liquid revealed that enzyme entrapment can increase its stability and decrease adsorption, which ensures easy preparation of the biocatalysts, low denaturation by protein unfolding.[18] Some of the biocatalysts pro­ production costs, and the possibility of reloading the support.[28] The duced are now used commercially at industrial scale, e.g., for the syn­ influence of the surface charge of the silica support on the loading ef­ thesis of L-aspartic acid, the production of acrylamide from acrylonitrile, ficiency of AbPPO4 was evaluated by functionalization of SBA-15 with a positively charged aminosilane, 3-aminopropyltriethoxysilane (APTS), introduced through a post-grafting strategy.[41] The prepared AbPPO4- based catalysts were successfully used for the catalytic oxidation of phenolic compounds, confirming the advantages of enzyme immobili­ zation and confinement, such as improved stability and reusability. The use of these biocatalysts would allow the design of an optimized bioreactor that mimics specific processes in peat soils, leading to the study of humic-derived iron complexes. 2. Materials and methods 2.1. Materials Scheme 1. o-Hydroxylation of monophenols and oxidation of catechols by Tetraethyl orthosilicate (TEOS, 98%), anhydrous toluene (99.8%), polyphenol oxidases. sodium n-dodecyl sulfate (SDS, 99%), sodium (meta)periodate (NaIO4, 414 C. Iriarte-Mesa et al. Journal of Colloid And Interface Science 646 (2023) 413–425 ≥99.8%) and poly(ethylene glycol)-block-poly(propylene glycol)-block- the latent enzyme [14] and 750 μL L-tyrosine (2 mM). Already after 3 poly(-ethylene glycol) (Pluronic P123, EO20PO70EO20, Mn ~ 5800) were min, a pronounced change in color was observed. After 15 min, the purchased from Sigma Aldrich. Trisodium citrate dihydrate (99%), so­ suspensions were centrifuged (11 770 × g, 2 min) and the supernatants dium hydroxide (NaOH, 1.0 N standardized solution), sodium chloride analyzed via UV/Vis, getting the maximum absorbance at 475 nm (L- (NaCl, 99.0%), 3-aminopropyltriethoxysilane (APTS, 98%), citric acid dopachrome). For the enzymatic activity determination of the control anhydrous (>99.5%), L-tyrosine (99%), tyramine (>98%), dopamine (free AbPPO4), L-tyrosine (750 μL, 2 mM), sodium citrate buffer (670 μL, hydrochloride (99%), 3,4-dihydroxy-L-phenylalanine (L-DOPA, >98%) 50 mM) and SDS (30 μL, 100 mM) were mixed in a quartz cuvette. Af­ and sodium hydrogen phosphate, (Na2HPO4, anhydrous, 99%) were terwards, 50 µL of free AbPPO4 (0.67 g⋅L-1) were added and the mixture purchased from Alfa Aesar. Hydrochloric acid (HCl, 37%) and 4-tert- was quickly shaken. The absorbance was measured at 475 nm (ε475 = butylcatechol (TBC, 99%) were purchased from VWR Chemicals and 3600 M− 1⋅cm− 1) [14] every 5 s during 5 min. A line was fitted to the Fisher Scientific, respectively. The functional mushroom tyrosinase, data using Origin Pro 2019 (OriginLab Corp., USA) software, and the Agaricus bisporus Polyphenol Oxidase 4 (AbPPO4, 65 kDa) was heterol­ slope was directly correlated to the enzymatic activity of the free ogously expressed at the Department of Biophysical Chemistry of the AbPPO4. The solid enzyme-loaded catalysts SBA-15-Tyr and SBA-15- University of Vienna, providing a single isoform of tyrosinase NH2-Tyr, as well as the free AbPPO4 (20 g⋅L-1, 50 mM sodium citrate (Figure S1).[14] buffer, pH 6.8), were stored at 4 ◦ C for stability determination over time. The residual enzymatic activity was determined once a week during the 2.2. Synthesis and functionalization of mesoporous SBA-15 first month and later after 24 weeks and one-year storage. SBA-15 silica used as enzyme support was synthesized following the 2.5. Oxidation of phenols by free and adsorbed AbPPO4 procedure described by Guillet-Nicolas et al.[37] Precisely, 8 g of Plur­ onic P123 were first dissolved in a solution of concentrated HCl (4.46 g, AbPPO4-adsorbed silica catalysts (SBA-15-Tyr or SBA-15-NH2-Tyr) 37%) and distilled water (146.25 g). This mixture was stirred at 35 ◦ C were dispersed in a mixture containing citrate buffer (2.4 mL, 50 mM, until complete dissolution of the polymer. When P123 was completely pH 6.8), SDS (100 μL, 100 mM) and 2.5 mL of the phenolic substrate dissolved, 13 g of tetraethyl orthosilicate (TEOS, Sigma-Aldrich, 98%) (dopamine, tyramine, L-tyrosine or TBC, 2 mM). The dispersions were were added to the mixture and incubated under stirring at 35 ◦ C. After placed on a mechanical shaker at room temperature for 20 min, taking 24 h, the mixture was heated to 100 ◦ C in a convection oven (Binder, 100 µL aliquots every minute for the first 10 min and at the end of the Germany) and kept under static conditions at 100 ◦ C for 48 h. The hot experiment. The enzyme present in the sample aliquots was inactivated solution was vacuum filtered without washing (Whatman® filter paper, by adding HCl (1 M, 500 µL) and citrate buffer (400 µL, 50 mM, pH 6.8). Grade 541, diameter 110 mm) and dried for 2 h at 100 ◦ C and then These acidic samples (pH = 3) were filtered (Puradisc™ Polyvinylidene overnight at 140 ◦ C. Pluronic P123 was removed by solvent extraction Difluoride Syringe Filters, diameter 4 mm) and then aliquots of 200 µL for 2 h in a solution of 200 mL of ethanol (96%) with two drops of were taken and diluted with water (800 µL) prior to HPLC/MS analysis. concentrated HCl (37%) followed by calcination at 550 ◦ C under air. The experiment was also performed with the free AbPPO4 (control) SBA-15 was functionalized with APTS by modifying the protocol using 200 µL of enzyme solution (0.5 mg⋅mL− 1 for the reaction with reported by von Baeckmann et al.