Design, synthesis and activity study of tyrosinase ... - Springer Link

J Sol-Gel Sci Technol (2011) 59:7–18 DOI 10.1007/s10971-011-2454-3

ORIGINAL PAPER

Design, synthesis and activity study of tyrosinase encapsulated silica aerogel (TESA) biosensor for phenol removal in aqueous solution Suriani Sani • Mohd Nazlan Mohd Muhid Halimaton Hamdan

•

Received: 15 December 2010 / Accepted: 22 March 2011 / Published online: 31 March 2011 Ó Springer Science+Business Media, LLC 2011

Abstract Tyrosinase encapsulated silica aerogel (TESA) was synthesized via an alcohol-free colloidal sol–gel route at room temperature and at neutral pH. Characterization on TESA indicated that 98% of enzyme was effectively loaded and located inside the aerogel network. TESA without solvent extraction showed higher tyrosinase activity than TESA extracted by amyl acetate/acetone (v/v:1/1). Stability of tyrosinase in TESA was enhanced towards extreme temperature, acidic and basic conditions. Optimization study indicates that 500 U enzyme/g silica aerogel; aged for 2 days, showed superior performance in the oxidation of catechol. The activity of TESA was remarkably enhanced; which was active at a wider temperature (up to 80 °C) and pH range (4–9). In contrast, free tyrosinase was totally inactive at these pH values and temperature[55 °C. TESA successfully removed about 90% of phenol in aqueous solution after 3 h of contact time with excellent reusability. Keywords Silica aerogel Encapsulation Enzyme Tyrosinase Phenol

1 Introduction Aromatic compounds including phenols, constitute one of the major classes of pollutants [1]. Due to its toxicity, the concentration of phenol which is greater than 50 ppb is harmful to some aquatic species and ingestion of 1 g

S. Sani M. N. Mohd Muhid H. Hamdan (&) Zeolite and Nanostructured Materials Laboratory, Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia e-mail: [email protected]

phenol in humans can be fatal [2]. Hence, the removal of phenol in water is important. Conventional methods for removing phenol and aromatic compounds from industrial waste are solvent extraction, microbial degradation, adsorption on activated carbon and chemical oxidation [3–6]. These methods, although effective and useful to remove phenol, suffer from serious drawbacks such as high cost, incompleteness of purification, formation of hazardous by-products, which are only applicable to limited phenol concentration range [2]. Enzymatic treatment has been proposed by many researchers as a convenient method for removing phenol [7]. Enzymes are highly selective and can effectively treat phenol even in dilute wastes [1]. In addition, enzymes operate over a broad aromatic concentration range and require low retention times with respect to other treatment methods [8]. Tyrosinase, also known as polyphenol oxidase, phenolase or catecholase has been demonstrated to remove phenols and aromatic amines from industrial effluents [1, 2, 7, 9]. Tyrosinase oxidizes numerous phenols, generating corresponding phenoxy radicals which diffuse from the active centre into solution to form polymeric substances that are much less water soluble. These insoluble polymers then precipitate out of the solution and can be separated by simple filtration or flocculation [9]. However, there has been almost no discussion about contamination due to remaining soluble enzyme and non-precipitated products in the aquatic solution after enzymatic treatment. It is very important to ensure that, especially in drinking water, after treatment, it is free of enzyme and such products. Furthermore, enzymes are relatively expensive reaction components which contribute to high cost of production. They are also extremely sensitive to environmental conditions and easily denatured. Free enzymes can be used only once and are generally soluble in

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aqueous solutions which may not be easily recovered in usable form [10]. In order to increase reusability and enhance stability, enzymes are often immobilized to the surface of insoluble supports by physical or chemical means [11, 12]. The appropriate matrix or support for encapsulation of enzyme is chosen based on several different properties which affect the production process [13]. One of the properties is that the materials need to have high surface area of up to 100 m2/g for high enzyme loadings and high porosity to provide access for the substrate. The immobilization matrix must also be resistant to chemical degradations and mechanical stability. Microbial resistance of matrix is also an important property that needs to be considered since a major concern to any immobilized enzyme process is the presence of microbes. Furthermore, the durability of the carrier is often determined by its resistance to microbial degradation [14]. Upon encapsulation in silica aerogel, the polymeric framework grows around the biomolecules, creates a cage and protects the enzyme either from aggregation and unfolding or from microbial attack [15]. Therefore, encapsulated biomolecules often retain some level of activity and functionality presumably because of sufficient retention of their native state conformations. Meanwhile, the matrix pores enable diffusion of reactant molecules and reaction with encapsulated biomolecules. As a result, encapsulation may improve activity and storage stability of the enzymes and facilitate its application, recovery and washing [16]. Silica aerogel is a potential host matrix for the encapsulation of enzymes. An aerogel is a porous solid material in which a very high percentage (95%) of its volume is filled with air. The most documented method for the preparation of silica aerogel is the sol–gel route [17]. The sol–gel process is a wet-chemical technique for the fabrication of materials starting either from a chemical solution or colloidal particles to produce an integrated network (gel) of silicon precursor through a change of interaction between the colloidal particles which is changing the systems characteristics from a liquid to a gel. The gel can be dried at very high pressure and temperature corresponding to the supercritical conditions of the dispersed liquid such as alcohol, in the gel to yield aerogels [18]. The extraction with alcohol is termed as the high temperature supercritical drying (SCD). Silica aerogel prepared by SCD typically consists of pores with diameter of 20–40 nm and surface area of 600–800 m2/g [19, 20]. However, the high level of sophistication instruments, high risks and high cost involves in the supercritical drying of the gel limits its application involving temperature sensitive biomolecules. Ambient pressure drying (APD) has been considered as a promising technique to be applied on a microbial system as

