w a t e r r e s e a r c h 5 7 ( 2 0 1 4 ) 2 9 5 e3 0 3

Available online at www.sciencedirect.com

ScienceDirect journal homepage: www.elsevier.com/locate/watres

Efficient immobilization of mushroom tyrosinase utilizing whole cells from Agaricus bisporus and its application for degradation of bisphenol A Markus Kampmann, Stefan Boll, Jan Kossuch, Julia Bielecki, Stefan Uhl, Beatrice Kleiner, Rolf Wichmann* Department of Biochemical and Chemical Engineering, TU Dortmund University, Emil-Figge-Str. 66, 44227 Dortmund, Germany

article info

abstract

Article history:

A simple and efficient procedure for preparation and immobilization of tyrosinase

Received 18 December 2013

enzyme was developed utilizing whole cells from the edible mushroom Agaricus bisporus,

Received in revised form

without the need for enzyme purification. Tyrosinase activity in the cell preparation

16 March 2014

remained constant during storage at 21
C for at least six months. The cells were

Accepted 18 March 2014

entrapped in chitosan and alginate matrix capsules and characterized with respect to

Available online 28 March 2014

their resulting tyrosinase activity. A modification of the alginate with colloidal silica enhanced the activity due to retention of both cells and tyrosinase from fractured cells,

Keywords:

which otherwise leached from matrix capsules. The observed activity was similar to the

Immobilization

activity that was obtained with immobilized isolated tyrosinase in the same material.

Tyrosinase

Mushroom cells in water were susceptible to rapid inactivation, whereas the immobilized

Mushroom cells

cells maintained 73% of their initial activity after 30 days of storage in water. Application

Bisphenol A

in repeated batch experiments resulted in almost 100% conversion of endocrine dis-

Degradation

rupting bisphenol A (BPA) for 11 days, under stirring conditions, and 50e60% conversion

Environmental water

after 20 days, without stirring under continuous usage. The results represent the longest yet reported application of immobilized tyrosinase for degradation of BPA in environmental water samples. ª 2014 Elsevier Ltd. All rights reserved.

1.

Introduction

Bisphenol A (BPA) is an important bulk chemical that is mainly used for fabrication of polycarbonate plastics and epoxy resins, which are common constituents of many household plastic products. BPA is also used, to a lesser extent, in the

* Corresponding author. Tel.: þ49 231 755 3205; fax: þ49 231 755 5110. E-mail address: [email protected] (R. Wichmann). http://dx.doi.org/10.1016/j.watres.2014.03.054 0043-1354/ª 2014 Elsevier Ltd. All rights reserved.

production of thermal paper. Due to its endocrine disrupting activity, BPA has received considerable attention (AlonsoMagdalena et al., 2006; Deutschmann et al., 2013; Howdeshell et al., 2003; Jobling et al., 2004; Kawai et al., 2003; Kubo et al., 2003; Markey et al., 2001; Oehlmann et al., 2006; Tarafder et al., 2013; vom Saal and Hughes, 2005), since it has been found in waste waters (Fu¨rhacker et al., 2000;

296

w a t e r r e s e a r c h 5 7 ( 2 0 1 4 ) 2 9 5 e3 0 3

Lagana et al., 2004; Lee and Peart, 2000; Rigol et al., 2002), surface waters (Bolz et al., 2001; Heemken et al., 2001; Stachel et al., 2003), food (Ballesteros-Go´mez et al., 2009; Biles et al., 1998) and mineral water (Toyo’oka and Oshige, 2000), as well as in human blood and urine (Dekant and Vo¨lkel, 2008; Vo¨lkel et al., 2008; Zhou et al., 2013). BPA may not be completely degraded in sewage treatment plants (Lagana et al., 2004; Lee and Peart, 2000; Rigol et al., 2002; Spring et al., 2007), hence there is a great demand for its removal from water bodies, for example, in waste water treatment (Kang et al., 2007). The enzymatic oxidation of BPA with tyrosinase has been suggested as a method for the degradation of this anthropogenic contaminant (Ispas et al., 2010; Yoshida et al., 2001). Tyrosinase is able to utilize molecular oxygen to oxidize phenolic compounds to o-diphenols and further to o-quinones. The o-quinones are colored and often toxic compounds, which can be removed via adsorption or binding to chitosan (Ispas et al., 2010; Tamura et al., 2010; Wada et al., 1993; Yamada et al., 2006). It has been shown that treatment of phenol solutions with tyrosinase and chitosan resulted in detoxified and colorless solutions (Ikehata and Nicell, 2000). Pure tyrosinase is expensive to produce on the scales required to be used for catalytic BPA degradation in waste water streams, therefore, cost reduction plays an important role with respect to an industrial application. Tyrosinase is present in the fruiting body of the edible mushroom Agaricus bisporus, which is produced in large amounts for human consumption, inexpensive, and readily available throughout the year. Some efforts have been made using semipurified tyrosinase preparations (Burton et al., 1993; Ensuncho et al., 2005; Labus et al., 2011; Marı´n-Zamora et al., 2006; Munjal and Sawhney, 2002) or whole mushroom tissue (Kameda et al., 2006; Silva et al., 2010). Since some enzyme activity may be lost during purification, and even simple purification strategies contribute significantly to overall process costs, it is a promising prospect to completely avoid enzyme purification prior to desired application. However, direct use of mushroom tissue may have disadvantages due to an inherent small surface to volume ratio, decreasing enzymatic reaction rate, as well as issues with respect to stability of the mushroom cells. These disadvantages may be mitigated by immobilization techniques. Immobilization of biocatalysts offers the possibility to protect these substances against deactivation as well as to facilitate their handling, separation, and reutilization. Nevertheless there are, to date, few reports regarding the immobilization of whole cells from A. bisporus in scientific literature (Friel and McLoughlin, 1999). In particular, information regarding immobilization of cells from the fruiting body of A. bisporus is currently non-existent. Immobilization of cells can be accomplished by entrapment in biopolymer materials, such as alginate or chitosan, both are inexpensive and commercially available, exhibit high biocompatibility, and have simple as well as mild immobilization methods (Smidsrød and Skja˚k-Bræk, 1990; Kaya and Picard, 1996). Immobilization has been demonstrated for purified tyrosinase (Ispas et al., 2010; Munjal and Sawhney, 2002). However, leaching of isolated enzyme from the biopolymer matrix capsules, including during their fabrication, is an issue which lowers immobilization efficiency, leading to high process costs.

