Synthetic applications of purified laccase from Pleurotus sajor caju MTCC-141

Article abstract of DOI:10.1134/S1070363215010302

The study is devoted to the role of laccase, purified from the liquid culture medium of the indigenous fungal strain Pleurotus sajor caju MTCC-141, in selective bioconversion of toluene derivatives into substituted benzaldehydes with high yield. Coupling reactions of 3-(3,4-dihydroxyphenyl)propionic acid with 4-aminobenzoic, 4-aminoacetophenone and 1-hexylamine proceeded at room temperature under the action of laccase to give the corresponding products with excellent yields.

Full text of DOI:10.1134/S1070363215010302

ISSN 1070-3632, Russian Journal of General Chemistry, 2015, Vol. 85, No. 1, pp. 173–175. © Pleiades Publishing, Ltd., 2015.
Synthetic Applications of Purified Laccase
from Pleurotus sajor caju MTCC-1411
Pankaj Kumar Chaurasia, Shashi Lata Bharati, Sunil Kumar Singh, and Sudha Yadava
Department of Chemistry, D.D.U. Gorakhpur University, Gorakhpur, Uttar Pradesh, 273009 India
e-mail: shashilatachem@gmail.com
Received October 29, 2014
Abstract—The study is devoted to the role of laccase, purified from the liquid culture medium of the
indigenous fungal strain Pleurotus sajor caju MTCC-141, in selective bioconversion of toluene derivatives into
substituted benzaldehydes with high yield. Coupling reactions of 3-(3,4-dihydroxyphenyl)propionic acid with
4-aminobenzoic, 4-aminoacetophenone and 1-hexylamine proceeded at room temperature under the action of
laccase to give the corresponding products with excellent yields.
Keywords: laccase, Pleurotus sajor caju, 3-(3,4-dihydroxyphenyl)propionic acid, ABTS
DOI: 10.1134/S1070363215010302
INTRODUCTION
The objective of this study was purification of laccase
from the liquid culture growth medium containing
natural lignin substrate bagasse particles of Pleurotus
sajor caju MTCC-141 [16] and its introduction in
selective oxidation of substituted toluene derivatives to
corresponding benzaldehydes in presence of ABTS as
a mediator and coupling of amines with 3-(3,4-
dihydroxyphenyl)propionic acid at room temperature.
In this communication the new active biocatalyst
suitable for the syntheses is presented.
Laccase (E. C. 1.10.3.2) belongs to a group of
multicopper containing oxidases [1, 2] that catalyze
[3–5] four electrons reduction of molecular oxygen to
water. Laccase was extracted for the first time from
Japanese lacquer tree Rhus vernicifera [6]. Little is
known about higher plant laccases, probably due to
their presence in cell walls. Laccases are the lignolytic
enzymes and abundantly occur in the fungal systems
[7]. Laccase is also reported in bacteria Azospirrullum
lipoferum [8] which was the first laccase producing
bacteria. It was also found in Streptomyces spec. [9,
10] and Anabaena azollae [11]. In addition to fungi,
plants and bacteria the presence of laccase was
detected in wasp venom [12] and in insects [13].
RESULTS AND DISCUSSION
Purity of the enzyme samples designed for the
synthesis was tested by SDS-PAGE. Selective bio-
oxidation of the toluene methyl group to the aldehyde
group is one of the most efficient reactions of laccases
in organic syntheses. Generally such conversion
requires tough conditions and is complicated by
uncontrollable formation of carboxylic acids along
with environmentally hazardous byproducts. In the
current study oxidation carried out with laccase
proceeded under mild conditions and high yield and
was ecologically friendly (Scheme 1).
Catalytic activity of laccases depends on Cu atoms
that are distributed among the three centres viz. type-1
or blue copper centre, type-2, or normal copper centre
and type-3 or coupled binuclear copper center
characterized by different electronic paramagnetic
resonance signals [14, 15]. The organic substrate is
oxidized by one electron at the active site of the
laccase generating a radical which reacts further non-
enzymatically. The electron is received at type-1 Cu
center and is shuttled to the trinuclear cluster where
oxygen is reduced to water. Fungal laccases are stable
and have high biocatalytic efficiency.
Syntheses of aryl substituted 3-(3,4-dihydroxy-
phenyl)propionic acid derivatives Va–Vc was carried
out by the corresponding coupling processes catalyzed
by purified laccase of Pleurotus sajor caju MTCC-141
(Scheme 2).
Progress of the reaction and purity of products were
monitored and tested by HPLC. Formation of the
1 The text was submitted by the authors in English.
173

