N-Oxidation of arylamines to nitrosobenzenes using chloroperoxidase purified from Musa paradisiaca stem juice

Article abstract of DOI:10.3109/10242422.2010.489943

N-Oxidation of arylamines to their corresponding nitrosobenzenes using a new chloroperoxidase purified from Musa paradisiaca stem juice has been examined. The enzymatic characteristics of the stem chloroperoxidase using 4-chloroaniline as substrate were determined. The Km values for 4-chloroaniline and H₂O₂ were 770 μM and 154 μM respectively, while the pH and temperature optima were 4.4 and 30°C respectively. The substrate specificities of the enzyme for the arylamines 3,4-dichloroamine, p-aminobenzoic acid, p-toluidine, p-anisidine, m-anisidine, p-aminophenol, o-aminophenol and m-aminophenol have been characterized. The feasibility of using concentrated M. paradisiaca stem juice for the specific conversion of 4-chloroaniline to 4-chloronitrosobenzene has been demonstrated. This enzyme can be used for the N-oxidation of other arylamines.

Full text of DOI:10.3109/10242422.2010.489943

Biocatalysis and Biotransformation, May–June 2010; 28(3): 222–226
ORIGINAL ARTICLE
N-Oxidation of arylamines to nitrosobenzenes using chloroperoxidase
purified from Musa paradisiaca stem juice
PRATIBHAYADAV1, JITENDRA K. SHARMA1, VINOD K. SINGH1 & KAPIL D. S.YADAV2
1Department of Chemistry, Udai Pratap College,Varansi, India and 2Department of Chemistry, D.D.U. Gorakhpur
University, Gorakhpur, India
Abstract
N-Oxidation of arylamines to their corresponding nitrosobenzenes using a new chloroperoxidase purified from Musa
paradisiaca stem juice has been examined.The enzymatic characteristics of the stem chloroperoxidase using 4-chloroaniline
as substrate were determined. The Km values for 4-chloroaniline and H2O2 were 770 μM and 154 μM respectively, while
the pH and temperature optima were 4.4 and 30°C respectively.The substrate specificities of the enzyme for the arylamines
3,4-dichloroamine, p-aminobenzoic acid, p-toluidine, p-anisidine, m-anisidine, p-aminophenol, o-aminophenol and m-ami-
nophenol have been characterized. The feasibility of using concentrated M. paradisiaca stem juice for the specific conver-
sion of 4-chloroaniline to 4-chloronitrosobenzene has been demonstrated. This enzyme can be used for the N-oxidation
of other arylamines.
Keywords: Peroxidase, N-oxidation, metalloenzyme, Musa paradisiaca, arylamine, nitrosobenzene
Introduction
et al. 2004; Ullrich & Hofrichter 2005). Although
chloroperoxidase has been detected in plants (Spe-
icher et al. 2003), the enzyme has not been purified
until recently. We have purified a chloroperoxidase
from Musa paradisiaca (plantain) stem juice (Yadav et
al. 2009) and have initiated studies on its potential as a
biocatalyst in organic synthetic reactions.The present
communication reports the N-oxidation of arylamines
to their corresponding nitrosobenzenes using the stem
juice chloroperoxidase.
