Stereospecificity of mushroom tyrosinase immobilized on a chiral and a nonchiral support

Article abstract of DOI:10.1021/jf0701178

Mushroom tyrosinase was immobilized from an extract onto glass beads covered with the cross-linked totally cinnamoylated derivates of D-sorbitol (sorbitol cinnamate) and glycerine (glycerine cinnamate). The enzyme was immobilized onto the support by direct adsorption, and the quantity of immobilized tyrosinase was higher for sorbitol cinnamate, the support with the higher number of esterified hydroxyls per unit of monosacharide, than for glycerine cinnamate. The results obtained from the stereospecificity study of the monophenolase and diphenolase activity of immobilized mushroom tyrosinase are reported. The enantiomers L-tyrosine, DL-tyrosine, D-tyrosine, L-dopa, DL-dopa, D-dopa, L-α-methyldopa, DL-α-methyldopa, L-isoprenaline, DL-isoprenaline, L-adrenaline, DL-adrenaline, L-noradrenaline, and D-noradrenaline were assayed with tyrosinase immobilized on a chiral support (sorbitol cinnamate), whereas L-tyrosine, DL-tyrosine, D-tyrosine, L-dopa, DL-dopa, D-dopa, L-α-methyldopa, and DL-α-methyldopa were assayed with tyrosinase immobilized on a nonchiral support (glycerine cinnamate). The same V_max^app values for each series of enantiomers were obtained. However, the K_m^app values were different, the L isomers showing lower values than the DL isomers, whereas the highest K _m^app value was obtained with D isomers. No difference was observed in the stereospecificity of tyrosinase immobilized on a chiral (sorbitol cinnamate) or nonchiral (glycerine cinnamate) support.

Full text of DOI:10.1021/jf0701178

J. Agric. Food Chem. 2007, 55, 4569−4575 4569
Stereospecificity of Mushroom Tyrosinase Immobilized on a
Chiral and a Nonchiral Support
†
†
MAR ´ı A ELISA MAR ´ı N-ZAMORA, FRANCISCO ROJAS-MELGAREJO,
FRANCISCO GARC ´ı A-C AÄ NOVAS,‡ AND PEDRO ANTONIO GARC ´ı A-RUIZ*,†
Grupo de Qu ´ı mica de Carbohidratos y Biotecnolog ´ı a de Alimentos (QCBA), Departamento de
Qu ´ı mica Org a´ nica, Facultad de Qu ´ı mica, Universidad de Murcia, E-30100 Espinardo, Murcia, Spain,
and Grupo de Investigaci o´ n de Enzimolog ´ı a (GENZ), Departamento de Bioqu ´ı mica y Biolog ´ı a
Molecular A, Facultad de Biolog ´ı a, Universidad de Murcia, E-30100 Espinardo, Murcia, Spain
Mushroom tyrosinase was immobilized from an extract onto glass beads covered with the cross-
linked totally cinnamoylated derivates of D-sorbitol (sorbitol cinnamate) and glycerine (glycerine
cinnamate). The enzyme was immobilized onto the support by direct adsorption, and the quantity of
immobilized tyrosinase was higher for sorbitol cinnamate, the support with the higher number of
esterified hydroxyls per unit of monosacharide, than for glycerine cinnamate. The results obtained
from the stereospecificity study of the monophenolase and diphenolase activity of immobilized
mushroom tyrosinase are reported. The enantiomers L-tyrosine, DL-tyrosine, D-tyrosine, L-dopa, DL-
dopa, D-dopa, L-R-methyldopa, DL-R-methyldopa, L-isoprenaline, DL-isoprenaline, L-adrenaline, DL-
adrenaline, L-noradrenaline, and D-noradrenaline were assayed with tyrosinase immobilized on a
chiral support (sorbitol cinnamate), whereas L-tyrosine, DL-tyrosine, D-tyrosine, L-dopa, DL-dopa, D-dopa,
L-R-methyldopa, and DL-R-methyldopa were assayed with tyrosinase immobilized on a nonchiral
app
max
support (glycerine cinnamate). The same V
values for each series of enantiomers were obtained.
app
However, the K m values were different, the L isomers showing lower values than the DL isomers,
whereas the highest K m value was obtained with D isomers. No difference was observed in the
app
stereospecificity of tyrosinase immobilized on a chiral (sorbitol cinnamate) or nonchiral (glycerine
cinnamate) support.