[41] Typically, 500 mg of calcined tyramine or L-tyrosine, and 12.5 µg⋅mL− 1 when the enzyme was added to SBA-15 were degassed overnight at 150 ◦ C and then dispersed in 80 mL the catechols dopamine or TBC). The conversion values (%) at specific of anhydrous toluene under stirring and argon atmosphere at 115 ◦ C. time-points (t) were expressed as a function of the substrate concen­ After 3 h, 0.6 mL of APTS (5 mmol⋅g-1 of SBA-15) were added and the tration in each sample (ct ) with respect to the initial concentration (c0 ) reaction was stirred overnight at 115 ◦ C. The amino-functionalized SBA- before the addition of the free or adsorbed AbPPO4 (t = 0 min): 15 (SBA-15-NH2) was recovered by centrifugation (9 000 × g, 20 min), ( ) washed once with toluene, twice with ethanol and dried at 80 ◦ C ct Conversion(%) = 1 − × 100 (1) overnight. c0 2.3. Loading of AbPPO4 on mesoporous silica 2.6. Adsorption of phenolic compounds and oxidized products within silica pores Agaricus bisporus Polyphenol Oxidase 4 (AbPPO4, 65 kDa) was introduced into the pores of SBA-15 and SBA-15-NH2 through physical Adsorption of oxidized phenolic compounds on mesoporous silica adsorption. First, enzyme solutions (concentration range from 1 µM to was carried out by dispersing calcined SBA-15 or SBA-15-NH2 (5 mg) in 100 µM) in Tris-HCl buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.1) 2 mL of a mixture containing the phenols tested (dopamine, DOPA or were prepared from a concentrated solution of AbPPO4 (20 g⋅L-1). For TBC; 2 mM; 1 mL), NaIO4 (100 µL, 200 mM), citrate buffer (860 µL, 50 obtaining the adsorption isotherms, 15 mg of SBA-15 or SBA-15-NH2 mM, pH 6.8) and SDS (40 µL, 200 mM). The dispersions were placed in a were slowly shaken in 2.5 mL of these enzyme solutions for 48 h at 4 ◦ C, mechanical rocker for 5 or 20 min at room temperature and then, the followed by centrifugation (9 000 × g, 30 min). The enzyme-loaded solid was centrifuged (11770 × g, 2 min) and the supernatants analyzed materials (SBA-15-Tyr or SBA-15-NH2-Tyr) were lyophilized and via UV/Vis. The isolated solids were washed continuously with SDS stored at 4 ◦ C for further evaluation. The supernatants were collected in (200 mM)/citrate buffer (50 mM) (1:24 v/v) or water, collecting the order to determine the remaining enzyme concentration via UV/Vis. For supernatants for UV/Vis analysis. The silica materials were lyophilized the phenol oxidation experiments and enzyme stability determinations, and stored at 4 ◦ C for further evaluation. the loading protocol was scaled up using 50 mg of SBA-15 or SBA-15- NH2 and a total concentration of AbPPO4 of 5 g⋅L-1 that ensured 2.7. Influence of SDS concentration on the enzyme activity of immobilized maximum loading efficiency. AbPPO4 2.4. Enzymatic activity determination The behavior of the confined enzyme with respect to the SDS con­ centration was carried out by dispersing the AbPPO4-loaded SBA-15 To verify that the enzyme preserved its activity once it is adsorbed on (SBA-15-Tyr, 2 mg, 38 wt% of AbPPO4) in citrate buffer (50 mM, pH the silica supports, 10 mg of the enzyme-loaded silica (SBA-15 or SBA- 6.8) and varying the volume of SDS (10 mM) added to the mixture. The 15-NH2) were suspended in 720 μL sodium citrate buffer at pH 6.8 (50 volumes of citrate buffer (50 mM, pH 6.8) and SDS (10 mM) were mM) followed by the addition of 30 μL SDS (100 mM) for activation of adjusted such that the final concentration of SDS after adding L-tyrosine 415 C. Iriarte-Mesa et al. Journal of Colloid And Interface Science 646 (2023) 413–425 (750 µL, 2 mM) was 0.1, 0.25, 0.5, 1.0, 2.0, and 4 mM in the total re­ EtOH deposited on a carbon-coated copper grid. Low-angle diffraction action volume of 1.5 mL. The experiment was also carried out in the data was recorded on a PANalytical Empyrean diffractometer (Malvern absence of SDS. The enzyme activity tests were performed as described PANalytical, United Kingdom) in a transmission geometry (Focusing in section 2.4. mirror) using Cu Kα1+2 radiation operated at a voltage of 45 kV, a tube current of 40 mA and with a fixed divergence slit of 0.76 mm. Mea­ 2.8. Thermal stability of AbPPO4 surements were performed in the continuous mode with a step size for 2θ of 0.013◦ and a data collection time per step of 50 s for the trans­ For the thermal stability determinations, six different dispersions of mission mode. Thermogravimetric (TGA) and differential scanning SBA-15-Tyr (2 mg, 38 wt% of adsorbed AbPPO4) in sodium citrate buffer calorimetry (DSC) analyses were executed using a Netzsch STA-449 F3 (720 µL, 50 mM; pH 6.8) were prepared and slowly stirred (80 rpm) at Jupiter instrument from 25 to 800 ◦ C under airflow of 20 mL⋅min− 1 as 10, 30, 40, 50, 60 and 70 ◦ C for 1 h. Once the samples were at room carrier gas with a heating rate of 10 ◦ C⋅min− 1. The mass losses (%) were temperature, the residual enzymatic activities were tested (section 2.4) estimated in the temperature range from 150 to 700 ◦ C. The zeta- after adding SDS (30 µL, 100 mM) and L-tyrosine (750 µL, 2 mM). The potential measurements of SBA-15 and SBA-15-NH2 were performed thermal stability of free AbPPO4 (50 mL, 0.67 g⋅L-1) was determined with a Malvern Nano Zetasizer ZS. To guarantee correct calibration following the same procedure and used as control. before zeta-potential measurements, a standard suspension (carboxylate modified polystyrene latex microspheres) with a zeta-potential of − 40 2.9. pH-stability of free and confined AbPPO4 (±6) mV was measured. Aqueous suspensions of the materials (0.7 mg⋅mL− 1) were obtained by performing three cycles of vortex shaking The stability of the adsorbed-AbPPO4 was determined in a pH range (10 min) and ultrasonic bath treatment (30 min). The supernatants were from 2 to 8. First, SBA-15-Tyr (2 mg, 38 wt% of loaded AbPPO4) was then analyzed to obtain zeta-potential values. Solid-state magic angle dispersed in 1 mL of McIlvaine buffers (pH = 2.2, 3, 5, 7, and 8) and spinning nuclear magnetic resonance spectroscopy (MAS NMR) was placed in an incubator (4 ◦ C) under gentle stirring (80 rpm) for 1 h. The performed on a Bruker Avance NEO 500 wide bore system (Bruker McIlvaine buffers were prepared from the mixture of Na2HPO4 (0.2 M) BioSpin, Rheinstetten, Germany). A 4 mm triple resonance magic angle and citric acid (0.1 M) at different proportions and subsequent pH spinning (MAS) probe was used. Cross-polarization (CP) was employed adjusting with HCl (0.25 M) or NaOH (0.2 M).[42,43] The catalysts were using a ramped contact pulse thereby sweeping the proton radio fre­ isolated by centrifugation (11770 × g, 2 min) and the respective enzyme quency field from 50 to 100%. For 29Si, the resonance frequency was activity tests were performed as described in section 2.4. The pH sta­ 99.38 MHz, the MAS spinning speed was 8 kHz, and the CP contact time bility of the free enzyme was carried out under the same conditions and was 5 ms. The resonance frequency for 13C NMR was 125.78 MHz, the used as control. The native AbPPO4 (60 µL; 0.67 g⋅L-1) was mixed with MAS rotor spinning was set to 14 kHz, and the CP contact time to 3 ms. 940 µL of McIlvaine buffers and stirred for 1 h at 4 ◦ C. Then, 25 µL of During acquisition, 1H was high-power decoupled using SPINAL with 64 these solutions were transferred to a quartz cuvette containing a mixture phase permutations.