Chloroperoxidase (EC 1.11.1.10) (Morris & Hager
1966; Kuhnel et al. 2006) is a versatile biocatalyst for
organic synthetic reactions (Spreti et al. 2004). In addi-
tion to halogenation (Hager et al. 1966) and classical
peroxidation reactions (Thomas 1970), it catalyzes
typical reactions of catalases and monooxygenases and
is the most promising enzyme for synthetic applica-
tions (Hofrichter & Ullrich 2006). Chloroperoxidase
is the catalyst of choice in oxygen transfer reactions
of a variety of organic compounds, e.g. N-oxidation
(Corbett et al.1980),S-oxidation (Colonna et al.1992;
Allenmark & Andersson 1996), epoxidation (Hager et
al. 1998; Zhu & Wang 2005), hydroxylation (Hu &
Hager 1999; Ullrich & Hofrichter 2007), oxidation
of alcohols (Kiljiunen & Kanerva 2000) and indole
(Van Deurzen et al. 1996). In all of these reactions,
chloroperoxidase from Caldariomyces fumago, a marine
fungus, has been used (Sanfilippo et al. 2004; La Rotta
Hernandez et al. 2005). Recently, a heme-containing
chloroperoxidase from Agrocybe aegerita has been puri-
fied and studies on its potential as a biocatalyst in syn-
thetic organic chemistry have been initiated (Ullrich
Materials and methods
Dimedone (1,1-dimethyl-3,5-cyclohexane) was from
Acros Organics (Geel, Belgium). All other chemicals
were either from Merck Ltd (Mumbai, India) or S.D.
Fine Chemicals Ltd (Mumbai, India) and were used
without further purification.The activity of the chlo-
roperoxidase was assayed spectrophotometrically
(Corbett et al 1978) using 4-chloroaniline as
the substrate and monitoring the formation of
4-chloronitrosobenzene by the absorbance change
at λꢁ320 nm, using a molar extinction coefficient
Correspondence: K. D. S. Yadav, Department of Chemistry, D.D.U. Gorakhpur University, Gorakhpur – 273 009, India. Tel: ꢀ91-551-2204943. Fax:
ꢀ91-551-2330767. E-mail: kds_chemistry@rediffmail.com
(Received 16 June 2009; revised 2 September 2009; accepted 8 March 2010)
ISSN 1024-2422 print/ISSN 1029-2446 online © 2010 Informa UK Ltd. (Informa Healthcare, Taylor & Francis AS)
DOI: 10.3109/10242422.2010.489943

N-Oxidation of arylamines using chloroperoxidase from M. paradisiaca 223
value of 10 300 M–1 cm–1. The assay solution con-
kDa) and lysozyme (14.3 kDa). The gel was run at
constant current of 20 mA.
sisted of 4 mM 4-chloroaniline, 0.6 mM H2O2 and
a suitable aliquot of the enzyme in 20 mM potas-
sium phosphate buffer pH 4.4 at 30°C.The reaction
was initiated by the addition of H2O2. One enzyme
unit is the amount of the enzyme which converts
1 μM of the substrate per minute into the product
under the specified assay conditions.
A Hitachi (Tokyo, Japan) U-2000 UV/VIS
spectrophotometer fitted with electronic tempera-
ture control was used for spectrophotometric mea-
surements. The lowest absorbance measurement
was 0.001 absorbance unit.
The pH optimum for N-oxidation activity of the
chloroperoxidase was determined by measuring the
steady-state velocity of the enzyme-catalyzed reac-
tion using 4-chloroaniline as substrate in solutions
ranging from pH 3.0 to 6.5 in 20 mM potassium
phosphate buffer at 30°C.The temperature optimum
was determined by measuring the steady-state veloc-
ity as above at temperatures in the range 20–45°C.
The substrate specificities of the purified enzyme
for the substituted arylamines were determined by
measuring the steady-state velocities of the enzyme-
catalyzed reactions using substituted arylamines as
the variable substrates and monitoring the formation
of the corresponding nitroso compounds spectro-
photometrically by absorbance measurement at
λꢁ320 nm. The Km values for various substituted
arylamines were determined from double-reciprocal
plots (Engel 1977).
In order to test the feasibility of larger-scale
enzymatic conversion of 4-chloroaniline to 4-chlo-
ronitrosobenzene, 200 mL of 2 mM 4-chloroaniline
in 0.1 M potassium phosphate buffer pH 4.4 at
25°C was treated with 200 μL of the concentrated
M. paradisiaca stem juice containing 4.7 U enzyme
mL–1 and H2O2 was added in four steps, 0.5 mM
in each step at intervals of 10 min. The reaction
solution was left for 1 h and then extracted twice
with 200 mL of diethyl ether. Diethyl ether was
removed by evaporation at room temperature and
the residual solid was recrystallized from methanol.