Modification of alginate matrix capsules with colloidal silica allows manipulation of capsule permeability (Pachariyanon et al., 2011) and can be utilized for more efficient immobilization, including better retention of enzyme. In this report, a simple procedure for preparation and immobilization of whole cells from the fruiting body of A. bisporus in alginate and chitosan matrix capsules is presented. The procedure is evaluated in terms of resulting tyrosinase activity. In order to reduce loss of tyrosinase due to release from fractured cells, a modification of this system with colloidal silica is also presented, demonstrating an efficient modification of the system for quantitative immobilization of mushroom cells, which maintain tyrosinase activity without the need for purification. These matrix capsules are described with respect to some of their characteristics as well as their application for degradation of BPA. Since most reports deal with BPA solutions prepared with laboratory water with relatively short reaction cycles (Ispas et al., 2010; Nicolucci et al., 2011; Yoshida et al., 2001), this report deals with real environmental water samples spiked with BPA and application of matrix capsules for several days in order to better simulate possible application in an industrial process.

2.

Materials and methods

2.1.

Materials

Mushrooms (Agaricus bisporus) at developmental stages 2e3 (Hammond and Nichols, 1976) (velum still closed) were acquired from a local supermarket and were used on the day of purchase. Tyrosinase from mushroom (product number T3824), alginic acid sodium salt from brown algae (suitable for immobilization of micro-organisms), chitosan from crab shells (highly viscous), Ludox HS-30 colloidal silica 30% (w/ w), sodium triphosphate pentabasic (NaTPP, 98% purity) and BPA (99% purity) were purchased from SigmaeAldrich GmbH, Steinheim, Germany. Acetonitrile (99.9% purity), CaCl2∙2H2O (99% purity), HCl (37%) and NaOH (99% purity) were obtained from Carl Roth GmbH & Co KG, Karlsruhe, Germany, acetic acid (glacial) from Merck KGaA, Darmstadt, (L-DOPA, Germany, 3,4-dihydroxy-L-phenylalanine 98% þ purity) from Alfa Aesar GmbH & Co KG, Karlsruhe, Germany and 2-morpholinoethanesulfonic acid (MES, molecular biology grade) from AppliChem GmbH, Darmstadt, Germany. Double distilled deionized water (ddH2O) was used for all solutions except BPA solutions, which were prepared with environmental water samples. Tyrosinase stock solution of 235 U/ml (according to the assay described in Section 2.5) was stored at 20
C and further diluted prior to use.

2.2.

Preparation of mushroom cells

The mushrooms were cut into small pieces and subsequently treated according to one of the following procedures. Procedure 1: Mushroom pieces were added to ddH2O (0.5 g/ ml) and crushed with a Philips HR2096 blender.

w a t e r r e s e a r c h 5 7 ( 2 0 1 4 ) 2 9 5 e3 0 3

Procedure 2: As an alternative to Procedure 1 mushroom pieces were lyophilized and ground mechanically with a Retsch S1 planetary ball mill (Retsch GmbH, Haan, Germany) to a fine powder. The obtained product was stored in a flask at 20
C, 4
C or 21
C. Cumulative volume undersize distribution (Q) of milled lyophilisate was examined with Cilas 715 laser diffraction spectrometer (Cilas, Orleans, France).

2.3.

Immobilization

2.3.1.

Equipment for fabrication of matrix capsules

Matrix capsules were fabricated using a self-designed droplet generator. A similar device is described in (Wolters et al., 1992). Briefly, the droplet generator is composed of a pressure vessel and an air jet nozzle. The pressure vessel serves as a polymer reservoir, from which the polymer solution is forced by aid of compressed air to a blunt cannula, positioned in the air jet nozzle. At the end of the cannula droplets are formed, whose sizes can be regulated by a coaxial air flow and which then fall into a gelling solution. Applying suitable cannulas, pressures, and air flow rates enables the manufacture of alginate and chitosan matrix capsules of the same size, although their gelling behavior, i.e. volume reduction of droplets, is different.

2.3.2.

Immobilization in alginate matrix capsules

Previous experiments have shown that the shape and mechanical stability of the matrix capsules depend on the alginate concentration. It was found in our laboratory that an alginate concentration of 2% (w/v) was sufficient for fabrication of mechanically stable matrix capsules that exhibit no destruction during handling or stirring. Therefore, this concentration was used for further investigations. First, 0.2 g sodium alginate was dissolved, by use of an agitator, in 9 ml ddH2O containing 0e11.1% (w/w) Ludox HS-30 with a certain pH value (5.5e7.5) adjusted to with HCl. Then, 1 ml of tyrosinase solution (2.35e47 U/ml) was added and slowly stirred for 15 min for homogenization as well as to allow bubbles to rise to the surface. The tyrosinase solution was not added until the alginate was completely dissolved to reduce exposure of the enzyme to surface tension stress. The volumetric ratio of the enzyme solution to the relatively viscous alginate solution was selected to enable fast homogenization. For immobilization of mushroom cells, 50e500 mg mushroom powder (cell dry weight, cdw) were added directly to 0.2 g sodium alginate and 10 ml ddH2O without or with 2.5% (w/w) LudoxHS-30 (pH 6.8) to avoid lump formation. Each alginate solution was then dropped into a gelation bath of 100 ml 2% (w/v) CaCl2 solution and kept submerged for 1 h. Both the volumetric ratio of alginate solution to CaCl2 solution and the gelation time were determined to accomplish an effective immobilization and to enable the comparison with the immobilization in chitosan matrix capsules (Section 2.3.3). After gelation, the matrix capsules were transferred to ddH2O, where they were stored until use to avoid drying and shrinkage from exposure to air. Therefore, all capsule masses reported below refer to their wet weight immediately after removal of water by filtration.