[41] Chemical shifts (δ) are reported in ppm and of citrate buffer (695 µL, 50 mM, pH 6.8) and SDS (30 µL, 100 mM). referenced externally for 13C to adamantane by setting its low field Enzyme activity was determined by following the oxidation of L-tyrosine signal to 38.48 ppm. For 29Si the shifts were referenced externally to (2 mM; 750 µL) for 5 min by UV/Vis spectroscopy (λ = 475 nm). The sodium trimethylsilylpropane sulfonate (DSS) by setting the signal to 0 enzyme activities of free and confined AbPPO4 (SBA-15-Tyr) were also ppm. 1H-liquid NMR spectra were recorded at a resonance frequency of measured after dispersing the respective samples for 1 h at 4 ◦ C in citrate 700.40 MHz on a Bruker Avance III HD 700 spectrometer (Bruker Bio­ buffer (50 mM; pH 6.8) and were used as reference. Spin, Rheinstetten, Germany) equipped with a helium cooled quadruple cryoprobe. All measurements were done at a temperature of 25 ◦ C using 2.10. Reusability of adsorbed AbPPO4 on mesoporous silica a mixture of 5% D2O/95% H2O as solvent. The water signal was sup­ pressed by presaturation and for referencing the carrier frequency was The catalyst SBA-15-Tyr (5 mg, 38 wt% of adsorbed AbPPO4) was set to 4.67 ppm. Attenuated total reflectance Fourier-transformed dispersed in sodium citrate buffer (720 µL, 50 mM, pH 6.8) followed by infrared (ATR-FTIR) spectra were recorded using a Bruker Vertex 70 addition of SDS (30 µL, 100 mM) and L-tyrosine (750 µL, 2 mM). After 15 FTIR spectrometer equipped with the Specac Golden Gate ATR acces­ min, the suspensions were centrifuged (11 770 × g, 2 min) and the su­ sory. The spectra were obtained from the acquisition of 72 scans at 4 pernatants analyzed via UV/Vis, getting the maximum absorbance at cm− 1 resolution in the range of 4000 to 500 cm− 1. Before each mea­ 475 nm (L-dopachrome). The solid was redispersed in citrate buffer and surement, a background spectrum was collected, gathered from the vortexed for 3 min before the next catalytic reaction with L-tyrosine. This acquisition of 72 scans at 4 cm− 1 resolution. UV/Vis absorption spectra procedure was repeated for 16 consecutive cycles. were collected on an Onda UV-30 SCAN spectrophotometer at room temperature (25 ◦ C). All samples were analyzed in 1 cm quartz cuvettes. 2.11. Materials characterization The quantification of phenolic compounds and oxidized products was performed using a Vanquish Horizon ultra-high-performance liquid N2-physisorption isotherms measurements were carried out at chromatography (UHPLC) system (Thermo Fisher Scientific, Germering, − 196 ◦ C (77 K) using an Autosorb-iQ3 sorption analyzer (Anton Paar, Germany) coupled to a LTQ Orbitrap Velos mass spectrometer (Thermo Boynton Beach, USA). Before the analysis, calcined SBA-15 and APTS- Fisher Scientific, Bremen, Germany) equipped with an electrospray ion modified SBA-15 (SBA-15-NH2) were outgassed overnight at 150 ◦ C source with the voltage set to 3 kV and an ion transfer capillary tem­ and 80 ◦ C, respectively. The enzyme loaded samples were outgassed at perature of 380 ◦ C. Separation of analytes was carried out on a C-18 35 ◦ C for 20 h prior to analysis. The surface area (SBET) was determined analytical column Acclaim 120 (2.1 × 150 mm, 3 μm, Thermo Fisher using the Brunauer-Emmet-Teller (BET) method [44] in the relative Scientific) at a flow rate of 0.4 mL⋅min− 1. The column oven temperature pressure range 0.05–0.3 P/Po. The pore size distributions (PSD) were was set to 30 ◦ C and the injection volume was 5 μL. The mobile phases estimated using the non-local density functional theory (NLDFT) were A: H2O (0.1% (v/v) formic acid) and B: ACN (0.1% (v/v) formic method on the equilibrium branch, considering an amorphous SiO2 acid). A linear gradient method was applied as follow: 0.0 – 8.0 min: (oxide) surface and a cylindrical pore model. The calculations were 5–53% B, 8.0–8.5 min: 53–95% B, 8.5–12.5 min: 95% B, 12.5–13.0 min: carried out using the ASiQwin 5.2 software provided by Anton Paar 95–5% B and re-equilibration was done until 17 min. The full-scan mass Quantatech Inc.[37] Transmission electron microscopy (TEM) images spectra (MS) were acquired in positive and negative ion mode in the were collected with a Philips CM200 microscope at an accelerating range of 80 – 500 m/z at a resolution of 60000. Samples containing TBC voltage of 200 kV, using suspensions of SBA-15 and SBA-15-NH2 in were measured in negative ion mode in order to detect the phenolic 416 C. Iriarte-Mesa et al. Journal of Colloid And Interface Science 646 (2023) 413–425 substrate. The correspondent oxidized product TBCox, was detected in hysteresis and a capillary condensation at a relative pressure (P/P0) positive ion mode like the rest of the analyzed compounds. SDS-PAGE range of 0.6–0.8 (Fig. S2c). NLDFT pore size distributions were fairly was performed using the classical discontinuous buffer system (stack­ narrow for both materials (Fig. S2d, Table S2), which is expected for ing and separating gel) and the single running buffer according to the mesoporous silica with well-defined and uniform mesopores.[38,48] Laemmli-protocol.[45] The gel used was cast with a linear gradient of From the NLDFT pore size distributions, it was found that the SBA-15 8%-15% acrylamide in the separating gel and 5% acryalmide in the had a pore size of 9.1 nm. Upon modification, the pore size of the stacking gel. Electrophoresis was performed for 30 min at 10 V⋅cm− 1 and SBA-15-NH2 was reduced to 8.1 nm. Likewise, both the specific surface 90 min at 15 V⋅cm− 1 using the Bio-Rad Mini-PROTEAN Tetra Vertical area and the pore volume decreased after functionalization, from 743 to Electrophoresis Cell. Gel staining with Coomassie brilliant blue G-250 403 m2⋅g− 1 and from 1.34 to 0.76 cm3⋅g− 1, respectively (Table S2). Such and Al3+ was carried out according to the protocol of Kang et al.