The purity of the product was confirmed by the
appearance of a single peak in HPLC (model M-600E
chromatograph and Spherisorb C18 column, 5 μm
The chloroperoxidase from M. paradisiaca stem
juice was purified by a method developed in our lab-
oratory (Yadav et al. 2009).This consisted of washing
the M. paradisiaca stem with MilliQ water, cutting it
into small pieces, crushing the pieces in a mortar with
a pestle, extracting the juice by placing the pieces in
four layers of cheese cloth and squeezing it.The juice
was centrifuged using a Sigma (Osterode, Germany)
refrigerated centrifuge model 3K30 at 4000g for
20 min at 4°C to remove the cloudiness of the juice.
The clear juice was concentrated using an Amicon
(Amicon Division, W.R. Grace & Co-Conn, Beverly,
MA, USA) model 8200 concentration cell and a PM
10 ultrafiltration membrane (molecular weight cut-off
of 10 kDa).The concentrated crude enzyme solution
was dialyzed against a 1000 times excess of 10 mM
sodium acetate buffer pH 6.0 for 24 h with three
changes of buffer.The dialyzed crude enzyme solution
was loaded on a DEAE cellulose column, size 1 cm
ꢂ 33 cm, equilibrated with 10 mM sodium acetate
buffer pH 6.0 at the flow rate of 16 mL h–1. The
bound protein was washed with the same buffer and
the protein eluted with a linear gradient of 0–1 M
NaCl in the same buffer (100 mL ꢀ 100 mL with 1
M NaCl). Fractions of 4.0 mL were collected and
analyzed for protein concentration (Lowry et al. 1951)
and chloroperoxidase activity. The active fractions
were combined, concentrated using an Amicon (Ami-
con Division,W.R. Grace & Co-Conn, Danvers, MA,
USA) model 8200 concentration cell and then a
model 3 concentration cell with PM 10 ultrafiltration
membranes. The concentrated enzyme sample was
stored at 4°C until required.
97.4 kDa
66.0 kDa
Chloroperoxidase
43.0 kDa
29.0 kDa
The homogeneity of the purified enzyme was
tested by SDS-PAGE analysis (Weber & Osborn
1969). The separating gel was 12% acrylamide in
0.375 M Tris–HCl buffer pH 8.8 and stacking gel
was 5% acrylamide in 0.063 M Tris–HCl buffer pH
6.8. The enzyme was visualized by staining with
Coomassie Blue R-250.The molecular weight mark-
ers were phosphorylase (97.4 kDa), bovine serum
albumin (66 kDa), ovalbumin (43 kDa), carbonic
anhydrase (29 kDa), soybean trypsin inhibitor (20.1
20.1 kDa
1
2
Figure 1. Results of SDS-PAGE analysis of the purified chloro-
peroxidase: lane 1, molecular weight markers; lane 2, purified
enzyme.

224
P. Yadav et al.
film, 4.6 mm ꢂ 250 mm (Waters, Milford, MA,
USA); methanol–water solvent (7:3, v/v) at a flow
rate of 1 mL min–1). The product was identified as
4-chloronitrosobenzene by IR (3100 FT-IR spec-
trometer; Varian Inc Corporate, Palo Alto, CA,
1
USA) and H and 13C NMR (AL300 FT NMR
spectrometer; JEOL Ltd, Tokyo, Japan).
Results and discussion
SDS-PAGE analysis of the purified enzyme is shown
in Figure 1. The appearance of a single polypeptide
band in lane 2 in which the purified chloroperoxi-
dase had been applied indicated that the enzyme was
pure. The Michaelis–Menten and double-reciprocal
plots for the purified chloroperoxidase using 4-
chloroaniline as the variable substrate at a fixed,
enzyme-saturating concentration of H2O2 are shown
in Figure 2. The equivalent plots using H2O2 as the
variable substrate at a fixed enzyme-saturating con-
centration of 4-chloroaniline are given in Figure 3.
