Purification and characterization of yellow laccase from Trametes hirsuta MTCC-1171 and its application in synthesis of aromatic aldehydes

Article abstract of DOI:10.1016/j.procbio.2014.06.016

A yellow laccase from the culture filtrate of Trametes hirsuta MTCC-1171 has been purified. The purification methods involved concentration of the culture filtrate by ammonium sulphate precipitation and an anion exchange chromatography on diethylaminoethyl cellulose. The sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and native polyacrylamide gel electrophoresis gave single protein band indicating that the enzyme preparation was pure. The molecular mass of the enzyme determined from SDS-PAGE analysis was 55.0 kDa. Using 2,6-dimethoxyphenol, 2,2′[azino-bis-(3-ethylbonzthiazoline-6-sulphonic acid) diammonium salt] and 3,5-dimethoxy-4-hydroxybenzaldehyde azine as the substrates, the K_m, k_cat and k_cat/K_m values of the laccase were found to be 420 μM, 13.04 s^-1, 3.11 × 10⁴ M^-1 s^-1, 225 μM, 13.03 s^-1, 1.3 × 10⁵ M^-1 s^-1 and 100 μM, 13.04 s^-1, 5.8 × 10⁴ M^-1 s^-1, respectively. The pH and temperature optima were 4.5 and 60 °C, respectively while pH and temperature stabilities were pH 4.5 and 50 °C. The activation energy for thermal denaturation of the enzyme was 18.6 kJ/mol/K. The purified laccase has yellow colour and does not show absorption band around 610 nm like blue laccases. The purified laccase transforms toluene, 3-nitrotoluene, 4-nitrotoluene, 3-chlorotoluene, 4-chlorotoluene and 3,4-dimethoxytoluene to benzaldehyde, 3-nitrobenzaldehyde, 4-nitrobenzaldehyde, 3-chlorobenzaldehyde, 4-chlorobenzaldehyde and 3,4-dimethoxybenzaldehyde in the absence of mediator molecules in high yields.

Full text of DOI:10.1016/j.procbio.2014.06.016

Accepted Manuscript
Title: Purification and characterization of yellow laccase from
Trametes hirsuta MTCC-1171 and its application in synthesis
of aromatic aldehydes
Author: Pankaj Kumar Chaurasia Rama S.S. Yadav Sudha
Yadava
PII:
S1359-5113(14)00344-4
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.procbio.2014.06.016
PRBI 10175
To appear in:
Process Biochemistry
Received date:
Revised date:
Accepted date:
16-4-2014
1-6-2014
19-6-2014
Please cite this article as: Chaurasia PK, Yadav RSS, Yadava S, Purification
and characterization of yellow laccase from Trametes hirsuta MTCC-1171 and
its application in synthesis of aromatic aldehydes, Process Biochemistry (2014),
http://dx.doi.org/10.1016/j.procbio.2014.06.016
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Purification and characterization of yellow laccase from Trametes hirsuta
MTCC-1171 and its application in synthesis of aromatic aldehydes
Pankaj Kumar Chaurasia, Rama S. S. Yadav* and Sudha Yadava
Department of Chemistry, D.D.U. Gorakhpur University, Gorakhpur 273009
Uttar- Pradesh, India.
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Correspondence to: R. S. S. Yadav*
Phone no: +91 9453608772, +91 0551 2204943
E-mail address: rssy_chemistry@rediffmail.com
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Highlights
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Purification and characterization of yellow laccase from new fungal strain.
Use of this purified laccase in syntheses of several aromatic aldehydes.
No mediator used for such syntheses.
Identification of products by HPLC and IR.
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Abstract A yellow laccase from the culture filtrate of Trametes hirsuta MTCC-1171
has been purified. The purification methods involved concentration of the culture
filtrate by ammonium sulphate precipitation and an anion exchange chromatography
on diethylaminoethyl cellulose. The sodium dodecyl sulphate polyacrylamide gel
electrophoresis (SDS-PAGE) and native polyacrylamide gel electrophoresis gave
single protein band indicating that the enzyme preparation was pure. The molecular
mass of the enzyme determined from SDS-PAGE analysis was 55.0 kDa. Using 2,6-
dimethoxyphenol,
2,2’
[Azino-bis-(3-ethylbonzthiazoline-6-sulphonic
acid)
diammonium salt] and 3,5-Dimethoxy-4-hydroxybenzaldehyde azine as the
substrates, the Km, kcat and kcat/Km values of the laccase were found to be 420 μM,
13.04 s-1, 3.11×104 M-1s-1, 225 μM, 13.03 s-1, 1.3×105 M-1s-1 and 100 μM, 13.04 s-1,
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5.8×104 M-1s-1, respectively. The pH and temperature optima were 4.5 and 60 C,
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respectively while pH and temperature stabilities were pH 4.5 and 50 C. The
activation energy for thermal denaturation of the enzyme was 18.6 kJ/mole/K. The
purified laccase has yellow colour and does not show absorption band around 610 nm
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like blue laccases. The purified laccase transforms toluene, 3-nitrotoluene,
4-nitrotoluene, 3-chlorotoluene, 4-chlorotoluene and 3,4-dimethoxytoluene to
benzaldehyde, 3-nitrobenzaldehyde, 4-nitrobenzaldehyde, 3-chlorobenzaldehyde,
4-chlorobenzaldehyde and 3,4-dimethoxybenzaldehyde in the absence of mediator
molecules in high yields.