[46] The decreases are consistent with the introduction of the organosilane on the destained gel was then photographed using a Bio-Rad Gel Doc XR Im­ pore surface, without major pore blocking, and still enough space in the aging System. pores of the functionalized silica for the later encapsulation of the AbPPO4 enzyme with a homogenous distribution within the mesopores 3. Results and discussion (Figure S3). Surface functionalization of SBA-15 was also confirmed by thermogravimetric analysis (TGA), from a weight loss of 8.9 wt% (SBA- 3.1. Synthesis and functionalization of mesoporous SBA-15 15-NH2) attributed to the thermal decomposition of the organic moieties on the functionalized silica between 150 and 700 ◦ C (Fig. S4a). The The SBA-15 used as enzyme support in this study was synthesized solid-state 29Si CP/MAS NMR spectrum of SBA-15-NH2 (Figure S5) according to established protocols that yield tunable and high-quality revealed the T2 and T3 signals, which indicates successful chemical silica materials.[47] Materials with different pore sizes could be ob­ modification of the SBA-15 silica surface. No T0-species were observed, tained by varying the temperature of the hydrothermal treatment during confirming the absence of non-covalently attached APTS (Table S1). the synthesis.[38] Based on the literature, the synthesis conditions of Moreover, in the solid-state 13C CP/MAS NMR spectrum of SBA-15-NH2 SBA-15 were chosen to ensure the formation of pores large enough for (Fig. 1a), three resonance signals were observed at 42.9, 25.5 and 9.0 enzyme encapsulation and keeping it confined within the mesoporous ppm, attributed to the carbons of the silane propyl chain, located in structure. The obtained SBA-15 was functionalized with a positively alpha, beta and gamma positions of the amine, respectively. charged amino-silane, 3-aminopropyltriethoxysilane (APTS), through a Additional confirmation of the successful surface modification of the classical post-grafting strategy.[41] SBA-15 was obtained through Zeta-potential measurements in nanopure The mesostructured ordering of SBA-15 and SBA-15-NH2 was veri­ water (Fig. 1b), whereas the amino-modified silica SBA-15-NH2 fied by low-angle XRD measurements (Fig. S2a). Three well-resolved exhibited a positive surface charge over the pH range tested, in contrast diffraction peaks were observed in the corresponding diffractograms to its all-silica precursor SBA-15. The Zeta-potential of both materials and were indexed as 100, 110, and 200 reflections, typical of two- was also measured in Tris-HCl buffer (50 mM, pH = 7.4), the same dimensional (2D) hexagonal mesostructures with a pore symmetry of medium in which the loading of the AbPPO4 enzyme was performed p6mm. The positions of three diffraction peaks for SBA-15-NH2 were later. The Zeta-potential values obtained in the buffer medium were similar to its all-silica precursor SBA-15, indicative of the retention of − 10.3 (±0.6) mV and +13.9 (±0.6) mV for SBA-15 and SBA-15-NH2, the ordered pore structure after surface functionalization. The TEM respectively. Considering that AbPPO4 is negatively charged under these images of both materials (Fig. S2b) revealed the periodic alignment of conditions,[49] this enabled the evaluation of the effect of surface the channel-like pores, which is in agreement with the low-angle XRD charge on the support–enzyme interactions and the catalytic perfor­ data. mance of the immobilized enzyme. Both materials exhibit a typical type IVa isotherm with a sharp H1 Fig. 1. (a) Solid-state 13C CP/NMR spectra of APTS-functionalized silica SBA-15-NH2 (b) Zeta-potential of SBA-15 (grey) and SBA-15-NH2 (blue), measured over the pH range 2–8 as well as in the buffer used for enzyme loading (50 mM Tris-HCl, pH = 7.4; red circle and dot). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 417 C. Iriarte-Mesa et al. Journal of Colloid And Interface Science 646 (2023) 413–425 3.2. AbPPO4 adsorption and enzyme activity increasing loaded enzyme concentration indicated that the pores are progressively filled by AbPPO4 and its confinement in the silica The polyphenol oxidase AbPPO4 was inserted into silica pores by mesopores. physical adsorption. Equilibrium enzyme adsorption isotherms were The same trend was observed for the low angle-patterns of different obtained by preparing solutions at different defined total concentrations SBA-15-NH2-Tyr samples (Figure S8), which suggested rather uniform of AbPPO4 that were put in contact with SBA-15 and SBA-15-NH2 in enzyme confinement within the amino-functionalized mesopores. To Tris-HCl buffer (pH 7.2) for 48 h at 4 ◦ C. The supernatants obtained after rule out the possible presence of adsorbed enzyme at the mouth of the the enzyme loading were analyzed via UV/Vis in order to determine the mesopores due to the interaction of AbPPO4 with amino groups of the remaining enzyme concentration (Figure S6). The enzyme quantifica­ external surface of SBA-15-NH2, a physical mixture was prepared by tion was performed following the band at 280 nm correspondent to the adding a solution of AbPPO4 (3.1 µmol⋅g− 1 of silica) to SBA15-NH2 absorbance from tryptophan, phenylalanine and tyrosine residues of the before freeze-drying. Subsequently to the drying, the low angle-XRD AbPPO4.[50] Using this absorption, the amount of AbPPO4 in the su­ analysis was performed and the intensity normalized relative to the pernatant was calculated. Subtraction of that value from the total silica amount. As observed in Figure S8, the intensity of the diffraction applied amount of AbPPO4 provided the adsorbed amount of AbPPO4. peaks of this sample (green dotted line) was similar to the control (SBA- The data shown in Fig. 2a indicates a steep linear increase of adsorption 15-NH2, black dotted line) without enzyme. These results support the with increasing enzyme concentration until reaching a plateau, which confinement of AbPPO4 within the silica mesopores. corresponds to the maximum amount of AbPPO4 absorbed on SBA-15 To verify that the tyrosinase preserved its catalytic performance (7.2 μmol⋅g-1of silica) and SBA-15-NH2 (3.2 μmol⋅g− 1 of silica). TGA throughout the immobilization process, enzymatic activity tests were of the enzyme-loaded materials revealed an increase in mass loss from performed following the oxidation of L-tyrosine (2 mM) by UV/Vis in 2.2 to 38 wt%, consistent with increasing equilibrium adsorbed enzyme citrate buffer at pH 6.8 (Fig. 3). concentrations in the isotherm (Fig. 2b, Figure S6, b-c), with the highest When testing the tyrosinase-loaded silicas (SBA-15-Tyr and SBA-15- mass loss for the highest concentrations of immobilized