Activity of Mushroom Polyphenol
Oxidase in Organic Medium
Stephanie G. Burton,'* John R. Duncan,' Perry T. Kaye? and Peter D. Rose'
Departments of 'Biochemistry and Microbiology and Chemistry, Rhodes
University, P.O. Box 94, Grahamstown, 6140, Republic of South Africa
Received October 26, 1992/Revised April 22, 1993/Accepted April 27, 1993
A kinetic study of the activity of mushroom polyphenol
oxidase in an organic system was carried out to obtain
detailed enzyme kinetic data in relation to optimization
of reaction conditions and substrate specificity. A simple
method for consistent measurement of reaction rates
in the heterogeneous enzymelorganic solvent system
(consisting of immobilized polyphenol oxidase and a
hydrated solution of the substrate in chloroform) was
designed. The aqueous content of the system was
optimized using pcresol as the substrate. With this
system, a crude extract of Agaricus bisporus was
used to hydroxylate and oxidize a range of selected
psubstituted phenolic substrates, yielding o-quinone
products. Michaelis-Menten kinetics were used to
obtain apparent KM and V,,, values with respect to each
of these substrates. Results from this analysis indicated
a correlation between the enzymic kinetic parameters
obtained and the steric requirements of the substrates,
which could be rationalized in terms of the restricted
flexibility of the enzyme when it is in chloroform and
also in terms of substrate and solvent hydrophobicity.
In the course of the investigation UV molar absorption
coefficients of several o-quinones were measured by a
novel method: 'H nuclear magnetic resonance (NMR)
spectroscopy was employed to determine component
concentrations in reaction mixtures resulting from the
transformation of phenols by polyphenol oxidase in
chloroform. Thus the UV molar absorption coefficients
could be obtained directly, avoiding the necessity to
isolate the water-sensitive, unstable o-quinones. 0 1993
John Wiley & Sons, Inc.
Key words: Polyphenol oxidase tyrosinase enzymes
in organic media o-quinones
INTRODUCTION
Polyphenol oxidase (EC 1.14.18.1) is a monooxygenase
which catalyzes the o-hydroxylation of phenols and the oxi-
dation of o-dihydric phenols to o-quinones using molecu-
lar oxygen.17 Since these products are often difficult to
synthesize by classical methods, the potential of this en-
zyme for catalyzing the reactions in vitro is clear, and
various investigations have shown that a wide range of
substrates can be transformed in conventional aqueous-
based enzyme ~ y s t e m s . ~ ~ ' ~The
- ' ~ drawbacks
~'~ associated
with the formation of o-quinones in aqueous systems, viz.,
polymerization yielding melanin-like, insoluble products,
can be overcome by the addition of reducing agents such
* To whom all correspondence should be addressed.
Biotechnology and Bioengineering, Vol. 42, Pp. 938-944 (1993)
0 1993 John Wiley & Sons, Inc.
*
as ascorbic acid,2 thus ensuring that the reaction products
are o-dihydric phenols.
Developments in the field of nonaqueous enzymology
have revealed the versatility of biocatalysts employed in
organic media, and an attractive option for the production
of o-quinones using polyphenol oxidase is its application in
nearly anhydrous organic solvents. This enzyme has been
shown to retain activity in various solvents, provided that,
as with the majority of enzymes in organic systems, a small
proportion of water is present in the system.22The water is
generally considered to maintain conformational flexibility
in the protein molecule^.^
The advantages afforded by the use of nonpolar organic
solvents include increased solubility of hydrophobic sub-
strates and reactants, which is of relevance in the case of
certain substituted phenols which are potential substrates of
polyphenol oxidase. Chloroform is a particularly suitable
medium for this enzyme since oxygen has a high solubility
in chloroform and thus the availability of oxygen for the
reaction need not be a limiting factor." Furthermore, the
enzyme is not soluble in chloroform and can be conve-
niently separated from the reaction mixture. The feasibility
of a purified polyphenol oxidase/chloroform system has
been demonstrated using gas liquid chromatography (GLC)
and high-performance liquid chromatography (HPLC) to
monitor the transformation of various p-substituted phe-
nolic substrates.'" This study reported enzyme kinetic data
for one substrate only, viz., p-cresol. An aim of the present
study has been to obtain further quantitative data on the
enzyme kinetics of polyphenol oxidase in a chloroform
medium as a function of substrate structure. Reports of
quantitative kinetic studies are relatively scarce in the
literature,I6 but they are essential in the development of
a theoretical basis for the application of enzymic catalysis
in organic media. Thus, the transformation by polyphenol
oxidase of a range of selected p-substituted phenols was ex-
amined under standardized conditions. Initial reaction rates
were measured spectrophotometrically using chloroform
(containing a specific quantity of aqueous buffer) as solvent.
Since application of the system to a larger scale process is
an objective of this research, a crude mushroom extract,
immobilized without further purification, was utilized as
a source of polyphenol oxidase. The level of enzyme
hydration in such systems would be governed by the