KEYWORDS: Mushroom tyrosinase; immobilization; cinnamic carbohydrate esters; tyrosinase extraction;
enzyme kinetics; stereospecificity
INTRODUCTION
peroxidase (12). However, no reference was found in the
literature to characterizing the stereospecificity of tyrosinase
immobilized on chiral or nonchiral supports. Furthermore,
because the way in which tyrosinase acts on its natural sources
might be influenced by chiral environnements, we considered
it to be of interest to determine whether immobilization on chiral
supports may affect its stereospecificity. Another reason for
determining the stereospecificity of immobilized tyrosinase was
because previous works (2, 8) had pointed to substantial changes
Tyrosinase or polyphenol oxidase is a copper enzyme that
catalyzes the ortho-hydroxylation of monophenols to o-diphenols
(monophenolase activity) and the oxidation of the o-diphenols
to o-quinones (diphenolase activity), using oxygen (1). It is of
central importance in such processes as vertebrate pigmentation
and browning of fruits and vegetables. Because tyrosinase is
an expensive enzyme, it is considered worthwhile to immobilize
it, so that it can be reused. Several authors have endeavored to
immobilize tyrosinase on different solid supports (2-5), using
several immobilization methods (6, 7), and have studied its
behavior, stability, and kinetic properties on different supports
using a variety of reaction substrates (3, 4, 8, 9).
app
in the K m values upon immobilization, and we wished to
app
know how this affected the K m values of the enantiomers and
the stereospecificity. The aim of the present work was to
systematically study the stereospecificity of mushroom tyrosi-
nase immobilized on a chiral (sorbitol cinnamate) and a
nonchiral (glycerine cinnamate) support in its action on several
chiral monophenolic and ortho-diphenolic substrates.
Catalytic stereospecificity has been reported for mushroom
tyrosinase in aqueous solution (10), for free pear and strawberry
tyrosinase (11), and for other free enzymes including horseradish
MATERIALS AND METHODS
*
Corresponding author (telephone 968364814; fax 968364147; e-mail
pagr@um.es).
Natural mushrooms (Agaricus bisporus) supplied by Mercadona
†
Grupo de Qu ´ı mica de Carbohidratos y Biotecnolog ´ı a de Alimentos
QCBA).
‡Grupo de Investigaci o´ n de Enzimolog ´ı a (GENZ).
(Murcia, Spain) were used to obtain fresh tyrosinase. L-Dopa, DL-dopa,
(
D-dopa, L-R-methyldopa, DL-R-methyldopa, L-isoprenaline, dl-isopre-
1
0.1021/jf0701178 CCC: $37.00
© 2007 American Chemical Society
Published on Web 05/09/2007

4570 J. Agric. Food Chem., Vol. 55, No. 11, 2007
Mar ´ı n-Zamora et al.
naline, L-adrenaline, DL-adrenaline, L-noradrenaline, D-noradrenaline,
L-tyrosine, dl-tyrosine, D-tyrosine, p-nitrophenol, and 4-tert-butylcat-
echol were purchased from Sigma (Madrid, Spain). A stock solution
of the phenolic substrate was prepared in 0.15 mM phosphoric acid to
prevent autoxidation. All other chemicals were of analytical grade and
supplied by Fluka (Madrid, Spain), Panreac (Barcelona, Spain), J. T.
Baker (Paris, France), and Sigma (Madrid, Spain). Ultrapure water from
a Milli-Q system (Millipore Corp., Madrid, Spain) was used throughout
this research.
