ANALYTICAL

BIOCHEMISTRY
Analytical Biochemistry 344 (2005) 224231
www.elsevier.com/locate/yabio
0003-2697/$ - see front matter 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.ab.2005.05.032
A spectrophotometric method for the quantiWcation of an enzyme
activity producing 4-substituted phenols: Determination of
toluene-4-monooxygenase activity
Louise C. Nolan, Kevin E. OConnor

Department of Industrial Microbiology, Centre for Synthesis and Chemical Biology, Conway Institute for Biomolecular and Biomedical Research,
National University of Ireland, University College Dublin, BelWeld, Dublin 4, Republic of Ireland
Received 11 March 2005
Available online 13 June 2005
Abstract
A spectrophotometric method for the quantitative determination of an enzyme activity resulting in the accumulation of 4-substi-
tuted phenols is described in this article. Toluene-4-monooxygenase (T4MO) activity in whole cells of Pseudomonas mendocina KR1
is used to demonstrate this method. This spectrophotometric assay is based on the coupling of T4MO activity with tyrosinase activ-
ity. The 4-substituted phenol, produced by the action of T4MO on the aromatic ring of a substituted arene, is a substrate for tyrosi-
nase, which converts phenols to o-quinones. The latter react with the nucleophile 3-methyl-2-benzothiazolinone hydrazone (MBTH)
to produce intensely colored products that absorb light maximally at diVerent wavelengths, depending on the phenolic substrate
used. The incubation of whole cells of P. mendocina KRI with Xuorobenzene resulted in the accumulation of 4-Xuorophenol. The
coupling of T4MO activity with tyrosinase activity in the presence of Xuorobenzene resulted in the formation of a colored product
absorbing maximally at 480 nm. The molar absorptivity () value for the o-quinoneMBTH adduct formed from 4-Xuorophenol was
determined experimentally to be 12,827 M
1
cm
1
with a linear range of quantiWcation between 2.5 and 75 M. The whole cell assay
was run as a continuous indirect assay. The initial rates of T4MO activity toward Xuorobenzene, as determined spectrophotometri-
cally, were 61.8 4.4 nmol/min/mg P. mendocina KR1 protein (using mushroom tyrosinase), 64.9 4.6 nmol/min/mg P. mendocina
KR1 protein (using cell extracts Pseudomonas putida F6), and, as determined by HPLC analysis, 62.6 1.4 nmol/min/mg P. mendo-
cina KR1 protein.
2005 Elsevier Inc. All rights reserved.
Keywords: Spectrophotometry; Tyrosinase; MBTH; 4-Substituted phenol; Toluene-4-monooxygenase
A broad range of bacteria capable of degrading aro-
matic hydrocarbons have been isolated and character-
ized extensively [19]. Many of these bacterial strains
possess enzymes capable of transforming a variety of
aromatic compounds to potentially valuable products
such as phenols, dihydrodiols, catechols, and epoxides
[1015]. One such microorganism, Pseudomonas mendo-
cina KR1, expresses a toluene-4-monooxygenase
(T4MO)
1
enzyme converting toluene to the phenol p-cre-
sol [1618]. In addition, P. mendocina KR1 is capable of
transforming a broad range of aromatic hydrocarbons
to 4-substituted phenols [18]. Although the ability to
synthesize value-added products such as phenols and
catechols has commercial potential, the low rate of
transformation often hinders the use of biocatalysts in
*
Corresponding author. Fax: +353 1 716 1183.
E-mail address: kevin.oconnor@ucd.ie (K.E. OConnor).
1
Abbreviations used: T4MO, toluene-4-monooxygenase; MBTH,
3-methyl-2-benzothiazolinone hydrazone; DMF, N,N-dimethylform-
amide; PHPA, p-hydroxyphenylacetic acid; DTT, dithiothreitol.