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J Sol-Gel Sci Technol (2011) 59:7–18

it does not require high pressure and high temperature conditions and use of alcohol unlike the supercritical drying technique [21–25]. In most of reported applications of silica aerogel, orthosilicate such as tetramethyl orthosilicate (TMOS) or tetraethyl orthosilicate (TEOS) has been used as the silica source in preparing the silica monoliths containing encapsulated proteins [26]. Despite the fact that these chemicals contain relatively pure silica, 70% reduction of enzymatic activity was reported when lipase was encapsulated into TMOS-based silica matrix, in the presence of 5% volume of methanol [27]. The presence of alcohol is known to be detrimental to the activity of proteins by causing chain unfolding, aggregation, destruction of secondary and tertiary protein structures to a significant extent [26]. Furthermore, such organic silicon precursors are in fact too expensive; hence not commercially viable. In order to overcome these problems, rice husk ash was used as silica precursor in preparing of sodium silicate required for the synthesis of silica aerogel [19]. This approach completely avoids the generation of alcohol and allows for encapsulation to be carried out at neutral pH in order to preserve biological activity of proteins. The organic based silica aerogel is chemically inert and exhibits higher mechanical strength, enhanced thermal stability and negligible swelling in organic solvents compared to most organic polymers. Aerogel encapsulation is expected to improve storage stability and protect the enzyme against deterioration by hydrophilic solvent [27–29]. Silica aerogel can also be tailored as a reservoir for water, thereby enhancing its ability to maintain the biological activity of entrapped enzymes.

2 Experimental procedure 2.1 Synthesis of TESA Rice husk ash was used as the silica source in producing sodium silicate solution according to established procedure [19]. Rice husk, calcined at 700 °C produces rice husk ash (RHA) containing 97 wt% amorphous silica, was reacted with NaOH to obtain the sodium silicate solution. The sodium silicate solution was prepared by dissolving RHA in sodium hydroxide (Merck; 99%) and distilled water with SiO2:Na2O ratio of 3.25. Sodium silicate was then stirred and heated in an oil bath at boiling water temperature of 100 °C for 24 h. After that, sodium silicate was filtered through Whatman filter paper (125 mm) to remove insoluble residues. In order to synthesize wet gel, silica sol was prepared by acidifying colloidal silica sol (Ludox LS-30; Sigma– Aldrich; 30 wt% suspensions in water; pH 8.2) with


9

sufficient amount of concentrated sulphuric acid (Merck; 97%) in a Teflon beaker. Glass vessels were avoided as glass participates in the reaction since silica would leach out from the glass [30, 31]. The sodium silicate solution prepared previously was added dropwise into the silica sol until pH 6.5 is achieved. Tyrosinase (Sigma; 5.00–30.00 mg/mL) in potassium phosphate buffer (GCE Laboratory Chemicals; 50 mM, pH 6.5) was then inserted into the silica sol under constant stirring for 1 h to form a transparent, solid-like aqueous tyrosinase-silica gel. The aqueous tyrosinase-silica gel was then aged at room temperature for 2 days in order to allow for formation of silica frameworks around the enzyme molecule. Solvent extraction was performed on the gel in order to remove water from the aqueous tyrosinase-silica gel before drying. In the solvent extraction process, water in the gel was extracted by solvent extraction technique. 100 mL of phosphate buffer (50 mM, pH 6.5) was added to the tyrosinase-silica gel followed by stirring to form a slurry. The tyrosinase-silica gel was left to soak for 2 days without stirring at room temperature and pressure. The slurry was then centrifuged for 10 min at 3,000 rpm. The clear supernatant liquid was discarded and the tyrosinase-silica gel was collected. Finally, the tyrosinase-silica gel was dried by ambient pressure drying technique (APD) at 36 °C until a constant weight of dried product was obtained. The dried TESA powder was then collected and stored in an airtight Teflon bottle at 5 °C. The entire preparation process of TESA is summarized in the flow chart shown in Fig. 1. 2.2 Assay of enzymatic activity The catecholase activity of tyrosinase (T) is based on the conversion of catechol (C) to o-benzoquinone (Q) per mg of enzyme. Unfortunately, it is difficult to accurately measure the amount of o-benzoquinone produced, due to its instability, which easily polymerizes to form stable compounds. In order to overcome this problem, the amount of o-benzoquinone was determined from the depletion of ascorbic acid with nicotinamide adenine dinucleotide (NADH) with a chemical formula of C21H29N7O14P2 as the chemical redactor. NADH is reduced into catechol (C) based on the standard method as represented in the following equations: [2, 9] T þ C þ 1=2 O2 $ TCO ! TQ þ H2 O