Preparation of Photoreactive Prepolymers. The preparation of
carbohydrate derivatives followed a modified version of the method
proposed by Van Cleve (13), in which 0.02 mol of carbohydrate was
dissolved in 100 mL of pyridine. The mixture was heated at 60 °C for
(DINKO D25V) was used to pump (50-55 mL min-1) the reaction
medium through the spectrophotometer, which contained a quartz
cuvette (1 cm). Syringes containing glass beads covered with the support
and bound tyrosinase were used as small packed-bed continuous reactors
with recirculation (8 and 10.5 mL in diphenol and monophenol assays,
respectively) and descending flow. Spectrophotometric measurements
were made as in a previous work (2) using as substrates l-tyrosine,
DL-tyrosine, D-tyrosine, L-dopa, DL-dopa, D-dopa, L-R-methyldopa, DL-
R-methyldopa, l-isoprenaline, DL-isoprenaline, L-adrenaline, DL-
adrenaline, L-noradrenaline, and D-noradrenaline at the desired con-
centration at room temperature in 50 mM sodium phosphate buffer
(pH 6.8). When tyrosine, dopa, R-methyldopa, isoprenaline, adrenaline,
noradrenaline, and 4-tert-butylcatechol were used as substrates, an
increase in absorbance was monitored at 302 nm (ꢀ302nm ) 9360/M
cm), 475 nm (ꢀ475nm ) 3600/M cm), 475 nm (ꢀ475nm ) 3200/M cm),
500 nm (ꢀ500nm ) 4300/M cm), 475 nm (ꢀ475nm ) 4000/M cm), 490 nm
(ꢀ490nm ) 3580/M cm), 400 nm (ꢀ400nm ) 1150/M cm), respectively. In
the case of diphenols the initial rate was measured between the first
10 and 70 s of the reaction. In the case of the monophenol, tyrosine,
the steady-state reaction rate was measured after the initial lag period,
which preceded the steady state (16). In the case of 4-tert-butylcatechol,
the absorbent species was the quinone generated, whereas in all other
cases the absorbent species was the aminochrome derivate of the
quinone generated (17).
1
h to ensure complete dissolution. After the mixture had cooled to
room temperature, 0.15 mol of cinnamic acid chloride was added for
sorbitol cinnamate and 0.07 mol for glycerine cinnamate. The reaction
was allowed to proceed at room temperature for 4 h, after which the
resulting mixture was poured into vigorously stirred water. The
precipitate obtained, after decanting and filtering of this mixture, was
dissolved in chloroform and purified by adding it, one drop at a time,
to vigorously shaken hexane. The solid obtained was redissolved and
reprecipitated before being dried on P
2 5
O at reduced pressure.
All hydroxyl groups were esterified with cinnamoyl groups, as can
1
13
be deduced from various experimental analyses: H NMR, C NMR,
distortionless enhancement by polarization transfer (DEPT) spectra,
different two-dimensional experiments (COSY and C/H ratio), and
infrared spectra of the prepared compound. All of these methods pointed
to full esterification (2).
Determination of Immobilized Tyrosinase. Fresh tyrosinase im-
mobilized on both types of glass bead (14 g) was released after a
treatment in which the immobilization support was dissolved. To each
sample of immobilized tyrosinase were added 10 mL of chloroform
and 5 mL of 400 mM sodium phosphate buffer, pH 6.8, before stirring
for 3 min. To assay the tyrosinase activity, 0.5 mL of 20 mM 4-tert-
butylcatechol ([4-tert-butylcatechol]final ) 4 mM) and 0.5 mL of 250
mM sodium phosphate buffer, pH 6.8, were added to a 1.5 mL aliquot
of the aqueous phase.
Tyrosinase Extraction. Mushroom tyrosinase was extracted as
previously described (8). Briefly, before use, the natural mushrooms
were lyophilized, ground mechanically, and stored at -18 °C. To extract
the fresh tyrosinase enzyme, 600 mg of lyophilized ground mushroom
was added to 16 mL of a 30 mM aqueous solution of p-nitrophenol
(
pH 7.0), to ensure minimal deterioration of the enzyme due to the
The quantity of immobilized enzyme was determined by extrapola-
tion from a straight line calibrated from the known concentrations of
purified enzyme (18) submitted to the same treatment; that is, 5 mL of
a solution of tyrosinase purified at a known concentration in 400 mM
sodium phosphate buffer, pH 6.8, 10 mL chloroform, and 14 g of beads
coated with the support (without enzyme) were stirred for 3 min, taking
a 1.5 mL aliquot of the aqueous phase to measure the activity.
Steady-State Kinetics and Kinetic Data Analysis. The steady-state
transformation of other substrates present in the extract (8), magnetically
stirred for 30 min at 4 °C, and finally centrifuged at 4000 rpm for 5
min. The supernatant (9 mL) containing the tyrosinase activity was
collected and equilibrated to pH 5.5 by adding 1 mL of a 0.9 M aqueous
solution of NaH PO and 0.1 M of H PO . The solids were totally
2 4 3 4
eliminated by means of a second centrifugation.
Tyrosinase Immobilization. JM-50 (1.7-2.4 mm diameter) and
Microperl Industrial (type A, 0.6-1.0 mm diameter) glass beads, both
manufactured by Sovitec Iberica S.A. (Barcelona, Spain) and supplied
by Jaque (Murcia, Spain), were used as inert matrix for tyrosinase
immobilization.