The calculated Km values for 4-chloroaniline and
H2O2 were 770 μM and 154 μM respectively. The
reported (Corbett et al. 1978) Km value for 4-chlo-
roaniline using C. fumago chloroperoxidase is 810
μM, which is similar to that calculated for the chlo-
roperoxidase from the M. paradisiaca stem juice.
In order to assess the potential of M. paradisiaca
stem juice chloroperoxidase in the transformation of
arylamines to their corresponding nitroso com-
pounds, the specificity towards 3,4-dichloroaniline,
p-aminobenzoic acid, p-toluidine, p-anisidine, m-ani-
sidine, p-aminophenol, o- aminophenol and m-amin-
ophenol was tested. Double-reciprocal plots were
drawn in all these cases and the Km and kcat values
Figure 3. Michaelis–Menten and double-reciprocal plot (insert)
for the chloroperoxidase of Musa paradisiaca stem juice using
H2O2 as the variable substrate.
calculated.The results are summarized inTable I. All
of the substituted arylamines tested were converted
into their corresponding nitroso compounds, which
were monitored spectrophotometrically by measur-
ing the absorbance change at λꢁ320 nm.
In order to find the optimum conditions for the
enzymatic transformations, the pH and temperature
optima of the M. paradisiaca stem chloroperoxidase
were determined using 4-chloroaniline as substrate
(Figures 4 and 5 respectively).This showed that the
pH and temperature optima for this transformation
were 4.4 and 30°C respectively.The pH optimum of
the chloroperoxidase from C. fumago also is also 4.4
(Corbett et al. 1978).
The feasibility of enzymatic transformation of
4-chloroaniline to 4-chloronitrosobenzene using the
crude preparation of chloroperoxidase of M. paradi-
siaca stem juice has been demonstrated.The product
extracted using diethyl ether gave a single peak
by HPLC analysis and IR (KBr, νmax (cm–1): 1622,
2924 cm (C-H aromatic),1622 (C=C),1492 (N=O),
1437 (C=C), 1279 (C-N), 910 (C-N), 822 (1,4
Table I. Km and kcat values of the purified enzyme for different
substrates.
Substrate
k
cat (s–1)
Km (μM)
H2O2
154
770
1000
44
0.36
0.35
0.56
0.57
0.59
0.48
0.4
4-Chloroaniline
3,4-Dichloroaniline
p-Aminobenzoic acid
p-Toluidine
50
p-Anisidine
133
132
36
p-Aminophenol
o-Aminophenol
m-Aminophenol
Figure 2. Michaelis–Menten and double-reciprocal plot (insert)
for the chloroperoxidase of Musa paradisiaca stem juice using
4-chloroaniline as the variable substrate.
0.54
0.29
125

N-Oxidation of arylamines using chloroperoxidase from M. paradisiaca 225
8.7
11.7
8.7
5.8
2.9
0
5.8
2.9
0
10
30
20
40
50
2
7
3
6
4
5
Temperature (°C)
pH
Figure 5.Variation of the activity of the chloroperoxidase of Musa
paradisiaca stem juice with reaction temperature.
Figure 4.Variation of the activity of the chloroperoxidase of Musa
paradisiaca stem juice with reaction pH.
disubstition in benzene). 1H NMR (300 MHz,
DMSO, δ (ppm): 6.48 (2H, d, Jꢁ8.4 Hz), 6.99 (2H,
d, Jꢁ8.4 Hz)) and 13C NMR (75 MHz, DMSO, δ
(ppm): 115.2, 118.7, 128.5, 147.7 (phenyl ring))
confirmed the identity of 4-chloronitrosobenzene.