2.3.3.

297

Immobilization in chitosan matrix capsules

Chitosan matrix capsules were fabricated according to a previously published protocol (Ispas et al., 2010) with slight modifications: 125 mg chitosan were dissolved in 9 ml 0.1 M acetic acid and stirred for 4 h. Then 1 ml of tyrosinase solution (23.5 U/ml) was added and stirred for 15 min. Immobilization of mushroom cells in chitosan was carried out in a similar manner as the alginate samples: 50 mg mushroom powder and 125 mg chitosan were dispersed in 10 ml 0.1 M acetic acid. Each chitosan solution was subsequently dropped into 100 ml of 1.5% (w/v) NaTPP solution and allowed to gel for 1 h. The addition of both tyrosinase solution and mushroom cells, the volumetric ratio of chitosan solution to NaTPP solution, gelation time, and storage were adopted from the protocol for immobilization in alginate matrix capsules (Section 2.3.2) in order to maintain consistency between experimental set-ups.

2.4.

Characterization of matrix capsules

The diameter of matrix capsules was determined utilizing an Axiostar plus microscope (Carl Zeiss Microimaging GmbH, Go¨ttingen, Germany) and a Canon PowerShot A640 digital camera or Traveler SU 1071 USB microscope with Ulead Video Studio 7 SE VCD software (Supra Foto-Elektronik-Vertriebs GmbH, Kaiserslautern, Germany). Photographs of matrix capsules were processed by image analysis software, ImageJ 1.46p. Reported diameters (d) represent the averages of 30 analyzed matrix capsules, taking into account their smallest diameter (dmin) and largest diameter (dmax) orthogonal to it. The aspect ratio AR ¼ dmin/dmax is at least 0.93. Standard deviations for d and AR are less than 5%. Scanning electron microscopy (SEM) analysis was performed with an S-4500 (Hitachi, Japan) at an accelerating voltage of 1 kV after the matrix capsules were lyophilized.

2.5.

Study of tyrosinase activity

The activity of tyrosinase was determined at 30
C using a colorimetric assay adapted from literature (Behbahani et al., 1993; Burton et al., 1993; Duckworth and Coleman, 1970; Fling et al., 1963; Horowitz et al., 1960; Lerch and Ettlinger, 1972) using a Libra S12 UV/Vis spectrophotometer (Biochrom Ltd., Cambridge, United Kingdom) at a wavelength of 475 nm. Substrate solution was prepared fresh daily by dissolving 10 mM L-DOPA in 0.1 M MES buffer (pH 6.0). Previous experiments have shown that lower concentrations of L-DOPA resulted in lower tyrosinase activity. Therefore, 10 mM was chosen to obtain higher sensitivity in determining lower activity ranges. In order to investigate the activity of free tyrosinase, 1 ml of substrate solution was added to 25 ml sample solution in a quartz cuvette. The reaction was followed measuring the absorbance at intervals of 10 s for 5 min. To study the activity of immobilized tyrosinase, 5 ml substrate solution was added to 100 mg matrix capsules in a glass vessel. The reaction was carried out for 7 min under stirring with a magnetic stirrer (300 rpm). Samples of 0.8 ml were withdrawn at intervals of 30 s, transferred into a quartz cuvette and absorbance was measured. After measurement, the analyzed solution was returned to the glass vessel

298

w a t e r r e s e a r c h 5 7 ( 2 0 1 4 ) 2 9 5 e3 0 3

immediately to ensure constant reaction volume and avoid enrichment of catalyst. Tyrosinase activity was determined by calculating the amount of produced dopachrome from the linear slope of absorbance increase using the extinction coefficient ε ¼ 3600 l/ (mol∙cm) (Mason, 1948). One Unit (U) reported here refers to one mmol dopachrome generated per min and is the average of three measurements with standard deviation of less than 7%, unless otherwise stated.

2.6. Application for degradation of BPA in environmental water samples To study the degradation of BPA, environmental water samples were collected from Ruhr river, Bochum, Germany and PhoenixSee, Dortmund, Germany, in August, 2013. Water samples were centrifuged for 10 min at 4000 rpm to remove suspended particles and spiked with BPA. A BPA concentration of 0.1 mg/l was used, as this concentration is relatively close to BPA concentrations which have been found in waste water samples (Fu¨rhacker et al., 2000), and this experiment is intended to simulate environmental conditions. A concentration of matrix capsules of less than 5% (v/v) was used as well to simulate possible industrial scale reaction conditions. Repeated batch experiments were carried out in glass vials by incubating 0.5 g matrix capsules with 10 ml BPA solution at 20
C with (300 rpm) or without stirring. BPA solution was changed every 24 h and residual BPA concentration was quantified by HPLC (Knauer Smartline series with detection at 227 nm, Eurospher 100-5 C18 (5 mm, 150  4 mm) column (Knauer GmbH, Berlin, Germany), mobile phase acetonitrile/water (ratio 1:1), flow rate 0.7 ml/min, 40
C) after filtration through 0.2 mm PTFE filter. The detection limit for BPA was 0.5 mg/l.

3.

Results and discussion

3.1.

