Colorimetric Determination of Sulfate via an Enzyme Cascade for High-Throughput Detection of Sulfatase Activity

Article abstract of DOI:10.1021/acs.analchem.7b03719

High-throughput screening (HTS) methods have become decisive for the discovery and development of new biocatalysts and their application in numerous fields. Sulfatases, a broad class of biocatalysts that hydrolyze sulfate esters, are involved in diverse r

Full text of DOI:10.1021/acs.analchem.7b03719

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Article
Colorimetric determination of sulfate via an enzyme
cascade for high-throughput detection of sulfatase activity
José G. Ortiz-Tena, Broder Rühmann, and Volker Sieber
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03719 • Publication Date (Web): 08 Jan 2018
Downloaded from http://pubs.acs.org on January 8, 2018
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Analytical Chemistry
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Colorimetric determination of sulfate via an enzyme cascade for
high-throughput detection of sulfatase activity
Jose G. OrtizꢀTenaa, Broder Rühmanna, Volker Siebera,b,c,d*
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aChair of Chemistry of Biogenic Resources, Technische Universität München, Straubing, Germany
bFraunhofer IGB, Straubing Branch BioCat, Straubing, Germany
cTUM Catalysis Research Center, ErnstꢀOttoꢀFischerꢀStraße 1, 85748, Garching, Germany
dThe University of Queensland, School of Chemistry and Molecular Biosciences, 68 Copper Road, St. Lucia 4072, Australia
*Phone: +49 (9421) 187ꢀ300. Eꢀmail: Sieber@tum.de
ABSTRACT: Highꢀthroughput screening (HTS) methods have become decisive for the discovery and development of new biocataꢀ
lysts and their application in numerous fields. Sulfatases, a broad class of biocatalysts that hydrolyze sulfate esters, are involved in
diverse relevant cellular functions (e.g., signaling and hormonal regulation) and are therefore gaining importance, particularly in the
medical field. Additionally, various technical applications have been recently devised. One of the major challenges in the field of
enzyme development is the sensitive and highꢀthroughput detection of the actual product of the biocatalyst of interest without the
need for chromophore analogues. Addressing this issue, a colorimetric assay for sulfatases was developed and validated for detectꢀ
ing sulfate through a twoꢀstep enzymatic cascade, with a linear detection range of 3.3 (limit of detection) up to 250 µM. The proceꢀ
dure is compatible with relevant compounds employed in sulfatase reactions, including coꢀsolvents, cations, and buffers. The assay
was optimized and performed as part of a 96ꢀwell screening workflow that included bacterial growth, heterologous sulfatase exꢀ
pression, cell lysis, sulfate ester hydrolysis, inactivation of cell lysate, and colorimetric sulfate determination. With this procedure,
the activity of an aryl and an alkyl sulfatase could be confirmed and validated. Overall, this assay provides a simple and fast alterꢀ
native for screening and engineering sulfatases from DNA libraries (e.g., using metagenomics) with medical or synthetic relevance
.
Current advances in enzyme discovery and engineering rely
highly on the analytical screening methods used. The deciꢀ
sion on how to screen large mutant or metagenomic libraries
is a crucial step for the success of any enzyme optimization
or search procedure.1 Microplate assays play a key role in the
screening phase because the existing compatible robotics
enables fast processing of a large number of enzyme variants.