174
PANKAJ KUMAR CHAURASIA et al.
Scheme 1. Oxidation of toluene and its derivatives by
purified laccase of Pleurotus sajor caju MTCC-141 in the
presence of ABTS as a mediator at room temperature
sterilized growth medium reported by Coll and co-
authors [18] and processed as presented in the
publication [16].
CH3
CHO
Purity of the prepared enzyme was tested by
sodium dodecyl sulphate-polyacrylamide gel electro-
phoresis [19]. Polyacrylamide gel electrophoresis was
run at the constant current 20 mA [20].
1
1
X
Y
X
Y
6
5
6
5
Purified laccase, ABTS
2
2
pH 4.5, 1−2 h, RT, 93−100%
3
3
4
4
Enzyme assay. The assay solution 1.0 mL for
DMP as the substrate [18] contained 1.0 mM DMP in
50 mM sodium malonate buffer, pH 5.0 (37°C). The
process was monitored by absorbance change at λ 468 nm
and ε = 49.6 × 103 M–1 cm–1. The UV–Vis spectro-
photometer Hitachi (Japan) model U-2900 fitted with
electronic temperature control unit was used in the
study. One enzyme unit produced 1 μmol of the pro-
duct per minute under the specified assay conditions.
Z
Z
Ia−Id
IIa−IId
I: X = H, Y = H, Z = H (a), X = Cl, Y = H, Z = H (b), X =
H, Y = NO2, Z = H (c), X = H, Y = H, Z = Cl (d). II: X = H,
Y = H, Z = H (a), X = Cl, Y = H, Z = H (b), X = H, Y =
NO2, Z = H (c), X = H, Y = H, Z = Cl (d).
enzymatically synthesized products was justified by
HPLC, IR, and 1H NMR data.
Bioconversion of toluenes to benzaldehydes under
the action of ABTS. The process was carried out in
100 mM sodium acetate buffer, pH 4.5, medium
containing 20 mM of toluene in 20 mL of dioxane,
0.1 mM of ABTS [21–24] and 500 µL of two times
diluted purified laccase (activity of concentrated
laccase 1.12 IU/mL) upon vigorous stirring for 60 min.
Completion of the reaction was monitored by UV–Vis
spectrophotometry. The reaction solution was extracted
by n-hexane (40 × 3 mL). 20 µL of n-hexane extract
was injected in Waters HPLC Model 600E using
spherisorb C18 5 UV, 4.5 × 250 mm column, eluent
methanol (flow rate 0.5 mL/min). Monitoring was
performed by Waters UV detector model 2487 at λ 254 nm.
EXPERIMENTAL
Materials. 4-Chlorotoluene and diethyl amino
ethyl cellulose were purchased from Sigma Chemical
Company (USA) and all other reagents from Fluka
(Switzerland). The chemicals used in gel electro-
phoresis of protein samples were obtained from
Bangalore Geni Pvt. Ltd. (India).
The fungal strain, its growth, and purification of
laccase The fungal strain was obtained from the
Microbial Type Culture Collection Center and Gene
Bank, Institute of Microbial Technology (India) and
was maintained on agar slant as reported in MTCC
Catalogue of strains-2000 [17]. For purification of the
laccase Pleurotus sajor caju MTCC-141was grown in
ten 100 mL culture flasks each containing 25 mL of
Biooxidation of 3-nitrotoluene, 2-chlorotoluene and
4-chlorotoluene were carried out according to the
method presented above. The reaction mixtures was
Scheme 2. Syntheses of 3-(3,4-dihydroxyphenyl)propionic acid derivatives
1
COOH
COOH
2
1
Purified laccase
X−NH
+ X−NH2
IVa−IVc
6
pH 5.0, 3−8 h, RT, 75−90%
2
5
OH
3
OH
4
OH
OH
III
Va−Vc
NH2
(IVa),
H3COC
NH2
(IVb),
(IVc).
X−NH2 =
H3C
NH2
HOOC
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 85 No. 1 2015