ð1Þ

TQ ! T þ Q

ð2Þ

Q þ NADH ! C

ð3Þ

Enzyme activity was calculated by determination of the amount of units of activity per milligram of enzyme left in the solution, obtained by dividing the slope of absorbance

RICE HUSK

Combustion 700 ° C

Silica Ash

NaOH water

TYROSINASE (5-30 mg/mL) in potassium phosphate buffer (50mM, pH 6.5)

Sodium silicate

Silica sol LUDOX LS30

Conc. Sulphuric Acid

COLLOIDAL SILICA SOL

Tyrosinasesilica hydrogel Ageing Solvent Extraction Tyrosinasesilica gel Ambient Pressure Drying

TYROSINASE ENCAPSULATED SILICA AEROGEL (TESA)

Fig. 1 Flow-chart for preparation process of encapsulated tyrosinasesilica gel (TESA)

(A) measurements with the mass of enzyme in the reaction mixture (Eq. 4). One unit of enzyme activity is defined as the change of absorbance detected in 3.0 mL reaction mixture at pH 7 and 25 °C. Units=mg enzyme ¼

DA265 nm = mintest DA265 nm = minblank ð0:001Þðmg enzyme=RMÞ ð4Þ

0.001 = the change in A265 nm per unit of tyrosinase in a 3.00 mL reaction mixture at pH 7 and 25 °C. RM = reaction mixture. 2.2.1 Free tyrosinase The substrate solution was prepared by mixing 0.1 mL catechol (Sigma–Aldrich; 5 mM; pH 7), 2.6 mL phosphate buffer (50.0 mM), 0.1 mL ascorbic acid (Acros Organics; 2.1 mM; pH 7) and 0.1 mL EDTA (Riedel-de-Haa¨nÒ; 0.065 mM; pH 7) in a cuvette and analyzed by UV–Vis spectrophotometer at 265 nm until the absorbance readings were constant. Then 0.10 mL of tyrosinase solution (Sigma; 5.00-30.00 mg/mL; pH 7) was added followed by the absorbance values recorded every 30 s for 5 min. Reference solution was prepared similar to the preparation of buffered substrate solution as stated previously but without the addition of tyrosinase. This was done in order to determine whether the substrate catechol is oxidized non-enzymatically at a perceptible rate.

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2.2.2 Tyrosinase encapsulated silica aerogel (TESA) The enzymatic activity of TESA was determined by measuring the amount of ascorbic acid depletion based on the formation of dehydro-ascorbic acid and o-benzoquinone from the reaction between catechol, oxygen and ascorbic acid, similar to method (i) but with some additional procedure. Substrate solution containing a mixture of buffer solution (50 mM; pH 7), catechol solution (5.0 mM; pH 7), ascorbic acid (2.1 mM; pH 7) and EDTA acid solution (0.065 mM; pH 7) was gently stirred and incubated at room temperature. Keeping the absorbance of the buffered substrate solution constant at 265 nm, TESA (5.00–30.00 mg/mL) was added in order to initiate oxidation of catechol (substrate). After 60 min, the suspension was filtered and analyzed by UV–Vis spectrophotometer. The procedure was repeated with silica aerogel without the presence of tyrosinase in order to confirm that the oxidation of catechol was initiated by the encapsulated tyrosinase rather than other materials present in the reaction beaker. The enzyme activity of TESA was calculated according to the dry weight of support. The unit of enzyme activity per gram support (A) is defined in Eq. 5. AðU=gÞ ¼

Uact Wdry

ð5Þ

Uact = activity of immobilized enzyme, Wdry = weight of dry support (g). In order to validate the accuracy of TESA assayed activity, the activity of encapsulated tyrosinase was assessed by comparing the enzyme activity in a freshlymade tyrosinase solution with the enzyme activity left in the supernatant after centrifugation in the gel phase. The fraction of residual of enzyme (R) is

R¼

to study the stability in acidic and basic conditions, tyrosinase and TESA were incubated in buffered substrate solution at 25 °C with a pH range of 4–9. 2.3.2 Effect of solvent extraction The effect of solvent extraction on the enzyme activity in TESA was studied by extracting TESA in different solvents at the final stage of the preparation of TESA. Acetone, amyl acetate and combination between acetone and amyl acetate were selected as solvents in the extraction of inside TESA during synthesis. The tyrosinase-silica gels were then dried at 37 °C until constant weight was obtained and the activity of enzyme in TESA was measured. 2.3.3 Effect of ageing period The effect of ageing period on activity of TESA during the synthesis was investigated for 1, 2, 4, 6, 8 and 12 days at 25 °C. Teflon beakers containing aqueous tyrosinase-silica gel were firstly sealed by parafilm during the aging period in order to avoid contamination of sample. After aging, the wet gels were then extracted using organic solvent. Finally, tyrosinase-silica gels were dried at 37 °C until constant weight of dried products was obtained. The dried product (TESA) was then collected and stored in an airtight Teflon bottle and the enzyme assay of the product was done according to 3.2(i). 2.3.4 Effect of enzyme loading The relation between enzyme loadings and TESA activities was examined by varying enzyme loading into silica aerogel. Various quantities of tyrosinase (5.00, 10.00,

Reaction rate of tyrosinase assay after gelation Reaction rate of tyrosinase assay in a stock solution of tyrosinase

The activity of encapsulated enzyme in silica matrix (I = 1 - R) was then calculated.