To determine diphenolase activity, the JM-50 glass beads were
washed and degreased (14) before use. A chloroform solution of the
corresponding immobilization support at 5 g/L was prepared, in which
the glass beads were immersed. A prepolymer film was formed on the
beads (0.2 mg per gram of glass beads) when the solvent was eliminated
by evaporation (8, 15). After drying, the prepolymer film was
polymerized by irradiation in the ultraviolet zone for 15 min using an
Osram HOL-125 W mercury vapor lamp providing a power of 1.6 mW/
app
kinetic constants, V max (apparent maximum steady-state rate) and
app
K m (apparent Michaelis constant) of the fresh immobilized mush-
room tyrosinase were obtained by measuring the initial rates of the
reaction with L-tyrosine (0.05-2 mM), DL-tyrosine (0.05-1.5 mM),
D-tyrosine (0.05-2 mM), L-dopa (0.1-7 mM), DL-dopa (0.1-18 mM),
D-dopa (0.1-7 mM), L-R-methyldopa (0.1-20 mM), DL-R-methyldopa
(0.1-20 mM), L-isoprenaline (0.1-35 mM), DL-isoprenaline (0.1-35
mM), L-adrenaline (0.1-10 mM), DL-adrenaline (0.1-12 mM), L-
noradrenaline (0.1-20 mM), and D-noradrenaline (0.1-10 mM) in 50
mM sodium phosphate buffer, pH 6.8, at room temperature from
0
triplicate measurements of V , as indicated above. To avoid the effect
of a second substrate, oxygen, on the enzyme activity, the concentration
of oxygen was kept constant in the assay medium (0.26 mM). The
2
cm , as determined by a Nover-Laser power/energy monitor (Ophir
Optronics Ltd.). To immobilize fresh tyrosinase, 3.5 mL of tyrosinase
extract (pH 5.5) was added to a syringe containing 1.5 g of glass beads
covered with the immobilization support, and the immobilization was
allowed to proceed for 1 h at 4 °C. After immobilization, the enzyme
solution was withdrawn and the immobilized enzyme was thoroughly
rinsed in distilled water.
reciprocals of the variances of V
nonlinear regression fitting of V
equation. The fitting was carried out using the Sigma Plot 8.0 program
for Windows.
NMR Assays. 13C NMR spectra of several substrates were obtained
in a Varian Unity spectrometer at 300 MHz. The spectra were obtained
0
were used as weighting factors in the
versus [substrate] to the Michaelis
0
To improve the spectrophotometric detection of monophenolase
activity, we used smaller glass beads than those used to detect
diphenolase activity. The 11 g of Microperl Industrial glass beads used
in this case improved the activity values obtained by a factor of 21.2
2
by using D O as solvent for the substrates. Chemical displacement (δ)
values were measured relative to those for tetramethylsilane (δ ) 0).
The maximum line width accepted in the NMR spectra was 0.06 Hz.
Therefore, the maximum error for each spectrum peak was (0.1 ppm.
(
0.5 with respect to 1.5 g of JM-50 glass beads (1.7-2.4 mm diameter)
(
results not shown). The immobilization process was the same as that
RESULTS AND DISCUSSION
described above.
Quantification of Immobilized Enzyme. To calculate the
apparent catalytic constant, k cat , the amount of tyrosinase
immobilized on glass beads covered with the cross-linked
sorbitol cinamate and glycerine cinnamate derivates (Figure 1)
Tyrosinase Activity Assay. Spectrophotometric measurements were
made with a Perkin-Elmer Lambda 35 UV-vis spectrophotometer
controlled by a PC running the software Lambda 35, KinLab, after
adjustment to the desired wavelength. A variable flow peristaltic pump
app

Stereospecificity of Immobilized Mushroom Tyrosinase
J. Agric. Food Chem., Vol. 55, No. 11, 2007 4571
Figure 1. Schematic representation of the structure of the immobilization supports of the enzyme and the substrates of the catalytic reaction.
was determined by measuring the activity of the enzyme released
after each support had been redissolved as described. These
activity values were interpolated in the calibration straight line
made for purified enzyme submitted to the same treatment. The
resulting equation was V0 ) 0.195x[Tyr] ( 0.0016 (V0 in ∆A
mobilized on each of the supports was 1.18 µgTyr/mgsupport in
the case of sorbitol cinamate derivate and 0.72 µgTyr/mgsupport
in the case of glycerine cinnamate derivate. The quantity of
enzyme immobilized on the cross-linked support derived from
a hexafunctional monomer (sorbitol cinnamate) was greater than
that obtained on the support derived from a trifunctional
-
1
min and [Tyr] in µg/mL). The quantity of enzyme im-

4572 J. Agric. Food Chem., Vol. 55, No. 11, 2007
Mar ´ı n-Zamora et al.