Preparation of mushroom cells

For disintegration of the mycelium of A. bisporus, two methods were investigated. First, a common household blender was used. Here, both speed and time demonstrated influence on the obtained tyrosinase activity (data not shown). The generated cell suspension could also be used for immobilization (data not shown). However, some difficulties arose from the mushroom quality: when mushrooms were stored longer than three days, tyrosinase activity declined and activity of fresh mushrooms sometimes varied with the package up to 50%, hampering sample consistency. To avoid time based variability issues, lyophilization and milling was considered as an alternative. During lyophilization, mushroom pieces lost 91% of their original weight. The obtained cumulative volume undersize distribution (Q) after milling is shown in Fig. 1 and presents the fraction smaller than stated sizes. The whole milled lyophilisate had a diameter smaller than 96 mm and 80% between 6.9 mm and 79.5 mm. Arithmetic mean was 27.3 mm and median was 35 mm. A cell suspension was prepared in ddH2O and tyrosinase activity was determined to 0.08 U/mg cdw. Considering the

Fig. 1 e Cumulative volume undersize distribution (Q) of lyophilised and milled mushroom cells.

weight loss during lyophilization, it was asserted that the obtained activity equaled the activity obtained from the blending process, suggesting that both simple methods are suitable for preparation of mushroom cells. Aliquots of the milled lyophilisate were stored at 20
C,
4 C and 21
C, activity was determined periodically. Within a period of six months, no loss of activity was observed regardless the storage temperature, indicating uncomplicated handling. All further investigations were conducted with the milled lyophilisate in order to assure sample consistency. To examine, whether there were any intact cells after milling, a cell suspension was passed through 0.2 mm filters and the activity of the filtrate was compared with the activity of the original cell suspension. Here, different membrane materials (PTFE, PET, cellulose acetate) were used to exclude adsorption of free tyrosinase. In each case, the filtrate maintained only 45% of the activity of the original cell suspension, indicating presence of whole cells or tyrosinase containing cell debris, that were retained in the filter. Processing only the filtrate would mean a remarkable waste of tyrosinase activity. Presence of shreds or fragments may even be advantageous for immobilization in biopolymer materials, as they are less prone to leaching than isolated enzymes. Therefore, the whole lyophilisate was used without any purification.

3.2.

Immobilization of isolated tyrosinase

The preparation of mushroom cells also caused cell destruction with release of tyrosinase. Since the immobilization of released enzyme in addition to whole cells could enhance the overall activity, preliminary experiments were conducted as a control to see if immobilization would work with this enzyme. In order to find a suitable immobilization system, isolated tyrosinase (2.35 U/ml polymer solution) was immobilized in different types of matrix capsules (d ¼ 1.35 mm) and studied for its resulting activity in the assay described in Section 2.5. This assay was chosen due to its fast reaction, enabling activity measurement of immobilized enzyme within a few minutes without distortion by activity of diffused enzyme, a common issue when reactions are allowed to run for an

w a t e r r e s e a r c h 5 7 ( 2 0 1 4 ) 2 9 5 e3 0 3

extended period of time. The obtained activities are presented in Table 1. Immobilization of tyrosinase in chitosan and alginate matrix capsules resulted in similar activities (0.24 U/g and 0.27 U/g, respectively). Taking into account that 60 g alginate and 10 g chitosan matrix capsules were obtained from 100 ml (approximately 100 g) of the corresponding polymer solution with appropriate amount of tyrosinase, the specified activities are relatively poor. To avoid misinterpretation arising from different retention of dopachrome generated within the matrix capsules, as well as to elucidate the immobilization efficiency, gelling solutions were studied for leaching of enzyme by tyrosinase activity assays. Both gelling solutions showed significant tyrosinase activity after matrix capsule formation (Table 1), which was attributed to enzyme leaching during the gelling process. Leaching resulted in low enzymatic activities in the matrix capsules. Increasing the alginate concentration (2.5e3.5%) in the polymer solution did not result in higher tyrosinase activities in the alginate matrix capsules (data not shown). In order to improve the immobilization, alginate matrix capsules were modified with 2.5% colloidal silica. Permeability of these and, therefore, also retention of entrapped enzyme, is affected by the size of the colloidal silica, requiring a sensitive adjustment of pH during preparation. A weak acidic medium was demonstrated to result in favorable silica particle aggregation (Pachariyanon et al., 2011). Moreover, mushroom tyrosinase exhibits maximal activity between pH 6e7 (tyrosinase product information from SigmaeAldrich). Therefore, immobilization experiments were carried out varying the pH in the range pH 5.5e7.5 to examine the effect of pH on enzyme activity. As shown in Table 1, at pH 6.8 tyrosinase activity was enhanced significantly to 0.89 U/g capsules, 220% higher activity compared to both unmodified alginate and chitosan matrix capsules. Moreover, no activity was found in the gelling solution, suggesting complete tyrosinase retention during fabrication. As demonstrated by SEM analysis, the size of the colloidal silica was smaller than 50 nm (Fig. 2). However, no activity dependence on pH during immobilization was observed in the investigated pH range. Also, higher silica content (5%, 10%) did not change the observed activity, suggesting that 2.5% was sufficient for tyrosinase retention. For these reasons, 2.5% silica with pH 6.8 was used for further investigations. To verify the suitability as an immobilization system, different concentrations of tyrosinase were used for immobilization in smaller matrix capsules (d ¼ 0.48 mm). These

299

Fig. 2 e Scanning electron micrograph of colloidal silica (indicated by arrows) immobilized in silica alginate matrix capsules at pH 6.8.

were fabricated to generate a larger surface area, through which more enzyme could diffuse during gelling process. The obtained activities are plotted in Fig. 3. Increasing concentrations of enzymes in the matrix capsules resulted in higher enzymatic activity. However, the activity does not increase proportionally to the amount of enzyme. For example, utilizing 0.235 U/ml polymer solution resulted in 0.38 U/g capsules, whereas 2.35 U/ml yielded 1.3 U/ g capsules, and 4.7 U/ml yielded 1.4 U/g capsules. Comparing the last two values, only a slight activity increase was observed, despite double the amount of enzyme. This can be explained by diffusion resistance of the matrix material. The substrate has to diffuse from the surrounding liquid into the matrix capsules before it can be converted by the immobilized tyrosinase. In matrix capsules with low tyrosinase content the diffusion rate is sufficient to supply the enzyme with substrate and to observe a certain activity. In matrix capsules with high tyrosinase content more substrate is converted by tyrosinase, located in the outer part of the matrix capsules, before it reaches the inner part. Therefore, enzyme located in

Table 1 e Obtained activities of immobilized tyrosinase (use of 2.35 U/ml polymer solution) in matrix capsules (d [ 1.35 mm) and their corresponding gelling solution. Capsule type

Activity [U/g capsules]

Activity in gelling solution [U/ml]

Chitosan Alginate Silica alginate (pH 6.8)

0.24 0.27 0.89

0.08 0.07 0

Fig. 3 e Activity of immobilized tyrosinase in silica alginate matrix capsules (d [ 0.48 mm) as a function of the used amount of enzyme.