QuantiWcation of enzyme activity producing 4-substituted phenols / L.C. Nolan, K.E. O'Connor / Anal. Biochem. 344 (2005) 224231 225
industry [19]. The in vitro or directed evolution of
enzymes for improved rates of reaction should result in
the use of enzymes as biocatalysts for value-added prod-
uct synthesis [20]. However, one of the limiting factors
for the directed evolution of enzymes is often the
absence of a rapid and convenient method for detection
of improved activity.
Methods for the quantiWcation of oxidoreductase
enzyme activities such as T4MO include HPLC, GC
MS, LCMS, and chromatographic separation of
metabolites followed by spectrophotometric or radio-
metric detection [13,1517]. These methods are sensitive,
but they are also time-consuming and do not lend them-
selves to rapid or inexpensive methods of detection [21].
Other procedures require carbon or radioactive labeling
to monitor enzyme activities and product formation
[16,2123]. These methods are expensive and require a
number of sample preparation steps. Spectrophotomet-
ric techniques have attracted much attention because
they are convenient and inexpensive [24]. Several spec-
trophotometric methods for the detection of phenols
have been described [21,2426]. Reagents such as Folin
Ciocalteau and p-toluidine have been employed in the
detection of phenols. However, these reagents can be
nonspeciWc and can react with a variety of compounds,
both phenolic and nonphenolic [25,27]. The use of an
enzyme, exhibiting a high degree of regioselectivity,
could allow the detection of a speciWc phenol in the pres-
ence of compounds that interfere with chemical-based
assays. Despite the array of diVerent methods to detect
phenols, none of these has been used to quantify enzyme
activity [25,27]. The aim of the current study was to cou-
ple T4MO activity with tyrosinase enzyme activity to
develop a rapid spectrophotometric assay for the quanti-
Wcation of T4MO activity (Fig. 1).
Tyrosinase enzyme activity converts 4-substituted
phenols to 4-substituted catechols (monophenolase)
and converts 4-substituted catechols to 4-substituted
o-quinones (diphenolase) (Fig. 2) [10,23,2833]. The
o-quinones react with 3-methyl-2-benzothiazolinone
hydrazone (MBTH) to produce intensely colored prod-
ucts where the intensity of color (optical density) corre-
lates with the concentration of phenol used [23,29].
Tyrosinase has a broad substrate range acting on a wide
range of 4-substituted phenols and catechols and does
not require the addition of exogenous cofactors [29,34].
These properties, together with the high value for
o-quinoneMBTH adducts, make this a versatile and
sensitive method for the quantiWcation of enzyme
activities that result in the formation of 4-substituted phe-
nols. In this article, we report on the coupling of T4MO
activity with tyrosinase activity in a rapid and sensitive
Fig. 1. Continuous indirect assay for the quantiWcation of T4MO activity by spectrophotometry and HPLC. Samples (1 ml) are taken from a 50-ml
T4MO activity assay and centrifuged. In the analysis, 200 l of the assay medium supernatant is analyzed spectrophotometrically using tyrosinase,
whereas 450 l is analyzed by HPLC.
226 QuantiWcation of enzyme activity producing 4-substituted phenols / L.C. Nolan, K.E. O'Connor / Anal. Biochem. 344 (2005) 224231
spectrophotometric assay for the quantiWcation of
T4MO activity in whole cells of P. mendocina KR1 using
Xuorobenzene as the substrate (Fig. 2) [23,30,32].
Materials and methods
Reagents
4-Fluorophenol, N,N-dimethylformamide (DMF),
and mushroom tyrosinase (3126 U/mg) were purchased
from Fluka. MBTH was purchased from Sigma
Aldrich. All other reagents were of analytical grade.
Tyrosinase enzyme source
Commercial mushroom tyrosinase was purchased
from Fluka and used without further puriWcation.
Mushroom tyrosinase (10mg) was dissolved in 1 ml of
50 mM potassium phosphate buVer (pH 7.0). Bacterial
tyrosinase was obtained from cell extracts of Pseudomo-
nas putida F6, known to possess tyrosinase activity
[10,34].