Recently, sulfatases (EC 3.1.6.X) have attracted the interest
of various disciplines from the scientific community owing to
both their biological relevance and the technical applications
devised in different fields.2 Sulfatases represent a very broad
family of enzymes that catalyze the hydrolysis of sulfate
esters from a wide spectrum of substrates. These enzymes are
involved in numerous cellular functions, such as cell signalꢀ
ing, cellular degradation, hormone regulation, and pathogeneꢀ
sis.2 As they play an essential role in several diseases, their
relevance in human health has been increasingly recognized.3
Given that the sulfation degree of their substrates is decisive
for, e.g., ligand activity (which appears to be strongly related
to certain types of cancer4), the inhibition and modulation of
sulfatases’ activity towards signaling molecules have been
increasingly studied.5,6 Furthermore, several technical appliꢀ
cations of sulfatases have been developed, such as the enantiꢀ
oselective production of secꢀalcohols from alkyl sulfates7 or
the regioselective cleavage of sulfate groups from different
types of carrageenan to control its gelling or texturizing propꢀ
erties.8 The increasing importance of this type of biocatalyst
is reflected in the recent creation of a sulfatase classification
database,9 which arranges enzymes according to their subꢀ
strate specificity into alkyl or aryl sulfatases. Given the
enormous diversity of sulfated substrates, enzyme HTS proꢀ
cedures are far from being straightforward. The substrates
used as standards to detect sulfatases’ activity include pꢀ
nitrophenylꢀ or pꢀnitrocatecholꢀsulfate for aryl sulfatases and
chromogenic substrate analogues for alkyl sulfatases.10 For
example, a very sensitive sulfatase activity assay was recently
proposed in which the fluorescent compound Nꢀ
methylisoindole is produced after sulfatase cleavage of a
corresponding sulfated chromophore.11 However, these asꢀ
says are only selective for sulfatases that are active on the
nonꢀnatural substrates that contain such proꢀfluorescent
probes. Although the easy colorimetric or UV detection of
the corresponding deꢀsulfated products makes their use very
convenient, this strategy does not necessarily guarantee activꢀ
ity on the actual substrate of interest, and the analogues are
usually expensive. If the deꢀsulfated product is not chromoꢀ
genic, instrumental analytical techniques (liquid or gas chroꢀ
matography, mass spectrometry and nuclear magnetic resoꢀ
nance) are required to detect the hydrolyzed product, which
impedes highꢀthroughput processing of large enzyme librarꢀ
ies. Direct sulfate detection would enable the screening and
discovery of sulfatases regardless of the substrate type. For a
wide range of common food and environmental applications
(e.g., sludge or drinking water analyses), sulfate is sensitively
detected (0.1 mg L−1) and quantified using ion chromatogꢀ
raphy or lead ionꢀselective electrodes.12,13 Given the long
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Analytical Chemistry
Page 2 of 9
experimental times, large eluent volumes, and tedious sample
preparation associated with such procedures, they are rather
unsuitable to realize HTS. Turbidimetric determination of
sulfate precipitates with barium chloride has become the
method of choice when large amounts of samples are measꢀ
ured because the procedure is straightforward and compatible
with the microplate format. Several variants of this method
have been developed for different applications, e.g., bacterial
cultures,14 human urine,15 or industrial effluents,16 and comꢀ
mercial kits are readily available. However, the precipitation
of barium sulfate for generating a turbidimetric signal is
strongly dependent on multiple factors, e.g., suspension stabiꢀ
lizing reagent used, ionic strength of the sample, and protein
concentration.14 Therefore, the application of turbidimetry
using barium chloride for screening sulfatases from cell lyꢀ
sates is somewhat inappropriate; as organic components, such
as glycosaminoglycans and peptides strongly inhibit the
precipitation of BaSO4.17 Recently, a study on a sensitive
colorimetric assay for sulfate detection was published in
which cysteamineꢀcoated gold nanoparticles aggregated in
the presence of SO42− ions, inducing a detectable absorption
shift in the range of 0.34–30 µM, with a sigmoidal sulfate
calibration curve.18 Also, highly sensitive phosphatase assays
have been recently developed based on the application of
various nanostructures. The output signal of these methods,
which in part involve substrateꢀcoordinated compounds,
ranges from colorimetry to fluorescence and voltammetry.
Unfortunately, their utilization for the analysis of sulfatases
has not been shown.19ꢀ21 In this study, addressing the need for
a reliable, sensitive, and inexpensive alternative for screening
sulfatase libraries with a broad detection range, a colorimetric
assay was developed and validated for sulfate determination
based on a twoꢀstep enzymatic cascade. After optimizing the
relevant reaction parameters of the enzymatic cascade, the
assay was validated and the influence thereon of pertinent
compounds was evaluated. Sample preparation was then
adjusted for optimal sulfate detection in bacterial lysates and
the application of the assay for HTS of sulfatase activity was
demonstrated by determining the activity of heterologously
expressed aryl and alkyl sulfatases in E. coli in microplate
format.
(SIB, Switzerland; Table S1). All purified enzymes and assay
reactants were aliquoted, stored at −20 °C and used only
once. 2ꢀheptyl sulfate (PISA1 substrate) was synthesized as
previously described25 with modifications (see supplementary
material).
