Removal of p-alkylphenols from aqueous solutions by combined use of mushroom tyrosinase and chitosan beads

Article abstract of DOI:10.1271/bbb.60205

Enzymatic removal of p-alkylphenols from aqueous solutions was investigated through the two-step approach, the quinone conversion of p-alkylphenols with mushroom tyrosinase (EC 1.14.18.1) and the subsequent adsorption of quinone derivatives enzymatically

Full text of DOI:10.1271/bbb.60205

Biosci. Biotechnol. Biochem., 70 (10), 2467–2475, 2006
Removal of p-Alkylphenols from Aqueous Solutions
by Combined Use of Mushroom Tyrosinase and Chitosan Beads
Kazunori YAMADA,y Tomoaki INOUE, Yuji AKIBA, Ayumi KASHIWADA,
Kiyomi MATSUDA, and Mitsuo HIRATA
Department of Applied Molecular Chemistry, College of Industrial Technology, Nihon University,
1-2-1 Izumi-cho, Narashino, Chiba 275-8575, Japan
Received April 10, 2006; Accepted June 9, 2006; Online Publication, October 7, 2006
[doi:10.1271/bbb.60205]
Enzymatic removal of p-alkylphenols from aqueous
solutions was investigated through the two-step ap-
proach, the quinone conversion of p-alkylphenols with
mushroom tyrosinase (EC 1.14.18.1) and the subsequent
adsorption of quinone derivatives enzymatically gener-
ated on chitosan beads at pH 7.0 and 45 ꢀC as the
optimum conditions. This technique is quite effective
for removal of various p-alkylphenols from an aqueous
solution. The % removal values of 97–100% were
obtained for p-n-alkylphenols with carbon chain lengths
of 5 to 9. In addition, removal of other p-alkylphenols
was enhanced by increasing either the tyrosinase
concentration or the amount of added chitosan beads,
and their % removal values reached >93 except for 4-
tert-pentylphenol. This technique was also applicable to
remove 4-n-octylphenol (4NOP) and 4-n-nonylphenol
(4NNP) as suspected endocrine disrupting chemicals.
The reaction of quinone derivatives enzymatically
generated with the chitosan’s amino groups was con-
firmed by the appearance of peaks for UV–visible
spectrum measurements of the chitosan films incubated
in the p-alkylphenol and tyrosinase mixture solutions. In
addition, 4-tert-pentylphenol underwent tyrosinase-cat-
alyzed oxidation in the presence of hydrogen peroxide.
tamination in their internal organs between 10 and
several hundred times greater than in the surrounding
environment.5) In addition, when alkylphenol ethoxy-
lates, which are widely used in manufacturing surfac-
tants and antioxidants in the plastics industry, are
discharged to wastewater treatment facilities or released
into the environment, primary degradation of them
generates alkylphenols as well as shorter-chain alkyl-
phenol ethoxylates. Further transformation produces
various intermediates such as alkylphenols, alkylphen-
oxy ethoxy acetic acid, and alkyl phenoxy acetic acid.5,6)
Alkylphenols have similar endocrine disrupting activ-
ities due to a phenyl ring in their chemical structures.7,8)
It has been reported that biodegradation of alkylphenol
ethoxylates and alkylphenols stimulates production of
biomarkers of estrogenic activity in male fish.5,9,10)
Many researchers have a great interest in oxidore-
ductases such as tyrosinase,11–15) laccase,16–18) and
peroxidase,19–23) because their industrial potentials are
recognized in degradation and detoxification of recalci-
trant environmental pollutions. The potential advantages
of the enzymatic treatment include operation at low
concentrations over wide pH and temperature ranges and
the easy control process. Tyrosinase is most used in
treating wastewater containing phenolic contaminants
and related compounds.11–15) Tyrosinase is a copper-
containing monooxygenase widely distributed in nature
and is responsible for melanization in animals and
browning in plants. It catalyzes two distinct reactions:
the hydroxylation of monophenols to o-diphenols (cre-
solase activity) and the oxidation of o-diphenols to o-
quinones (catecholase activity), which undergo a non-
enzymatic polymerization.11)
Key words: mushroom tyrosinase; alkylphenol; chito-
san; quinone formation; adsorption
In recent years, there has been a growing concern that
some chemicals, known as endocrine disrupting chem-
icals, may have adverse effects on humans and wild-
life.1,2) If endocrine disrupting chemicals are released
into the environment, they may bioaccumulate and
become biomagnified in the food chain. In addition, they
have been confirmed to disturb sexual characteristics in
wildlife populations such as fish and amphibians3,4) and
may be responsible for increases in cancer. Alkylphe-
nols have received considerable attention due to high
production and widespread usage. They are concentrated
by organisms such as fish and birds, leading to con-
In addition, many studies have been done on the
enzymatic oxidation of phenol compounds by tyrosinase
to the corresponding o-quinone derivatives in addition
to the kinetic mechanism of action of tyrosinase,24–26)
but, little has been reported on the removal of phenol
compounds. In addition, the successful use of cationic
polymers such as chitosan and polyethylenimine to
y
To whom correspondence should be addressed. Tel: +81-47-474-2571; Fax: +81-47-474-2579; E-mail: k5yamada@cit.nihon-u.ac.jp