300

w a t e r r e s e a r c h 5 7 ( 2 0 1 4 ) 2 9 5 e3 0 3

Table 2 e Obtained activities of immobilized mushroom cells (0.08 U/mg cdw) in matrix capsules (d [ 1.35 mm) and their corresponding gelling solution. Capsule type Chitosan Alginate Silica alginate Silica alginate

Mushroom concentration [mg cdw/ml polymer solution]

Activity [U/g capsules]

Activity in gelling solution [U/ml]

5 5 5 50

0.44 0.44 0.86 1.38

0.03 0.07 0 0

the center of the capsules might have only limited access to the substrate and might not significantly contribute to the observed activity. Nevertheless, since no activity was found in any gelling solution after matrix capsule formation, it was concluded that the use of silica modified alginate would be suitable for efficient immobilization of tyrosinase.

3.3.

Immobilization of mushroom cells

In analogous experiments to those conducted with isolated tyrosinase, mushroom cells (5 mg/ml polymer solution) were immobilized in different types of matrix capsules (d ¼ 1.35 mm) and studied for activity. As presented in Table 2, chitosan and alginate matrix capsules showed identical activities (0.44 U/g capsules) suggesting that immobilization of mushroom cells was successful and not hindered by components from disrupted cells. Both gelling solutions exhibited a certain tyrosinase activity after matrix capsule formation (Table 2), likely due to leaching of tyrosinase from disrupted cells. The activity of 0.07 U/ml in the gelling solution is similar to the activity observed in the gelling solution after immobilization of isolated tyrosinase in chitosan or alginate (Table 1). However, the corresponding activity of the immobilized cells (0.44 U/g capsules) was significantly higher than the corresponding activity obtained with immobilized isolated tyrosinase (0.27 U/ g capsules). Thus, lower tyrosinase activity loss occurred in the gelling solution when whole cells were immobilized. Therefore, it was concluded that the entrapment of cells was more efficient than the entrapment of isolated tyrosinase in chitosan and alginate. Addition of 2.5% colloidal silica to the alginate resulted in increased enzymatic activity of 0.86 U/g capsules, 95% higher activity compared to immobilized cells in both unmodified alginate and chitosan, and no activity was detected in the gelling solution. This was attributed to effective retention of both cells and tyrosinase from fractured cells in the silica alginate matrix capsules. Even when increasing the cell concentration to 50 mg/ml polymer solution, no activity was found in the gelling solution, whereas the activity of silica alginate matrix capsules was enhanced to 1.38 U/g capsules. Comparing the data given in Table 1, Fig. 3 and Table 2, it can be concluded that the immobilized cells achieve similar activities as the immobilized isolated tyrosinase, despite the larger diameter of the applied matrix capsules. Since the mushroom cell preparation was easily obtained without purification, this finding may be very useful to reduce the cost of enzyme preparation.

3.4.

Tyrosinase stability in immobilized mushroom cells

To characterize the stability of various enzyme preparations over time, free and immobilized mushroom cells were stored in ddH2O at 21
C and tyrosinase activity was determined at certain intervals. The residual activities are illustrated in Fig. 4. Mushroom cell suspensions exhibited rapid loss of initial tyrosinase activity after only a few days, indicating its susceptibility to inactivation, inherent protein degradation from proteases or microbial digestion, since experiments were carried out under non-sterile conditions. The residual activity in the first days was less reproducible and may also be a consequence of microbial contamination. In comparison to cell suspensions, immobilized cells in alginate matrix capsules retained approximately 63% of initial activity after ten days and 35% after 30 days. This remaining tyrosinase activity was considerably enhanced by immobilization in silica alginate matrix capsules: 83% after ten days and 73% after 30 days, approximately twice the residual enzymatic activity observed in alginate. This is likely due to the stabilizing effect of immobilization and different retention of tyrosinase in the various matrix capsules. Immobilization likely protects the tyrosinase and cells from rapid inactivation and microbial digestion as well as inherent protease degradation by minimizing kinetic interactions in the solution. This is likely the case for immobilization in both alginate and silica alginate matrix capsules. The higher remaining activity in silica alginate matrix capsules can be attributed to better retention of tyrosinase released from fractured cells (Pachariyanon et al., 2011). As presented in Table 1, the addition of silica to the alginate reduces enzyme leaching during fabrication of the matrix capsules. Even after immobilization,

Fig. 4 e Residual tyrosinase activity in free and immobilized A. bisporus cells, stored in bidistilled water at 21
C.

w a t e r r e s e a r c h 5 7 ( 2 0 1 4 ) 2 9 5 e3 0 3

301

the entrapped enzyme is better retained in silica alginate compared to alginate matrix capsules.