enzyme, due to NH2-Tyr) containing different amounts of adsorbed AbPPO4, a clear the plateau regime (38 wt% and 16 wt% for enzyme-loaded SBA-15 and color change was observed already after 3 min interaction with L-tyro­ SBA-15-NH2, respectively). The lower adsorption capacity of SBA-15- sine (Fig. 3, inserts) due to the formation of the oxidation product L- NH2 in comparison to SBA-15 was attributed to its lower pore size and dopachrome (Fig. 3, Figure S11). After 15 min, the suspensions were overall lower porosity, which prevented the adsorption of higher centrifuged and the supernatants analysed via UV/Vis spectroscopy. The amounts of AbPPO4. Although the introduction of amino-functionalities spectra shown in Fig. 3 correspond to mixtures of 200 μL of supernatants changed the external surface charge of SBA-15 to positive values as solutions with 800 μL of citrate buffer. The enzyme concentration observed in Fig. 1b, favoring the electrostatic interaction of SBA-15-NH2 dependence of the reaction was clearly observed since the highest with the negatively charged AbPPO4, the presence of the hydrophobic amount of L-dopachrome (absorption band centred at 475 nm) could be carbon chain of the APTS could also have decreased the polarity of the detected for the highest concentrations of immobilized enzyme for both grafted silica compared to the non-functionalized support.[41] This SBA-15-Tyr (38 wt%, Solution 7, Fig. 3a) and SBA-15-NH2-Tyr (16 wt%, could additionally limit the amount of enzyme loaded on SBA-15-NH2- Solution 6, Fig. 3b). These observations provided a strong first indica­ Tyr. tion that the latent enzyme retained its activity after immobilization. Furthermore, N2-physisorption analysis at 77 K performed on loaded The enzyme activity test was also carried out using a solution of free samples, SBA-15-Tyr and SBA-15-NH2-Tyr (Figure S7; Table S2) showed AbPPO4 at the same maximum concentration of immobilized tyrosinase a substantial decrease in pore volume and surface area of the materials on SBA-15 (7.2 μmol⋅g− 1 of SBA-15) and under the same conditions after the enzyme adsorption, leading to the assumption that most of the (solution labelled as T, Fig. 3a). The maximum absorption at 475 nm enzyme was indeed located within the pores. This could also be (Fig. 3a, dotted black line) was similar to the maximum absorbances corroborated by low angle-XRD measurements (Fig. 2c). In the dif­ obtained when the samples with the highest loaded amount of AbPPO4 fractograms of the AbPPO4-loaded SBA-15-Tyr samples, the diffraction were tested. This constitutes strong evidence that the enzymatic activity peaks indexed as 100, 110, and 200 reflections were observed as a result of AbPPO4 is not significantly affected when it is adsorbed on silica. For of retention of ordered structure after enzyme loading, as expected. tyrosinase-loaded silica SBA-15-Tyr (7.2 μmol⋅g-1 of SBA-15), the Moreover, their systematic broadening and decrease in intensity with enzymatic activity experiment was performed without the addition of Fig. 2. Enzyme adsorption isotherms of SBA-15 and SBA-15-NH2 silica at 4 ◦ C obtained by (a) UV/Vis and (b) TGA. (c) Low-angle XRD patterns of the different tyrosinase-loaded samples on non-functionalized SBA-15 (SBA-15-Tyr). Scattering intensities were normalized with respect to the amount of silica in each sample. 418 C. Iriarte-Mesa et al. Journal of Colloid And Interface Science 646 (2023) 413–425 Fig. 3. UV/Vis spectra of the diluted supernatants (5 times dilution) after 15 min of reaction of L-tyrosine with (a) AbPPO4-loaded SBA-15 silica and (b) AbPPO4- loaded SBA-15-NH2. Inserts: corresponding photo of the samples. (c) Concentration of L-dopachrome produced in the catalytic reaction as a function of concentration of AbPPO4 loaded on SBA-15-Tyr (grey line) or SBA-15-NH2-Tyr (blue line). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) substrate (Control, solution C) and no activity was observed in these interaction between the positively charged carrier and the immobilized conditions. The supernatant obtained after separating the solid was tyrosinase in the pores. analysed via UV/Vis spectroscopy. The negligible adsorption observed at 280 nm (Fig. 3a, dotted red line) confirmed that the immobilized enzyme is not desorbed from silica during enzyme activity assays and 3.3. Oxidation of phenolic compounds hence the observed enzyme activity in the presence of substrate origi­ nates from the immobilized enzyme. The oxidation of L-tyrosine and other phenolic compounds (TBC, To further confirm that the immobilized AbPPO4 is not released in dopamine and tyramine) was followed individually by HPLC/MS using solution from the silica support an additional leaching test was per­ SBA-15-Tyr (38 wt% of latent AbPPO4) and SBA-15-NH2-Tyr (13.2 wt% formed (Figure S9). The conditions for the enzymatic activity test were of AbPPO4), as represented in Scheme S1. adjusted for SBA-15-Tyr but without adding substrate (L-tyrosine). The At different time-points of the reactions, 100 μL aliquots were taken catalyst was shaken in the presence of citrate buffer and SDS, and after and the reaction was stopped by acidifying the medium with HCl (0.1 M) 15 min the solid was isolated by centrifugation. The negligible absorp­ to inactivate the enzyme (pH = 3). The samples were then centrifuged tion observed at 280 nm in the supernatant suggested the absence of and filtered for subsequent HPLC/MS measurements. The MS analysis desorbed enzyme in solution (grey line), as previously described. After allowed elucidating the structure of the oxidized products and deter­ adding L-tyrosine (final concentration 1 mM), only the band at 275 nm mining their relative proportion with respect to the initial concentration corresponding to the absorbance from the added substrate was observed of the phenolic substrate (Fig. 4). For each phenolic compound studied, (blue line). However, after 15 min of interaction of the isolated solid mixtures of several oxidation products were detected according to the with L-tyrosine, the mixture was centrifuged and the corresponding su­ gradual decrease in the relative abundance of the substrate (Scheme S2). pernatant analyzed by UV/Vis. An intense absorption band at 475 nm For dopamine, L-tyrosine and tyramine, also intense signals corre­ was observed, indicating the oxidized product L-dopachrome (red line). sponding to deamination ions (M − 17) + were detected as result of the The intensity of this band was similar to the control (dotted black line), i. NH3-loss.