Bacterial strains and culture conditions
Pseudomonas mendocina KR1 was originally isolated
by Richardson and Gibson [35]. P. putida F6 was iso-
lated from soil on a mineral medium with p-hydroxyphe-
nylacetic acid (PHPA) as the sole source of carbon and
energy, as described previously [34]. Stock cultures of
both bacteria were stored at 80 C in 15% glycerol. E2
culture medium supplemented with a vitamin mix, trace
elements, and magnesium sulfate (1 mM) was used for
growth of P. mendocina KR1 and P. putida F6 [36].
Growth and harvesting cells
Overnight starter cultures of P. mendocina KR1 were
grown on toluene in 50ml E2 supplemented with vita-
mins, trace elements, and magnesium sulfate. Overnight
cultures were used to inoculate (2%) larger Xasks of batch
culture growth medium (800ml) with toluene as the sole
source of carbon and energy supplied in the vapor phase
at 30 C for 18h. Cells were harvested at an optical den-
sity (540nm) of 0.70.8. Cells were placed on ice and cen-
trifuged at 16,200g for 10min. Cell pellets were washed
with ice-cold 50mM potassium phosphate buVer (pH 7.0)
and centrifuged again as above, after which 20ml of
50mM potassium phosphate buVer (pH 7.0) was used to
resuspend the cell pellets. P. mendocina KR1 cell suspen-
sion was maintained on ice prior to the determination of
T4MO activity. For growth of non-induced P. mendocina
KR1 cells, glutamate was used as the sole source of car-
bon and energy. These cells were treated as above during
cell preparation and experimental analysis.
Overnight starter cultures of P. putida F6 were grown
on 5 mM phenylacetic acid in 4 ml E2 supplemented with
vitamins, trace elements, and magnesium sulfate. P. put-
ida F6 overnight cultures were used to inoculate batch
Fig. 2. Coupling of T4MO activity with tyrosinase activity for a spectrophotometric assay to quantify T4MO activity. T4MO activity converts
Xuorobenzene (A) to 4-Xuorophenol (B). Based on previous reports, tyrosinase converts 4-Xuorophenol (B) to 4-Xuorocatechol (C) [10]. As described
by Espn and co-workers, catechols (C) are then converted to quinones (D), which react with MBTH (E) to form the catecholMBTH adduct (F),
which is then converted to the o-quinoneMBTH adduct (G). Adapted from [30].
QuantiWcation of enzyme activity producing 4-substituted phenols / L.C. Nolan, K.E. O'Connor / Anal. Biochem. 344 (2005) 224231 227
culture growth medium (800 ml) with 5 mM tyrosine as
the sole source of carbon and energy at 30 C for 16 h.
Cells were refreshed with 1 mM tyrosine for a further 2 h
and harvested at an optical density (540 nm) of 0.91.0.
Cells were placed on ice and centrifuged at 16,200g for
10 min at 4 C. Pellets were washed once with ice-cold
50 mM potassium phosphate buVer (pH 7.0) before
being centrifuged again as above. Cell suspensions for
disruption were resuspended in 1 ml of 50 mM potas-
sium phosphate buVer (pH 7.0) containing 10% (w/v)
glycerol and 1.0 mM dithiothreitol (DTT). These cells
were passed twice through a precooled French pressure
cell and centrifuged at 38,700g for 30 min. The resulting
cell extract was collected and stored on ice prior to use
or was stored at 20 C [10]. Cell extracts were stored at
20 C for a maximum of 1 month without loss of tyros-
inase activity.
Protein determination
Protein concentration in cell extracts of P. putida F6
and P. mendocina KR1 were determined using the
method of Lowry et al. [37].
4-Fluorophenol determination by HPLC
All samples were analyzed by HPLC using a C18
Hypersil ODS 5- column (125 3 mm, Hypersil, Run-
corn, UK) and a HewlettPackard HP1100 instrument
equipped with an Agilent 1100 series diode array detec-
tor. The samples were eluted isocratically using a (0.1%)
phosphoric acidmethanol mix (70:30) at a Xow rate of
0.5 ml/min [10]. 4-Fluorophenol standards were acidiWed
and treated in the same way as assay samples for HPLC
analysis. The injection volume was 20l, and the elution
was monitored at 210 nm.