initial water content of the solvent and that of the protein
CCC QQQ6-3592/93/Q8Q938-Q7
preparation as well as the extent to which the solvent
“strips” water from the protein6 The optimal amount of
water to be added would depend on these factors. While it
is desirable to optimize the rate of catalysis by providing
sufficient water to maximize protein hydration and hence
conformational flexibility, any excess water would lead to
phase separation and o-quinone polymerization. The iden-
tity of the active enzyme was confirmed by electrophoresis
under nondenaturing conditions using specific substrates for
staining.s
In conjunction with the kinetic measurements, ‘H nu-
clear magnetic resonance (NMR) spectroscopy was used
to confirm the nature of the products of the reaction as
well as to facilitate measurement of the absorption coef-
ficients of o-quinones in cases where these values were
not available from the literature. All the p-substituted phe-
nolic substrates tested were found to be successfully trans-
formed to o-quinones, with no other products or side
reactions. However, considerable variation in the kinetic
parameters was observed, and rates of reaction were found
to be substantially different from those reported for aqueous
systems.’
MATERIALS AND METHODS
Materials
Mushroom polyphenol oxidase was extracted from fresh
mushrooms (Agaricus bisporus) generously donated by
Tongaat Mushrooms, Natal, South Africa. Glass beads (250
p M acid washed) were purchased from Supelco. Phenolic
substrates were obtained from commercial suppliers. Chlo-
roform was distilled prior to use, and its water content was
determined by Karl Fischer titration.
Preparation of Biocatalyst
Fresh mushrooms (1 kg) were frozen and then homog-
enized in cold acetone (2.5 L) using a Waring blender.
The resulting slurry was filtered rapidly on a Buchner
funnel, and the residual pulp was air dried briefly before
freezing with liquid N2 and stirring into H2O (500 mL).
The mixture was kept overnight at 4”C, filtered through
cheesecloth, and then allowed to stand in ice while N2
was bubbled gently through the solution to remove residual
acetone. The aqueous extract was freeze dried to yield a
pale brown powder (9.5 g) which was kept frozen until
required.
Immobilization was effected by precipitation onto non-
porous glass beads. A mixture of the crude, freeze-dried
extract (6 g) with phosphate buffer (50 mM; pH 7; 12 mL)
and dry glass beads (36 g) was stirred gently in a petri dish
and allowed to stand at room temperature with adequate
ventilation and occasional stirring until no visible liquid
remained. Portions of the (slightly sticky) biocatalyst were
kept frozen in closed containers and thawed as required.
Samples were not refrozen or reused.
BURTON ET AL.: ACTIVITY OF MUSHROOM POLYPHENOL OXIDASE
Assay of Immobilized Polyphenol Oxidase
The activity of the polyphenol oxidase after immobilization
was assayed using an adaptation of the “dopachrome”
m e t h ~ d .A~ small sample of biocatalyst (approximately
12-15 mg) was added to a solution of L-DOPA (10 mM;
3 mL) in phosphate buffer (50 mM; pH 6.0) at 25°C in
a cuvette. The mixture was briefly but rapidly shaken
to dissolve the enzyme off the beads, and the increase
in absorbance due to dopachrome at 475 nm was moni-
tored for 1 min using a Beckman Spectronic 2000 spec-
trophotometer. The relative activity (as concentration of
L-DOPA utilized per minute per milligram of protein) of
each sample of biocatalyst was measured when it was
thawed and before it was used for kinetic measurements.
The protein content of the biocatalyst was measured by
the Folin-Lowry method, again utilizing small portions
of biocatalyst (6-10 mg) in H20 (1 mL) to give sam-
ple solutions and using bovine serum albumin as the
standard.
Electrophoresis
Polyacrylamide disc gel electrophoresis was carried out
using 3% stacking and 7.5% resolving gels in Tris-glycine
buffer (pH 8.3), but in the absence of sodium dodecyl
sulfate (SDS), using a Hoefer “Mighty Small” apparatus.
Two identical gels were loaded with 100-130 p g of crude
extract and run simultaneously. One of these was then
stained with Coomassie blue using a standard procedure.21
The second gel was placed in a solution of L-DOPA (5 mM;
100 mL) in phosphate buffer (50 mM; pH 7) at room
temperature for 0.5 h, after which the reaction was halted
by placing the gel in 15% trichloroacetic acid solution
for 15 min and then washing with 10% glycerol solution.
This electrophoretic procedure was repeated using solu-
tions (5 mM) of each of the phenols listed in Table I and
L-tyrosine, in place of the L-DOPA to stain the second gel
in each case.
Table I. Nondenaturing electrophoresis of crude mushroom extract.
Mobility of protein components detected
Stain by staining of gela
Coomassie blue 0.26 0.41 0.60 0.75
L-DOPA 0.40 0.75
L-Tyrosine 0.40
p-Cresol 0.41
p-Ethylphenol 0.40
p-Isopropylphenol 0.40
p-t-Butylphenol 0.40
p-Methoxyphenol 0.40
p-Fluorophenol 0.40
p-Chlorophenol 0.40
p-Bromophenol 0.40
a Calculated as a fraction of the distance moved in the resolving gel by
bromothymol blue indicator during the electrophoresis.
939
Optimization of Water Requirement
p-Cresol was adopted as a “standard” phenolic substrate
and, for the determination of optimal hydration conditions,
was used at an initial concentration of 25 mM. Biocatalyst
(40 mg, containing 1.1 mg protein) and chloroform (9 mL)