ð6Þ

20.00 and 30.00 mg/mL) was added into the silica sol suspension before gelation. Tyrosinase-silica gel was processed in the same manner as described in (ii).

2.3 Optimization of enzyme activity in TESA 2.3.5 Leaching study 2.3.1 Enzymatic ability test The enzymatic ability of TESA and free enzyme at various temperatures and pH ranges were studied. Free tyrosinase and TESA were incubated in buffered substrate solution with pH 7 and temperature range of 5 °C–70 °C. In order

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Control studies were designed to confirm that the activity was due to TESA rather than other species present in the reaction vessel. It was undertaken to assess the oxidation of catechol upon exposure to the support alone and to determine if tyrosinase was desorbed from the support and


contributed to the oxidation of catechol. Sample from TESA was analysed in the same way using UV–Vis Spectroscopy. The filtrate was analyzed 48 h later at 265 nm. 2.4 Removal of phenol TESA and phosphate buffer (0.1 M, pH7) were placed in artificial phenolic waste water (2.0 mM). The reaction mixture was incubated under aerobic conditions using a stirrer. After the prescribed time, the sample was withdrawn and assayed for phenols by UV–Vis Spectroscopy and the disappearance absorption spectra of reaction solutions were measured. 2.4.1 Reusability In order to test for reusability, TESA was recycled for a different number of batches. For each batch, TESA was immersed in artificial phenolic waste water and the phenol degradation for each batch was monitored using UV–Vis Spectrophotometer. Samples were collected at regular intervals for each batch. 2.5 Characterizations X-Ray diffraction (XRD) of samples were recorded using a Bruker AXS diffractometer with a CuKa radiation of ˚ at 40 kV and 20 mA in the range of k = 1.5418 A 2h = 5°–45° with a scanning speed of 0.05° per second. The analysis was performed at the 2h scale of 1.5–10° with a step interval of 0.025° and counting time of 1 s per step. Fourier Transformed Infrared (FTIR) spectra were recorded on Shimadzu FTIR-8300, Spectrometer using KBr method. Approximately 0.001 g of the solid sample was used as a representative amount of the overall sample, thoroughly mixed with KBr with sample:KBr ratio of 1:100. The mixture was then pressed at a pressure of 7 tonnes to form a KBr disk; recorded in the wavenumber range of 350–4,000 cm-1. Absorbance readings at 265 nm of samples were measured using Perkin Elmer, model Lambda 25 UV–Vis Spectrophotometer. The mixture in the cuvette was mixed by inversion and subsequently, absorbance values were taken every 30 s for 5 min. The cuvette was washed before and after each run with denatured alcohol and rinsed with distilled water to ensure that it was free from enzyme contamination. Reference solution was prepared similar to the preparation of buffered substrate solution as stated previously but without the addition of tyrosinase. Field Emission Scanning Electron micrographs (FESEM) were recorded using a JEOL microscope model JSM

11

6701F at an emission current of 2.00 kV with working distance of 3.0 mm and probe current of 8 kV. Elemental analysis of samples was determined using Energy Dispersive X-ray (EDX) spectrometer (model EX2300 BU, JEOL) with emission current of 15.0 kV with working distance of 8.0 mm and probe current of 14 kV. Thermal analysis was performed using TG Analyzer (model TA 4000, Mettler Toledo) consisting of an electronic microbalance, furnace, temperature controller and the recorder. A few mg of silica aerogel and TESA were weighed in a standard alumina crucible and sealed. The empty sample and reference pans were put into an analyzer chamber and were heated at an initial temperature of 30 °C at an increasing rate of 10 °C/min to 1,000 °C. The power (energy per unit time) differential between the sample and reference was measured during the programmed heating and cooling periods.