Table 1. Kinetic Constants for the Diphenolase Activity of Mushroom Tyrosinase Immobilized on Sorbitol Cinnamate (*) and Glycerine Cinnamate (**)
Using Several Ortho-diphenolic Enantiomers as Enzymatic Reaction Substrates
app
max
10 mM min-1)
3
app
cat
(s-1)
app
app
max
app
10 min-1)
2
app
cat
/K m (s- mM-1)
app
1
o-diphenol
-dopa
DL-dopa
V
(
×
k
K m (mM)
V
/K m
(
×
k
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
L
276
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
3
3
3
5
5
5
2
2
5
5
5
5
6
4
10
6
6252
6275
6256
10226
10190
10190
6312
6323
10518
10370
5980
5930
6782
6669
4516
4770
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
118
7.4
9.3
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.6
0.3
1.5
0.6
0.3
1.5
0.10
0.10
0.3
0.4
0.3
0.3
0.10
0.10
0.3
0.3
3.73
3.01
1.82
2.90
2.50
1.64
6.38
5.45
6.04
4.12
3.53
3.48
10.35
8.22
4.21
4.20
±
0.3
0.1
0.2
0.2
0.1
0.2
0.2
0.1
0.4
0.3
0.2
0.2
0.4
0.3
0.3
0.3
844.86
674.73
411.57
1076
926
610
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
70
25
41
98
66
68
41
32
205
134
37
37
100
64
78
64
278
276
276
275
275
279
279
284
280
265
261
299
295
200
210
118
118
673
671
671
108
108
691
681
146
145
171
137
236
154
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
D
-dopa
15.2
9.5
*
*
*
L-dopa
DL-dopa
11.0
16.7
4.37
5.12
4.7
D
-dopa
-methyldopa
L
-
R
1444.39
1234.96
2237
DL
-R
-methyldopa
*
*
L-R-methyldopa
DL
-R
-methyldopa
-isoprenaline
DL-isoprenaline
-adrenaline
DL-adrenaline
-noradrenaline
-noradrenaline
6.8
1525
L
7.5
797.33
790.66
2347
1857
950
7.5
L
2.89
3.59
4.75
5.0
L
D
954
Table 2. Kinetic Constants Published by Esp ´ı n et al. (10) for the Diphenolase and Monophenolase Activity of Free Mushroom Tyrosinase Using
Several Diphenolic and Monophenolic Enantiomersa
app
max
M min-1)
app
cat
(s-1)
app
app
max
app
10 min-1)
2
app
cat
/K m (s- mM-1)
app
1
substrate
-dopa
DL-dopa
-dopa
-methyldopa
-methyldopa
-isoprenaline
DL-isoprenaline
-tyrosine
DL-tyrosine
-tyrosine
V
(µ
k
K m (mM)
V
/K m
(
×
k
L
24.5
24.5
24.5
10.1
10.1
6.7
±
±
±
±
±
±
±
±
±
±
1
1
1
0.5
0.5
0.3
0.3
0.05
0.06
0.06
107.4
107.0
107.4
44.3
44.3
29.4
29.4
7.9
±
±
±
±
±
±
±
±
±
±
44
0.8
1.4
±
±
±
±
±
±
±
±
±
±
0.04
0.06
0.15
0.28
0.41
0.31
0.61
0.01
0.01
0.02
3.1
1.7
0.54
0.15
0.13
0.10
±
±
±
±
±
±
±
±
±
±
0.28
0.14
0.04
0.014
0.013
0.0092
0.0074
0.045
0.0074
0.0043
134.25
76.43
23.86
6.51
4.4
4.4
1.8
2.2
1.2
1.2
0.2
0.3
0.3
D
4.5
L-
R
6.8
DL
-
R
8.0
5.53
4.45
3.03
L
6.6
6.7
9.7
0.069
0.67
0.2
L
1.80
1.85
1.82
0.27
0.90
1.86
29.26
9.00
8.1
8.0
D
0.098
4.30
a
Conditions: 50 mM phosphate buffer, pH 6.8, 2% (v/v) DMF, saturating MBTH concentration, differing monophenol concentrations, and 3.8 nM mushroom tyrosinases.