were placed in a stoppered flask in a water bath at 25”C, and
phosphate buffer (50 mM; pH 7; 25-40 pL) was added.
The mixture was magnetically stirred for 2 min at constant
speed to allow spreading of the beads in the flask. The
reaction was started by addition of a chloroform solution of
p-cresol (250 mM; 1 mL) to give the required initial con-
centration. To measure the progress of the reaction, aliquots
(2 mL) were removed from the flask, their absorbance at
395 nm due to the o-quinone produced was measured, and
the aliquots were returned to the flask as rapidly as possible.
This procedure was carried out every 2 min for a period
of 10 min. Flasks were checked for the absence of any
visible aqueous droplets and precipitated quinone after the
10-min assays. Assays were repeated several times for each
proportion of buffer.
Kinetics Measurements
For each substrate, five different concentrations were cho-
sen (depending on solubility) to give measurable reaction
rates while avoiding substrate inhibition. Reactions were
carried out using exactly the same procedure as for the
optimization of hydration, with 30 p L of aqueous buffer
being used in each case. Absorbances (due to o-quinone
products) were measured at wavelengths determined as de-
scribed below. Assays for each concentration were repeated
at least five times to obtain mean rates with standard errors
of less than 10%. Linear regression analysis of the results
from each assay gave initial rates in terms of increase in
absorbance per minute, and these results were converted to
increase in concentration of product per minute using the
molar absorption coefficients measured as described below.
Kinetic constants were determined using Hanes plots of
mean initial rates with linear regression analysis.
Determination of Wavelengths of Maximum
Absorbance for A h i n o n e s
For each substrate, biocatalyst (10 mg) and buffer (8 p L )
were added to a chloroform solution of substrate (25 mM,
2 mL), and the mixture was shaken for 2-4 h. Thereafter,
the UV spectrum (800-300 nm) of the decanted chloroform
solution was measured and compared with the spectrum of
a solution of the substrate (25 mM) in CHC13, allowing
identification of the absorbtion peak due to the o-quinone
product and determination of its wavelength at maximum
absorbance (Amax).
H NMR Measurement of eQuinone
Concentrations in Reaction Mixtures
In a typical experiment, the substrate (3-10 mg) was dis-
solved in CDC13 (2.5 mL), and biocatalyst (200-400 mg)
940 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 42, NO. 8, OCTOBER 1993
and D20 (15-50 p L ) were added. The mixture was shaken
rapidly in a closed flask, on a bench flask shaker, for
4-8 h. The UV absorbance of the CDC13 solution was
measured at the appropriate wavelength before and after
reaction, dilution being required in some cases. The CDC13
solution was then decanted and used undiluted for ‘H
NMR spectroscopy on a Bruker 400 MHz AMX NMR
spectrometer. Integrals for product and substrate peaks were
measured to obtain relative concentrations and hence to
ascertain the extent of reaction and the UV molar absorption
coefficients.
RESULTS AND DISCUSSION
Activity of Polyphenol Oxidase in Biocatalyst
The activity of the immobilized polyphenol oxidase in
the biocatalyst was assayed in aqueous medium to ob-
tain a measure of the activity of different samples of
biocatalyst, independent of any variations which might
arise from the heterogeneity of the organic system. In
kinetic measurements using biocatalyst samples which had
been stored frozen and then thawed, rates of substrate
transformation in the organic system could be corrected
for small losses in activity by calculating the “dopachrome
activity” and multiplying by a correction factor, C, equal to
the dopachrome activity of the freshly prepared biocatalyst
divided by the dopachrome activity of the present sample.
The polyphenol oxidase activity in the freshly prepared bio-
catalyst was found to be 1.15 pmol dopachrome produced
/min. mg protein (for dopachrome, E = 3600 M-’ cm-l)?
and storage for several weeks was found to cause reduction
of activity to 0.83 pmol/min * mg protein. The protein
content of the biocatalyst was found to be 27.5 pg/mg
prepared beads.
Electrophoresis
Electrophoresis, under nondenaturing conditions, and stain-
ing specifically for polyphenol oxidase activity7 permitted
unambiguous identification of the active enzyme in the
crude extract while obviating the need for extensive purifi-
cation procedures. The same technique was utilized, with
the relevant p-substituted phenolic substrates being used
as stains, to confirm that the transformation of phenols to
o-quinones could be attributed exclusively to the polyphe-
no1 oxidase in the crude extract. Four main protein com-
ponents were found to be present in the crude mushroom
extract, and two of these showed polyphenol oxidase activ-
ity with L-DOPA as a substrate. Of these two proteins, only
the major one showed activity when phenolic substrates
were used for staining (see Table I), indicating that this
component possessed both “cresolase” and “catecholase”
activity. The same protein band was shown to be respon-
sible for color changes indicating o-quinone formation in
the cases of all the phenolic substrates. The minor active
component may indicate the presence in the mushroom
extract of a subunit or an isoenzyme with predominantly
:::
Absorbance