2468
K. YAMADA et al.
Scheme 1. Removal of p-Alkylphenols by the Quinone Conversion with Mushroom Tyrosinase and the Subsequent Adsorption of Quinone
Derivatives on the Chitosan Beads.
remove tyrosinase-generated quinone derivatives has
been reported by some researchers.11,27,28) Their results
indicate that the combination of tyrosinase and cationic
polymer coagulants is an effective procedure in remov-
ing carcinogenic phenol compounds from aqueous
solutions. But, one of the major problems in this
procedure is that there is an optimum concentration
range of the coagulant for maximum flocculation. When
the dosage is less or more than the optimum concen-
tration range, flocculation does not occur. We have
reported on the enzymatic removal of phenol com-
pounds by combined use of mushroom tyrosinase and
chitosan in the form of beads.12) Quinone derivatives
enzymatically generated from p-alkylphenols can under-
go either Schiff base or Michael’s-type addition reaction
with chitosan’s amines as shown in Scheme 1. The
quantities of phenol compounds of >90% were effec-
tively removed by this procedure at pH 7.0 and 45 ꢀC as
the optimum conditions.
The goal of this study was to investigate the removal
of various p-alkylphenols containing 4-n-octylphenol
(4NOP) and 4-n-nonylphenol (4NNP) through their
enzymatic oxidation and the subsequent adsorption of
quinone derivatives formed on the chitosan beads.
Specifically, we followed the effect of the addition of
chitosan in the form of beads, and the capability of this
two-step approach for removing various p-alkylphenols.
in a pH 7.0 phosphate buffer (ionic strength, 0.01 M).
All p-alkylphenols used were purchased from Wako
Pure Chemicals (Tokyo, Japan) and Tokyo Kasei Kogyo
(Tokyo, Japan), and used without further purification.
A chitosan solution (1 w/v%) was prepared using 1.0 g
chitosan flakes (chitosan 1000, Wako Pure Chemi-
cals).12) The chitosan films were prepared by pipetting a
chitosan solution of 4.0 g into a Petri dish of diameter
3.2 cm. The chitosan solution was allowed to dry in an
oven at 60 ꢀC. The chitosan films (average thickness,
31.4 mm) removed from the Petri dishes were thoroughly
washed with 1 M NaOH and water to neutralize the
amino groups, and then dried under reduced pressure.
