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Efficient immobilization of mushroom tyrosinaseutilizing whole cells from Agaricus bisporus and itsapplication 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

a r t i c l e i n f o

Article history:

Received 18 December 2013

Received in revised form

16 March 2014

Accepted 18 March 2014

Available online 28 March 2014

Keywords:

Immobilization

Tyrosinase

Mushroom cells

Bisphenol A

Degradation

Environmental water

* Corresponding author. Tel.: þ49 231 755 32E-mail address: wichmann@bci.tu-dortm

http://dx.doi.org/10.1016/j.watres.2014.03.0540043-1354/ª 2014 Elsevier Ltd. All rights rese

a b s t r a c t

A simple and efficient procedure for preparation and immobilization of tyrosinase

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

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

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

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

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,

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

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

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

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

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

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

after 20 days, without stirring under continuous usage. The results represent the longest

yet reported application of immobilized tyrosinase for degradation of BPA in environ-

mental water samples.

ª 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Bisphenol A (BPA) is an important bulk chemical that ismainly

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

05; fax: þ49 231 755 5110.und.de (R. Wichmann).

rved.

production of thermal paper. Due to its endocrine disrupting

activity, BPA has received considerable attention (Alonso-

Magdalena 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 (Furhacker et al., 2000;

wat e r r e s e a r c h 5 7 ( 2 0 1 4 ) 2 9 5e3 0 3296

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-Gomez 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 Volkel, 2008; Volkel

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 anthropo-

genic 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-qui-

nones. The o-quinones are colored and often toxic com-

pounds, 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 tobeused for catalytic BPAdegradation inwastewater

streams, therefore, cost reduction plays an important role with

respect to an industrial application. Tyrosinase is present in the

fruitingbodyof theediblemushroomAgaricus bisporus,which is

produced in large amounts for human consumption, inexpen-

sive, 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;

Marın-Zamora et al., 2006; Munjal and Sawhney, 2002) or

wholemushroom tissue (Kameda et al., 2006; Silva et al., 2010).

Since someenzymeactivitymaybe lostduringpurification, 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. How-

ever, 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 sta-

bility 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. Never-

theless there are, to date, few reports regarding the immobi-

lization 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 Skjak-Bræk, 1990; Kaya and Picard, 1996).

Immobilization has been demonstrated for purified tyros-

inase (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 effi-

cient 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 pre-

sented. 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 immo-

bilization of mushroom cells, which maintain tyrosinase

activity without the need for purification. These matrix

capsules are described with respect to some of their char-

acteristics 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 ac-

quired 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,

Germany, 3,4-dihydroxy-L-phenylalanine (L-DOPA,

98% þ purity) from Alfa Aesar GmbH & Co KG, Karlsruhe,

Germany and 2-morpholinoethanesulfonic acid (MES, molec-

ular 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.

wat e r r e s e a r c h 5 7 ( 2 0 1 4 ) 2 9 5e3 0 3 297

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, Ger-

many) 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 capsulesMatrix 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 pres-

sure 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 manufac-

ture of alginate and chitosanmatrix capsules of the same size,

although their gelling behavior, i.e. volume reduction of

droplets, is different.

2.3.2. Immobilization in alginate matrix capsulesPrevious experiments have shown that the shape and me-

chanical stability of the matrix capsules depend on the algi-

nate concentration. It was found in our laboratory that an

alginate concentration of 2% (w/v) was sufficient for fabri-

cation of mechanically stable matrix capsules that exhibit no

destruction during handling or stirring. Therefore, this con-

centration 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 mush-

room 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) Ludox�HS-30 (pH 6.8) to avoid lump formation.

Each alginate solution was then dropped into a gelation

bath of 100ml 2% (w/v) CaCl2 solution and kept submerged for

1 h. Both the volumetric ratio of alginate solution to CaCl2solution and the gelation timewere 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. Immobilization in chitosan matrix capsulesChitosan matrix capsules were fabricated according to a pre-

viously 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 1ml 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 125mg chitosan were dispersed in 10ml 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, gela-

tion 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,

Gottingen, 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 de-

viations for d and AR are less than 5%.

Scanning electron microscopy (SEM) analysis was per-

formed 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 experi-

ments 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 ac-

tivity ranges.

In order to investigate the activity of free tyrosinase, 1ml 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 sub-

strate solution was added to 100mgmatrix 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

Fig. 1 e Cumulative volume undersize distribution (Q) of

lyophilised and milled mushroom cells.

wat e r r e s e a r c h 5 7 ( 2 0 1 4 ) 2 9 5e3 0 3298

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 ε¼ 3600 l/

(mol∙cm) (Mason, 1948). One Unit (U) reported here refers to

one mmol dopachrome generated permin and is the average of

three measurements with standard deviation of less than 7%,

unless otherwise stated.