No other products were detected and 53 mg of
4-chloronitrosobenzene was obtained from 87 mg of
4-chloroaniline using 0.94 U enzyme, which was
equivalent to 0.16 mg of the pure enzyme.
Elucidating the detailed kinetics and mechanism
of the transformation of 4-chloroaniline to 4-chlo-
ronitrosobenzene catalyzed by chloroperoxidase from
M. paradisiaca stem juice requires extensive studies.
However, the mechanism suggested for the transfor-
mation of 4-chloroaniline to 4-chloronitrosobenzene
catalyzed by chloroperoxidase from C. fumago (Cor-
bett et al. 1978, 1980) may be considered operative.
According to this mechanism, the enzyme-bound
oxygen of chloroperoxidase (compound I) is directly
incorporated into the arylamine substrate as shown in
Scheme 1. A further two-electron abstraction from
hydroxylamine leads to the arylnitroso compound.
This proposal is based on the peroxidation mecha-
nism for chloroperoxidase-catalyzed N-oxidation of
arylamines (Doerge & Corbett 1991) and is sup-
ported by the accepted mechanism for cytochrome
P450-catalyzed oxygenation of numerous substrates
(Urlacher & Eiben 2006; Urlacher & Schmid 2006).
In conclusion, this communication reports the
transformation of arylamines into their correspond-
ing nitroso compounds using a new chloroperoxi-
dase from M.paradisiaca stem juice. A single product
is formed using even the crude enzyme preparation,
which indicates the synthetic significance of the
chloroperoxidase isolated from a convenient source
using a simple procedure.
Acknowledgements
Pratibha Yadav is thankful to the Head, Depart-
ment of Chemistry, D.D.U. Gorakhpur University,
Gorakhpur for providing laboratory facilities.
Scheme 1. Proposed mechanism for halide-independent N-oxidation (Corbett et al. 1980). (a) Tetravalent iron porphyrin π-cation radical
resonant form of chloroperoxidase (compound I); (b) trivalent iron oxene resonant form of chloroperoxidase compound I; (c) arylamine bound
to chloroperoxidase compound I. (d) arylhydroxyamine ligand of compound I. P represents the sixth axial ligand of chloroperoxidase.

226
P. Yadav et al.
modified by reductive alkylation, amidation and cross-linking.
Enzyme Microb Technol 37:582–588.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. 1951. Protein
measurement with the Folin phenol reagent. J Biol Chem
193:265–275.
Declaration of interest: The authors report no
conflicts of interest. The authors alone are respon-
sible for the content and writing of the paper.
Morris DR, Hager LP. 1966. Chloroperoxidase. I. Isolation
and properties of crystalline glycoprotein. J Biol Chem 241:
1763–1768.
References
Sanfilippo C, D’Antona N, Nicolosi G. 2004. Choloroperoxidase
from Caldariomyces fumago is active in the presence of an ionic
liquid as co-solvent. Biotechnol Lett 26:1815–1819.
Speicher A, Heisel R, Kolz J. 2003. First detection of a chloroper-
oxidase in bryophytes Phytochemistry 62:679–682.
Spreti N, Germani R, Incani A, Savelli G. 2004. Stabilization of
chloroperoxidase by polyethylene glycols in aqueous media: kinetic
studies and synthetic applications. Biotechnol Prog 20:96–101.
Thomas JA. 1970. Chloroperoxidase: classical peroxidatic cata-
latic and halogenating forms of the enzymes. J Biol Chem
245:3129–3134.
Ullrich R, Hofrichter M. 2005. The haloperoxidase of the agaric
fungus Agrocybe aergerita hydroxylates toluene and naphtha-
lene. FEBS Lett 579:6247–6250.
Ullrich R, Hofrichter M. 2007. Enzymatic hydroxylation of aro-
matic compounds. Cell Mol Life Sci 64:271–293.
Ullrich R, Nuske J, Scheibner K, Spantzel J, Hofrichter M. 2004.