Batch reactions. Enzymatic removal of p-alkylphe-
nols was carried out in the batch systems. One of the p-
alkylphenols used (0.5 mM) and tyrosinase (50 U/cm3)
were dissolved in a pH 7.0 phosphate buffer. A given
amount of chitosan beads were added to the mixture
solutions (20 cm3) containing one of the p-alkylphenols
used and tyrosinase at pH 7.0 and 45 ꢀC.12)
Quantitative assay of p-alkylphenols. The concentra-
tion of remaining p-alkylphenols was determined by
Hitachi L-7000 high performance liquid chromatogra-
phy combined with a spectrophotometer and an integra-
tor (Hitachi L-7420) or a colorimetric assay with 4-
aminoantipyrine (4-AAP).14,29)
Materials and Methods
For the colorimetric assay, the reagents were added to
an aliquot of 0.4 cm3 taken from the reaction solutions in
the following order: 3.0 cm3 of 0.25 M sodium bicar-
bonate, 0.4 cm3 of 20.8 mM 4-AAP, and 0.4 cm3 of 83.4
mM potassium ferricyanide. After vigorous stirring for
5 min, the concentration of p-alkylphenols was deter-
mined from absorbance measured at wavelengths de-
pending on the type of phenol compound.24,30)
Chemicals. Mushroom tyrosinase (EC 1.14.18.1) of
the specific activity of 2,590 U/mg-solid (activity
determined by the supplier) was purchased from Sigma
(St. Louis, MO). A chitosan bead, Chitopearl AL-01,
from Fuji Spinning (Tokyo, Japan). (particle size, 70–
200 mm; specific surface area, 70–100 m2/g; water
content, 92.5%) was used as an adsorbent, and stored
The concentration of remaining p-alkylphenols,

Enzymatic Water Purification
2469
which had no color development by 4-AAP, was
determined by the HPLC method. The reverse phase
columns, Inertsil ODS-2 and C8-3 (5 mm, 4.6 mm i.d. ꢁ
15 cm), were used, and the ratio of the mobile phase
with a flow rate of 1.0 cm3/min consisting of acetonitrile
and water depended on the carbon-chain lengths of p-
alkylphenols used. After a prescribed time, 0.010 cm3 of
reaction solution was injected. The absorption spectra of
the reaction solutions were determined by the spectro-
photometer mentioned above.12)
Estimation of removal of p-alkylphenols. The %
conversion values were calculated from the remaining
p-alkylphenol concentration determined by the 4-AAP
and HPLC methods, Crem, and the initial concentration
of the p-alkylphenols used, C0, according to eq. 1:
Fig. 1. Changes in the Absorbance at 400 nm of a 4-n-Pentylphenol
(0.5 mM) Solution Containing Tyrosinase of 50 U/cm3 with the
Reaction Time in the Absence ( ) and Presence ( ) of Chitosan
Beads of 0.025 cm3/cm3 at pH 7.0 and 45 ꢀC.
C0 ꢂ Crem
C0
% conversion ¼
ꢁ 100
ð1Þ
The % adsorption values were calculated from the
absorbances at the wavelengths assigned to the gener-
ated quinone derivatives in the mixture solutions
containing the chitosan beads, Abschi, and the maximum
absorbance at the same wavelength in the absence of
chitosan beads, Absmax, according to eq. 2:
Absmax ꢂ Abschi
% adsorption ¼
ꢁ 100
ð2Þ
Absmax
In addition, the % removal values were obtained from
eq. 3:12)
C0 ꢂ Crem
C0
% removal ¼
ꢃ % adsorption
ð3Þ
Results and Discussion
Fig. 2. Changes in % Conversion ( ), % Adsorption ( ), and %
Removal ( ) with the Reaction Time for 4-n-Pentylphenol (0.5 mM)
at pH 7.0 and 45 ꢀC in the Presence of Tyrosinase at 50 U/cm3 and
Chitosan Beads of 0.025 cm3/cm3.
Removal of p-n-alkylphenols
For 4-n-pentylphenol
In our previous paper, removal of p-n-alkylphenols
such as p-cresol, 4-ethylphenol, 4-n-propylphenol, and
4-n-butylphenol was investigated by the combined use
of tyrosinase and chitosan beads, and the % removal
values of 93–97% were obtained.12) Here, we began
with enzymatic removal of other p-n-alkylphenols. The
enzymatic quinone conversion of 4-n-pentylphenol
(4NPenP) was carried out at pH 7.0 and 45 ꢀC. Figure 1
shows the changes in the absorbance at 400 nm with the
reaction time in the absence and presence of the chitosan
beads for 4NPenP. The absorbance at 400 nm of a
4NPenP solution containing tyrosinase (50 U/cm3)
sharply increased in the absence of chitosan beads.
derivatives enzymatically generated were adsorbed on
the chitosan beads.31–34) Figure 2 shows the changes in
the % conversion, % adsorption, and % removal values
with the reaction time for 4NPenP. The quinone
derivatives enzymatically generated from 4NPenP were
effectively adsorbed on the chitosan beads through
nonenzymatic chemical reaction of the quinone deriv-
atives with the chitosan’s amino groups. The % removal
value rapidly increased and 4NPenP was completely
removed at 40 min.