3.5. Application for degradation of BPA in environmental water samples The fabricated silica alginate matrix capsules with immobilized mushroom cells were also analyzed for their capacity for the degradation of BPA. Environmental water samples, spiked with BPA, were used as substrate in order to examine the enzymatic activity in complex systems, i.e. in presence of naturally occurring microorganisms. Repeated batch experiments were carried out with 24 h cycles using BPA concentration of 0.1 mg/l, which is comparable to concentrations of BPA found in some waste water samples (72 mg/l, Fu¨rhacker et al., 2000). Figs. 5 and 6 depict BPA conversion after each reaction cycle. In water samples from Ruhr river, BPA conversion was approximately 95% in the first three reaction cycles without stirring (Fig. 5). In further reaction cycles, BPA conversion decreased gradually to approximately 60% in the 8th and remained between 50 and 60% until the 20th cycle. To enhance BPA conversion, further experiments were carried out under stirring conditions. BPA conversion in stirred batches was almost 100% for 11 reaction cycles (Fig. 5), demonstrating that the matrix capsules could be successfully applied for degradation of BPA even in concentrations in the lower mg/l range. Further reaction cycles were hindered due to the instability of matrix capsules, likely due to combined effects of shear stress and microbial digestion, as water samples were intentionally not sterilized. However, loss of capsule stability is not to be interpreted as undesirable, as biodegradability of matrix capsules may be advantageous in environmental remediation concepts. The release of mushroom cell debris in the environment after destruction of matrix capsules is similarly not an issue of concern, because the cells originate from a non-toxic biodegradable product. It is likely, however, that some degree of investigation is required into any potential ecological effects of the components of this system entering water sources.

Fig. 5 e BPA conversions in repeated batch experiments (24 h cycles) with immobilized mushroom cells (50 mg/ml polymer solution, 0.5 g silica alginate matrix capsules) in BPA enriched (0.1 mg/l) water from Ruhr river (10 ml).

Fig. 6 e BPA conversions in repeated batch experiments (24 h cycles) with immobilized mushroom cells (50 mg/ml polymer solution, 0.5 g silica alginate matrix capsules) in BPA enriched (0.1 mg/l) water from Phoenixsee (10 ml).

In water samples from Phoenixsee, BPA conversion was approximately 80% for 9 reaction cycles, 70% for two more reaction cycles, and remained constant at 50e60% until the 20th reaction cycle (Fig. 6). Under stirring conditions, high BPA conversion of 98% was maintained for 11 reaction cycles, until it decreased from 93% to approximately 10% from the 12th to the 16th cycle. In contrast to experiments with Ruhr water, no destruction of matrix capsules was observed here, demonstrating good mechanical stability and suggesting that reusability could depend on different factors, potentially including microbial contaminants. In absence of mushroom cells, no BPA conversion was observed in any sample tested, suggesting that any microorganisms present in water samples did not catalyze the degradation of BPA. Thus considerable catalytic activity of immobilized mushroom cells could be demonstrated for at least 20 days (without stirring) during constant reactions. These results represent the longest application of continuous catalytic activity from immobilized tyrosinase based treatment of BPA in environmental water samples reported in literature to date. In parallel studies (data not shown) with higher BPA concentrations (10 mg/l) it was observed that the color of the matrix capsules changed from light brown to dark brown. This may be indicative of accumulated reaction products in the matrix capsules. The o-quinones formed in tyrosinase catalyzed BPA degradation are colored compounds, which can undergo further reactions. Thus the dark coloring observed in these experiments may be explained by secondary products formed and retained in the matrix capsules. This would mean, at least in part, a simultaneous removal of the formed o-quinones derived from BPA. However, the products from this reaction have not been characterized in this work and may require further investigation. Another option for removal of the formed o-quinones could be the use of chitosan. When chitosan matrix capsules (without catalyst) were added to the BPA solution the BPA concentration did not change, suggesting that BPA did not adsorb or bind to chitosan. However, when chitosan matrix capsules (without catalyst) were added to the reaction

302

w a t e r r e s e a r c h 5 7 ( 2 0 1 4 ) 2 9 5 e3 0 3

mixture, consisting of BPA solution and mushroom cells immobilized in silica alginate, it was observed that these changed their color from white to dark blue green. These findings are in accordance with observations reported by other authors (Ispas et al., 2010), who worked with chitosan and isolated tyrosinase. The color change has been attributed to binding of the formed o-quinones to chitosan (Ispas et al., 2010). Moreover, it was also observed that the peaks for the reaction products in the HPLC chromatograms became smaller when chitosan matrix capsules were added to the reaction mixture. Therefore, it was concluded that the use of cells from the fruiting body of A. bisporus instead of isolated tyrosinase for degradation of BPA results in similar reaction products, which are likely removed by the use of chitosan.

4.

Conclusion

A simple method for preparation and immobilization of mushroom cells in silica alginate matrix capsules has been developed. The procedure also allows simultaneous immobilization of tyrosinase, released from fractured cells. The developed catalyst system is suitable for treatment of BPA in environmental water samples and, therefore, may be useful for waste water treatment. Since no enzyme purification was applied and tyrosinase containing cell extracts were completely immobilized without leaching, the presented immobilization strategy offers great potentials for reducing the cost of enzyme catalyzed bioremediation processes.

Acknowledgements The research leading to these results has received funding from the Ministry of Innovation, Science and Research of North Rhine-Westphalia in the frame of CLIB-Graduate Cluster Industrial Biotechnology, contract no. 314 e 108 001 08. The authors are grateful to Gerhard Schaldach for measurement with the laser diffraction spectrometer and Monika Meuris for SEM analysis.

references

Alonso-Magdalena, P., Morimoto, S., Ripoll, C., Fuentes, E., Nadal, A., 2006. The estrogenic effect of bisphenol a disrupts pancreatic b-cell function in vivo and induces insulin resistance. Environ. Health Perspect. 114 (1), 106e112. Ballesteros-Go´mez, A., Rubio, S., Pe´rez-Bendito, D., 2009. Analytical methods for the determination of bisphenol A in food. J. Chromatogr. A 1216 (3), 449e469. Behbahani, I., Miller, S.A., O’Keeffe, D.H., 1993. A comparison of mushroom tyrosinase dopaquinone and dopachrome assays using diode-array spectrophotometry: dopachrome formation vs ascorbate-linked dopaquinone reduction. Microchem. J. 47 (1e2), 251e260. Biles, J.E., McNeal, T.P., Begley, T.H., Hollifield, H.C., 1998. Determination of bisphenol-A in reusable polycarbonate foodcontact plastics and migration to food-simulating liquids. J. Agric. Food Chem. 46 (7), 2894.