Results are means SD for three separate experiments.
±
Table 3.
pH 6.8
δ Values for C-3 and C-4 of Several Phenolic Compounds at
monomer (glycerine cinnamate), probably due to the lower
molecular size of the latter and the different structure arising
from the cross-linking (14).
substrate
-dopa
DL-dopa
-dopa
-methyldopa
-methyldopa
-isoprenaline
DL-isoprenaline
δ
3
δ
4
substrate
δ
3
δ
4
Kinetic Assay. In all cases (o-diphenols and monophenols)
L
146.9
146.9
146.9
146.7
146.7
146.9
146.9
146.1
146.1
146.1
146.2
146.2
146.8
146.8
L
-adrenaline
143.7
143.7
144.0
144.0
143.7
143.7
144.0
144.0
158.8
158.8
158.8
a Michaelis-Menten behavior was observed. With regard to
DL-adrenaline
-noradrenaline
-noradrenaline
-tyrosine
DL-tyrosine
-tyrosine
app
max
the kinetic constants for each series of isomers, the V
and
D
L
app
cat
app
L
-
R
D
k
values were the same, but the K m values differed. The
app
DL
-
R
L
sequence of K m values was D isomers > DL isomers > L
L
isomers (demostrating the greater affinity for the natural L
D
app
app
isomer), the inverse sequence from the V /K m and
max
app
cat
app
k
/K m values.
(10, 11). This nucleophilic power can be measured by 13C NMR
assays and is related with the highest electron donor capacity
of the side chain of the substrate, which corresponds to the
highest electronic charge and the lowest δ values for C-3 (δ3)
and C-4 (δ4) in o-diphenols. As the substituent is the same for
enantiomers, the δ values were similar for the whole series (L,
Diphenolase Activity. Enantiomers of several diphenols
dopa, R-methyldopa, isoprenaline, adrenaline, and noradrena-
line, Figure 1) were assayed with tyrosinase immobilized on
glass beads covered with the chiral support sorbitol cinnamate
Table 1). To ascertain whether the chirality of the immobiliza-
tion support affects the stereospecificity of immobilized mush-
room tyrosinase, enantiomers of dopa and R-methyldopa were
assayed with tyrosinase immobilized on glass beads covered
(
(
DL, and D isomers) of each o-diphenol (Table 3). Therefore,
app
max
the corresponding V
values for the three enantiomers must
with the nonchiral support glycerine cinnamate (Table 1).
be the same, as can be seen for immobilized enzyme (Table 1)
and for free enzyme (Table 2).
app
Maximum Steady-State Rate (V ). The results (Table 1)
max
showed that the apparent maximum steady-state rate values,
For immobilized mushroom tyrosinase we observed similar
app
app
V
, of mushroom tyrosinase immobilized on a chiral (sorbi-
V
values when the o-diphenol δ values increased, perhaps
max
max
4
tol cinnamate) or a nonchiral (glycerine cinnamate) support were
because these δ values were very similar and not sufficiently
4
app
max
the same for each series of isomers. The V
values of free
different from to observe differences in immobilized mushroom
mushroom tyrosinase (Table 2) were also the same for each
series of isomers and were related with the same nucleophilic
power of the oxygen from the aromatic hydroxy group of the
isomers to attack the copper atoms of the enzyme active site
tyrosinase and due to the influence of other factors, such as
enzyme-support interactions, which do not exist in the case of
the free enzyme (10, 11). Of note are the higher apparent
catalytic constant values of the immobilized enzyme (Table 1)

Stereospecificity of Immobilized Mushroom Tyrosinase
J. Agric. Food Chem., Vol. 55, No. 11, 2007 4573
Figure 2. Effect of enzyme quantity on (A) lag time and (B) tyrosinase
activity. Support, sorbitol cinnamate; substrate, L-tyrosine, 2 mM.
Figure 4. Effect of nature of monophenol enantiomers on lag time.
Spectrophotometric recordings were taken of the monophenolase activity
of immobilized fresh mushroom tyrosinase (7.81
buffer (pH 6.8). -Tyrosine (L, 1 mM), DL-tyrosine (DL, 1 mM), and
-tyrosine (D, 1 mM) were used as substrates.