For 4-n-hexylphenol and 4-n-heptylphenol
´
According to Jimenez et al., a gradual decrease in the
The concentrations of 4NHexP and 4NHepP solutions
prepared with a pH 7.0 buffer were 0.3 and 0.1 mM,
respectively, due to their low solubility. Figure 3 shows
the changes in the % conversion, % adsorption, and %
removal values with the reaction time for 4NHexP and
4NHepP at 50 U/cm3 in the presence of chitosan beads
of 0.025 cm3/cm3. Both 4NHexP and 4NHepP were
absorbance indicates that some of the generated quinone
derivatives undergo the water addition reaction.26) On
the other hand, the absorbance of the 4NPenP + tyro-
sinase mixture solution with the chitosan beads of
0.025 cm3/cm3 sharply decreased 5 min after the enzy-
matic reaction started. This indicates that quinone

2470
K. YAMADA et al.
Fig. 3. Changes in % Conversion ( ), % Adsorption ( ), and %
Removal ( ) with the Reaction Time for (a) 4-n-Hexylphenol (0.3
mM) and (b) 4-n-Heptylphenol (0.1 mM) at pH 7.0 and 45 ꢀC in the
Presence of Tyrosinase at 50 U/cm3 and Chitosan Beads of 0.025
cm3/cm3.
Fig. 4. Changes in the Absorbance at 400 nm of the (a) 4-n-
Octylphenol (0.05 mM) and (b) 4-n-Nonylphenol (0.05 mM) Solu-
tions Containing Tyrosinase at 50 U/cm3 with the Reaction Time in
the Absence ( ) and Presence ( ) of Chitosan Beads of 0.025 cm3/
cm3 at pH 7.0 and 45 ꢀC.
quinone-converted within 10 min. The quinone adsorp-
tion successfully occurred in a short time, and the %
removal values for 4NHexP and 4NHepP reached 100
and 98.7% at 60 min, respectively.
Figure 5 shows the changes in the % conversion, %
adsorption, and % removal values with the reaction time
in the presence of chitosan beads of 0.025 cm3/cm3 for
4NOP and 4NNP. 4NOP was enzymatically oxidized for
40 min in the presence of chitosan beads. Although the
reaction of quinone derivatives with chitosan was slower
than that for other p-n-alkylphenols, the % removal
value reached 100% at 120 min. Either nonenzymatic
adsorption of quinone derivatives or enzymatic quinone
conversion was slow for 4NNP, and the % removal
value went up to 97.2% at 180 min. When the quinone
derivatives generated from 4NPenP, 4NHexP, and
4NHepP were highly adsorbed on the chitosan beads,
the chitosan beads were colored dark brown, but no
distinct color development of the chitosan beads was
observed for 4NNP and 4NOP. Therefore, the UV–
visible spectra were measured of the chitosan films
incubated in 4NOP and 4NNP solutions containing
tyrosinase for 18 hr to confirm the reaction of quinone
derivatives with chitosan’s amino groups. The chitosan
films turned light brown due to quinone tanning.31–34)
Figure 6 shows the UV–visible spectra of the chitosan
films incubated in the 4NOP and 4NNP solutions
For 4-n-octylphenol and 4-n-nonyllpehnol
4NOP and 4NNP solutions of 0.05 mM were prepared
with a pH 7.0 buffer. Figure 4 shows the changes in the
absorbance at 400 nm with the reaction time at 50 U/
cm3 in the absence and presence of chitosan beads of
0.025 cm3/cm3 for 4NOP and 4NNP. 4NOP and 4NNP
were enzymatically oxidized for 10 and 120 min,
respectively, in the absence of chitosan beads. Quinone
conversion was very fast, and the quinone derivatives
generated from 4NOP were relatively unstable in the
reaction solution. Therefore, the absorbance at 510 nm
gradually increased concurrently with the decrease in
the absorbance at 400 nm. The increase in the absorb-
ance at 510 nm for 4NOP indicates that the water
addition reaction occurs.26) On the other hand, the
absorbance at 400 nm gradually increased and then
leveled off for 4NNP. It is thought that the quinone
derivatives generated from 4NNP undergo the water
addition reaction only under extreme conditions.