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Assay optimization: The colorimetric assay was subsequentꢀ
ly optimized by evaluating a K2SO4 calibration curve conꢀ
structed in the range 2.5–250 µM. Since reaction 2 of the
assay (Scheme 1) is already established for determining pyꢀ
rophosphate and pyruvate,22,26 the optimization was focused
on reaction 1 and each optimization round served as basis for
the next. Optimization reactions were performed in duplicate
by mixing 50 µL of K2SO4 standard and 50 µL of master mix
1. After incubation at 25 °C for varying times depending on
the optimization round (see below), 100 µL of master mix 2
(K2HPO4 100 mM pH 6.5, DAꢀ64 100 µM, thiamine pyroꢀ
phosphate (TPP) 50 µM, MgCl2 100 µM, phosphoenolpyꢀ
ruvate (PEP) 500 µM, adenosine monophosphate (AMP)
500 µM, PPDK 50 mU, POX 50 mU, and HRP 200 mU;
concentrations in reaction) was added to the abovementioned
solution. The mixture was then incubated at 37 °C for 30 min.
In all cases, the absorbance (A) was measured at 727 and
540 nm (Varioskan, Thermo Scientific), and A727–540 was
computed for each standard and a sulfate blank. The calibraꢀ
tion curve was then calculated by subtracting the sulfate
blank absorbance value from each calibration point (ꢁA727–
540). The first optimization round focused on finding the best
enzymatic concentrations for reaction 1 in HEPES (4ꢀ2ꢀ
hydroxyethylꢀ1ꢀpiperazineethanesulfonic acid) 12.5 mM pH
7.8, GTP 2 mM, ATP 1 mM, and MgCl2 3 mM (final concenꢀ
trations, incubation time 45 min). Next, GTP and ATP conꢀ
centrations were varied (HEPES 12.5 mM pH 7.8, MgCl2
3 mM, ATPs 0.46 µM, and APSk 5.3 µM; final concentraꢀ
tions, incubation time 45 min). The optimal reaction time was
determined from four equal intervals between 15 and 90 min
using the previously found optimal concentrations (HEPES
12.5 mM pH 7.8, GTP and ATP 1 mM, MgCl2 3 mM, ATPs
0.46 µM, and APSk 5.3 µM, concentrations in reaction).
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Assay validation and characterization: All the data obꢀ
tained were verified for normal distribution via standard
skewness and kurtosis tests. For intraꢀday repeatability and
interꢀday reproducibility validation, the standard deviation
(SD) and precision were calculated using the Student’s tꢀtest
(P = 95%, n = 4 and 6, respectively). The limit of detection
(LOD) was calculated as b ± 3σBlank and the limit of quantifiꢀ
cation (LOQ) was b ± 10σBlank, where b is the yꢀintercept of
the calibration curve. The effect of 100, 50, and 25 mM
HEPES, TRIS (2ꢀaminoꢀ2ꢀ(hydroxymethyl) propaneꢀ1,3ꢀ
diol), MOPS (3ꢀmorpholinopropaneꢀ1ꢀsulfonic acid), citrate,
and phosphate buffers at the corresponding pKa was evaluatꢀ
ed by first spiking the samples with a K2SO4 solution (100
µM) and then adding the buffers to a complete calibration
curve. The influence of various metal ions at different conꢀ
centrations (1.0, 0.1, and 0.01 mM) was also investigated by
spiking with 100 µM K2SO4 solutions and calculating the
recovery. Different solvents at 5% v/v (final concentration in
assay) were spiked with 100 µM sulfate as well.
