Efficient Assay for the Detection of Hydrogen Peroxide by Estimating Enzyme Promiscuous Activity in the Perhydrolysis Reaction

Article abstract of DOI:10.1002/cbic.202000770

Hydrogen peroxide is an ideal oxidant in view of its availability, atom economy, or green aspects. Furthermore, it is produced by the cell mitochondria and plays a meaningful role in controlling physiological processes, but its unregulated production leads to the destruction of organs. Due to its diverse roles, a fast and selective method for hydrogen peroxide detection is the major limitation to preventing the negative effects caused by its excess. Therefore, we aimed to develop an efficient assay for the detection of H₂O₂. For this purpose, we combined the enzymatic method for the detection of hydrogen peroxide with the estimation of the promiscuity of various enzymes. We estimated the activity of an enzyme in the reaction of p-nitrophenyl esters with hydrogen peroxide resulting in the formation of peracid. To our knowledge, there is no example of a simple, multi-sensor demonstrating the promiscuous activity of an enzyme and detecting hydrogen peroxide in aqueous media.

Full text of DOI:10.1002/cbic.202000770

Combining Chemistry and Biology
Accepted Article
Title: Efficient assay for the detection of hydrogen peroxide
via estimation of the enzyme promiscuous activity in the
perhydrolysis reaction
Authors: Ryszard Ostaszewski and Monika Wilk
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemBioChem 10.1002/cbic.202000770
Link to VoR: https://doi.org/10.1002/cbic.202000770
01/2020

10.1002/cbic.202000770
ChemBioChem
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Efficient assay for the detection of hydrogen peroxide via
estimation of the enzyme promiscuous activity in the
perhydrolysis reaction
Monika Wilk and Ryszard Ostaszewski*[a]
Abstract: Hydrogen peroxide is an ideal oxidant in the view of
availability, atom-economy, or green aspects. Furthermore, it is
produced by the cell mitochondria and plays a meaningful role in
controlling the physiological processes, but unregulated production
leads to the destruction of organs. Due to its diverse roles, a fast and
selective method for hydrogen peroxide detection is the major
limitation to prevent the negative effects caused by its excess.
Therefore, we aimed to develop an efficient assay for the detection
of H2O2. For this purpose, we combined the enzymatic method for
the detection of hydrogen peroxide via the estimation of the
promiscuity of various enzymes. We estimated the activity of an
enzyme in the reaction of p-nitrophenyl esters with hydrogen
peroxide resulting in the peracid formation. According to our
major limitation on way to prevent the negative effects caused
by hydrogen peroxide as well as diagnoses of relative diseases.
Furthermore, hydrogen peroxide can be transformed into a more
potent oxidant- respective peroxy acid which also can inactivate
several regulatory enzymes such as protein tyrosine
phosphatase.[10]
The literature data shows several examples wherein hydrogen
peroxide was used as a reagent on way to produce peroxy acids,
which due to the synthetical point of view are important reagents,
used in organic chemistry as well as diverse kinds of industries.
Nevertheless, due to its unstable nature and strong oxidative
properties, the mild and safe methods for synthesis are
significant. Therefore, enzymatic manner found a wide range of
applications.[11] In particular, the perhydrolysis reaction allows
forming these valuable products in mild condition. Generally, in
the presence of hydrogen peroxide enzyme is used for the
production of peracid via perhydrolysis of acids or carboxylic
acid esters. Importantly, the transformation relies on the enzyme
promiscuous activity. This concept is defined as the capability of
an enzyme to mediate more than one reaction, which is different
from its natural transformation. Moreover, the enzyme
promiscuity plays a key role to understand the astonishing ability
of Nature for building complex molecules, especially in the
context of Darwinian evolution [12].