Enzymatic Water Purification
2471
Fig. 7. Changes in % Conversion ( ), % Adsorption ( ), and %
Removal ( ) with the Reaction Time for 4-Isopropylphenol (0.5
mM) at pH 7.0 and 45 ꢀC in the Presence of Tyrosinase at 50 U/cm3
and Chitosan Beads.
Fig. 5. Changes in % Conversion ( ), % Adsorption ( ), and %
Removal ( ) with the Reaction Time for (a) 4-n-Octylphenol
(0.05 mM) and (b) 4-n-Nonylphenol (0.05 mM) at pH 7.0 and 45 ꢀC
in the Presence of Tyrosinase at 50 U/cm3 and Chitosan Beads of
0.025 cm3/cm3.
Amount of chitosan beads (cm3/cm3): (a) 0.025, (b) 0.100.
tives enzymatically generated from 4NOP and 4NNP
react with chitosan’s amino groups.
Removal of other p-alkylphenols
Since p-n-alkylphenols were removed as described
above, we tried to remove other p-alkylphenols enzy-
matically with a nonlinear carbon chain such as 4-
isopropylphenol (4IProP), 4-sec-butylphenol (4SBP),
and 4-tert-pentylphenol (4TPenP). Of these, 4SBP and
4TPenP were suspected to be the endocrine disrupting
chemical. 4IProP and 4SBP were oxidized at pH 7.0 and
45 ꢀC with tyrosinase. The optimum conditions were
determined for enzymatic removal of these three kinds
of p-alkylphenols.
Fig. 6. UV–Visible Spectra of the Chitosan Films Incubated in (a)
4-n-Octylphenol and (b) 4-n-Nonylphenol Solutions Containing
Tyrosinase at 50 U/cm3 for 18 h at pH 7.0 and 45 ꢀC.
Removal of 4-isopropylphenol
Figure 7(a) shows the changes in the % conversion, %
adsorption, and % removal values with the reaction time
for 4IProP (0.5 mM) at pH 7.0 and 45 ꢀC in the presence
of chitosan beads of 0.025 cm3/cm3. 4IProP was
completely converted into the corresponding quinone
derivative at 40 min, but, the increase in the %
adsorption value was more gradual than the increase in
the % conversion value because some of the quinone
containing tyrosinase of 50 U/cm3. A broad peak
emerged at 350 nm for 4NOP. A small peak at 300 nm
was observed for 4NNP. No browning and increase in
UV–visible adsorption were observed for the chitosan
films immersed in the 4NOP or 4NNP solution without
tyrosinase. These results indicate that quinone deriva-

2472
K. YAMADA et al.
Fig. 8. Changes in % Conversion ( ), % Adsorption ( ), and %
Removal ( ) with the Reaction Time for 4-sec-Butylphenol (0.5
mM) at pH 7.0 and 45 ꢀC in the Presence of Tyrosinase at 50 U/cm3
and Chitosan Beads of 0.025 cm3/cm3.
Fig. 9. Changes in % Conversion with the Reaction Time for 4-sec-
Butylphenol (0.5 mM) at pH 7.0 and 45 ꢀC in the Presence of
Tyrosinase at 50 ( ), 75 ( ), and 100 ( ) U/cm3 and Chitosan
Beads of 0.025 cm3/cm3.
derivatives generated from 4IProP underwent the water
addition reaction,26) and the % removal value was
limited to 86.7% at 120 min. Therefore, the amount of
added chitosan beads was further increased, while the
tyrosinase concentration was kept unchanged. The %
removal values went up to 93.3% as shown in Fig. 7(b),
since the increase in the amount of chitosan beads added
to the 4IProP + tyrosinase mixture solution led to the
increase in the reaction of the quinone derivatives with
chitosan’s amino groups.