Bolz, U., Hagenmaier, H., Ko¨rner, W., 2001. Phenolic xenoestrogens in surface water, sediments, and sewage sludge from Baden-Wu¨rttemberg, south-west Germany. Environ. Pollut. 115 (2), 291e301. Burton, S.G., Duncan, J.R., Kaye, P.T., Rose, P.D., 1993. Activity of mushroom polyphenol oxidase in organic medium. Biotechnol. Bioeng. 42 (8), 938e944. Dekant, W., Vo¨lkel, W., 2008. Human exposure to bisphenol A by biomonitoring: methods, results and assessment of environmental exposures. Toxicol. Appl. Pharmacol. 228 (1), 114e134. Deutschmann, A., Hans, M., Meyer, R., Ha¨berlein, H., Swandulla, D., 2013. Bisphenol A inhibits voltage-activated Ca2þ channels in vitro: mechanisms and structural requirements. Mol. Pharmacol. 83 (2), 501e511. Duckworth, H.W., Coleman, J.E., 1970. Physicochemical and kinetic properties of mushroom tyrosinase. J. Biol. Chem. 245 (7), 1613e1625. Ensuncho, L., Alvarez-Cuenca, M., Legge, R.L., 2005. Removal of aqueous phenol using immobilized enzymes in a bench scale and pilot scale three-phase fluidized bed reactor. Bioprocess Biosyst. Eng. 27 (3), 185e191. Fling, M., Horowitz, N.H., Heinemann, S.F., 1963. The isolation and properties of crystalline tyrosinase from neurospora. J. Biol. Chem. 238 (6), 2045e2053. Friel, M.T., McLoughlin, A.J., 1999. Immobilisation as a strategy to increase the ecological competence of liquid cultures of Agaricus bisporus in pasteurised compost. FEMS Microbiol. Ecol. 30 (1), 39e46. Fu¨rhacker, M., Scharf, S., Weber, H., 2000. Bisphenol A: emissions from point sources. Chemosphere 41 (5), 751e756. Hammond, J.B.W., Nichols, R., 1976. Carbohydrate metabolism in Agaricus bisporus (Lange) Sing.: changes in soluble carbohydrates during growth of mycelium and sporophore. J. General. Microbiol. 93 (2), 309e320. Heemken, O.P., Reincke, H., Stachel, B., Theobald, N., 2001. The occurrence of xenoestrogens in the Elbe River and the North Sea. Chemosphere 45 (3), 245e259. Horowitz, N.H., Fling, M., Macleod, H.L., Sueoka, N., 1960. Genetic determination and enzymatic induction of tyrosinase in Neurospora. J. Mol. Biol. 2 (2), 96e104. Howdeshell, K.L., Peterman, P.H., Judy, B.M., Taylor, J.A., Orazio, C.E., Ruhlen, R.L., vom Saal, F.S., Welshons, W.V., 2003. Bisphenol A is released from used polycarbonate animal cages into water at room temperature. Environ. Health Perspect. 111 (9), 1180e1187. Ikehata, K., Nicell, J.A., 2000. Color and toxicity removal following tyrosinase-catalyzed oxidation of phenols. Biotechnol. Prog. 16 (4), 533e540. Ispas, C.R., Ravalli, M.T., Steere, A., Andreescu, S., 2010. Multifunctional biomagnetic capsules for easy removal of phenol and bisphenol A. Water Res. 44 (6), 1961e1969. Jobling, S., Casey, D., Rodgers-Gray, T., Oehlmann, J., SchulteOehlmann, U., Pawlowski, S., Baunbeck, T., Turner, A.P., Tyler, C.R., 2004. Comparative responses of molluscs and fish to environmental estrogens and an estrogenic effluent. Aquat. Toxicol. 66 (2), 207e222. Kameda, E., Langone, M.A.P., Coelho, M.A.Z., 2006. Tyrosinase extract from Agaricus bisporus mushroom and its in Natura tissue for specific phenol removal. Environ. Technol. 27 (11), 1209e1215. Kang, J.H., Aasi, D., Katayama, Y., 2007. Bisphenol A in the aquatic environment and its endocrine-disruptive effects on aquatic organisms. Critical Rev. Toxicol. 37 (7), 607e625. Kawai, K., Nozaki, T., Nishikata, H., Aou, S., Takii, M., Kubo, C., 2003. Aggressive behavior and serum testosterone concentration during the maturation process of male mice: the effects of fetal exposure to bisphenol A. Environ. Health Perspect. 111 (2), 175e178.