Keywords: 2, 6- dimethoxyphenol, copper containing enzymes, lignolytic enzymes,
Trametes hirsuta, yellow laccases
Introduction
Laccase [benzenediol: oxygen oxidoreductase; E.C. 1.10.3.2] is a polyphenol
oxidase, which belongs to the superfamily of multicopper oxidases and catalyzes the
four electron reduction of molecular oxygen to water [1-5]. Laccases are dimeric or
tetrameric glycoproteins. To perform their catalytic functions, laccases depend on Cu
atoms that are distributed at the three different copper centres viz. type-1 or blue
copper centre, type-2 or normal copper centre and type-3 or coupled binuclear copper
centres, differing in their characteristics electronic paramagnetic resonance (EPR)
signals [6]. The organic substrate is oxidized by one electron at the active site of the
laccase generating a reaction radical which further reacts non-enzymatically. The
electron is received at type1 Cu and is shuttled to the trinuclear cluster where oxygen
is reduced to water.
More than 100 fungal laccases have been purified and characterized. Their
different physiochemical properties like temperature optima, pH optima, Km and
molecular masses for different substrates have also been studied [4]. Molecular
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masses of fungal lccases have been found in the range of 43-383 kDa [4]. Most of the
studies reported so far are on blue laccases. Yellow or white laccases have rarely been
studied [7, 8]. Yellow or white laccases differs from blue laccases in two aspects.
Blue laccases have the absorption band around 610 nm [7] while yellow or white
laccases lack it. Yellow or white laccases oxidize non-phenolic substrates in absence
of mediator molecules [8] which are required in case of blue laccases. Thus, yellow or
white laccases are better biocatalysts than blue laccases. Ortho and para diphenols,
aminophenols, polyphenols, polyamines, lignins, and arylamines and some of the
inorganic ions are the substrates for laccases. The ability of laccases to catalyze the
oxidation of various phenolic as well as non-phenolic compounds, coupled to the
reduction of molecular oxygen to water makes it valuable from the point of view of
their different applications [4, 9-12]. The biotechnological importance of laccases
have increased after the discovery that oxidizable reaction substrate range could be
further extended in the presence of small readily oxidizable molecules called
mediators [13, 14]. During the last two decades, laccases have turned out to be the
most promising enzymes for industrial uses [11, 12] having applications in food,
pulps, paper, textile and cosmetic industries and in synthetic organic chemistry like
selective oxidation of aromatic methyl group, novel penicillins synthesis, coupling of
amines, oxidation of amides etc [9, 15-18].
Laccases purified from different sources exhibit different properties and are
suitable for different applications. Keeping these points in view, authors have initiated
studies on purification, characterization and biotechnological applications of laccases
from different sources. It is already reported in the literature that some fungal strains
which secrete blue laccases in submerged liquid cultures, secrete yellow laccases in
the presence of culture media containing natural lignin substrates [7-8, 11, 19-21].
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Moreover, blue laccases transform toluene to benzaldehyde in the presence of
mediator molecules [22, 23] whereas some of the yellow laccases perform same
reactions in the absence of mediator molecules [24]. Only conversion of toluene to
benzaldehyde was done previously [24]. Thus, the objective of this communication
was to purify and characterize a novel yellow laccase from T. hirsuta MTCC-1171
and to demonstrate the conversion of toluene, 3-nitrotoluene, 4-nitrotoluene,
3-chlorotoluene, 4-chlorotoluene and 3,4-dimethoxytoluene to benzaldehyde,
3-nitrobenzaldehyde,
4-nitrobenzaldehyde,
3-chlorobenzaldehyde,
4-chlorobenzaldehyde and 3,4-dimethoxybenzaldehyde in the absence of mediator
molecules.
Materials and methods
Materials
3,5-Dimethoxy-4-hydroxybenzaldehyde azine (syringaldazine), 3-chlorotoluene,
4-chlorotoluene and diethyl amino ethyl (DEAE) cellulose were from Sigma
Chemical Company, St. Louis (USA). 2,2’-Azino-bis (3-ethylbenzthiazoline-6-
sulphonic acid) diammonium salt (ABTS) and 2,6-dimethoxy phenol (DMP) were
from Fluka, Chemi new Ulm (Switzerland). All other chemicals used in these
investigations were either from Himedia laboratory Ltd. Mumbai (India) or from E.
Merck Ltd. Mumbai (India) and were used without further purifications. The
chemicals used in the gel electrophoresis of the protein samples were from Bangalore
Geni Pvt. Ltd., Bangalore (India). DEAE Cellulose column used in purification of
enzyme was from Pharmacia, Upssala, Sweden.
The fungal strain and its growth
The fungal strain was procured from the Microbial Type Culture Collection
Center and Gene Bank, Institute of Microbial Technology, Chandigarh (India) and
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was maintained on agar slant as reported in MTCC Catalogue of strains-2000 [25].
The growth medium for the fungal strain T. hirsuta MTCC-1171 consisted of malt
extract 20.0 g, and agar 20.0 g in 1.0 L Milli-Q water. The pH of the growth medium
was adjusted to 6.5.
In order to detect the extracellular secretion of laccase by T. hirsuta, the liquid
culture growth medium reported by Coll et al. [26] was used. This medium consisted
of glucose 10.0 g, asparagine 1.0 g, yeast extract 0.5 g, MgSO4∙7H2O and
FeSO4∙7H2O, 0.01 g in 1.0 L of Milli-Q water. This liquid culture growth medium
containing natural lignin substrates like coir dust, corn cob, wheat straw, saw dust and
bagasse particles were separately prepared by adding 0.5 g of one of the natural lignin
substrates to 25 mL of growth medium in 100 mL culture flasks which were
sterilized. The sterilized growth media were inoculated with small pieces of mycelia
(0.5 cm  0.5 cm) under aseptic condition and the fungal cultures were grown under
stationary culture conditions at 25 °C in a biological oxygen demand (BOD)
incubator. In order to monitor the production of laccase in the liquid culture medium,
0.5 mL aliquots of the growth medium were withdrawn at the regular intervals of 24
hrs and filtered through sterilized Millipore filter 0.22 m. The filtered extract was
analyzed for the activity of laccase using DMP as substrate by the method [26] given
in assay section. Extracellular secretion of laccase in the liquid culture medium by
T. hirsuta MTCC-1171 was determined by plotting enzyme unit/mL of the growth
medium against the number of days after inoculation of the fungal mycelia. Each
point on the curve is an average of three measurements. The growth medium for
control experiment has the same composition except that no natural lignolytic
substrate has been added. In order to optimize the conditions for maximum production
of laccase by T. hirsuta MTCC-1171 in the liquid culture medium, the amount of the
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best inducer wheat-straw particles were varied from 100 mg to 1000 mg in 25 mL of
the growth medium. The amount of inducer in the growth medium which gave the
maximum height of the enzyme activity peak was taken as the optimal amount of the
inducer.