Determination of molar extinction coeYcients
The incubation of 4-Xuorophenol with tyrosinase
activity resulted in the formation of a colored product.
The relationship between optical density at lambda maxi-
mum (
max
) and 4-Xuorophenol concentration (molar
extinction coeYcient) was determined as follows. A series
of 1ml spectrophotometric reactions were carried out in
triplicate at diVerent concentrations of 4-Xuorophenol.
All spectrophotometric assays were carried out using a
UVVis Unicam Helios spectrophotometer. Tempera-
ture was controlled at 30C using a Grant circulating
water bath. The standard 1-ml reaction mixture con-
tained 50mM potassium phosphate buVer (pH 7.0), 2%
DMF, mushroom tyrosinase (0.2mg/ml), 6mM MBTH
(prepared fresh with Millipore H
2
O), and 4-Xuorophenol.
All reactions were started with the addition of 200 l of
4-Xuorophenol. Reference cuvettes contained all reaction
components except 4-Xuorophenol, which was replaced
with 50mM potassium phosphate buVer (pH 7.0). Nega-
tive controls containing all assay components without
substrate or enzyme failed to either produce a color reac-
tion or show 4-Xuorophenol consumption by HPLC.
Commercial mushroom tyrosinase was used as the
source of enzyme for molar extinction coeYcient value
determinations. However, transformation of 4-Xuor-
ophenol by cell extracts of P. putida F6 resulted in a
product with the same
max
(480 nm) for 4-Xuorophenol.
The Wnal absorbance of all reaction samples was noted,
and 100l of 1 N HCl was added to a 900-l sample
from the reaction medium. This medium was then centri-
fuged, Wltered using a nylon Wlter (0.2 m, Labquip), and
analyzed for substrate depletion using HPLC. The molar
extinction coeYcient values for the product of tyrosinase
activity with 4-Xuorophenol was calculated within the
linear part of the curve using the BeerLambert law,
ADcl, where A is absorbance, is the molar extinction
coeYcient, c is the concentration, and l is the path length.
QuantiWcation of T4MO activity in whole cells of
P. mendocina KR1
In a 50-ml whole cell assay, 15mM Xuorobenzene was
added to whole cells of P. mendocina KR1 at an absor-
bance (540nm) of 1.0 and was incubated at 30C with
shaking (200rpm). Aliquots (1ml) were withdrawn period-
ically from the whole cell assay medium over time and
were centrifuged at 23,400g for 10min at 4C to stop the
reaction (Fig. 1). To determine the concentration of phenol
product present in the whole cell assay spectrophotometri-
cally, 200l of the supernatant was incubated with either
mushroom tyrosinase (0.2mg/ml) or cell extracts of P. put-
ida F6 (0.50.7mg/ml), 2% (v/v) DMF, 6mM MBTH, and
50mM potassium phosphate buVer (pH 7.0) (total volume
1ml) (Fig. 1). The increase in optical density (480nm) was
monitored with a spectrophotometer over time until the
value reached a maximum and then a plateau. This took
less than 5min to occur for all concentrations of 4-Xuor-
ophenol. In subsequent replicates, the optical density at
480nm was noted after 5min. In parallel with the spectro-
photometric measurements, 450l of the supernatant,
acidiWed with 50l of 1N HCl, was centrifuged, Wltered
using a nylon Wlter (0.2m, Labquip), and analyzed using
HPLC (Fig. 1). The initial T4MO rate of reaction was
determined by carrying out the above assay in triplicate
and sampling within the Wrst 10min of the reaction.