µg) in 50 mM phosphate
L
D
cinnamate (chiral support) and glycerine cinnamate (nonchiral
app
support). However, the differences between the K m values of
the enantiomers were smaller for immobilized tyrosinase than
for free tyrosinase, and even substrates such as isoprenaline and
app
noradrenaline showed very similar K m values between iso-
mers in the case of immobilized tyrosinase (Table 1). The
app
sequence of K m values obtained for the different enantiomers
(L < DL < D) could be explained by the effect of the spatial
orientation of the side chain, which would affect one of several
rate constants in the Michaelis constant expression for the
diphenolase activity of tyrosinase (10, 11).
The effect of introducing a methyl group in the side chain of
the substrate had the opposite effect on the K m values in
app
Figure 3. Effect of substrate concentration on (A) lag time and (B)
tyrosinase activity. Support, sorbitol cinnamate.
immobilized enzyme than in free enzyme. In the case of free
app
enzyme, K m values increased when the molecular mass of the
compared with those of the free enzyme (Table 2), which may
have been due to conformational changes to more unfolded
states (19) provoked by the strong hydrophobic interactions
taking place during immobilization, although perhaps other
factors derived from the use of a crude mushroom material could
also have had some influence. In an aqueous solution, the
enzyme would find itself in a state in which the ionic groups
are in contact with the solution and the innermost hydrophobic
part would remain protected; when immobilized on the hydro-
phobic supports, the enzyme would open out, binding to the
support by means of the hydrophobic part, whereas the ionic
part would remain in contact with the solution. The substrate
would then be able to reach the active center of the immobilized
tyrosinase more easily than the active center of free enzyme,
substituent side chain of o-diphenols was raised by adding a
methyl group (10, 11). The immobilized enzyme showed lower
app
K m values for methyldopa than for dopa and lower values for
adrenaline than for noradrenaline (Table 1). This effect could
be due to the increase in substrate hydrophobicity caused by
adding a methyl group, because the more hydrophobic the
substrate, the greater the affinity for the hydrophobic im-
mobilization support, and the higher the concentration of
substrate in the microenvironnement of the enzyme, the lower
app
the K m values (20). However, we found that when the
molecular mass of the substituent was quite high (introduction
app
of two methyl groups), the K m values of the immobilized
enzyme increased, as in the case of free enzyme; for example,
app
app
thus explaining the higher k cat values obtained for immobi-
isoprenaline showed a higher K m value than noradrenaline
app
lized tyrosinase and the similar values obtained for substrates
with similar δ4.
(Table 1). The absolute values of K m were higher for
tyrosinase immobilized on glycerine cinnamate than for tyro-
sinase immobilized on sorbitol cinnamate, perhaps due to
different support-substrate interactions resulting from the
structural differences in this immobilization support. There are
Michaelis Constant. The apparent Michaelis constant values,
app
K m , of immobilized mushroom tyrosinase differed between
isomers of the same series of o-diphenols (Table 1). When
app
app
immobilized tyrosinase acted on L isomers, K m was lower
two kinds of factors that affect K m values for immobilized
than when immobilized tyrosinase acted on DL isomers, which
was lower than in the case of D isomers, like free mushroom
tyrosinase (Table 2). The same sequence of K m values was
tyrosinase. The first is the hydrophobicity of the substrate and
the support, which is responsible for substrate-support and
enzyme-support interactions, and conformational changes in
the enzyme; the second is the steric effect produced by the
app
observed for mushroom tyrosinase immobilized on sorbitol

4574 J. Agric. Food Chem., Vol. 55, No. 11, 2007
Mar ´ı n-Zamora et al.
Table 4. Kinetic Constants for the Monophenolase Activity of Mushroom Tyrosinase Immobilized on Sorbitol Cinnamate (*) and Glycerine Cinnamate
**) Using Several Monophenolic Enantiomers as Enzymatic Reaction Substrates
(
app
max
3
10 mM min-1)
app
cat
(s-1)
app
app
max
app
2
10 mM min-1)
app
cat
/K m (s- mM-1)
app
1
monophenol
V
(
×
k
K m (mM)
V
/K m
(×
k
*
*
*
*
*
*
L
-tyrosine
34.08
33.97
34.12
30.07
30.98
31.83
±
±
±
±
±
±
0.80
1.29
1.61
0.95
1.45
2.15
45.94
45.79
46.00
68.01
68.48
70.37
±
±
±
±
±
±
1.35
0.32
0.51
1.5
0.55
1.06
1.71
±
±
±
±
±
±
0.02
0.02
0.10
0.05
0.15
0.20
10.65
6.66
2.27
5.46
2.92
1.86
±
±
±
±
±
±
0.71
0.36
0.19
0.52
0.44
0.25
143.6
89.8
30.6
123.6
64.6
37.83 ± 5.6
±
±
±
±
±
9.9
5.2
2.6
14.3
10.9
DL-tyrosine
1.95
2.37
4.85
5.42
6.54
D
-tyrosine
-tyrosine
DL-tyrosine
*
*
*
L
D
-tyrosine
different spatial orientations of different isomers or by big
substituents in the substrate.