Enzyme assay
The assay solution using DMP as the substrate [26] contained 1.0 mM DMP in 50
mM sodium malonate buffer (pH 4.5) at 37°C, using ABTS as the substrate [27]
contained 0.5 mM ABTS in 0.1 M sodium acetate buffer (pH 5.0) at 25°C and using
syringaldazine as the substrate [28] contained 0.1 mM syringaldazine in 50 mM
sodium phosphate buffer (pH 6.0) at 50°C. In case of DMP, the reaction was
monitored by measuring the absorbance change at =468 nm (ɛ = 49.6 ×103 M-1cm-1)
while in case of ABTS and syringaldazine, the reactions were monitored by
measuring the absorbance change at =420 nm (ɛ = 36.0 ×103 M-1cm-1) [27] and
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=530 nm (ɛ =65.0 mM-1 cm-1) [28], respectively. The UV/Vis spectrophotometer
Hitachi (Japan) model U-2900 fitted with electronic temperature control unit was used
for absorbance measurement. The least count of absorbance measurement was 0.001
absorbance unit. One enzyme unit produced 1 mol of the product per minute under
the specified assay conditions.
Purification of laccase
For the purification of laccase, T. hirsuta MTCC-1171 was grown in ten 100 mL
culture flasks each containing 25 mL sterilized growth medium with optimal amount
600 mg of the inducer, wheat-straw particles, under stationary culture condition in a
BOD incubator at 30°C. The maximum activity of laccase appeared on 7th day of the
inoculation of fungal mycelia. On 7th day, all the cultures in 10 flasks were pooled,
mycelia were removed by filtration through four layers of cheese cloth. The culture
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filtrate was saturated up to 30% with ammonium sulphate and centrifuged using
refrigerated centrifuge Sigma (Germany) model 3K-30 at 5480×g for 20 min. at 4°C.
The precipitate was discarded and the supernatant was saturated up to 90% by further
addition of ammonium sulphate. The resulting suspension was centrifuged by
repeating the same process of centrifugation and the supernatant was discarded. The
precipitate was dissolved in 100 mM sodium acetate buffer pH 4.5 and dialyzed
against 10 mM sodium acetate buffer pH 4.5 in volume ratio 1:1000 with three
changes at the intervals of 8 hrs. The dialyzed enzyme sample 15 mL containing 1.4
mg/mL protein was loaded on to DEAE column (size 0.75 cm x 18.0 cm) which was
pre-equilibrated with 10 mM sodium acetate/acetic acid buffer (pH 4.5) and the flow
rate was 15 mL/hr. Column was washed with 100 mL of the same buffer. The enzyme
was eluted by applying linear gradient of NaCl in the range of 0.23 to 0.84 M in same
buffer (35 mL buffer and 35 mL buffer). The fractions of 5.0 mL size were collected
and analyzed for laccase activity [26]. The protein estimation was done by Lowry
method [29]. All laccase active fractions were combined and concentrated by Amicon
concentrator cell model 8200 and then by model-3 to 3 mL. The enzyme was stored in
refrigerator in 10 mM sodium acetate/acetic acid buffer pH 4.0. The enzyme does not
loose any activity for one month under these conditions.
Copper content analysis
This is based on the study of complex formation of 2, 2'-biquinoline with copper
present in protein, spectrophotometrically, at 546 nm [30, 31]. Purified protein was
first diluted with 0.1 M phosphate buffer (pH 6.0) to 2mL and then this solution was
diluted to 5 mL with 2, 2’-biquinoline solution in glacial acetic acid. A blank solution
was also prepared using similar method with 2 ml of buffer. Absorbances were
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measured at 546 nm and the copper content was estimated by reported method
[30, 31].
SDS-PAGE analysis
Purity of the enzyme preparation was checked by using sodium dodecyl
sulphate-polyacrylamide gel electrophoresis [32]. The molecular mass markers were
phosphorylase (97.4 kDa), bovine serum albumin (66.0 kDa), ovalbumin (43.0 kDa),
carbonic anhydrase (29.0 kDa), soyabean trypsin inhibitor (20.1 kDa) and lysozyme
(14.3 kDa), and were procured from Bangalore Genei Pvt. Ltd. Bangalore (India). Gel
was run at a constant current 20 mA Polyacrylamide Gel Electrophoresis [33]. The
molecular mass was determined by Weber and Osborn method [34].
Native-PAGE and zymogram analysis
The native polyacrylamide gel electrophoresis of the purified enzyme was done
using the reported method [35]. The composition of resolving and stacking gel was
similar to that used in SDS-PAGE except SDS was absent. Native molecular mass
protein markers used were similar to SDS-PAGE analysis. Two sets of native gel
were done. One set was strained with Coomassie Brilliant Blue R-250 and the other
set was used for zymogram [36] preparation. For the preparation of zymogram 100
mM DMP solution was made in 10 mM sodium tartrate buffer pH 5.0. The native gel
was dipped in zymogram solution for five minutes and a brown band appeared. The
zymogram was removed from DMP solution and was washed thrice with 10 mM
sodium tartrate buffer pH 5.0 at the interval of five minutes.
Steady state enzyme kinetics
The steady state enzyme kinetics of the purified laccase was studied using DMP,
ABTS and syringaldazine as the substrates following the methods as mentioned in
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assay section and kinetic parameters were calculated for different substrates from
Lineweaver-Burk plots by incorporating the data obtained during enzymatic assay.