[54] e., the same amount of SBA-15-Tyr where the enzyme activity was Interestingly, upon oxidation of tyramine (m/z = 138), the product evaluated by adding L-tyrosine at the beginning of the experiment. These obtained as a result of o-hydroxylation of monophenol (dopamine, m/z results confirm that the enzyme remains confined in the mesopores = 154) seemed to stabilize and took longer to produce the corresponding during the catalytic reaction. dopachrome derivative (m/z = 150) when the reaction was catalyzed by From the enzymatic activity studies, it was possible to calculate the confined AbPPO4. When the free enzyme was used to catalyze the same concentration of L-dopachrome produced after 15 min of reaction of L- reaction, the relative proportions of the dopamine intermediate were tyrosine with the different catalysts obtained (Fig. 3c). The maximum negligible and the major oxidation product was the dopachrome- concentration of L-dopachrome obtained (0.39 mmol⋅L-1 and 0.71 derivative (m/z = 150), the same major product that was determined mmol⋅L-1 for the reaction catalysed by SBA-15-Tyr and SBA-15-NH2-Tyr, when the reaction was carried out with dopamine (m/z = 154) as the respectively) was corresponding to the maximum amount of immobi­ substrate. The corresponding o-quinone (m/z = 152), obtained from the lized AbPPO4 and showed an enhanced biocatalytic performance with oxidation of dopamine, could also be detected. The relative abundance respect to other tyrosinase-immobilized silica catalysts.[51–53] It of this o-quinone intermediate was the highest in the reactions catalyzed should be noted here that the enzyme immobilized on SBA-15-NH2 was by the adsorbed enzyme (SBA-15-Tyr and SBA-15-NH2-Tyr), suggesting able to produce higher concentrations of L-dopachrome in comparison the stabilization of the intermediate within the silica pores. In the case of with equivalent enzyme concentrations in SBA-15-Tyr. Although the the reactions with TBC, the substrate was detected in negative ion mode, adsorption capacity of SBA-15-NH2 was lower than that of SBA-15, the in contrast to the other phenolic compounds investigated. No interme­ immobilized AbPPO4 on the amino-functionalized material showed diate products were observed in positive ion mode during the oxidation better catalytic performance, possibly as a result of a more efficient of the catechol and the formation of the corresponding o-quinone (m/z = 165) was corroborated. 419 C. Iriarte-Mesa et al. Journal of Colloid And Interface Science 646 (2023) 413–425 In the supernatants corresponding to reactions with L-tyrosine (m/z 15-Tyr and SBA-15-NH2-Tyr, respectively. A similar catalytic perfor­ = 182), the monophenol hydroxylation product (DOPA, m/z = 198) and mance was observed during the oxidation of L-tyrosine, where the EA of the correspondent dopaquinone (m/z = 196) were undetectable. The SBA-15-Tyr was 2-fold decreased relative to the free AbPPO4, and was relative levels of dopachrome (m/z = 194) were very low. However, the even enhanced (up to 20%) when the reaction was catalyzed by SBA-15- product of the decarboxylation of dopachrome (m/z = 150) was found, NH2-Tyr. These results demonstrate the positive effect of the amino- as well as the product of the subsequent α-deprotonation (m/z = 148). functionalization of the silica support. Although for both SBA-15-Tyr The dopachrome transformation through oxidation of L-tyrosine by and SBA-15-NH2-Tyr it was verified that the enzyme is confined SBA-15-Tyr, SBA-15-NH2-Tyr and the free AbPPO4 was followed by 1H within the mesopores, AbPPO4 loading on the amino-functional SBA-15 NMR over time (Figure S10). The formation of dopachrome in the early could result in more favorable orientation of the loaded enzyme, arising stages of the reaction was corroborated from the increase in the signal 3 times higher EA values for the amino-functionalized catalyst in com­ intensities at 6.5 ppm and 5.7 ppm. However, when the free AbPPO4 was parison to SBA-15-Tyr, where the AbPPO4 only interacts with the –OH used for the catalysis, a gradual decrease in these signal intensities was moieties of the silanol groups of the non-functionalized support. observed after 10 min, and at the same time the signal at 6.3 ppm was increased (α-deprotonated derivative). The gradual decrease in dop­ 3.4. Adsorption of phenolic compounds and oxidized products within achrome concentration was also observed during the oxidation by SBA- silica pores 15-Tyr and SBA-15-NH2-Tyr, but only after 35 min from the start of the reaction. These results suggested the stabilization of the dopachrome The apparent drop in enzyme activity after immobilization has been due to its adsorption within the silica pores, resulting in its slower previously reported, as well as the alteration of the conformation of the transformation into the α-deprotonated product. Fig. 5 shows the con­ enzyme with respect to its native state.[56–58] Furthermore, the version (%) obtained from the HPLC/MS measurements and calculated decrease in the enzymatic activity could also be attributed to the from the decrease in the initial concentrations of the phenolic substrates. adsorption of the components of the buffer used for catalysis (i.e., citrate For all phenolic compounds tested, conversions between 25% and 92% and SDS) in the silica mesopores. This was corroborated by studying the were achieved after 20 min of reaction, with similar and even improved adsorption of standard solutions of phenolic compounds on SBA-15 and biocatalytic performances compared to free AbPPO4 (control). SBA-15-NH2. The studied compounds (dopamineox, DOPAox and TBCox) Table 1 includes the specific enzyme activity values EA (U⋅mg− 1) were previously oxidized with sodium periodate and put in contact for 5 obtained for each of the reactions studied. The EA was expressed as the or 20 min with SBA-15 and SBA-15-NH2, in the same conditions under amount of free or adsorbed AbPPO4 that catalyzed the reaction of 1 µmol which the enzyme activity studies were performed with the enzyme- of substrate per minute (U), reported per mg of protein.