3 Results and discussion 3.1 Physical characterization Semi transparent and fluffy powder of silica aerogel was obtained after Ambient Pressure Drying (APD). The density and surface area of silica aerogel is 0.1084 g/cm3 and 600 m2/g, respectively. After encapsulation, the sample (TESA) was more opaque and denser with about 3% increment in density of 0.1113 g/cm3. The surface area decreased to 400 m2/g. Upon reaction with substrate, the white colour of TESA changed to yellowish, due to the adsorption of coloured quinones or polyphenolics species on its surface [26]. Silica aerogel was completely amorphous as indicated by the featureless XRD pattern and appearance of diffused maximum at 2h = 21°. Silica aerogel remained amorphous after addition of tyrosinase, which suggests that tyrosinase was highly dispersed into the silica aerogel networks and the amount of tyrosinase is compatible to the quantity of silica aerogel. FTIR spectra of free tyrosinase, silica aerogel and TESA in the wavenumber range of 1,800–1,000 cm-1 are shown in Fig. 2a–c, respectively. IR spectra of all samples exhibit intense and broad peak beyond 1,650 cm-1 which corresponds to O–H stretching and O–H bending mode; possibly derived from physically adsorbed water. IR spectrum of tyrosinase (Fig. 2a) shows an intense broad peak at 1,550 cm-1; assigned to N–H2 bending, indicative of the presence of tyrosinase. IR spectrum of silica aerogel in Fig. 2b shows a strong and significant peak at 1,250–1,020 cm-1 which corresponds to Si–O–Si asymmetric stretching. Typical silicate absorption peaks are also observed at 800 and 470 cm-1 attributed to Si–O–Si

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J Sol-Gel Sci Technol (2011) 59:7–18 Table 1 Major elemental analysis (mass% ± SD) of free tyrosinase, silica aerogel and TESA by EDX

(a)

NH2

Transmittance (%)

Element (mass% ± SD) (b)

(c)

Tyrosinase

Aerogel

TESA

C

57.30 ± 0.95 7.85 ± 0.51

4.06 ± 0.39

N

12.23 ± 0.23 nd

nd

O Na

25.37 ± 0.20 45.83 ± 0.35 48.92 ± 0.32 nd 1.83 ± 0.17 4.39 ± 0.34

Mg

0.55 ± 0.01

nd

Si

nd

44.50 ± 0.53 42.63 ± 0.87

Ca

1.10 ± 0.18

nd

nd

Cu

3.45 ± 0.45

nd

nd

nd

nd not detected 1800

1700

1600

1500

1400

1300

1200

1100 1000.0

Wavenumber (cm-1) Fig. 2 Infrared spectra of (a) free tyrosinase, (b) silica aerogel, (c) TESA (10 mg/mL of tyrosinase in 30 g of silica)

symmetric stretching and Si–O–Si bending modes, respectively. An additional absorption at 970 cm-1 corresponds to surface Si–OH vibrations [20, 32, 33]. IR spectrum of TESA in Fig. 2c exhibits a significant peak at 1,550 cm-1 assigned to N-H2, which confirms the existence of tyrosinase [34]. However, the intensity of this peak is lower than the peak in the IR spectrum of free tyrosinase probably due to the interference effects of silica aerogel to the encapsulated tyrosinase. Likewise, the IR spectra for TESA show the same features as in the spectra of silica aerogel but with different relative intensities; signifying that the silica aerogel network does not collapse upon the encapsulation process. The surface morphology of silica aerogel and TESA are similar, as seen in the SEM image in Fig. 3a, b, respectively. The surface morphology of silica aerogel shows homogeneous aggregates of spherical particles. On the other hand, surface morphology of TESA is observed as rough and homogeneous, with denser aggregates of

spherical particles of *20 nm. This effect might be due to the loss of liquid phase that is essential to the enzyme located in the silica aerogel network during the solvent extraction process. The absence of any secondary phase in the SEM image of TESA suggests that most of tyrosinase was encapsulated inside the silica aerogel, and not being adsorbed on the surface of silica aerogel. Energy dispersive X-ray (EDX) analysis in Table 1 lists the mass percentage of major elements measured in the samples. It is important to bear in mind that EDX data is an average value measured from several spots on the surface of sample, not the bulk of sample. In this case, it is useful to relatively compare the presence of element on the surface of tyrosinase, silica aerogel and TESA. The data indicates that nitrogen; the major element in tyrosinase, is relatively not detectable on the surface of TESA. This further supports the surface morphology analysis by FESEM on the encapsulation process described earlier, which indicates that tyrosinase molecule in TESA is located inside the silica aerogel network, not at the surface of silica aerogel. TEM micrographs of silica aerogel and TESA in Fig. 4a, b, respectively, show that particle size of silica

Fig. 3 FESEM micrographs showing the surface morphology of (a) silica aerogel and (b) TESA (10 mg/mL of tyrosinase in 30 g of silica)

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Fig. 4 TEM micrographs showing the surface morphology of (a) silica aerogel and (b) TESA (10 mg/mL in 30 g of silica aerogel)

almost identical to silica aerogel, it is concluded that low concentration of encapsulated tyrosinase in TESA (10 mg/mL of tyrosinase in 30 g of silica) is stable and not desorbed or denatured upon direct heating treatment. This directly demonstrates the protecting effect of silica aerogel on tyrosinase; which were located inside the silica aerogel network as a result of encapsulation. With a low thermal conductivity of 0.05 Wm-1K-1, silica aerogel evidently is an excellent insulator that can protect tyrosinase molecules which are sterically confined in its network, from direct heating treatment. UV–Vis spectra of TESA and free tyrosinase in Fig. 5 show similar absorptions centered at 290 nm. Apparently, the absorption band of TESA did not change after encapsulation, suggesting that the enzyme conformation was well-maintained. It further suggests that use of an aqueous sol–gel encapsulation route does provide a promising approach for enzymes stabilization. Identical absorbance spectra of ascorbic acid in contact with free tyrosinase in solution and TESA proved that encapsulation of tyrosinase 1.00 0.9 0.8 0.7