Km and kcat values for the enzyme were determined from linear regression of double
reciprocal plots. The pH and temperature optima of enzyme were determined by
measuring the steady state velocities of enzyme catalyzed reaction in the solutions of
varying pH/temperature keeping the other parameter fixed and drawing graphs of
steady state velocities versus the variable parameter.
Thermal and pH stabilities
Thermal stability of the enzyme was tested by incubating an enzyme aliquot at
○
particular temperature ranges from 30-70 C for 60 minutes, assaying its residual
activity and plotting the residual activity against temperature. For thermal
denaturation of enzyme, the rate constants for denaturation of enzyme at different
temperatures were determined by keeping the enzyme aliquots at different fixed
temperatures and assaying the enzyme activity at regular intervals of time and plotting
residual activity against time. The rate constants were calculated from t1/2 values using
the equation k
0.693/t1/2. Energy of activation for thermal denaturation was
=
calculated from Arrhenius plot.
pH stability of enzyme was tested by incubating an enzyme aliquot at particular
pH value ranges from 3.0-5.0 for 60 minutes, assaying its residual activity and
plotting the residual activity against pH.
Bioconversions in the absence of mediators
The bioconversions of toluene to benzaldehyde [22-24] was done in 15 mL of
100 mM sodium acetate buffer pH 4.5 containing 20 mM toluene in 20 mL of dioxane
and 60 µg of purified laccase kept in a 100 mL conical flask which was stirred
vigorously for 30 minutes. The reaction solution was extracted thrice with 40 mL of
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n-hexane. 20 µL of the n-hexane extract was injected in Waters HPLC Model 600E
using spherisorb C18 5 UV, 4.5×250 mm column at 25○C. The eluant phase was
methanol at the flow rate of 0.5 mL/min. The detection was made using Waters UV
detector model 2487 at λ=254 nm.
The bioconversions of 3-nitrotoluene, 4-nitrotoluene, 3-chlorotoluene,
4-chlorotoluene
and
3,4-dimethoxytoluene
3-chlorobenzaldehyde,
to
3-nitrobenzaldehyde,
and
4-nitrobenzaldehyde,
4-chlorobenzaldehyde
3,4-dimethoxybenzaldehyde, respectively were also studied using the same method
described above except time of stirring the reaction solutions were 90, 90, 120, 120
and 90 minutes, respectively.
Since only small amounts of chemical auxiliaries are applied which remain in
the aqueous phase after extraction of the aldehydes with a suitable organic solvent
(hexane/ethyl acetate), very pure compounds are obtained requiring no further
purification. During these oxidations, no side reactions occur because of the high
specificity of laccase as biocatalyst. Thus, authors have used hexane/ethyl acetate as
organic solvent for the extraction of products and found the almost pure
benzaldehydes and substituted benzaldehydes in high yields (average yield was 96%).
All the synthesized products were identified and characterized by HPLC and IR
techniques.
High performance liquid chromatography (HPLC)
Retention time for reactants toluene, 3-nitrotoluene, 4-nitrotoluene,
3-chlorotoluene, 4-chlorotoluene, 3,4-dimethoxytoluene were 2.2, 3.0, 4.2, 7.3, 3.3
and 6.6 minutes, respectively while retention time for standard of products
benzaldehyde, 3-nitrobenzaldehyde, 4-nitrobenzaldehyde, 3-chlorobenzaldehyde,
4-chlorobenzaldehyde and 3,4-dimethoxybenzaldehyde were 3.2, 3.3, 3.6, 6.2, 3.7 and
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5.9 minutes, respectively. Retention time for synthesized products benzaldehyde,
3-nitrobenzaldehyde, 4-nitrobenzaldehyde, 3-chlorobenzaldehyde,
4-chlorobenzaldehyde and 3, 4-dimethoxybenzaldehyde were approximately same as
for standard: 3.2, 3.3, 3.6, 6.2, 3.7 and 5.9 minutes, respectively.
Infrared (IR) spectroscopy
Major bands obtained from spectral analysis for different enzymatically
synthesized compounds were: For benzaldehyde: ~3064, ~2819, ~2737, ~1918,
~1702, ~1391, ~1287, ~746, ~714 cm-1. For 4-nitrobenzaldehyde and
3-nitrobenzaldehyde: ~3087, ~2858, ~2759, ~2045, ~1694, ~1572, ~1384, ~815,
~701 cm-1. For 3-chlorobenzaldehyde and 4-chlorobenzaldehyde: ~3075, ~2840,
~2740,
~2016,
~1697,
~1315,
~833,
~689,
~602
cm-1.
For
3,4-dimethoxybenzaldehyde: ~3021, ~2981, ~1701, ~1376, ~1223, ~751 cm-1.
Results and discussion
Fig. 1(a) shows the secretion of laccase in the liquid culture growth medium
amended with various lignin containing natural substrates like corn cob, coir dust, saw
dust, wheat straw and bagasse particles and inoculated with T. hirsuta MTCC-1171.
The control experiment has similar medium composition except that the natural lignin
containing substrate was absent. It is obvious from the figure that lignin containing
natural substrates when present in the liquid culture growth medium enhanced the
secretion of laccase. This enhancement of extracellular secretion of laccase is
maximum in case of the growth medium containing wheat-straw particles. In order to
optimize the secretion of laccase in the presence of wheat-straw particles, secretion of
laccase in presence of different amounts of wheat-straw particles were studied. The
results are shown in fig. 1(b). The maximum level of laccase was secreted in the
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liquid culture medium containing 600 mg of the wheat-straw particles per 25 mL of
the culture medium.
The purification procedure of laccase from the liquid culture filtrate of
T. hirsuta MTCC-1171 is summarized in table-1. The enzyme bound to DEAE
cellulose in 10 mM sodium acetate/acetic acid buffer pH 4.5 and was eluted by the
linear gradient of NaCl in the range 0.23 to 0.84 M. The fraction numbers 4-8 were
combined and concenterated to 3 mL by using the Amicon concentrator cells. The
method gave 10.42 fold purification with 12.57% recovery of the enzyme activity.