The field of hydrogen peroxide detection is highly active and
several methods have been published including electrochemistry,
chemiluminescence, spectrophotometry, or other.[13] Although
the reported techniques deliver sensitive methods for H2O2
detection, the requirements of expensive instrumentation, long
analysis time, or reaction media limit its application in medicinal
chemistry. From the diagnostic point of view, the control of
hydrogen peroxide and peroxy carboxylic acid concentrations
require the be carried out in the aqueous media. Hence, we
aimed to develop an efficient assay to hydrogen peroxide
detection based on the enzyme promiscuity revealed in the
perhydrolysis reaction running in the buffer solution. Uncovering
the new role of enzyme which is different from their natural
properties is highly active [14], but the development of fast and
simple screening assay for enzymatic divergent is still
demanding. In particular, spectrophotometric methods are well
suited to the enzymatic assay, because they are sensitive and
allow to avoid the background signals. Previously, we reported
revised studies on the fluorogenic-based versatile probes for the
detection of lipases and esterases activities toward hydrolysis
reaction. Also, the application of chiral fluorogenic substrates
proved the enantiopreference of investigated enzymes.[15]
However, to the best of our knowledge, no suitable and direct
assay for enzymatic promiscuous activity is known so far. Thus,
we decided to adopt the previously described technique [15] for
knowledge, there is no example of
a simple, multi-sensor
demonstrating the promiscuous activity of an enzyme and detecting
the hydrogen peroxide in the aqueous media.
Introduction
The development of sustainable oxidations processes continues
to have outstanding importance in organic syntheses. Several
industrially useful transformations are performed in the presence
of hydrogen peroxide. Hydrogen peroxide is an ideal oxidant in
the view of availability, atom-economy, or green aspects.[1]
H2O2 has comparatively mild reactivity and is believed to play a
meaningful role in the reversible oxidation in biological
systems.[2] Besides, its oxidant properties are widely applied in
the pharmaceutical and biochemical industries.[3] H2O2 is also
known as the major species among reactive oxygen (ROS),
produced by cell mitochondria [4] and has been emerged as an
indispensable messenger of cellular signaling cascades.[5]
However, the presence of hydrogen peroxide influences the
activity of certain enzymes.[6] Therefore, despite its essential
role in controlling various processes, the unregulated production
or mismanagement of these oxygen-containing molecules[2]
leads to the destruction of tissue and organs.[7] The excess of
hydrogen peroxide can damage nucleic acids or proteins.[8] Due
to diverse physiological and pathological roles of hydrogen
peroxide [9] efficient and selective method for its detection is the
[a]
M. Wilk, Prof. Ryszard Ostaszewski
Institute of Organic Chemistry Polish Academy of Sciences
Kasprzaka 44/52, 01-224 Warsaw (Poland)
E-mail: r.ostaszewski@icho.edu.pl
Supporting information for this article is given via a link at the end of
the document.((Please delete this text if not appropriate))
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10.1002/cbic.202000770
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the rapid determination of enzyme promiscuity revealed in the
perhydrolysis reaction. For this purpose, we estimated the
promiscuous activity of an enzyme in the reaction of p-
nitrophenyl esters with hydrogen peroxide resulting in the
peracid formation. The designed system combined the efficient
and selective enzymatic method for the detection of hydrogen
peroxide via estimation of the promiscuity for various enzymes.
Moreover, the proposed protocol delivers essential knowledge of
the enzyme-mediated peracid production which is profitable in
both the biological and chemical industry. According to our
knowledge, there is no example of a simple, versatile multi-
sensor demonstrating the promiscuity activity of an enzyme and
detecting the hydrogen peroxide in the aqueous media.
Scheme 1. Schematic representation of enzyme-promoted hydrolysis and
perhydrolysis reaction.
Results and Discussion
The rate of the perhydrolysis contains the native activity (V1) and
the rate of promiscuous reaction (V2). However, the comparison
of two rates delivers revised knowledge of the influence of
hydrogen peroxide on the enzyme activity. If the value (V2) is
higher than (V1) we can observe the promotion of the
promiscuous perhydrolysis reaction. In the case of lower (V2)
than (V1), we have the inhibition of enzymatic activity by the
molecules of hydrogen peroxide. The unchanged rate of
perhydrolysis (V2) to native reaction (V1) suggests, that the
specific enzyme does not take part in the perhydrolysis of p-
nitrophenol esters.
Initially, we designated the reaction rates (Vn0E) in the absence
of an enzyme. The obtained results showed a low influence of
spontaneous hydrolysis and perhydrolysis reactions. In further
experiments, among forty tested enzymes, we have selected
several which showed activity toward perhydrolysis reaction. We
measured and compared the kinetic rates of hydrolysis (V1) and
perhydrolysis (V2) of p-nitrophenyl acetate (1), thereby proving
the essential role of an enzyme.