catecholase activity, as suggested elsewhere.'

Optimization of Water Requirements

Initial rates for the conversion of p-cresol to 4-methyl-1,2-
benzoquinone in the presence of different proportions of
water are summarized in Table 11. All data were obtained
by the method described in the experimental section, the
system being designed to maximize contact between en-
!!i&sSl
0.1

0 2 4 8
Time (minuted
8 10 12

zyme and substrate and to allow saturating aeration, thus - p-Cresol * p-Chlorophenol
* p-Methoxyphenol + p-Ethylphenol
obviating the possibility of oxygen depletion slowing the
reaction rates. The highest initial reaction rate corresponded Figure 1. Typical time courses of polyphenol oxidase-catalyzed
to the system where the chloroform had an aqueous content oxidations of some phenolic substrates. Substrate concentration for
these reactions is 50 mM,
of 0.338% (v/v). In addition, the freshly prepared biocata-
lyst was found to contain 2.83% water (w/w), determined
by weighing before and after freeze-drying and heating at
large error margins for this substrate are attributable to the
100°C for 12 h and thus, assuming that no water is stripped
very slow conversion rates measured and to some turbidity
from the protein by the hydrophobic s ~ l v e n t , 'the
~ volume
produced in reaction mixtures as the product was formed.
of water available to hydrate the protein was 5.9% (w/w)
The substrates chosen represent a series in which
with respect to protein or 0.65% (v/v) with respect to
the steric bulk and the electronic characteristics of the
chloroform.
p-substituent vary gradually. The kinetic parameters
obtained show trends which correlate to some extent
Enzyme Kinetics in Chloroform with the steric parameters. Thus a correlation between
the efficiency of substrate transformation by polyphenol
The choice of p-cresol as a standard substrate arises from
the wide range of concentrations over which its trans-
formation rate remains constant, indicating no substrate
or product inhibition under these conditions. Thus the S/v (rntd/min.mg)
3000
reaction rates for p-cresol could be compared with the
other substrates whether these were used in relatively 2500 -
high concentrations (in cases of slow reactions) or low 2000 -
concentrations (in cases of low solubility).
Results of the enzyme kinetic analysis of polyphenol 1500 -
oxidase using a range of phenolic substrates are shown in 1000 -
Figures 1-3 and Table 111. It is clear from Figures 1 and 2
that the method used, albeit a simple one, provides consis-
tent data within acceptable error margins. (For the purpose 0
0 50 100 150 200 250
of observing trends from the data obtained, standard errors Conc. S (mM)
of less than 10% in experimental data and calculated
parameters were regarded as acceptable.) The data obtained - p-Cresol