The concentrated enzyme sample was analyzed by SDS-PAGE, native PAGE and
zymogram preparation. The results of SDS-PAGE, native PAGE and zymogram
preparation are shown in Fig. 2(a), (b) and (c), respectively. In Fig. 2(a), reported lane
1 contains molecular mass markers and lane 2 contains the purified enzyme. The
appearance of single protein band in SDS-PAGE indicated that the enzyme sample
was pure. In Fig. 2(b), lane 1 contains native molecular mass markers similar to SDS-
PAGE and lane 2 contains the purified enzyme. The appearance of single protein band
in native-PAGE (lane 2), also indicated that the enzyme sample was pure. The
position of purified enzyme in the zymogram Fig. 2(c) of native PAGE coincides with
the position of purified laccase in the native PAGE. The molecular mass of purified
laccase determined from the analysis [31] of SDS-PAGE was 55.0 kDa. Same
molecular mass was also obtained from native-PAGE analysis. The molecular mass of
purified laccase is well compared to the molecular masses of laccases reported in the
literature which were in the range 43-383 kDa [4, 37].
Since laccases are the copper containing enzymes, it is common to determine the
copper contents in the newly purified laccase. The copper content of the newly
purified laccase was determined spectrophotometrically by 2, 2'-biquinoline at 546
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nm [30, 31] which was found to be 2.1 copper ions per enzyme. The uncertainties in
determining copper concentration and protein concentration could affect the results.
The standard error for each measurement was estimated to be less than 5%.
The Michaelis-Menten and double reciprocal plots for purified enzyme using
DMP as the variable substrate are shown in Fig. 3. Similar types of Michaelis-Menten
and double reciprocal plots were obtained in case of syringaldazine and ABTS, hence
have not been given here. The Km, kcat and kcat/Km values determined from the double
reciprocal plot using DMP, syringaldazine and ABTS as the substrates of the enzyme
with standard error were given in table 2. The range of Km values reported [4] for
fungal laccases using DMP, ABTS, and syringaldazine as the substrates are 8-14720
μM, 4-770 μM and 3-3400 μM, respectively. However, the Km value for this laccase
for DMP compares well with the Km of laccase purified from Trametes gallica Lac-1
[4].The Km value for ABTS compares well with the Km of laccase purified from
Pleurotus sajor-caju IV and Marasmius quercophilus [4]. The Km value for
syringaldazine compares well with the Km of laccase purified from Pleurotus
ostreatus POXA 1b [4]. Low values of Km and high values of Kcat and Kcat/km (table 2)
of purified laccase from T. hirsuta MTCC-1171 for different substrates like DMP,
ABTS and syringaldazine, clearly demonstrate that this purified enzyme has good
catalytic efficiency and can be utilized as effective biocatalyst.
The results of the variation of the activity of the purified laccase with the
variation of pH of the reaction solution are shown in Fig. 4 by dotted line. The
determined pH optimum of enzyme was 4.5. The pH optima reported in the literature
[4] for laccases using DMP as the substrate are in the range 3.0-8.0 pH units. The pH
optimum of the purified laccase compares well with pH optima of laccases of Chalara
paradoxa, Gaeumannomyces graminis, Pleurotus ostreatus POXA 1b, Pleurotus
14
Page 14 of 35

349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
ostreatus POXA 1W, Pleurotus ostreatus POXC and Volvarielia volvacea [4]. The
results of the studies on the pH stability of laccase are shown in Fig. 4 by solid line
where the residual activities of enzyme have been plotted against the different pHs for
which enzyme has been exposed for 1 h. It follows that the enzyme has maximum
stability at pH 4.5.
The results of variation of the activity of the purified laccase as a result of
variation of the temperature of the enzyme catalyzed reaction solution are shown in
Fig. 5 by dotted line. The temperature optimum was 60 °C. The range of temperature
optima reported for other laccases are 25-80 °C. The purified laccases of Chaetomium
termophilum and Coniothyriem minitans also have temperature optima at 60 °C [4].
The result of the studies on the thermal stability of the purified laccase is shown in
Fig. 5 by solid line where the residual activities of enzyme have been plotted against
the different temperatures at which enzyme has been exposed for 1 h.
The purified laccase does not possess blue colour as visually observed in cases
of blue laccases. Instead, it has yellow colour. The purified laccase lacked absorption
band around 610 nm generally observed in cases of blue laccases, which indicated
that it was a yellow laccase. It has also been reported that yellow laccase [7, 8]
oxidizes non-phenolic substrates in the absence of electron transfer mediators which
is essential for the oxidation of non-phenolic substrates by blue laccases. The purified
laccase oxidized veratryl alcohol to veratraldehyde in the absence of mediator
molecules confirming that it is a yellow laccase [7, 8].
One of the applications of the laccases in organic synthesis is selective oxidation
of the aromatic methyl groups to the corresponding aldehydes. The chemical routes of
this conversion are inconvenient because the reaction has to be stopped at the
aldehyde stage so that carboxylic acid could not be formed. Moreover, they require
15
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375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
drastic reaction conditions which pollute the environment. The conversion done with
laccase occurs under milder conditions, yield is > 95% and the process is ecofriendly.
Laccases are the preferred enzymes because they cycle on oxygen and generate no
side products except water. The use of blue laccases for this purpose has been studied
but they require mediator molecules like ABTS [22] and 1-hydroxy-1H-benzotrizole
(HBT) [23]. But the laccase purified by authors has strong capability to oxidize
toluene as well as different substituted toluenes to respective aromatic aldehydes
selectively without using any mediator molecules. This was tested using toluene,
3-nitrotoluene,
4-nitrotoluene,
3-chlorotoluene,
4-chlorotoluene
and
3,4-dimethoxytoluene as the substrates, successfully. All the reactions are ecofriendly
and good examples of green chemistry. They always occur at room temperature and
pressure without any side reactions. In all reactions, water is only the byproduct.