Perhydrolysis is one of the non-conventional reaction mediated
by hydrolases. Since the first precedence of enzymatic peracid
formation [16], many applications have been presented [17].
However, the characterization of these reactions is limited by the
analysis of products of the subsequent transformation. Herein,
we decided to estimate the promiscuous activity of the enzyme
revealed directly in the perhydrolysis reaction. Hydrolases are
very versatile enzymes that can be used at various
biotransformation. Due to their mild reaction conditions and
availability are often considered more valuable than classical
catalysts. To justify the balance of enzymatic potential and the
hazardous nature of hydrogen peroxide we decide to adopt the
previously described technique [14] for the rapid determination
of the perhydrolytic activity of an enzyme.
Samples were prepared in aqueous phosphate buffer (pH 7.0) at
37 0C as a representative of optimal conditions for most
hydrolases. As a model substrate, we chose p-nitrophenyl
acetate (1). In general, an ester is hydrolyzed by lipase to the
formation of carboxylic acid and alcohol. The amount of released
p-nitrophenol can be measured in time by UV-Vis spectroscopy.
Hydrogen peroxide reacts as the nucleophile instead of water in
the deacylation step according to the classical mechanism of
enzymatic hydrolysis. Reaction proceeds in the aqueous solvent
wherein the concentration of water continues unchanged.
Because hydrogen peroxide is a stronger nucleophile, we can
observe the competition between native and promiscuous
reactions mediated by the same enzyme. As an effect of
promiscuous behavior, the peroxy acid is formed together with a
p-nitrophenol molecule (Scheme 1.).
The data in Table 1 show the net reaction rates for native and
promiscuous reactions catalyzed by the same enzymes.
Noteworthy, all tested enzymes were able to catalyze the
perhydrolysis reaction faster than the hydrolysis of substrate 1.
Table 1. Summary of net rates for the native and promiscuous activity of
enzymes.
Vnet [nMs-1] [a]
[b]
[c]
Entry
Enzyme
Novozym 435
C.cylindracea L.
C. rugosa L.
V1
V2
90
1
2
3
4
5
6
7
76
901
280
900
850
540
725
949
473
P. cepacia L.
P. fluorescens L.
PPL
1480
1117
1198
733
Rhizopus niveus L.
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10.1002/cbic.202000770
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FULL PAPER
mediated by Novozym 435 proceeded with the lowest speed, we
conducted a series of experiments in time, for Novozym 435 and
increased the amount of hydrogen peroxide. A detailed analysis
of the activity as a function of nucleophile concentration is
shown in Figure 2.
8
Acylase I
1110
1117
Conditions: 0.06 mM substrate in phosphate buffer (0.1 M, pH 7.0), 37 ⁰C,
enzyme 17 µg mL-1, 0.06 mM H2O2 [a] Vnet = Vobs – Vn0E; [b] V1 calculated for
hydrolysis reaction ; [c] V2 calculated for perhydrolysis reaction.
Reaction mediated by Pseudomonas cepacia lipase (Table 1,
entry 4) has been characterized by the highest reaction rate of
perhydrolysis reaction. The rate determined for hydrolysis and
perhydrolysis promoted by Rhizopus niveus lipase (Table 1,
entry 7) is almost identical. Moreover, there is no difference
between native and promiscuous reactions catalyzed by Acylase
I (Table 1, entry 8). The lowest reaction rates have been
designated for Novozym 435 (Table 1, entry 1).
The obtained results show the preference of promiscuous
activity of examined enzymes concerning the perhydrolysis of p-
nitrophenyl acetate (1), but every transformation is described by
different contributions of enzyme promiscuous activity. Thus, we
decided to normalized the impact of divergent properties of a
single enzyme in perhydrolysis reaction with the sum of activities
observed in the perhydrolysis reaction. For this purpose, we
calculated the percentage contribution from equation: % of
enzyme promiscuity = (V2 – V1) / V2 ∙ 100 %, for example of
Novozym 435: (90 – 76) / 90 ∙ 100 % = 16 %.
Figure 2. The influence of hydrogen peroxide on the perhydrolysis of p-
nitrophenyl acetate mediated by Novozym 435.