for p-tert-butylphenol, although less precise, is included for

(b)
the sake of completeness and is regarded as reasonable W v (rnM/rnln.mg)
1800
in view of its consistency with the observed trends. The
1600 -
1400 -
Table 11. Effect of varying hydration on o-hydroxylation and oxidation 1200 -
of p-cresol by crude mushroom polyphenol oxidase in chloroform. 1000 -

Volume Percent H 2 0 a Initial rate 800 -

of buffer in reaction (pmol o-quinone
(PL) mixture product/min . mg protein)

20 0.238 0.079 ? 0.008

0
25 0.288 0.096 ? 0.009 0 10 20 30 40 50 60
30 0.338 0.118 2 0.007 Conc. S (mM)
35 0.388 0.114 ? 0.006
40 0.438 0.110 ? 0.008 -.- p-Methoxyphenol

a Karl Fischer titration indicated that the original H 2 0 content of CHC13 Figure 2. Examples of Hanes plots to obtain KM and V,,, values
was 0.038%, amounting to 3.8 pL/lO mL using (a) p-cresol and (b) p-methoxyphenol.

BURTON ET AL.: ACTIVITY OF MUSHROOM POLYPHENOL OXIDASE 941

 concerning the functioning of enzymes in hydrophobic
solvents.16 The hydrophobicity of the phenolic substrates
used in this investigation may well affect their partitioning
between the chloroform and the enzyme active site, decreas-
ing the amount of substrate available to the active site, and

:i
I I
20
F OMe CI Br Me Et
Substrate
V l K m values = E8 (size) [7Km
Es value8 x -10, V l K m value8 x 10
Figure 3. Correlations between substrate p-substituent size and
reaction kinetic constants. (Es values times -10, V m a X / Kvalues
~ not been fully established, and it is a matter for conjecture
times 10, to show trends relative to K M ) .
oxidase and the steric bulk of the p-substituent is suggested
by Table 111 and Figure 3 since the E, values are indicative
of steric size.12 The KM value reflects the ability of an
enzyme to bind a substrate, and V m a X / K the ~ , catalytic
efficiency,16 indicates the ability of the enzyme to transform
this substrate. An inverse relationship between V,,,/KM
and ES can be observed for the various substrates, regarding
them in two groups. In the p-alkylphenols, as the steric
size of the alkyl group increases from methyl to tert-butyl,
the V m a x / Kvalues
~ decrease. Similarly, for the remaining
phenolic substrates, taken in the order fluoro-, methoxy-,
chloro-, and bromo-, there is a decrease in V,,,/KM as
the steric size increases. (It should be noted that a large
negative E, value correlates with a large steric volume.)
One effect of hydrophobic solvents on enzymes is a
reduction in conformational fle~ibility,~ and these results
support this. In aqueous systems, polyphenol oxidase is
known to be capable of hydroxylating molecules of con-
siderable size such as steroids,' but in chloroform, access
of bulky substrates such as p-tert-butylphenol to the active
site may well be restricted by the rigidity of the protein
pocket of the enzyme.
A further factor, the hydrophobicity of the substrate,
should also be considered in rationalizing observations
hence giving rise to the high apparent KM values obtained.
The trend of increasing KM with increasing hydrophobicity
is reflected in Table 111. Thus, the present investigation
concurs with the findings of Ryu and Dordick16 in their
investigation of peroxidase activity.
i-Pr t-But On the other hand, the relationship between substrate
utilization by polyphenol oxidase and the electronic char-
acteristics of the p-substituent are less clear. Although the
mechanism of the reaction is regarded as a nucleophilic at-
tack by the phenolic aromatic ring, on a polarized peroxide
bridge in the enzyme active centre,'* this mechanism has
as to whether or not electron donation by p-substituents
facilitates the reaction.
Determination of Molar Absorption
Coefficients of o-Quinones
The polyphenol oxidase/chloroform system lends itself to
investigation of the composition of reaction mixtures be-
cause the organic solvent phase can be separated from the
biocatalyst by simple decantation. Use of CDC13 and D20
in place of CHC13 and H20 permitted examination of the
reaction mixtures by 'H NMR spectroscopy to determine
the nature and the extent of reaction, as illustrated by the
example shown in Figure 4. The calculation of UV molar
absorption coefficients was carried out by correlating UV-
visible absorbances with NMR-determined concentrations
of o-quinone products in the reaction mixtures, as shown
in Table IV. The E values obtained correspond well to
literature values where these are available.2o This method
provides a means of calculating E values without the
necessity of isolating the o-quinone products, which is a
major advantage for compounds which are air sensitive and
have a strong tendency to polymerize. The NMR spectra
also show unambiguously that under these experimental
conditions the reactions proceed cleanly and that only the
o-quinone products are formed.