Since, there is no need of mediators, drastic reaction conditions, costly reagents etc
use of this laccase as biocatalyst may be more valuable in the synthesis of these
compounds economically as well as environmentally. This laccase may be used in the
industrial process for future large scale production of these substituted benzaldehydes
by purifying laccase and enhancing the amount of substrate according to requirements
using same method as given in experimental section.
In order to demonstrate the advantage of this purified laccase over other fungal
laccases, comparative catalytic experiments have been done by authors. Authors have
found that laccase from T. hirsuta MTCC-1171 selectively oxidizes the several
substituted toluenes to corresponding benzaldehydes within <2 hrs in the absence of
mediator while several other laccases do not oxidize the same substrates in the
absence of mediator molecules. Laccase from Trametes hirsuta MTCC-11397 has
poor oxidizing capability and takes very long time in the absence of mediator
16
Page 16 of 35

399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
molecule but causes rapid oxidation of the same substrates in presence of ABTS as
mediator molecule within 2 hrs. Other laccase from T. versicolor, Coriolus versicolor
MTCC-138 [38], Pleurotus ostreatus MTCC-1803 [39], Xylaria polymorpha MTCC-
1100 [40, 41] and Phellinus linteus MTCC-1175 [42] failed to oxidize the above
described substrates in the absence of mediator molecule. In all the above
comparative experiments, laccases used were obtained from liquid culture growth
media of respective fungal sources and reactions were studied spectrophotometrically.
These catalytic comparative experiments have been given in table 3.
In above enzyme catalyzed biotransformations, all products formed are easily
available and simple. HPLC technique has been used to confirm the actual product
formation by comparing the HPLC chromatograms of standard aldehyde compounds
with the enzymatically transformed compounds. The retention time of the standard
samples of toluene, 3-nitrotoluene, 4-nitrotoluene, 3-chlorotoluene, 4-chlorotoluene,
3,4-dimethoxytoluene, benzaldehyde, 3-nitrobenzaldehyde, 4-nitrobenzaldehyde,
3-chlorobenzaldehyde, 4-chlorobenzaldehyde and 3,4-dimethoxybenzaldehyde were
2.2, 3.0, 4.2, 4.3, 3.3, 6.6, 3.2, 3.3, 3.6, 6.2, 3.7 and 5.9, respectively. Thus, the
retention time of the products of the enzyme catalyzed reactions (3.2, 3.3, 3.6, 6.2,
3.7, and 5.9) revealed that the products of the enzyme catalyzed reactions were
benzaldehyde, 3-nitrobenzaldehyde, 4-nitrobenzaldehyde, 3-chlorobenzaldehyde,
4-chlorobenzaldehyde and 3,4-dimethoxybenzaldehyde (table 4). Chromatograms for
the conversion of toluene to benzaldehyde are given in Fig. 6.
In IR-spectra, bands between 2819-2858 cm-1 were due to aldehydic C-H
-1
stretching while bands between 1695-1705 cm were due to conjugated aldehydic
C=O stretching which prove the conversion of aromatic methyl group into aromatic
aldehydic group. One IR-spectrum for benzaldehyde and one for substituted
17
Page 17 of 35

424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
benzaldehyde i.e. 3-nitrobenzaldehyde are given in Fig. 7 and 8, respectively for
proof.
Conclusions
In this communication, a novel yellow laccase from a new fungal strain
T. hirsuta MTCC-1171 has been purified and characterized. The use of this enzyme in
synthesis of aromatic aldehydes from toluene and different substituted toluenes in the
absence of mediator molecules has been demonstrated. Thus, this purified laccase is a
better biocatalyst than blue laccases in which the use of mediator molecule is
necessary. All the laccase catalyzed reactions in this communication are good
examples of green chemistry.
Acknowledgements
Mr. P. K. Chaurasia is thankful to CSIR, New Delhi for the award of JRF and
SRF award no. 09/057(0201)2010-EMR-I. Authors are also thankful to Department of
Chemistry, DDU Gorakhpur University for providing facilities for research. The
authors are thankful to Prof. K.D.S. Yadav, Emeritus Scientist, CSIR, New Delhi for
his helpful discussions.
18
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451
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463
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465
466
467
468
469
470
471
472
References
1. Hoegger PJ, Kilaru S, James TY, Thacker JR., Kuees U. Phylogenetic comparison
and classification of laccase and related multicopper oxidase protein sequences.
FEBS J 2006; 273:2308-2326.
2. Messerschmidt A. Multi-Copper Oxidases. World Scientific, Singapore, 1997 .
3. Riva S. Laccase: blue enzymes for green chemistry. Trends in Biotechnol 2006;
24(5):219-226.
4. Baldrian P. Fungal laccases – occurrence and properties. FEMS Microbiol Rev
2006; 30:215-242.
5. Dwivedi UN, Singh P, Pandey VP, Kumar A. Structure-function retationship among
bacterial, fungal and plant laccases. J. Mol. Cat. B: Enzymatic 2011; 68(2):117- 128.
6. Solomon EI, Baldwin MJ, Lowery MD. A 1H NMR NOE study on Co(II) stellacyanin:
Some clues about the structure of the metal site. Chem Rev 1992; 92:521-542.
19
Page 19 of 35

473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
495
496
7. Leontievsky AA, Vares T, Lankinen P, Shergill JK, Pozdnyakova N, Myasoedova N
M et al. Blue and yellow laccases of ligninolytic fungi. FEMS Microbiol Lett 1997;
156:9-14.
8. Leontievsky A, Myasoedova N, Pozdnyakova N, Golovleva L. `Yellow' laccase of
Panus tigrinus oxidizes non-phenolic substrates without electron-transfer mediators.