more, the spectrophotometric recordings of the monophenolase
activity of immobilized fresh mushroom tyrosinase on a chiral
support (sorbitol cinnamate) with L-, DL-, and D-tyrosine (Figure
4) showed different lag periods (τ). The largest τ corresponded
Monophenolase Activity. The monophenolase reaction mech-
anism of tyrosinase is coupled to diphenolase activity (1, 21).
When tyrosine was used as substrate (Figure 1), immobilized
mushroom tyrosinase showed a lengthened transient phase
before reaching the steady state (22), as did free tyrosinase (16).
When the product is plotted versus the time of reaction, the lag
time (τ) can be estimated by extrapolation of the linear portion
of the curve to the abscissa. When we determined the behavior
of τ versus immobilized enzyme quantity and versus substrate
concentration (Figures 2A and 3A), τ decreased with increasing
quantity of enzyme and increased with increasing concentrations
of substrate, as has been described for free mushroom tyrosinase
app
to the isomer having the lowest K m , and the smallest τ
app
corresponded to the isomer having the hightest K m (Table 4).
The same behavior was obtained for tyrosinase immobilized
on a nonchiral support (glycerine cinnamate) (results not shown).
app
max
app
That the substrate with the highest catalytic power (V /K m
)
showed a longer lag period might seem to be contradictory,
but it could be explained in terms of the reaction mechanism
(10, 11).
To sum up, the results concerning the diphenolase and
monophenolase activity of immobilized mushroom tyrosinase
suggest that this enzyme shows stereospecificity in its affinity
(23). In the range of enzyme quantity assayed, the activity versus
the quantity of immobilized mushroom tyrosinase acting on
L-tyrosine as substrate showed a linear relationship, the activity
increasing with the enzyme quantity (Figure 2B). The activity
of immobilized mushroom tyrosinase versus substrate concen-
tration also increased with higher L-tyrosine concentrations
app
toward the substrates (K m ), but does not show stereospecific-
app
max
ity in the maximum steady-state rate (V ) of these substrates.
This behavior is the same for mushroom tyrosinase immobilized
on a chiral or nonchiral support and for free tyrosinase,
demonstrating that the process of immobilization has no effect
on the stereospecificity of mushroom tyrosinase. These results
suggest that when tyrosinase is adsorbed on natural supports,
such as cell menbranes, its stereospecificity will be maintained,
as it is on the hydrophobic supports described above. Moreover,
knowledge of this property could facilitate the use of im-
mobilized tyrosinase in the food industry as a sensor of phenols
in food solutions and for their possible elimination.
(Figure 3B). The chirality of the support did not affect the
behavior of τ or activity versus immobilized enzyme quantity
or versus substrate concentration, and the same behavior was
obtained for tyrosinase immobilized on a chiral support (sorbitol
cinnamate) (Figures 2 and 3) as for tyrosinase immobilized on
a nonchiral support (glycerine cinnamate) (results not shown).
app
Maximum Steady-State Rate (V ). The results showed that
max
app
max
the V
of mushroom tyrosinase immobilized on a chiral
(sorbitol cinnamate) or a nonchiral (glycerine cinnamate) support
LITERATURE CITED
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the case of o-diphenols (Table 1) and free tyrosinase (Table
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app
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Received for review January 15, 2007. Revised manuscript received
March 28, 2007. Accepted March 28, 2007. This work was supported
by a grant from the Ministerio de Educaci o´ n y Cultura (Spain) Project
MAT2004-01893, Consejer ´ı a de Ciencia, Tecnolog ´ı a, Industria y Com-
ercio (Murcia, Spain) Project 2103 SIU0043, FCG:MEC (Madrid,
Spain) Project B102006-15363, Conserjeria de Educaci o´ n (BioCARM,
Murcia, Spain) Project Bio-BMC 0601-0004, and the Fundaci o´ n S e´ neca
1
455-1464.
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(
Spain) Project o4691/BPS/06. M.E.M.-Z. gratefully acknowledges a
working contract with the University of Murcia and DOMCA S.A.
JF0701178