2.6. Application for degradation of BPA inenvironmental water samples

To study the degradation of BPA, environmental water samples

were collected fromRuhr river, Bochum,GermanyandPhoenix-

See, Dortmund, Germany, in August, 2013.Water sampleswere

centrifuged for 10 min at 4000 rpm to remove suspended parti-

cles and spiked with BPA. A BPA concentration of 0.1 mg/l was

used, as this concentration is relatively close to BPA concen-

trations which have been found in waste water samples

(Furhacker 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 10ml BPA solution at 20 �Cwith (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 0.7ml/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 themyceliumofA. bisporus, twomethods

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 gener-

ated 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 lyophiliza-

tion, 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

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 uncompli-

cated 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 sus-

pension, indicating presence of whole cells or tyrosinase

containing cell debris, that were retained in the filter. Pro-

cessing 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 destruc-

tion 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 workwith 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 ac-

tivity 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

Fig. 2 e Scanning electron micrograph of colloidal silica

(indicated by arrows) immobilized in silica alginate matrix

capsules at pH 6.8.

wat e r r e s e a r c h 5 7 ( 2 0 1 4 ) 2 9 5e3 0 3 299

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 avoidmisinterpretation 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

capsulesweremodifiedwith 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 aggre-

gation (Pachariyanon et al., 2011). Moreover, mushroom

tyrosinase exhibits maximal activity between pH 6e7 (tyrosi-

nase 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 ac-

tivity compared to both unmodified alginate and chitosan

matrix capsules. Moreover, no activity was found in the gel-

ling 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 immobili-

zationwas 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 immo-

bilization in smaller matrix capsules (d ¼ 0.48 mm). These

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/gcapsules]

Activity in gellingsolution [U/ml]

Chitosan 0.24 0.08

Alginate 0.27 0.07

Silica alginate

(pH 6.8)

0.89 0

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 cap-

sules resulted in higher enzymatic activity. However, the ac-

tivity 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. Inmatrix capsuleswith low tyrosinase content the

diffusion rate is sufficient to supply the enzyme with sub-

strate 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

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.

Table 2 e Obtained activities of immobilized mushroom cells (0.08 U/mg cdw) in matrix capsules (d [ 1.35 mm) and theircorresponding gelling solution.

Capsuletype

Mushroom concentration [mg cdw/ml polymersolution]

Activity [U/gcapsules]

Activity in gelling solution [U/ml]

Chitosan 5 0.44 0.03

Alginate 5 0.44 0.07

Silica alginate 5 0.86 0

Silica alginate 50 1.38 0

wat e r r e s e a r c h 5 7 ( 2 0 1 4 ) 2 9 5e3 0 3300

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 effi-

cient immobilization of tyrosinase.

Fig. 4 e Residual tyrosinase activity in free and

immobilized A. bisporus cells, stored in bidistilled water at

21 �C.

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

(d¼ 1.35mm) and studied for activity. As presented in Table 2,

chitosan and alginate matrix capsules showed identical ac-

tivities (0.44 U/g capsules) suggesting that immobilization of

mushroom cells was successful and not hindered by compo-

nents from disrupted cells.

Both gelling solutions exhibited a certain tyrosinase ac-

tivity 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 iso-

lated 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 obtainedwith 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 activitywas 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 immobi-

lized isolated tyrosinase, despite the larger diameter of the

appliedmatrix capsules. Since themushroom 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, freeand immobilizedmushroomcellswerestored 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 sus-

ceptibility 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

alginatematrix capsules retained approximately 63% of initial

activity after ten days and 35% after 30 days. This remaining

tyrosinase activity was considerably enhanced by immobili-

zation 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 degra-

dation byminimizing 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 addi-

tion of silica to the alginate reduces enzyme leaching during

fabrication of the matrix capsules. Even after immobilization,

wat e r r e s e a r c h 5 7 ( 2 0 1 4 ) 2 9 5e3 0 3 301

the entrapped enzyme is better retained in silica alginate

compared to alginate matrix capsules.

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).

3.5. Application for degradation of BPA inenvironmental water samples

The fabricated silica alginate matrix capsules with immobi-

lizedmushroom 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 experi-

ments were carried out with 24 h cycles using BPA concen-

tration of 0.1 mg/l, which is comparable to concentrations of

BPA found in some waste water samples (72 mg/l, Furhacker

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 stir-

red 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 ef-

fects of shear stress andmicrobial digestion, as water samples

were intentionally not sterilized. However, loss of capsule

stability is not to be interpreted as undesirable, as biode-

gradability of matrix capsules may be advantageous in envi-

ronmental 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).

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%wasmaintained 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, demon-

strating good mechanical stability and suggesting that reus-

ability 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 microor-

ganisms 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 contin-

uous 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 con-

centrations (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 cata-

lyzed 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 thematrix capsules. This wouldmean,

at least in part, a simultaneous removal of the formed o-qui-

nones 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

wat e r r e s e a r c h 5 7 ( 2 0 1 4 ) 2 9 5e3 0 3302

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 immobi-

lization 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 Clus-

ter Industrial Biotechnology, contract no. 314e 108 001 08. The

authors are grateful to Gerhard Schaldach for measurement

with the laser diffraction spectrometer andMonika Meuris for

SEM analysis.

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