FEBS Lett 1997; 413:446-448.
9. Chaurasia, P.K., Yadav, R.S.S., Yadava, Purification and Characterization of
Laccase Secreted by Phellinus linteus MTCC-1175 and its role in the selective
oxidation of aromatic methyl group. Appl Biochem Microbiol 2013; 49(6):592–
599.
10. Mogharabi M, Faramarzi, MA. Laccase and laccase-mediated systems in the
synthesis of organic compounds. Adv Synth Cat 2014; 56(5):897-927.
11. Couto SR, Harrera JLT. Industrial and biotechnological applications of laccase: A
review. Biotechnol Advan 2006; 24:500-513.
12. Xu F. Applications of oxidoreductases: recent progress. Ind Biotechnol 2005; 1:38-
50.
13. Acunzo DF, Galli C. First evidence of catalytic mediation by phenolic compounds in
the laccase induced oxidation of lignin models. J Europ Biochem 2003; 270:3634-
3640.
14. Morozova OV, Shumakovich GP, Shleev SV, Yaropolov YI. Laccase-mediator
systems and their applications: A review. Appl Biochem Microbiol 2007; 43:523-
535.
15. Coniglio A, Galli C, Gentili P. Oxidation of amides by laccase-generated aminoxyl
radicals. J Mol Cat B: Enzymatic 2008; 50(1):40-49.
20
Page 20 of 35

497
498
499
500
501
502
503
504
505
506
507
508
509
510
511
16. Mikolasch A, Niedermeyer THJ, Lalk M, Witt S, Seefeld S, Hammer E, Schauer F,
Gesell M, Hessel S, Julich WD, Lindoquist U. Novel penicillins synthesized by
biotransformation using laccase from Trametes spec. Chem Pharma Bull 2006;
54(5):632-638.
17. Mikolasch A, Niedermeyer THJ Lalk M, Witt S, Seefeldt S, Hammer E, Schauer F,
Salazar MG, Hessel S, Julich WD, Lindequist U. Novel cephalosporins synthesized
by amination of 2,5- dihydroxybenzoic acid derivatives using fungal laccases II.
Chem Pharma Bull 2007; 55(3):412-416.
18. Mikolasch A, Hammer E, Jonas U, Popowski K, Stielow A, Schaner F. Synthesis of
3- (3,4-dihydroxyphenyl)-propionic acid derivatives by N-coupling of amines using
laccase. Tetrahedron 2002; 58:7589-7593.
19. Osma J, Toca-Herrera JL, Rodriguez-Couto S. Preparation of
immobilized Trametes pubescens laccase on a cryogel-type polymeric carrier and
application of the biocatalyst to apple juice phenolic compounds oxidation. Enz Res
2010; doi: 10, 4061/2010/918761. ID: 918761, pp. 8.
512 20. Brijwani K, Rigdon A, Vadlani PV. Fungal Laccases: Production, Function, and
513
514
Applications in Food Processing, Enz Res 2010; doi: 10.4061/2010/149748. ID:
149748, pp. 10.
515 21. Desai SS, Nityanand C. Microbial Laccases and their Applications: A Review. Asian
516 J Biotechnol 2011; 3(2):98-124.
517 22. Potthast A, Rosenau T, Chen C-L, Gratzl JS. Selective Enzymic Oxidation of
518 Aromatic Methyl Groups to Aldehydes. J Org Chem 1995; 60:4320-4321.
519 23. Fritz-Langhals, Kunath B. Synthesis of aromatic aldehydes by laccase-mediator
520 assisted oxidation, Tetrahedron Lett 1998; 39:5955-5956.
21
Page 21 of 35

521 24. Sahay R, Yadav RSS, Yadava S. A laccase of Fomes durissimus MTCC-1173 and its
522
523
role in the conversion of methylbenzene to benzaldehyde, Appl Biochem Biotechnol
2012; 166:563-575.
524 25. Catalogue of strains-2000 (5th edition), Microbial Type Culture Collection and Gene
525 Bank Institute of Microbial Technology, Chandigarh p.60.
526 26. Coll MP, Fernandez-Abalos JM, Villanueva JR, Santamaria R, Perez P. Purification
527
528
and characterization of a phenoloxidase (laccase) from the lignindegrading
Basidiomycete PM 1 (CECT2971). Appl Env Microbiol 1993; 59:2607-2613.
529 27. Bourbonnais R, Paice MG. Demethylation and delignification of Kraft Pulp by
530
531
Trametes versicolor laccase in the presence of 2,2’-azinobis-(3-ethyl benzthiozoline-
6-sulphonate), Appl Microbiol Biotechnol 1992; 36:823-827.
532 28. Chefetz B, Chen Y, Hador Y. Purification and characterization of laccase from
533
534
Chaetomium thermophilium and its role in humification, Appl Env Microbiol 1998;
64:3175-3179.
535 29. Lowry OH, Rosebrough NJ, Farrand AL, Randall RJ. Protein measurement with the
536 folin-phenol reagent. J Biol Chem 1951; 193(1):265-275.
537 30. Felsenfeld G. The determination of cuprous ion in copper proteins. Arch Biochem
538 Biophys 1960; 87:247-251.
539 31. Hanna PM, Tamilarasan R, McMillin DR. Cu(I) analysis of blue copper proteins.
540 Biochem J 1988; 256:1001-1004.
541 32. Laemmli UK. Cleavage of Structural Proteins during the Assembly of the Head of
542 Bacteriophage T 4. Nature 1970; 227:680-685.
543 33. Polyacrylamide Gel Electrophoresis: (1984) Laboratory Techniques, Pharmacia,
544 Laboratory Separation Division, Uppsala Sweden.
22
Page 22 of 35

545 34. Weber K, Osborn M. The reliability of molecular weight determination by dodecyle
546 sulphate polyacrylamide Gel-electrophoresis. J Biol Chem 1969; 244:4406-4412.