The increasing concentration of hydrogen peroxide results in
increased reaction rate (Vobs) for enzymatic perhydrolysis of
substrate 1. The application of 0.18 mM hydrogen peroxide has
the highest preference toward enzymatic process and good
resistance to the spontaneous reaction. Therefore, we chose
this result for further studies. It is worth noting that after the
exceed this optimal concentration, auto-perhydrolysis of ester 1
was promoted. This observation prompted us to check reactions
in the presence of greater excess of hydrogen peroxide to the
substrate (Figure S6, Supporting information). In contrast to
published reports, we did not observe the inhibition of enzyme
activity with the high concentration of hydrogen peroxide.
Moreover, the difference in released p-nitrophenol between
reactions with and without enzyme was not detected, which
suggests that the spontaneous reaction proceeds faster at a
higher concentration of hydrogen peroxide. Therefore, the
proposed method is an excellent example of a simple probe for
hydrogen peroxide detection. The utility of the enzymatic method
allows the detection of a low amount of hydrogen peroxide (up to
1 ppm), while a higher concentration can be estimated based on
the spontaneous perhydrolysis of investigated ester.
Next, we decided to estimate the enzyme promiscuity regarding
a substrate with a long alkyl chain. We performed a series of
experiments in time for p-nitrophenyl dodecanoate using various
enzymes. Reactions were conducted in an aqueous phosphate
buffer (pH 7.0) at 37 0C. Perhydrolysis was conducted in the
presence of 3 equivalents of hydrogen peroxide. During these
studies, we compared the reaction rates of hydrolysis and
perhydrolysis of substrate 2 in the presence of enzyme including
the influence of spontaneous processes running in the examined
system. Furthermore, based on these results (Table S1,
Supporting information), we calculated the percentage
100
80
60
40
20
0
1
2
3
4
5
6
7
8
Enzyme
Figure 1. The contribution of enzyme promiscuity for substrate 1. Enzyme: 1-
Novozym 435, 2- Candida cylindracea L., 3- Candida rugosa L., 4-
Pseudomonas cepacia L., 5- Pseudomonas fluorescens L., 6- PPL, 7-
Rhizopus niveus L., 8- Acylase I.
As can be seen in Figure 1. the impact of the enzymatic
promiscuous activity is characteristic of the type of enzyme. The
abilities of Novozym 435 (1) and Candida cylindracea lipase (2)
toward perhydrolysis reaction have been considerably lower,
compared to all lipases tested. The most powerful enzyme was
PPL (6), which shows more than 50 % promiscuity at the
investigated reaction. Rhizopus niveus lipase (7) and Acylase I
(8) nearly do not show promiscuous activity toward the
perhydrolysis of p-nitrophenyl acetate (1).
Interestingly, for all tested biocatalyst, no inhibition by hydrogen
peroxide was observed. Nevertheless, hydrogen peroxide is
known for its strong inhibition effects.[6] Therefore, we decided
to check its influence on enzyme activity. Since the reaction
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10.1002/cbic.202000770
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Figure 4. The impact of enzyme promiscuity revealed in the perhydrolysis of
p-nitrophenyl phenylacetate (3). Enzyme: 1- Novozym 435, 2- Candida
cylindracea L., 3- Candida rugosa L., 4- Pseudomonas cepacia L., 5-
Pseudomonas fluorescens L., 6- PPL, 7- Rhizopus niveus L., 8- Acylase I.
contribution of enzyme promiscuity estimated for specific
enzyme and substrate.
The results obtained for p-nitrophenyl dodecanoate are
presented in Figure 3.
Surprisingly, Acylase I (8) shows the highest promiscuous
activity toward the perhydrolysis of p-nitrophenyl phenylacetate
(3). The influence of promiscuous property for Candida rugosa
lipase (3) is estimated at 86 %. However, we noticed also the
lower activity of several enzymes. The maximal inhibition effect
was estimated for Candida cylindracea lipase (2). Further, chiral
esters 4S and 4R were used as probes for the determination of
the enantiopreference of a single enzyme in the promiscuous
reaction. Samples were incubated at 37 0C in the PBS buffer (pH
7.0). We measured and compared the relatives reaction rates of
hydrolysis and perhydrolysis for two single enantiomers and
different enzymes (Table S3 and S4, Supporting information).