(a) λ max = 289

(b)

0.6

A

aerogel are in nano-size range of 20–25 nm. Hence, the synthesis of silica aerogel via alcohol-free aqueous colloidal sol–gel route was found to be successful in producing nano-sized particles of silica aerogel. In addition, TEM micrograph of silica aerogel in Fig. 4a is apparently different from micrograph of TESA in Fig. 4b, whereby TESA is observed to be more opaque than that of silica aerogel. This is because the networks of silica aerogel surround the tyrosinase molecules in TESA which resulted in the increment of the thickness of silica aerogel. Hence, it proves that tyrosinase is located inside the silica aerogel network rather than at the surface. Evidently, enzyme-sol interaction which resulted from the addition of tyrosinase into the sol before the aging process promotes encapsulation of enzyme into the growing silica network. The large number of hydrogen bonding groups on the surface of tyrosinase serves as nuclei that enabled condensation and polymerization of sol with silicate polymer containing Si–O(H)-Si and Si–OH fragments during the initial stages; acting as a template which eventually formed a porous inorganic polymer cage surrounding tyrosinase [27]. TGA analysis of silica aerogel and TESA samples, carried out at temperatures ranging from 50 °C to 900 °C indicates that upon heating, free tyrosinase lost a few percent of weight at 50 °C and eventually reached 100% weight loss at 900 °C due to the release of amino acids and organic residuals. Silica aerogel was more thermally stable where it lost about 19 wt% at 50 °C.and 21 wt% at 900 °C. The weight loss of silica aerogel and free tyrosinase at this temperature may be attributed to the thermo-desorption of physically adsorbed water in free tyrosinase and silica aerogel pores as well as some silanol groups present at the surface of silica aerogel. In comparison, TESA lost a total of 11 wt% after heating to 900 °C. Since the thermal transition of TESA is

λ max = 290

0.5 0.4 0.3 0.2 0.1 0.00 280

285

290

295

300

305

310

Wavelength (nm) Fig. 5 Optical absorption spectra of ascorbic acid in the presence of (a) free tyrosinase, kmax = 289 nm, (b) TESA, kmax = 290 nm

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into silica aerogel matrix via sol–gel process was successful and occurred without any denaturation of enzyme. Almost 98% of the tyrosinase was successfully encapsulated into the polymeric silica aerogel gel network, due to uniform distribution of tyrosinase in the sol which formed before gelation process during the synthesis of TESA. 3.2 Effect of solvent extraction Solvent extraction has a significant effect on the activity of encapsulated tyrosinase. Selection of suitable organic solvent as a solvent extractor in the synthesis of TESA depends on several factors such as toxicity, cost, solvent hydrophobicity and polarity index. Polarity index is the guiding solvent parameter for the enzyme stability in aqueous-organic co-solvent mixture, in which solvents having polarity indexes of 5.8 and above are suitable for tyrosinase because it does not denature the tyrosinase. Hydrophobicity of the solvent is represented as the logarithm of the partition coefficient; log P, where P is defined as the partitioning of a given solvent between water and 1-octanol in a two phases system. Solvents with a log P value between 2 and 2.5 show higher activity of tyrosinase since they did not interfere with the essential water coating surrounding the tyrosinase molecule in its active state [35]. The effect of amyl acetate and acetone as solvent extractor in the synthesis of TESA to the enzymatic activity of tyrosinase were investigated. These two types of organic solvents were chosen because of their ability to evaporate up to 100% (by volume) at 21 °C. In addition, both amyl acetate and acetone are known cheap organic solvents. Table 2 shows that the activity of TESA was influenced by the type of solvents used during the extraction process. The result demonstrates that subsequent extraction of water in silica gel network by acetone after the extraction process by amyl acetate produced a significant increase of 15% in activity of tyrosinase. It indicates that sufficient amount of acetone was required in the hydrophobic medium of amyl acetate in order to produce tyrosinase of higher activity. The addition of sufficient amount of hydrophilic solvent into hydrophobic solvent prevented the