[55] For each of loaded catalysts. the phenolic substrates studied, a considerable drop in the EA of the The mass losses (%) of the solid samples obtained after the adsorp­ immobilized AbPPO4 was observed when compared to the free enzyme, tion were found between 20 wt% and 37 wt% (Fig. 6a). Those values decreasing up to 30-fold for SBA-15-Tyr and 10-fold for SBA-15-NH2-Tyr were similar or even lower than those obtained for SBA-15 and SBA-15- in the reactions with dopamine and TBC (catechols). In the catalytic NH2 samples that did not interact with oxidized phenols but were pre­ reaction with tyramine, the EA decreased only 4-fold and 2-fold for SBA- pared under the same conditions (control samples represented with grey Fig. 4. MS characterization of the products obtained from the oxidation of phenolic compounds at different time periods in correlation with their respec­ tive proportion. 420 C. Iriarte-Mesa et al. Journal of Colloid And Interface Science 646 (2023) 413–425 Fig. 5. Oxidation of phenolic compounds followed by HPLC/MS. The biocatalytic performances of AbPPO4 loaded on non-functionalized SBA-15 (SBA-15-Tyr; 38 wt %) and on the APTS-modified silica (SBA-15-NH2-Tyr; 13.2 wt%) were tested and compared with free AbPPO4 (control). Table 1 Reaction rate (μM⋅min− 1) and specific activity (U⋅mg− 1) of free and adsorbed AbPPO4 during the oxidation of phenolic compounds. Substrate Catalyst AbPPO4 concentration Reaction rate EA (mg⋅L-1) (μM⋅min− 1) (U⋅mg− 1) L-tyrosine SBA-15-Tyr 131.9 25.0 0.19 (46.6%) (1 mM) SBA-15-NH2-Tyr 59.7 29.2 0.49 (120.1%) free AbPPO4 (Control) 20.0 8.2 0.41 tyramine SBA-15-Tyr 134.5 22.7 0.17 (23.5%) (1 mM) SBA-15-NH2-Tyr 61.6 26.4 0.43 (59.8%) free AbPPO4 (Control) 20.0 14.3 0.72 dopamine SBA-15-Tyr 134.5 374.4 2.78 (3.5%) (1 mM) SBA-15-NH2-Tyr 59.4 476.1 8.01 (10.2%) free AbPPO4 (Control) 0.5 39.3 78.6 TBC SBA-15-Tyr 128.2 201.5 1.57 (3.2%) (1 mM) SBA-15-NH2-Tyr 55.3 275.1 4.97 (10.2%) free AbPPO4 (Control) 0.5 24.5 49.0 bars in Fig. 6). These results confirmed that most of the organic matter 15-Tyr and SBA-15-NH2-Tyr compared to the free AbPPO4 may be adsorbed here on the silica corresponds to the citrate buffer and/or SDS, associated with excess of citrate adsorbed within silica pores. which could block the pores, preventing the access of substrate mole­ The samples obtained after the interaction of SBA-15 and SBA-15- cules to the catalytic sites during the loaded-enzyme reaction, with a NH2 with oxidized phenols were further analyzed using solid-state 13C negative impact on the reaction rate and the apparent enzyme activity. CP/MAS NMR, which confirmed the presence of citrate adsorbed in the The UV/Vis spectra of the supernatants obtained after 20 min- silica pores (Fig. 7a). The signals corresponding to the citrate molecules interaction of dopamineox, DOPAox and TBCox with SBA-15 were similar were found in all the samples analysed at 177–185 ppm, 75 ppm and to the spectra of standard solutions prepared under the same conditions 42–46 ppm, and were maintained even after the sixth washing with the but without adding silica (Figure S11). These results confirmed the same medium in which the experiments were carried out (Fig. 7b). negligible adsorption of oxidized phenols within silica pores. The sig­ Despite citrate being the compound mainly adsorbed in the pores of nificant decrease in the intensity of the adsorption maximum (λ = 470 SBA-15 some weak signals in the sample washed with water after the nm, 468 nm and 400 nm for Dopamineox, DOPAox and TBCox, respec­ adsorption of oxidized dopamine could be observed in the 13C CP/MAS tively) compared to freshly prepared standards (Figure S11, black lines) NMR spectra (Fig. 7b, insert). Additionally, the ATR-FTIR spectra of the was attributed to the low stability of these oxidized phenols due to samples obtained after the oxidation of L-tyrosine by SBA-15-Tyr and overoxidation caused by the 10-fold excess of sodium periodate. In SBA-15-NH2-Tyr (Figure S13) showed C– – O and C–N stretching bands contrast, better defined maxima but of low intensity were observed in at 1700 cm− 1 and 1430 cm− 1, respectively, as well as N–H bending at the spectra of the supernatants correspondent to the SBA-15-NH2 sam­ 1137 cm− 1 (Table S3), which also suggested the adsorption of the ples, suggesting the possible adsorption and stabilization of the oxidized oxidized product L-dopachrome after the catalytic reaction. phenols. With the purpose of analysing the reversibility of the observed The oxidation of L-tyrosine was also carried out using SBA-15-Tyr as adsorption, the SBA-15 and SBA-15-NH2 obtained after 20 min-inter­ catalyst but varying the SDS concentration (Figure S12) in order to study action with 1 mM oxidized phenols (i.e., dopamineox, DOPAox and the influence of the surfactant adsorption on the catalytic performance. TBCox) were washed with a solution containing citrate (50 mM) and SDS A gradual increase in the concentration of the dopachrome-derivative (2 mM). After 3 and 6 washings, the mass losses of the washed solids produced was observed when the SDS concentration increased up to remained in a range between 27 and 37 wt% (Fig. 6b, grey background), 4 mM. This suggested that even when the anionic surfactant is adsorbed at the same time the presence of phenols or oxidized products could not in the pores of the silica, its presence does not affect the reaction rates. be detected via UV/Vis in the collected supernatants (Figure S11, dashed Thus, despite that the adsorption of catechols is not ruled out (Fig. 7, lines). It was only possible to remove the citrate buffer and SDS adsorbed insert), the major cause of the lower apparent enzymatic activity of SBA- on the silica after washing three times with water, obtaining mass losses 421 C. Iriarte-Mesa et al. Journal of Colloid And Interface Science 646 (2023) 413–425 Fig. 6. (a) Adsorption of phenolic compounds on SBA-15 and SBA-15-NH2 materials. The controls (grey bars) correspond to silica that did not interact with oxidized phenols (1 mM) but only with citrate buffer (50 mM) and SDS (2 mM). (b) Desorption of phenolic compounds from silica after washing the samples with citrate buffer/SDS or only with distilled water. The correspondent mass losses are highlighted with a gray or blue background, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Fig. 7. Solid-state 13C CP/MAS NMR spectra of samples obtained after (a) adsorption experiments and (b) six consecutive