Figure 5 presents the comparison of enantiopreference revealed
by diverse enzymes in the promiscuous reaction.
100
50
0
1
2
3
4
5
6
7
8
-50
-100
-150
Enzyme
Enzymes from Candida cylindracea lipase (2) showed
a
Figure 3. The impact of enzyme promiscuity revealed in the perhydrolysis of
p-nitrophenyl dodecanoate (2). Enzyme: 1- Novozym 435, 2- Candida
cylindracea L., 3- Candida rugosa L., 4- Pseudomonas cepacia L., 5-
Pseudomonas fluorescens L., 6- PPL, 7- Rhizopus niveus L., 8- Acylase I.
preference for only (S)-ester. The higher enantiomeric
preference of 4S was detected for Pseudomonas fluorescens (5)
and Rhizopus niveus lipase (7). Enantiomer (R)-ester was
catalyzed more likely by Novozym 435 (1), Candida rugosa
lipase (3), and Acylase I (8). Pseudomonas cepacia lipase (4)
did not show the promiscuous activity in the perhydrolysis of
esters 4S and 4R. We observed the inhibition of the natural
function of PPL (6) for both enantiomers, whereas the higher
inhibition effect was shown toward R-enantiomer.
Among tested enzymes, Rhizopus niveus lipase (7) and
Candida rugosa lipase (3) show the highest promiscuous activity
at perhydrolysis of substrate 2. For the reactions catalyzed by
Pseudomonas fluorescens lipase (5) the impact of the divergent
ability of enzymes are significantly low. Surprisingly we observed
the inhibition of native function for PPL (6), whereas this enzyme
shows the highest perhydrolytic activity toward p-nitrophenyl
acetate (1). Moreover, the highest 86 % inhibition of natural
function was appointed for Pseudomonas cepacia L. (4).
Next, we investigated the enzymes promiscuous activity for p-
nitrophenyl phenylacetate (3). All probes were prepared at the
same condition and the corresponding time-courses measures
were recorded for single enzymes. It is useful to judge the
influence of promiscuous activities of biocatalyst over native
properties via its percentage contribution in the whole process.
Thus, we transformed the obtained results (Table S2,
Supporting information) and summarized in Figure 4.
110
90
70
4S
4R
50
30
10
-10
-30
-50
1
2
3
4
5
6
7
8
Enzyme
.
100
50
0
Figure 5. The impact of enzyme promiscuity revealed in the stereoselective
perhydrolysis of chiral p-nitrophenyl 2-phenylpropanoate. Enzyme: 1-
Novozym 435, 2- Candida cylindracea L., 3- Candida rugosa L., 4-
Pseudomonas cepacia L., 5- Pseudomonas fluorescens L., 6- PPL, 7-
Rhizopus niveus L., 8- Acylase I.
1
2
3
4
5
6
7
8
-50
-100
-150
-200
-250
-300
-350
Conclusions
Enzyme
In conclusion, we have developed an efficient and selective
enzymatic method for the detection of hydrogen peroxide via the
estimation of the promiscuity for various enzymes. Moreover, for
the first time, we estimated the numerical contribution of
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1
(s). All the resonances of H and 13C NMR spectra were consistent with
promiscuous activity characteristic for a single enzyme through a
comparison of the reaction rates for enzymatic hydrolysis and
perhydrolysis reactions. We connected the substrate promiscuity
of lipases revealed in the promiscuous reaction. The most
suitable substrate for all tested enzymes is p-nitrophenyl acetate,
which can be explained by the relatively small size of this
molecule. Interestingly, p-nitrophenyl phenylacetate turned out
to be the most demanding substrate. However, surprisingly the
highest level of promiscuous efficiency (91 %) among all
experiments was estimated for Acylase I, catalyzed
perhydrolysis of p-nitrophenyl phenylacetate. As expected the
application of chiral esters, unfolded the stereoselectivity of
tested biocatalysts. The reactions mediated by C. cylindracea is
enantioselective with S-preference, whereas C. rugosa lipase
showed a preference toward R-ester. The proposed protocol
combines the parallel probe for hydrogen peroxide, which can
be detected in a wide range of concentrations (1 − 7.8 ∙ 103ppm),
using enzymatic or non-enzymatic manner. To the best of our
knowledge, for the first time, we have demonstrated a simple,
fast, and versatile multi-sensor based on the enzyme
promiscuity. Besides, the obtained results deliver direct
knowledge of enzyme-promoted peracid formation, which is
profitable in the biological and chemical industry.
reported values.[15] UV/Vis (phosphate buffer, pH 7.0) λmax = 271-272
nm.