limitation diffusion of substrate to the enzyme as enzyme is insoluble in hydrophobic media. The use of acetone as a solvent extractor decreased the activity of TESA by 10% as compared to amyl acetate. Acetone is a dipolar solvent and very soluble in water with log P and polarity index value of -0.2 and 5.4, respectively. In contrast, amyl acetate is a non-polar solvent and partially soluble in water which have log P and polarity index value of 2.3 and 3.5, respectively. In this case, when acetone was solely used as a solvent extractor, the hydrophilic organic solvent interacts with the essential water layer of tyrosinase which is required for stabilizing and maintaining the proper polypeptide conformation of tyrosinase. This phenomenon induces partial unfolding and inactivation of tyrosinase. On the other hand, hydrophobic solvent such as amyl acetate was incapable to strip away substantial amount of water from tyrosinase. Subsequent suspension in amyl acetate did not disrupt its interactions with essential water molecules of tyrosinase due to its nonpolarity properties. Therefore, the number of water molecules that surround the tyrosinase was sufficient to provide high flexibility to the polypeptide conformation. The resulting relative structural rigidity of tyrosinase reduces the risks of denaturation and inactivation [36]. 3.3 Effect of aging period The effect of aging period to the activity of encapsulated tyrosinase was examined in order to identify the optimal aging time for the synthesis of TESA. The results are presented in Table 3. It is shown that TESA exhibits the highest enzymatic activities of 84% after 2 days aging at room temperature. A significant drop in activities of encapsulated tyrosinase to 68% was observed after 4 days aging followed by a constant activity afterwards. The initial decrease in the activities of encapsulated tyrosinase was probably due to the formation and changes within the tyrosinase-silica gel network which resulted in a faster hydrolysis and condensation reaction, hence caused deterioration to the native structure of encapsulated enzyme. The shorter aging period

Table 3 Enzymatic activities of TESA at different aging periods Table 2 Effect of solvent extraction to the enzymatic activity of TESA Solvent

Activity of tyrosinase (%)

Aging period (days)

Enzymatic activity (%)

1

75

2

84 68 55

Amyl acetate/acetone (v/v:1/1)

70

4 6

Acetone

45

8

54

Amyl acetate

55

12

55

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may not allow for complete formation of the silica aerogel framework around the encapsulated enzyme. Consequently, the encapsulated enzyme could easily diffuse out of the silica aerogel network during reaction. Lower activities for encapsulated tyrosinase after 4 days of aging period was observed, probably due to denaturation of tyrosinase.

tyrosinase were sterically confined inside the silica aerogel network. Since tyrosinase was added prior to gelation, it is possible that a silica aerogel network was formed around the tyrosinase; hence most of the tyrosinase was completely loaded.

3.4 Effect of enzyme loading Activity of encapsulated tyrosinase as a function of different enzyme loadings in 30 g of silica aerogel demonstrated the highest activity for encapsulated tyrosinase with enzyme loading of 10.00 mg/mL (500 U enzyme/g silica aerogel). After maximum enzymatic activity, further increase in enzyme concentration resulted in a gradual decrease in activity of encapsulated tyrosinase due to aggregation of tyrosinase molecules in the solution. In a highly concentrated solution, tyrosinase molecules tend to aggregate with each other, thus tyrosinase molecules become less soluble resulting in less interaction between enzyme and substrate.

The activity of free tyrosinase and TESA as a function of temperature subjected to 60 min of reaction time are presented in Fig. 6. Concentration of tyrosinase was 10 mg/mL in 30 g of silica aerogel. It shows that a long-term thermostability of tyrosinase was enhanced after the encapsulation process. The optimum temperature for both free tyrosinase and TESA was 25 °C. TESA was stable up to 70 °C at all contact periods; with loss of some activity observed at 80 °C. For free tyrosinase, incubation at temperature above 40 °C was detrimental to enzyme activity; which began to decrease sharply beyond 35 °C. The enhancement in the thermostability of the TESA is attributed to the tight confinement of tyrosinase in the silica aerogel matrix which acts as an insulator to tyrosinase.

3.5 Enzymatic activity

3.5.4 Influence of pH

3.5.1 Assay of free tyrosinase and tyrosinase encapsulated silica aerogel (TESA) activity

The pH profiles for enzymatic activity of free tyrosinase with the concentration of 10 mg/mL and TESA with tyrosinase concentration of 10 mg/mL in 30 g of silica aerogel, in the pH range of 4–9 are depicted in Fig. 7. It reveals that TESA and free tyrosinase achieved their maximum activity level at pH 7. This finding is in agreement with previous study which reported that the optimum activity of tyrosinase occurred near pH 7 [7]. However, the enzymatic activity of tyrosinase was slightly decreased

3.5.2 Leaching study It is essential to ensure that no dissolved or free tyrosinase existed in the assay system when evaluating the immobilized enzyme activity or else, the dissolved enzyme would give higher and false remaining activity. No enzymatic activity was observed when silica aerogel alone was immersed in the substrate, confirming that the identified activity was solely due to tyrosinase inside TESA. In contrast, the filtrate from TESA showed negligible enzymatic activity (0.2% after 60 min reaction) due to oxidation of catechol. Therefore, it suggests that almost 98% of

100 90

Activity or Tyrosinase (%)

Study on the enzymatic activity upon oxidation of catechol at pH 7 indicates that free tyrosinase has a higher enzymatic activity compared to TESA. This finding can be explained by the diffusion resistance of catechol which acts as a substrate in the silica aerogel network. Since the diffusion rate of the substrate was sufficiently slow compared to enzymatic catalysis, tyrosinase molecules which were encapsulated close to the surface of silica aerogel could easily oxidize the catechol due to denaturation of small amount of the tyrosinase by solvent extractor during the synthesis process, leading to the lower activity of the encapsulated tyrosinase.