Table 111. Kinetic parameters determined for formation of o-quinones from p-substituted phenols by polyphenol oxidase in chloroform.

Concentration range Vmax a KM VmaxIKMa

p-Substituent used (mM) (Xl02) (mM) (~10-3) E!*
Me 5 -200 8.12 ? 0.5 24.81 3.27 -1.24
Et 10-75 4.52 2 0.2 38.64 1.17 -1.31
Pr' 25-100 0.94 2 0.02 12.47 0.75 - 1.75
Bu' 10-50 4.4 2 1.0 83.29 0.53 -2.78
OMe 5-50 5.1 2 0.09 21.76 2.34 -0.55
F 5-50 1.16 ? 0.02 3.56 3.26 -0.46
C1 25 -200 0.68 ? 0.01 5.95 1.43 -0.97
Br 50-200 0.99 ? 0.01 52.30 0.19 -1.30

a ~ (min . mg protein)-'
The units of V,, are mM/min. mg protein and units of V m a X / Kare
Apparent K M values.

942 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 42, NO. 8, OCTOBER 1993


i 6 A i i i i i
Figure 4. A 400-MHz ‘H NMR spectrum of a CDC13 solution of the
reaction mixture from the o-hydroxylation and oxidation of p-ethyl
phenol by polyphenol oxidase.
CONCLUSION
The results of this investigation show that the crude mush-
room extract provides an accessible and viable source of
polyphenol oxidase with a sufficiently high activity for
it to be a feasible biocatalyst in chloroform. The kinetic
study of this system has provided quantitative data which
is consistent with and confirms current general theories on
the effects of organic systems on the catalytic functioning
of enzymes. The NMR analysis of the reaction mixtures
has given clear evidence that in chloroform, polyphenol
oxidase catalyzes the same reaction as it does in aqueous
systems, without interfering secondary reactions, and that
the reactions proceed to a significant extent for a range of
phenolic substrates.
The authors thank Rhodes University, the Foundation for Research
and Development, and AECI (Pty) Ltd., South Africa, for generous
financial support.
References
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of mushroom tyrosinase. J. Biol. Chem. 238: 1699-1707.
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pounds by polyphenol oxidase in organic solvents and in water.
Biotechnol. Bioeng. 32: 716-718.
3. Dordick, J. S. 1989. Enzymic catalysis in monophasic organic sol-
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Copper proteins, vol. 3. Wiley, New York.

Table IV. Determination of UV molar absorption coefficientsa of o-quinone products.

Substrate Initial substrate o-Quinone Percent Time Absorbance

p-substituent concentration (mM) concentrationb ( m ~ ) reaction taken (h) increase‘ E (mol-’ . dm3 .cm-’ )

Me 0.0533 1.43 X lo-’ 26.8 20.94 1460 2 100

Et 0.0171 8.77 x 51.3 10.44 1190 ? 50
Ptl 0.0167 4.6 x 10-3 27.5 7.27 1580 ? 30
Bu’ 0.0115 1.22 x 10-~ 10.6 2.02 1660 2 100
OMe 0.0261 9.54 x 10-~ 36.6 17.24 1800 ? 100
F 0.0282 4.67 x 10-3 16.6 15.50 3320 2 30
CI 0.0290 6.12 x 10” 21.1 23.27 3800 2 300
Br 0.0130 3.05 x 10-3 23.5 10.96 3600 2 400

a At wavelengths of maximum absorbance determined as described under Methods and Materials.

After reaction, calculated from mean integral ratios for peaks corresponding to substrate and product, respectively.
Calculated from measured absorbances of suitably diluted samples taken after reaction time.

BURTON ET AL.: ACTIVITY OF MUSHROOM POLYPHENOL OXIDASE 943

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944 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 42, NO. 8, OCTOBER 1993