EXPERIMENTAL SECTION
Chemicals and enzymes: All chemicals were of analytical
grade and were purchased from Sigma Aldrich, Merck
KGaA, and Carl Roth GmbH. Pyruvate oxidase (POX) and
horseradish peroxidase (HRP) were obtained from Sigma
Aldrich. The leuco dye Nꢀ(carboxymethylaminocarbonyl)ꢀ
4,4′ꢀbis(dimethylamino)diphenylaminesodium salt (DAꢀ64)
was acquired from Wako. The genes for pyruvate phosphate
dikinase from Propionibacterium freudenreichii (PPDK) and
APS kinase (APSk) from S. cerevisiae were purchased from
Genscript in pETꢀ28a(+) expression vectors; they were heterꢀ
ologously produced in E. coli BL21 DE3 and purified using
affinity chromatography according to published protocols.22,23
The cysDN gene encoding ATP sulfurylase (ATPs) with
GTPase activity was amplified from E. coli genomic DNA
using designed primers (see supplementary information),
cloned into pETꢀ28a(+), and purified via affinity chromatogꢀ
raphy with a NiꢀNTA column (Aekta, GE). Aryl sulfatase
from P. aeruginosa (PAS) and alkyl sulfatase from Pseudoꢀ
monas sp. DSM6611 (PISA1) were purified as previously
described.7,24 The concentrations of the purified enzymes
were determined using a nanophotometer (Implen, Pꢀ330) by
employing the parameters from the online tool ProtParam
Assay application in sulfatase reactions: First, the applicaꢀ
bility of the developed assay for screening sulfatase activity
was assessed using purified PAS and PISA1. The reaction for
PAS (3.0 nM) was performed using pꢀnitrophenyl sulfate
(250 µM) as the model substrate in TRIS 100 mM (pH 8, 1
mL reaction volume) at 30 °C overnight. The concentration
of pꢀnitrophenol produced was determined from the absorbꢀ
ance at 400 nm, quantifying against a standard calibration
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Analytical Chemistry
curve. The reaction for PISA1 (1.8 µM) was performed with
2ꢀheptylꢀsulfate as previously described.27 The concentration
of sulfate released by both enzymes was quantified using the
optimized colorimetric assay.
Owing to their outstanding specificity, enzymes offer unique
advantages as molecular recognition elements in analytical
chemistry.30 As sulfate ion is the analyte of interest in this
case, an enzyme that utilizes it as a substrate is required as
the recognition element. However, the unreactive nature of
sulfate makes the selection of this enzyme challenging. ATP
sulfurylase (ATPs, EC 2.7.7.4) catalyzes the universal startꢀ
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The influence of different cell lysis methods on the assay was
tested. For this, E. coli BL21 DE3 bearing the pETꢀ28a(+)
empty vector were cultured overnight at 37 °C. The cells
were centrifuged and reꢀsuspended in HEPES (100 mM,
pH 8) containing either BꢀPerTM (Thermo Scientific) or Bugꢀ
Buster® (Merck) according to manufacturer’s instructions,
lysozyme (2.5 mg mL−1), and DNase (10 µg mL−1). The cells
were incubated for 1 h at 37 °C, and the sulfate content was
measured in spiked dilutions prior to and after a 5ꢀmin incuꢀ
bation step at 70 °C. In a second experiment, different inactiꢀ
vation times were evaluated over the whole calibration range.
As large signal variability was observed in bacterial lysates
with encoding sulfatases, the stabilizing effect of bovine
serum albumin (BSA) at different concentrations in the lysis
solution was evaluated.
2−
ing step in the sulfur metabolism in vivo, “activating” SO4
by transferring it to adenosine triphosphate (ATP) through a
highly endergonic reaction (ꢁGº′ = 19.5 kcal mol−1).31 Adenꢀ
osineꢀ5′ꢀphosphosulfate (APS) and inorganic pyrophosphate
(PPi) are formed as the products in a reaction whose equilibꢀ
rium favors the reactant side (K = 10−8).32 This issue is solved
in vivo by many organisms through channeling complexes, in
which APS is immediately transferred from the active site of
the ATPs to an embedded APS kinase (APSk, EC 2.7.1.25),
which in turn phosphorylates APS, shifting the equilibrium to
the product side.33 A promising alternative for performing
this reaction in vitro, without the need of such a complex, is
the ATPs/GTPase from E. coli.34 This enzyme can couple the
chemical potential of guanosine triphosphate (GTP) hydrolyꢀ
sis with the unfavorable activation of sulfate to produce APS,
PPi, and GDP. This particular feature is not displayed by
ATPs from other organisms, e.g., S. cerevisiae.35 Given this
advantage, the ATPs/GTPase reaction was selected as the
starting step of the proposed cascade for sulfate detection
(Scheme 1). This reaction can be further driven towards
completion by the immediate phosphorylation of APS to
afford 3′ꢀphosphoadenosine 5′ꢀphosphosulfate (PAPS) using
APSk from S. cerevisiae. Of all the products formed to this
point, the resulting equimolar production of PPi from sulfate
enables the possibility of connection to a transducer element.