Removal of 4-sec-butylphenol
Enzymatic removal of 4SBP was carried out at pH 7.0
and 45 ꢀC. As Fig. 8 shows, quinone conversion sharply
increased against the reaction time in the initial stage
of the enzymatic reaction, and the % conversion value
leveled off at 80 min in the presence of chitosan beads of
0.025 cm3/cm3. Quinone adsorption gradually increased
with the reaction time. However, since the quantities of
4SBP of 4.1% remained in the reaction solution and the
% removal value was limited to 83.9% at 120 min, both
tyrosinase concentration and the amount of added
chitosan beads were further increased for removal of
4SBP. Figure 9 shows the changes in the % conversion
value with the reaction time at different tyrosinase
concentrations. Quinone conversion more sharply in-
creased at shorter reaction times at higher tyrosinase
concentrations. 4SBP was completely converted to the
quinone derivative at 75 and 100 U/cm3. Since the time
required to convert 4SBP enzymatically was shorter at
100 U/cm3, a further increased amount of chitosan
beads were added to the 4SBP solution containing
tyrosinase of 100 U/cm3. Figure 10 shows the effect of
the amount of added chitosan beads on 4SBP removal.
The absorbance at 400 nm gradually decreased 60 min
after the enzymatic reaction started, and decreased with
an increase in the amount of added chitosan beads.
Quinone adsorption on the chitosan beads takes place in
competition with water addition reaction of the quinone
Fig. 10. Changes in the Absorbance at 400 nm of 4-sec-Butylphenol
(0.5 mM) Solutions Containing Tyrosinase at 50 U/cm3 with the
Reaction Time in the Absence ( ) and Presence of Chitosan Beads
of 0.025 ( ), 0.050 ( ), and 0.100 ( ) cm3/cm3 at pH 7.0 and
45 ꢀC.
derivatives generated.26) Since an intermediate gener-
ated from the quinone derivatives through water addition
reaction is not considered to react with chitosan, water
addition reaction more preferentially occurs at lower
amounts of chitosan beads. This reaction results in the
decrease in the % adsorption value. The results of 4SBP
removal at 100 U/cm3 are shown in Fig. 11. In the case
where chitosan beads of 100 cm3/cm3 were added to
a 4SBP solution containing tyrosinase at 100 U/cm3,
4SBP was completely converted to quinone derivatives
at 60 min. The % removal value gradually increased
with an increase in the reaction time, and went up to
95.2% at 120 min.
For 4-tert-pentylphenol
No enzymatic oxidation of 4TPenP occurred at pH 7.0
in the absence of hydrogen peroxide (H2O2). In the
presence of H2O2 of 0.5 mM at pH 6.0 as the optimum

Enzymatic Water Purification
2473
Fig. 12. Changes in % Conversion ( ), % Adsorption ( ), and %
Removal ( ) with the Reaction Time for 4-tert-Pentylphenol (0.5
mM) at pH 6.0 and 45 ꢀC in the Presence of Tyrosinase at 50 U/cm3,
H2O2 (0.5 mM), and Chitosan Beads of 0.025 cm3/cm3.
Fig. 11. Changes in % Conversion ( ), % Adsorption ( ), and %
Removal ( ) with the Reaction Time for 4-sec-Butylphenol (0.5
mM) at pH 7.0 and 45 ꢀC in the Presence of Tyrosinase at 100
U/cm3 and Chitosan Beads of 0.100 cm3/cm3.
conditions for 4-tert-butylphenol (4TBP) as determined
in our previous study,12) the % conversion value of
4TPenP gradually increased. Since some tyrosinase
molecules are transformed from met into oxy forms by
the addition of H2O2, enzymatic oxidation proceeds
under the above-mentioned conditions.26) Oxy-form
tyrosinase is complexed with 4TPenP, leading to the
corresponding quinone derivative. In the absence
of H2O2, the portion of oxy form tyrosinase is so small
that the efficiency of the enzymatic oxidation is very
slow, and oxy-form tyrosinase is recovered in the
turnover. In addition, since the peak position of the
product enzymatically generated from 4TPenP was in
fair agreement with one of 4-tert-butyl-o-benzoquinone
concentration and the amount of added chitosan beads
had a favorable influence on removal of 4TPenP like
4TBP.