washings with citrate buffer and SDS. Insert: Solid-state 13C CP/MAS NMR spectra of the SBA-15 washed three times with water after adsorption of oxidized dopamine. that did not exceed 4 wt% in the washed samples (Fig. 6b, blue back­ buffers) or temperature regimes for 1 h before the enzymatic assays. The ground). The effectiveness of water washing in the removal of adsorbed same concentrations of free AbPPO4 were tested under the same con­ citrate/SDS within the silica pores might have a positive impact on ditions and used as controls (Fig. 8). After 1 h-incubation, activity tests catalysts regeneration and their potential applications in column-type were performed in citrate buffer at pH 6.8 and 25 ◦ C. For the residual reactors. enzymatic activities obtained from UV/Vis measurements, the calcu­ lated standard deviations were below 5% and corroborate the repro­ 3.5. Stability of immobilized AbPPO4 on SBA-15 ducibility of the experiments performed. The observed gains in enzymatic activity (i.e., 120–130%) with respect to the reference value To evaluate the stability of the immobilized AbPPO4, enzymatic (i.e., 100% at pH 6.8 and 25 ◦ C) could be attributed to a secondary activity tests were performed as described above, but keeping the activation of the latent enzyme. There may be some rearrangements in enzyme-loaded SBA-15 (38 wt% of AbPPO4) at different pH (McIlvaine the C-terminal domain of the protein or a repositioning of the 422 C. Iriarte-Mesa et al. Journal of Colloid And Interface Science 646 (2023) 413–425 Fig. 8. (a) Effect of temperature on the residual enzymatic activity of SBA-15-Tyr (blue bars) compared to the free enzyme (grey bars). (b) Dependence of the enzymatic activity of SBA-15-Tyr (blue) and free AbPPO4 (grey) on pH. (c) Cycles of reuse of SBA-15-Tyr. (d) Stability of dry SBA-15-Tyr over time. Standard deviation of the residual enzymatic activity data was calculated from triplicates. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) immobilized AbPPO4 within the mesopores that allow for slightly better enzyme-loaded silica could be stored dry at 4 ◦ C, which is only attain­ substrate access.[60] The buffer might also influence the enhanced able for the free AbPPO4 after lyophilization, but induces the inactiva­ enzymatic activity. For the latent Dimocarpus longan PPO, [61] a pro­ tion of the latent enzyme. Such behaviour constituted an important nounced increase of enzymatic activity was observed when the enzyme improvement in comparison to other reports of tyrosinase immobilized was contacted with 2-(N-morpholino)ethanesulfonic acid buffer at 4 ◦ C on solid supports or by other methods of immobilization.[59,63,65] and then brought back to Tris buffer at room temperature. This could The reusability of SBA-15-Tyr was also studied for 15 consecutive explain the increased activity observed for the immobilized AbPPO4 cycles of substrate oxidation/centrifugation/redispersion (Fig. 8c). Re­ when the enzyme was transferred from McIlvaine buffers (e.g., pH 7 and sidual enzymatic activity after each cycle was determined using L-tyro­ 8, 4 ◦ C) to citrate buffer for subsequent measurements of enzymatic sine (1 mM) as substrate for the enzyme. After 6 cycles of reutilization, activity at standard conditions (25 ◦ C; pH 6.8). 91% of the initial enzymatic activity of the enzyme-loaded SBA-15 was SBA-15-Tyr exhibited enhanced stability over a wide range of pH and retained. Under those conditions, the biocatalyst still showed levels of temperatures compared to the free AbPPO4, preserving 22%, 20% and enzymatic activity comparable to free AbPPO4 in solution. The enzy­ 25% of its initial enzymatic activity after keeping the material for 1 h at matic activity of SBA-15-Tyr represented 63% of the initial enzymatic 70 ◦ C (Fig. 8a), as well as at pH 2.2 and pH 3 (Fig. 8b) respectively, activity after 15 consecutive cycles. This decrease was attributed to the conditions in which the free enzyme was completely deactivated. stress suffered by the enzyme after each separation step due to the for­ Strongly acidic pH can cause most tyrosinases to denature, e.g., the mation of compact aggregates. There is likely also a purely chemical activated AbPPO4 retains>50% of its activity at the optimal pH 6.8 only contribution derived from the reactive oxidation products that can also in the pH-range from 5 to 10, but precipitates at pH values below 3 [14], react with various amino acids of the enzyme.[12,66] However, the while our studies showed that the latent AbPPO4 is not stable below pH possibility of reusing the biocatalyst, maintaining high values of residual 4.5. These results confirm the advantages of enzyme confinement within enzymatic activity, added to its enhanced stability due to the effect of the silica pores, which protected AbPPO4 from drastic conditions, enzyme confinement, point towards the possible use of SBA-15-Tyr for expanding the useful range of the catalyst.[62–64] the preparation of a column-type bioreactor to mimic specific processes The stability of SBA-15-Tyr over time was studied by measuring its in the soil and allow structure elucidation of humic-derived iron- residual EA several weeks after the immobilization (Fig. 8d) and storage complexes. of the dried catalyst at 4 ◦ C. After the first week of storage, the residual EA was found to be 98% of its initial activity. Furthermore, SBA-15-Tyr 4. Conclusions retained 66% of its initial EA after 6 months and 21% after one year of storage at 4 ◦ C, while the free AbPPO4 was almost completely inacti­ In this study, the tyrosinase AbPPO4 was successfully immobilized vated at those times (i.e., only 3% of its initial EA was observed). The on mesoporous SBA-15-type silica. Liquid adsorption proved to be an 423 C. Iriarte-Mesa et al. Journal of Colloid And Interface Science 646 (2023) 413–425 effective immobilization method, not only because of its reproducibility, References but also due to the encapsulation of high amounts of AbPPO4 within silica mesopores, capable of efficiently hosting the enzyme with minimal [1] P.W. Boyd, M.J. Ellwood, The biogeochemical cycle of iron in the ocean, Nat. Geosci. 3 (2010) 675–682, https://doi.org/10.1038/ngeo964. risk of desorption. The adsorbed enzyme in amino-functionalized SBA- [2] A. Tagliabue, A.R. Bowie, P.W. Boyd, K.N. Buck, K.S. 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