Synthesis of substrate 2-4:
To the stirred solution of 1 eq of p-nitrophenol and 1 eq of the
corresponding
acid
(2-4)
in
5
mL
of
ethyl
acetate,
dicyclohexylcarbodiimide (1.2 eq) was added. The mixture was mixed in
the cooling bath for a period of 1 h and later overnight at room
temperature. The reaction was monitored by TLC chromatography
(hexane : ethyl acetate, 9:1) and after the reaction was completed the
precipitate was filtered off and the excess of solvent was evaporated.
The product was purified by the column chromatography, using
hexane:ethyl acetate (9:1) as an eluent. Corresponding esters were
obtained as solid products with an 85-95 % yield.
p-nitrophenyl dodecanoate (2): White solid, 1H NMR (CDCl3, 400
MHz): δ 8.28 – 8.23 (m, 2H), 7.30 – 7.24 (m, 2H), 2.59 (t, J = 7.5 Hz, 2H),
1.76 (dt, J = 15.1, 7.5 Hz, 2H), 1.46 – 1.17 (m, 17H), 0.88 (t, J = 6.8 Hz,
3H).; 13C NMR (CDCl3, 100 MHz): δ 171.28 (s), 155.54 (s), 145.26 (s),
125.16 (s), 122.40 (s), 34.33 (s), 31.88 (s), 29.70 – 28.93 (m), 24.74 (s),
22.65 (s), 14.07 (s). All the resonances of H and 13C NMR spectra were
consistent with reported values.[16] UV/Vis (phosphate buffer saline, pH
1
7.0) λmax = 279 nm.
p-nitrophenyl phenylacetate (3): 1H NMR (CDCl3, 400 MHz): δ 8.27 –
8.22 (m, 1H), 7.40 – 7.31 (m, 2H), 7.28 – 7.22 (m, 1H), 3.90 (s, 1H).; 13
C
Experimental Section
NMR (CDCl3, 100 MHz): δ 169.03 (s), 155.42 (s), 145.39 (s), 132.66 (s),
129.27 (s), 128.89 (s), 127.67 (s), 125.16 (s), 122.33 (s), 41.33 (s). All
the resonances of 1H and 13C NMR spectra were consistent with reported
values.[17] UV/Vis (phosphate buffer saline, pH 7.0) λmax = 272 nm.
1
General: H NMR and 13C NMR spectra were recorded with Bruker 400
MHz spectrometer in CDCl3 and TMS as a standard (0 ppm). Reactions
were performed under a normal atmosphere and monitored by TLC on
silica gel 60 F254 aluminum sheets using UV light as a visualizing agent.
UV/Vis spectra time profiles were recorded in 1 cm quartz cuvettes at 37
(S)-p-nitrophenyl 2-phenylpropanoate (4S): 1H NMR (CDCl3, 400
MHz): δ 8.26 – 8.18 (m, 2H), 7.42 – 7.29 (m, 5H), 7.20 – 7.15 (m, 2H),
3.99 (q, J = 7.1 Hz, 1H), 1.64 (d, J = 7.1 Hz, 3H).; 13C NMR (CDCl3, 100
MHz): δ 172.09 (s), 155.57 (s), 145.33 (s), 139.33 (s), 128.99 (s), 127.70
(s), 127.47 (s), 125.12 (s), 122.27 (s), 45.69 (s), 18.34 (s). All the
⁰C on
a U-1900 spectrophotometer (Hitachi). Acetyl chloride was
purchased from Fluka, the rest of the substrates were purchased from
Sigma Aldrich and used without additional purification. All enzymes used,
except Novozym 435 (Novo Nordisk), were purchased from Sigma.
Hydrogen peroxide solution (50 wt. % in H2O, stabilized) was purchased
from Chempur company. Buffer was prepared using the anhydrous
sodium dihydrogen phosphate and di-sodium hydrogen phosphate, which
were purchased from Chempur company. UV/Vis for p-nitrophenol
recorded at phosphate buffer (pH 7.0): λmax = 400-401 nm.