The removal of p-alkylphenols by the combined use
of tyrosinase and chitosan beads is summarized in
Table 1. This procedure is quite useful for removal of
various p-alkylphenols from aqueous solutions. The %
removal values of 97–100% were obtained for p-n-
alkylphenols. In addition, the % removal value increased
by adjusting either the tyrosinase concentration or the
amount of added chitosan beads for some of the p-
alkylphenols shown in Table 1. In conclusion, removal
of p-alkylphenols by this procedure is much more
effective than other methods repeated previously. This
method is an easy-to-operate procedure, and the chitosan
beads are readily separable from the reaction solutions
after removal of p-alkylphenols. It should be empha-
sized that this procedure can also be applied to remove
4NOP and 4NNP suspected endocrine disrupting chem-
icals. But, one of the problems that must be solved is
low chemical resistance to organic solvents, since
tyrosinase was deactivated by the use of ethanol. In
addition, the repeated use of tyrosinase is a significant
challenge in applying this procedure for practical
purposes. One way to solve the problem is to immobilize
tyrosinase on a water-insoluble support. Retention of
the activity of immobilized tyrosinase is required. On
this point, the covalent immobilization of tyrosinase on
hydrophilic polymer supports with a water-soluble
carbodiimide is an effective procedure, and this is our
goal for the future.
generated from 4TBP under the same condition,12,26)
a
product from 4TPenP in our system is considered to be
4-tert-pentylbenzoquinone (4TPenBQ).
When H2O2 was added to the 4TPenP + tyrosinase
(50 U/cm3) mixture solutions, the enzymatic oxidation
of 4TPenP was enhanced and reached the maximum at
an H2O2 concentration of 0.5 mM, but the % conversion
and % adsorption values were 40 and 38%, respectively,
at 120 min. Therefore, the % removal value was limited
to 16%. It was found from these results that enzymatic
quinone conversion and the subsequent quinone adsorp-
tion were very slow under the above-mentioned con-
ditions, and that removal of 4TPenBQ was carried out
at 150 U/cm3 in the presence of chitosan beads of 0.100
cm3/cm3.
Figure 12 shows the changes in the % conversion, %
adsorption, and % removal values with the reaction time
at an H2O2 concentration of 0.5 mM and a tyrosinase
concentration of 150 U/cm3 in the presence of chitosan
beads of 0.100 cm3/cm3 at pH 6.0. The % conversion
value increased to 92.3% at 180 min. The increase in
chitosan beads led to the increase in the reaction of
generated 4TPenBQ with chitosan. The % removal
value went up to 71.3%. The increase in the tyrosinase
Acknowledgment
This work was partly supported by a Grant for High
Technology Research Projects from the Ministry of
Education, Culture, Sports, Science and Technology of
Japan.

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K. YAMADA et al.
Table 1. Removal of p-Alkylphenols by the Combined Use of Tyrosinase and Chitosan Beads at 45 ꢀC
Phenol Tyrosinase
concentration concentration
Chitosan
beads
(cm3/cm3)
Reaction
time
(min)
Phenol
compound
H2O2
(mM)
pH % Conversion % Adsorption % Removal
(mM)
(U/cm3)
4-n-pentylphenol
4-n-hexylphenol
4-n-heptylphenol
4-n-octylphenol
4-n-nonylphenol
4-isopropylphenol
0.5
0.3
0.1
0.05
0.05
0.5
0.5
0.5
0.5
0.5
0.5
50
50
0.025
0.025
0.025
0.025
0.025
0.025
0.025
0.025
0.100
0.025
0.100
40
40
7.0
7.0
7.0
7.0
7.0
7.0
7.0
7.0
7.0
6.0
6.0
100
100
100
100
100
100
100
100
100
50
50
40
98.7
100
97.2
98.7
100
97.2
120
180
120
120
120
180
120
180
50
50
99.9
86.7
93.3
87.5
98.2
40.7
77.2
86.6
93.2
83.9
98.2
17.5
71.3
50
50
99.9
95.9
4-sec-butylphenol
4-tert-pentylphenol
100
50
100
0.5
0.5
43.0
92.3
150
estrogenic activities of bisphenol A and nonylphenol
by oxidative enzymes from lignin-degrading basidiomy-
cetes. Chemosphere, 42, 271–276 (2001).
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