Its detection is possible owing to a second set of enzymeꢀ
catalyzed reactions, which rely on the previously described
assays for amino acids based on PPi detection using pyruvate
phosphate dikinase (PPDK) from Propionibacterium
freudenreichii.36 The PPi released by ATPs/GTPase reacts
with adenosine monophosphate (AMP) and phosphoenolpyꢀ
ruvate (PEP) to produce pyruvate, which is in turn oxidized
by pyruvate oxidase (POX) to afford H2O2. In the last reacꢀ
tion of the cascade, horseradish peroxidase (HRP) catalyzes
the oxidation of the dye DAꢀ64 to produce Bindschedler’s
green using the H2O2 produced, yielding a colorimetric signal
at 727 nm, which is proportional to the sulfate concentration
in the sample. The measurement at this particular wavelength
avoids detection in the yellow region of the spectra, in which
several disturbing compounds or matrices exhibit absorbance
(e.g., bacterial culture media or aromatic compounds).
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Sulfatase screening procedure: E. coli BL21 DE3 colonies
bearing the expression vectors pASKꢀIBA5+_PAS, and pETꢀ
21(+)_PISA1 and a corresponding empty vector were picked
from lysogeny broth (LB) agar plates (RapidPick CPꢀ7200,
Hudson). The colonies were then inoculated into a preꢀculture
deep well plate (Greiner) containing 1.2 mL of LB medium
with carbenicillin (100 µg mL−1) and grown at 37 °C overꢀ
night. 20 µL of the preꢀculture was then used to inoculate an
expression plate, and the cells were incubated for 2 h at
37 °C. The expression of PAS was induced with anhydrotetꢀ
racycline (200 µg L−1) at 30 °C for 2 h.7 The PISA1ꢀbearing
cells were induced with IPTG (0.5 mM), expression conductꢀ
ed at 20 °C for 10 h.28 After collecting the cell pellet by cenꢀ
trifugation (3000 g, 15 min), 500 µL of lysis solution containꢀ
ing HEPES 100 mM, Bugbuster® protein extraction reagent
(according to manufacturers’ instructions), lysozyme
(2.5 mg mL−1), DNase (10 µg mL−1), and BSA (2.5 g L−1)
was added to each well, and the plate was incubated for 1 h at
37 °C and 1000 rpm. After centrifugation of cell debris (4000
g, 15 min), 450 µL of cell lysates were transferred to a new
Uꢀbottom plate (Riplate, Ritter) and incubated at the respecꢀ
tive Topt for each sulfatase (57 °C and 1 h for PAS cpNPꢀSulfate
=
1 mM29 and 25 °C and 10 h for PISA1 c2ꢀheptylꢀSulfate = 5 mM).
At the end of the reaction, cell lysates were inactivated in a
water bath at 70 °C for 30 min. The concentration of the pꢀ
nitrophenol produced by PAS was determined at 400 nm and
quantified against a standard calibration curve in micro titer
plates (Greiner) using a 20ꢀµL sample. The content of the
sulfate released by both enzymes was quantified using the
optimized assay with 5ꢀµL samples.
Scheme 1. Enzymatic cascade for colorimetric detection
of inorganic sulfate.
Optimized assay: The final assay was performed by mixing
50 µL of the sample or standard with 50 µL of master mix 1
(HEPES 12.5 mM pH 7.8, GTP and ATP 1 mM, MgCl2
3 mM, ATPs 0.46 µM, APSk 5.3 µM). An incubation step at
25 °C for 45 min followed and then 100 µL of master mix 2
was added (K2HPO4 100 mM pH 6.5, DAꢀ64 100 µM, TPP
50 µM, MgCl2 100 µM, PEP 500 µM, AMP 500 µM, PPDK
50 mU, POX 50 mU, HRP 200 mU). The plate was then
incubated for 30 min at 37 °C and A727–540 was computed.
RESULTS AND DISCUSSION
For any assay aiming to identify an analyte of interest, a
recognition element is required in the detection system, folꢀ
lowed by a transducer element, e.g., colorimetric signal,
whose intensity correlates to the analyte’ s concentration.
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