Novel haloperoxidase from the agaric basidiomycete Agrocybe
aegerita oxidizes aryl alcohols and aldehydes. Appl Environ
Microbiol 70:4575–4581.
Urlacher VB, Eiben S. 2006. Cytochrome P450 monoxygenases:
perspective for synthetic application. Trends Biotechnol 24:
324–330.
Urlacher VB, Schmid RD. 2006. Recent advances in oxyge-
nase catalysed biotransformations. Curr Opin Chem Biol
10:156–161.
Van Deurzen MPJ, Van Rantwijk, Sheldon RA. 1996. Synthesis
of substituted oxindole by chloroperoxidase catalysed oxidation
of indols. J Mol Catal B: Enzym 9:163–173.
Allenmark SG, Andersson MA. 1996. Chloroperoxidase catalysed
asymmetric synthesis of a series of aromatic cyclic sulfoxides.
Tetrahedron Asymm 7:1089–1094.
Colonna S, Gaggers N, Casella L, Carrea G, Pasta P. 1992.
Chloroperoxidase and hydrogen peroxide: an efficient system
for enzymatic and enantioselective sulfoxidations. Tetrahedron
Asymm 3:95–106.
Corbett MD, Chipco BR, Baden DG. 1978. Chloroperoxidase
catalysed oxidation of 4-chloroaniline to 4-chloronitrosobenzene.
Biochem J 175:353.
Corbett MD, Chipko BR, Batchelor AO. 1980. The action of
chloride peroxidase on 4- chloroaniline: N-oxidation and ring
halogenation. Biochem J 187:892–903.
Doerge DR, Corbett MD. 1991. Peroxygenation mechanism for
chloroperoxidase catalyzed N-oxidation of arylamines. Chem
Res Toxicol 4:556–560.
Engel PC. 1977. Enzyme kinetics: The steady-state approach.
London: Chapman and Hall.
Hager LP, Morris DR, Brown FS, Eberwein H. 1966. Chloroper-
oxidase. II. Utilization of halogen anions. J Biol Chem 241:
1769–1777.
Hager LP, Lakner FJ, Basavapathruni A. 1998. Chiral synthons via
chloroperoxidase catalysis. J Mol Catal B: Enzym 5:95–101.
Hofrichter M, Ullrich R. 2006. Hemethiolate haloperoxidases:
versatile biocatalysts with biotechnological and environmental
significance. Appl Microbiol Biotechnol 71:276–288.
Hu S, Hager LP. 1999. Highly enantioselective propargylic
hydroxylations catalyzed by chloroperoxidase. J Am Chem Soc
121:872–873.
Weber K, Osborn M. 1969. The reliability of molecular weight
determinations by dodecyl sulfate–polyacrylamide gel electro-
phoresis. J Biol Chem 244:4406–4412.
Kiljiunen E, Kanerva LT. 2000. Chloroperoxidase catalysed
oxidation of alcohols to aldehydes. J Mol Catal B: Enzym
6:163–172.
Yadav P, Sharma JK, Singh VK, Yadav M, Yadav KDS. 2010.
Purification of chloroperoxidase from Musa paradisiaca stem
juice. Process Biochem (in press).
Zhu G, Wang P. 2005. Novel interface-binding chloroperoxi-
dase for interfacial epoxidation of styrene. J Biotechnol 117:
195–202.
Kuhnel K, Blankenfeldt W, Terner J, Schlichting I. 2006. Crystal
structure of chloroperoxidase with its bound substrates and
complexed with formate, acetate and nitrate. J Biol Chem
281:23990–23998.
La Rotta Hernandez CE, Lutz S, Liese A, Bon EPS. 2005. Activ-
ity and stability of Caldariomyces fumago choloroperoxidase

Copyright of Biocatalysis & Biotransformation is the property of Taylor & Francis Ltd and its content may not
be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written
permission. However, users may print, download, or email articles for individual use.