w a t e r r e s e a r c h 5 7 ( 2 0 1 4 ) 2 9 5 e3 0 3

Kaya, V.M., Picard, G., 1996. Stability of chitosan gel as entrapment matrix of viable Scenedesmus bicellularis cells immobilized on screens for tertiary treatment of wastewater. Bioresour. Technol. 56 (2e3), 147e155. Kubo, K., Arai, O., Omura, M., Watanabe, R., Ogata, R., Aou, S., 2003. Low dose effects of bisphenol A on sexual differentiation of the brain and behavior in rats. Neurosci. Res. 45 (3), 345e356. Labus, K., Turek, A., Liesiene, J., Bryjak, J., 2011. Efficient Agaricus bisporus tyrosinase immobilization on cellulose-based carriers. Biochem. Eng. J. 56 (3), 232e240. Lagana, A., Bacaloni, A., De Leva, I., Faberi, A., Fago, G., Marino, A., 2004. Analytical methodologies for determining the occurrence of endocrine disrupting chemicals in sewage treatment plants and natural waters. Anal. Chim. Acta 501 (1), 79e88. Lee, H.B., Peart, T.E., 2000. Determination of bisphenol A in sewage effluent and sludge by solid-phase and supercritical fluid extraction and gas chromatography/mass spectrometry. J. AOAC Int. 83 (2), 290e297. Lerch, K., Ettlinger, L., 1972. Purification and characterization of a tyrosinase from Streptomyces glaucescens. Eur. J. Biochem. 31 (3), 427e437. Marı´n-Zamora, M.E., Rojas-Melgarejo, F., Garcı´a-Ca´novas, F., Garcı´a-Ruiz, P.A., 2006. Direct immobilization of tyrosinase enzyme from natural mushrooms (Agaricus bisporus) on D-sorbitol cinnamic ester. J. Biotechnol. 126 (3), 295e303. Markey, C.M., Luque, E.H., Munoz de Toro, M., Sonnenschein, C., Soto, A.M., 2001. In utero exposure to bisphenol A alters the development and tissue organization of the mouse mammary gland. Biol. Reprod. 65 (4), 1215e1223. Mason, H.S., 1948. The chemistry of melanin: III. Mechanism of the oxidation of dihydroxyphenylalanine by tyrosinase. J. Biol. Chem. 172 (1), 83e99. Munjal, N., Sawhney, S.K., 2002. Stability and properties of mushroom tyrosinase entrapped in alginate, polyacrylamide and gelatin gels. Enzyme Microb. Technol. 30 (5), 613e619. Nicolucci, C., Rossi, S., Menale, C., Godjevargova, T., Ivanov, Y., Bianco, M., Mita, L., Bencivenga, U., Mita, D.G., Diano, N., 2011. Biodegradation of bisphenols with immobilized laccase or tyrosinase on polyacrylonitrile beads. Biodegradation 22 (3), 673e683. Oehlmann, J., Schulte-Oehlmann, U., Bachmann, J., Oetken, M., Lutz, I., Kloas, W., Ternes, T.A., 2006. Bisphenol A induces superfeminization in the ramshorn snail Marisa cornuarietis (Gastropoda: Prosobranchia) at environmentally relevant concentrations. Environ. Health Perspect. 114 (Suppl. 1), 127e133. Pachariyanon, P., Barth, E., Agar, D.W., 2011. Enzyme immobilization in permselective microcapsules. J. Microencapsul. 28 (5), 370e383. Rigol, A., Latorre, A., Lacorte, S., Barcelo´, D., 2002. Determination of toxic compounds in paper-recycling process waters by gas chromatography-mass spectrometry and liquid

303

chromatography-mass spectrometry. J. Chromatogr. A 963 (1e2), 265e275. Silva, L.M.C., Salgado, A.M., Coelho, M.A.Z., 2010. Agaricus bisporus as a source of tyrosinase for phenol detection for future biosensor development. Environ. Technol. 31 (6), 611e616. Smidsrød, O., Skja˚k-Bræk, G., 1990. Alginate as immobilization matrix for cells. Trends Biotechnol. 8, 71e78. Spring, A.J., Bagley, D.M., Andrews, R.C., Lemanik, S., Yang, P., 2007. Removal of endocrine disrupting compounds using a membrane bioreactor and disinfection. J. Environ. Eng. Sci. 6 (2), 131e137. Stachel, B., Ehrhorn, U., Heemken, O.P., Lepom, P., Reincke, H., Sawal, G., Theobald, N., 2003. Xenoestrogens in the River Elbe and its tributaries. Environ. Pollut. 124 (3), 497e507. Tamura, A., Satoh, E., Kashiwada, A., Matsuda, K., Yamada, K., 2010. Removal of alkylphenols by the combined use of tyrosinase immobilized on ion-exchange resins and chitosan beads. J. Appl. Polym. Sci. 115 (1), 137e145. Tarafder, P., Sarkar, K., Nath, P.P., Paul, G., 2013. Inhibition of heart ventricular function of rat by bisphenol A through oxidative stress induced injury of ventricular tissue. Int. J. Pharma. Bio. Sci. 4 (2(B)), 811e820. Toyo’oka, T., Oshige, Y., 2000. Determination of alkylphenols in mineral water contained in PET bottles by liquid chromatography with coulometric detection. Anal. Sci. 16 (10), 1071e1076. vom Saal, F.S., Hughes, C., 2005. An extensive new literature concerning low-dose effects of bisphenol A shows the need for a new risk assessment. Environ. Health Perspect. 113 (8), 926e933. Vo¨lkel, W., Kiranoglu, M., Fromme, H., 2008. Determination of free and total bisphenol A in human urine to assess daily uptake as a basis for a valid risk assessment. Toxicol. Lett. 179 (3), 155e162. Wada, S., Ichikawa, H., Tatsumi, K., 1993. Removal of phenols from wastewater by soluble and immobilized tyrosinase. Biotechnol. Bioeng. 42 (7), 854e858. Wolters, G.H.J., Fritschy, W.M., Gerrits, D., van Schilfgaarde, R., 1992. A versatile alginate droplet generator applicable for microencapsulation of pancreatic Islets. J. Appl. Biomat. 3 (4), 281e286. Yamada, K., Inoue, T., Akiba, Y., Kashiwada, A., Matsuda, K., Hirata, M., 2006. Removal of p-alkylphenols from aqueous solutions by combined use of mushroom tyrosinase and chitosan beads. Biosci. Biotechnol. Biochem. 70 (10), 2467e2475. Yoshida, M., Ono, H., Mori, Y., Chuda, Y., Onishi, K., 2001. Oxidation of bisphenol A and related compounds. Biosci. Biotechnol. Biochem. 65 (6), 1444e1446. Zhou, F., Zhang, L., Liu, A., Shen, Y., Yuan, J., Yu, X., Feng, X., Xu, Q., Cheng, C., 2013. Measurement of phenolic environmental estrogens in human urine samples by HPLCMS/MS and primary discussion the possible linkage with uterine leiomyoma. J. Chromatogr. B 938, 80e85.