Results and discussion
HPLC analysis showed that whole cells of P. mendo-
cina KR1 transformed Xuorobenzene to a single product
4-Xuorophenol. The addition of either mushroom tyrosi-
nase or cell extracts of P. putida F6 and MBTH to spent
whole cell assay media resulted in the formation of a
228 QuantiWcation of enzyme activity producing 4-substituted phenols / L.C. Nolan, K.E. O'Connor / Anal. Biochem. 344 (2005) 224231
dark redbrown color. This is indicative of the transfor-
mation of 4-Xuorophenol by tyrosinase activity resulting
in the formation of colored o-quinoneMBTH adducts
(Fig. 2) [23,2833]. To quantify the 4-Xuorophenol
formed by P. mendocina KR1 spectrophotometrically,
the molar extinction coeYcient values for the o-qui-
noneMBTH adducts formed by the action of tyrosinase
were determined.
Determination of the molar extinction coeYcient values of
o-quinoneMBTH adducts
An authentic standard of 4-Xuorophenol (0.01mM)
was incubated with mushroom tyrosinase and MBTH. A
dark color formed in the reaction medium. The wave-
length at which the o-quinoneMBTH adducts absorb
maximally was determined by scanning the reaction mix-
ture at wavelengths between 400 and 600nm (Fig. 3).
Subsequently, various concentrations of 4-Xuorophenol
(0.00250.1mM) were incubated with mushroom tyrosi-
nase (0.2mg/ml) in the presence of 6mM MBTH, and the
absorbance at 480nm was determined. The concentration
of phenol transformed by tyrosinase was determined by
HPLC. A linear relationship between phenol depletion
and color formation was observed for 4-Xuorophenol at
concentrations below 0.075mM (Fig. 4). The molar
extinction coeYcient value of the o-quinoneMBTH
adduct for 4-Xuorophenol was determined from the
BeerLambert law. Control samples of either authentic
4-Xuorophenol or spent whole cells assay medium
incubated in a tyrosinase assay without tyrosinase did
not produce any color. Furthermore, HPLC analysis
revealed that 4-Xuorophenol in whole cell assay medium
was stable and that the concentration remained stable
over time.
Quantifying T4MO activity in whole cells of P. mendocina
KR1
The initial rate of T4MO activity was monitored by
determining the concentration of 4-Xuorophenol accu-
mulating in the media over a period of 10 min. In the
development of this assay, cell suspensions of P. mendo-
cina KR1 were originally incubated with Xuorobenzene,
MBTH, and tyrosinase. However, under these condi-
tions, color formation was not reproducible (data not
shown). Following various experimental approaches, it
was decided that the optimal conditions for quantiWca-
tion of T4MO activity were achieved by continuous indi-
rect sampling assay [38].
At various time points, 1-ml samples were withdrawn
from the whole cell assay (50 ml) and centrifuged to pro-
duce a cell pellet and a supernatant. The supernatant
(200 l) was incubated with either mushroom tyrosinase
(0.2 mg/ml) or cell extracts of P. putida F6 (0.50.7mg/
ml), 2% (v/v) DMF, 6 mM MBTH, and 50 mM potas-
sium phosphate buVer (pH 7.0) (total volume 1ml) for
5 min, as described in Materials and methods. The Wnal
absorbance at 480 nm (
max
) was determined. HPLC
analysis of the spent spectrophotometric assay medium
showed that all of the 4-substituted phenol was used by
tyrosinase during this time period. Consequently, the con-
centration of 4-substituted phenol in the whole cell assay
medium was calculated using the molar extinction coeY-
cient value (Fig. 5). In parallel with the spectrophotometric
assay, 450l of the remaining whole cell assay medium was
Fig. 3. Wavelength scan of the o-quinoneMBTH product formed when 10 M of 4-Xuorophenol is incubated with tyrosinase in the presence of
MBTH.
QuantiWcation of enzyme activity producing 4-substituted phenols / L.C. Nolan, K.E. O'Connor / Anal. Biochem. 344 (2005) 224231 229
added to 50l of 1N HCl, centrifuged, Wltered, and ana-
lyzed by HPLC. The concentrations of 4-Xuorophenol
from the whole cell assays as determined by HPLC were
nearly identical to those of samples determined spectro-
photometrically (Fig. 5).