SYNTHETIC APPLICATIONS OF PURIFIED LACCASE
175
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stirred for 75 or 90 min. The compounds isolated
required no further purification. In the course of
oxidation no side reactions occured due to high
specificity of laccase. Thus, extraction of products by
ethyl acetate gave almost pure benzaldehyde and
substituted benzaldehydes (yields >93%).
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Synthesis of 3-[6-(4-carboxyphenyl)amino-3,4-
dihydroxyphenyl] propanoic acid (Va), 3-[6-(4-
acetophenyl)amino-3,4-dihydroxyphenyl]propanoic
acid (Vb), and 3-(6-hexylamino-3,4-dihydroxy-
phenyl) propanoic acid (Vc) [25]. The pure enzyme
(1.12 IU/mL) was diluted twice with 20 mM sodium
acetate buffer, pH 5.0. 3-(3,4-Dihydroxyphenyl)pro-
pionic acid (1 mM) and 4-aminobenzoic acid (1 mM)
were added to 2 mL of the solution. The reaction
mixture was incubated for 4.15 h at room temperature
upon vigorous stirring. The reaction was monitored by
UV–Vis spectrophotometry. The reaction solution was
extracted thrice with ethyl acetate. The ethyl acetate
(20 µL) extract was injected in 4.5 × 250 mm column
(Waters HPLC Model 600E, spherisorb C18 5 UV),
eluent methanol, flow rate 0.5 mL/min. The similar
method was used for coupling of 3-(3,4-dihyd-
roxyphenyl)propionic acid with 4-aminoacetophenone
n-hexylamine, stirring time 4.30 h (IVb) and 6 h (IVc).
Yields: 90% (Va), 86% (Va), and 75% (Vc).
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CONCLUSIONS
The methyl group of toluene and its substituted
derivatives were biooxidized into the aldehyde group
under the action of ABTS as a mediator. The reaction
of 3-(3,4-dihydroxyphenyl)propionic acid with amines
leading to substitution in the aromatic ring of the acid
III was carried out under the action of purified laccase
of sajor caju MTCC-141 at room temperature and
atmospheric pressure with high yield.
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ACKNOWLEDGMENTS
20. Polyacrylamide Gel Electrophoresis: Laboratory Tech-
niques, Pharmacia, Laboratory Separation Division,
Uppsala Sweden, 1984.
The authors acknowledge the financial support of
CSIR-HRDG, New Delhi for the award of JRF (NET)
and SRF (NET), award no. 09/057(0201)2010-EMR-I
to Dr. Pankaj Kumar Chaurasia. Dr. S.K Singh and Dr.
S.L. Bharati are thankful to CSIR and UGC Delhi for
award of RA and UGC-Post Doctoral Fellowship,
respectively.
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J. Org. Chem. 1995, vol. 60, p. 4320.
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Synth. Comm., 2014, vol. 44, no. 17, 2535.
23. Chaurasia, P.K., Yadav, A., Yadav, R.S.S., and Yadava, S.,
J. Chem. Sci., 2013, vol. 125, no. 6, pp. 1395–1403.
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