1
resonances of H and 13C NMR spectra were in accordance with values
recorder for racemate.[18] UV/Vis (phosphate buffer saline, pH 7.0) λmax
= 272 nm; [훼]2퐷0 = 115 (c 1.5, CHCl3).
(R)-p-nitrophenyl 2-phenylpropanoate (4R): 1H NMR (CDCl3, 400
MHz): δ 8.24 – 8.19 (m, 2H), 7.41 – 7.29 (m, 5H), 7.18 (d, J = 9.2 Hz, 2H),
3.99 (q, J = 7.1 Hz, 1H), 1.63 (d, J = 7.2 Hz, 3H); 13C NMR (CDCl3, 100
MHz): δ 172.11 (s), 155.57 (s), 145.33 (s), 139.33 (s), 128.99 (s), 127.70
(s), 127.47 (s), 125.12 (s), 122.27 (s), 45.69 (s), 18.33 (s). All the
Synthesis of substrate 1:
1
resonances of H and 13C NMR spectra were in accordance with values
To the stirred mixture of 1 eq of p-nitrophenol in 5 mL of THF, 1.2 eq of
triethylamine was added. The mixture was kept in the cooling bath
(water/ice) for 15 min, followed by the dropwise addition of 1.5 eq of acyl
chloride. After the addition was completed, the reaction was left for c.a. 2
h at room temperature. The reaction was monitored by TLC (hexane :
ethyl acetate, 9:1). Then the solvent was evaporated under reduced
pressure. Later the crude product was dissolved in DCM (80 mL) and
subsequently extracted with an aqueous solution of NH4Cl (1 x 50 mL)
and water (1 x 50 mL). The organic layer was dried over MgSO4. After
filtration, DCM was evaporated and p-nitrophenyl acetate was purified by
the column chromatography: hexane: ethyl acetate, 9:1), to give light
yellow solid with 86 % yield.
recorder for racemate.[18] UV/Vis (phosphate buffer, pH 7.0) λmax = 272
nm; [훼]2퐷0 = − 110 (c 1.5, CHCl3).
Enzyme assay for hydrolysis reaction:
p-nitrophenyl esters (1-4) were prepared as 5 mM stock solutions in
acetonitrile. Enzymes were diluted from 10 mg mL-1 stock solutions in
phosphate buffer saline (0.1 mM, pH 7.0). 1 mg of Novozym 435 was
added directly to the reaction mixture. To the 3 mL of PBS in the 1 cm
quartz cuvettes, 5 µL of enzyme solution was added. Assays were
followed by the addition of a 40 µL substrate solution to the mixture
containing enzyme. Time profiles were recorded at 37 ⁰C on a U-1900
spectrophotometer (Hitachi). The obtained data was converted to p-
nitrophenol concentration by using a calibration curve. The indicated
reaction rates were derived from the linear portion of every curve.
p-nitrophenyl acetate (1): 1H NMR (CDCl3, 400 MHz): δ 8.26 (d, J = 9.2
Hz, 2H), 7.28 (d, J = 9.2 Hz, 2H), 2.34 (s, 3H).13C NMR (CDCl3, 100
MHz): δ 168.33 (s), 155.37 (s), 145.34 (s), 125.17 (s), 122.41 (s), 21.06
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10.1002/cbic.202000770
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FULL PAPER
Enzyme assay for perhydrolysis reaction:
c.) T. Finkel, M. Serrano, M.A. Blasco, Nature 2007, 448, 767–774, d.)
E.W. Miller, O. Tulyathan, E.Y. Sacoff, C.J. Chang, Nat. Chem. Biol.
2007, 3, 263-267.
The procedure is the same as above, but to the stock solution of a
substrate, the corresponding amount of hydrogen peroxide (50 % wt. %
H2O, stabilized) was added.
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Keywords: enzyme • kinetic studies • perhydrolysis •
promiscuity assay • hydrogen peroxide detection
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10.1002/cbic.202000770
ChemBioChem
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Title Efficient assay for the detection
of hydrogen peroxide via estimation
of the enzyme promiscuous activity
in the perhydrolysis reaction.
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