An initial rate of T4MO activity, as determined spec-
trophotometrically, using mushroom tyrosinase
(61.8 4.4 nmol/min/mg P. mendocina KR1 protein) or
cell extracts of P. putida F6 (64.94.6nmol/min/mg
P. mendocina KR1 protein) was similar to the initial rate
of T4MO activity as determined by HPLC (62.6
1.4 nmol/min/mg P. mendocina KR1 protein)
(Fig. 5). Spent whole cell (T4MO) assay medium or
authentic 4-Xuorophenol incubated in a tyrosinase
assay but without enzyme did not produce a color
reaction. When glutamate grown cells of P. mendocina
KR1 (noninduced) were used in the assay, no forma-
tion of 4-Xuorophenol was observed in HPLC samples
and no color formation was observed in spectrophoto-
metric samples.
The coupling of T4MO with tyrosinase activity
allows a rapid and simple spectrophotometric method
Fig. 4. Linear relationship between concentration of 4-Xuorophenol and absorbance at
max
of the o-quinoneMBTH product.
Fig. 5. Initial rates of T4MO activity as determined by monitoring the increase in concentrations of 4-Xuorophenol over time by HPLC () and spec-
trophotometrically (480 nm) using mushroom tyrosinase () or cell extracts of P. putida F6 ().
230 QuantiWcation of enzyme activity producing 4-substituted phenols / L.C. Nolan, K.E. O'Connor / Anal. Biochem. 344 (2005) 224231
for the quantiWcation of T4MO activity in whole cells.
The molar extinction coeYcient of the o-quinone
MBTH adduct formed in the assay makes this a sensitive
method with a linear range of quantiWcation for 4-Xuor-
ophenol from 2.5 to 75 M (Fig. 4). The sensitivity of the
method described here compares favorably with previ-
ously reported phenol detection methods [18,23,27,39].
The method of Gupta et al. [25], employed by Yen and
Blatt [18] to detect the formation of phenolic com-
pounds that result from T4MO activity, has a 26-fold
higher detection limit (65M). Gupta and coworkers
employed the FolinCiocalteau reagent to produce a
stable color complex with the phenol products [25].
However, the FolinCiocalteau reagent is nonspeciWc
and reacts with a variety of compounds, both phenolic
and nonphenolic, resulting in false positives and overes-
timation of the product [25]. Other methods, such as that
described by Parke [27], employed p-toluidine to detect
catechol and protocatechuate formation in solid media.
However, the detection limit of this method was not
described. The method of Wackett and Gibson [21],
employing tetrazolitized o-dianisidine for the detection
of 1-naphthol, is sensitive (detection limit 4M) and
easy to use. However, the speciWcity or sensitivity of this
method for other phenols is not clear. A simple colori-
metric method based on the coupling of phenols with
4-aminoantipyrine determination is also available, but
this method is also nonspeciWc reacting with nonphen-
olic compounds [39]. Although a broad range of spectro-
photometric phenol detection methods have been
reported, none of these methods has been employed for
the quantiWcation of enzyme activities such as T4MO
[18,21,23,25,27,39].
Tyrosinase from mushroom and P. putida F6 react
with a broad range of 4-substituted phenols and cate-
chols, and the lower limit for reaction of tyrosinase with
4-substituted phenols is in the low micromolar range
[23,2830]. In addition, tyrosinase does not require the
addition of expensive exogenous cofactors for activity
[34]. The use of bacterial cell-free extracts makes the
source of the enzyme for this assay inexpensive and easy
to obtain. We envisage that this method, used as a contin-
uous indirect sampling assay, could easily be employed as
a rapid and convenient method for quantifying improved
enzymatic activity in microtiter plate assays for directed
evolution experiments. Furthermore, the use of this
method may be extended to the screening of large num-
bers of wild-type and mutant strains of microorganisms
for their ability to accumulate phenolic and diphenolic
compounds from aromatic hydrocarbons.
Acknowledgment
This work was supported in part by the Enterprise
Ireland Research Scholarship Programme.
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