3.5.3 Influence of temperatures

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o

Temperature ( C) Fig. 6 Enzymatic activities at different temperatures of (a) free tyrosinase, (b) TESA (10 mg/mL of tyrosinase in 30 g of silica)

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Fig. 7 Enzymatic activities at different pH of (a) free tyrosinase and (b) TESA (10 mg/mL of tyrosinase in 30 g of silica)

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3.6 Removal of phenol by TESA The efficiency of TESA is tested in the removal of phenol since tyrosinase is well known having the capability to degrade phenol in aqueous solution. Free tyrosinase is capable to remove numerous phenol compounds through the oxidization of phenols by phenoxy radicals. These phenoxy radicals are diffused from the active centre of tyrosinase into the reaction solution to react with phenol and subsequently form substances that is much less water soluble. These insoluble polymers can then be separated by simple filtration or flocculation. Figure 8 shows the percentage removal of phenol by free tyrosinase and TESA as a function of contact time. Almost 70% of phenol was removed after an hour contact with TESA compared to free tyrosinase which removed phenol up to 80%. The removal of phenol after 80 min of contact time with both TESA and free tyrosinase was not much increased probably due

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Fig. 8 Percent removal of phenol by (a) free tyrosinase, (b) TESA (10 mg/mL of tyrosinase in 30 g of silica)

excess generation of quinones formed during phenol degradation. The accumulation of the quinones on the support may have prevented TESA from undergoing further reaction. 3.6.1 Reusability The average percentage removal of phenol by TESA over 10 batches of recycle was 60%. It is very interesting to observe that the percent removal of phenol was only slightly reduced even after 10 times reuse as shown in Fig. 9. Therefore, it can be concluded that encapsulated tyrosinase in TESA was well protected from extreme temperature and pH conditions, making it much more stable since each cycle resulted in essentially the same activity. Such phenomena is not observed in free tyrosinase. Upon filtration of TESA from the substrate solution of each cycle, brown particles were observed on the recovered TESA probably due to production of quinones or polyphenolics species which are known to be coloured.

Removal of Phenol (%)

upon encapsulation into silica aerogel network due to the diffusion limitation. Only 50% of activity was detected at pH 8 from free tyrosinase while 75% of activity was achieved by TESA. This can be explained by the fact that some of the free enzymes were denatured at pH 8. Besides, the decrease in activity at pH 8 may be attributed to the polymerization of quinones [9]. During polymerization process, more stable and insoluble intermediates were produced. Accumulation of intermediates could deactivate free tyrosinase, thus, limit further activity of tyrosinase. At pH 5, enzymatic activity of free tyrosinase was further decreased to 10% and it became totally inactive at pH 4. In contrast, up to 15% of enzymatic activity was achieved when tyrosinase was encapsulated into silica aerogel. At pH 4 and pH 9, TESA achieved activity of 15–25%, respectively, while free tyrosinase was completely inactive at these pH values. This demonstrates that the stability of tyrosinase towards acidic and basic conditions is significantly enhanced after encapsulation.

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17 OH

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active with 60% phenol removal. In conclusion, the study indicates that the stability of tyrosinase towards extreme temperature as well as acidic and basic conditions is significantly improved after encapsulation in silica aerogel, thus making TESA an excellent nanosensor for removal of phenol in water.

O OH O OH

+

O2

Acknowledgments The authors would like to thank Ministry of Science Technology and Innovation for funding.

Tyrosinase

Fig. 10 Oxidation of phenol catalyzed by tyrosinase

3.6.2 Mechanism for removal of phenol The oxidation of phenol catalyzed by tyrosinase is shown in Fig. 10. In the presence of proton, the oxidation process generates phenoxy radicals which diffuse from the active centre into solution to react with phenol and form substances that are much less water soluble known as quinones. These quinones are reactive and slowly undergo non-enzymatic conversion, oligomerization reactions, which ultimately yield high molecular weight; insoluble polyphenolics [37–39] and consequently inactivate tyrosinase for further reaction. Finally, these insoluble polymers can be precipitated out of the solution and separated by simple filtration or flocculation.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

4 Conclusions

15.

Tyrosinase encapsulated silica aerogel (TESA) is synthesized via an alcohol-free colloidal sol–gel route at room temperature and at neutral pH. Characterization on TESA indicates that 98% of tyrosinase is effectively loaded and located inside the silica aerogel network. XRD analysis reveals tyrosinase molecules are well-dispersed into silica aerogel as the amorphous structure remains even after the encapsulation process. TESA without solvent extraction shows higher tyrosinase activity than TESA extracted by amyl acetate/acetone (v/v:1/1). Optimization study indicates that 500 U enzyme/g silica aerogel; aged for 2 days, exhibits superior performance in the oxidation of catechol. Significant catalytic activity of TESA is observed at pH range of 4–9, with the maximum activity at pH 7 and a temperature range of 5–80 °C. In contrast, free tyrosinase is totally inactive at these pH values and temperature exceeding 55 °C. TESA successfully removes 80% of phenol in aqueous solution after 3 h of contact time. It indicates excellent reusability, whereby after 10 times of use, TESA remains

16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.

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