547 35. Schagger, Jajow H. Blue native electrophoresis for isolation of membrane protein
548 complexes in enzymatically active form. Anal Biochem 1991; 199:223-231.
549 36. Zouri-Mechichi H, Mechichi T, Dhouib A, Sayadi S, Martinez AT, Martinez MJ.
550
551
552
Laccase purification and characterization from Trametes trogii isolated in Tunisia:
decolorization of textile dyes by the purified enzyme. Enz Microb Technol 2006;
39(1):141-148.
553 37. Dhakar K, Pandey A. Laccase production from a temperature and pH tolerant fungal
554
555
strain
of
Trametes
hirsuta
(MTCC
11397).
Enz
Res
2013;
http://dx.doi.org/10.1155/2013/869062, ID: 869062, pp 9.
556 38. Chaurasia PK, Singh SK. Role of laccase from Coriolus versicolor MTCC-138 in
557
558
559
560
561
562
563
564
565
566
567
568
569
selective oxidation of aromatic methyl group. Russ J Bioorg Chem, 2014; 40(3):288-
292.
39. Chaurasia PK, Yadav RSS, Yadava S. Selective biotransformation of aromatic
methyl groups to aldehyde groups using crude laccase of Pleurotus ostreatus
MTCC-1803. Int J Res Chem Environ 2013; 3(1):188-97.
40. Chaurasia PK, Yadav RSS, Yadava S. Application of crude laccase of Xylaria
polymorpha MTCC-1100 in selective oxidation of aromatic methyl group to
aldehyde group. Biochem: An Indian journal 2012; 6(7):237-242.
41. Chaurasia PK, Yadava S, Bharati SL, Singh SK. Selective oxidation and N-
coupling of amines by purified laccase of X. polymorpha MTCC-1100. Russ J
Bioorg Chem 2014; 40(4):453-458 [In press].
42. Chaurasia PK, Yadav A, Yadav RSS, Yadava S. Purification and characterization
of laccase secreted by Phellinus linteus MTCC-1175 and its role in the selective
23
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579
580
581
582
583
584
585
586
587
588
589
590
591
592
593
594
oxidation of aromatic methyl group. Appl Biochem Microbiol, 2013; 49(6):592–
599.
Legends to the figures:
Fig. 1 (a) Secretion of laccase by T. hirsuta MTCC-1171 in the liquid culture
medium supplemented with different natural lignin containing substrates:
wheat straw(♦), corn cob(■), bagasse(*), saw dust(×), coir dust(▲),
control(●)
(b) Optimization of laccase secretion by T. hirsuta MTCC-1171 in liquid
culture medium supplemented with different amount of wheat straw : 100
mg (●), 200 mg(■), 400 mg (▲) 500mg(×), 600 mg (*), 800 mg (∆) ,
1000 mg (♦).
Fig. 2 SDS-PAGE (a), native-PAGE (b) and zymogram (c) of the purified
laccase.
24
Page 24 of 35

595
596
597
598
599
600
601
602
603
604
605
606
607
608
609
610
611
(a) Molecular mass markers (lane 1), Purified laccase (lane 2).
(b) Molecular mass markers (lane 1), Purified laccase (2).
(c) Pure enzyme stained with DMP.
Fig. 3 Michaelis-Menten curve (solid line curve) and double reciprocal plot (dotted
line as insert figure) for the laccase of T. hirsuta MTCC-1171 using DMP as
the variable substrate.
In both cases, 1 mL reaction solution contained 0.0-1.4 mM DMP, 50 μL of
the enzyme in 100 mM sodium malonate buffer pH 4.5 at 37 0C.
Fig. 4 Determination of pH optimum (dotted line) and pH stability (solid line) of the
purified enzyme.
1 mL of reaction solution contained 1.0 mM DMP in 100 mM sodium
malonate buffer with varying pH from 3.0 to 5.5 in case of pH optimum while
3.0 to 5.0 in case of pH stability.
Fig. 5 Determination of temperature optimum (dotted line) and thermal stability (solid
line) of the purified enzyme.
1 mL of reaction solution contained 1.0 mM DMP in 100 mM sodium
⁰
malonate buffer pH 4.5 with varying temperature from 20 to 90 C in case of
⁰
612
temperature optimum and 30 to 70 C in case of thermal stability.
613
614
615
616
617
618
619
620
Fig. 6 Transformation of toluene to benzaldehyde by the laccase of T. hirsuta MTCC-1171 in
the absence of mediator.
(a) The chromatogram of the pure toluene.
(b) The chromatogram of the pure benzaldehyde.
(c) The chromatogram of the product of enzymatic reaction with Toluene.
The reaction solution contained 20 mM of methylbenzene, 20 ml dioxane and 15
mL sodium acetate buffer (pH 4.5) at room temperature was stirred for 30 minutes
and extracted thrice with ethyl acetate and 20 μL was injected in HPLC.
25
Page 25 of 35

621
622
623
Fig. 7 IR-spectrum for enzymatically synthesized product benzaldehyde.
Fig. 8 IR-spectrum for enzymatically synthesized product 3-nitrobenzaldehyde.
26
Page 26 of 35

Table(s)
Table 1 Different steps involved in purification procedure of laccase from the
liquid culture filtrate of T. hirsuta MTCC-1171
Steps
Volume Specific activity Total protein Total activity Purification
Yield
%
(mL)
(IU/mg)
(mg)
(IU)*
fold
Culture
filtrate
100
0.36
100.0
35.80
1.00
100.00
50.00
Ammonium
sulphate
precipitation
10
15
3
1.28
1.33
3.75
14.0
13.5
1.2
17.90
18.00
4.50
3.56
3.70
Dialysis with
10 mM Sodium
acetate buffer
50.28
12.57
DEAE cellulose
10.42
column chromatography
*IU is international unit/enzyme unit which is unit for the amount of enzyme that
catalyzes the conversion of one mole of the substrate per minute under the specified
assay conditions
Table 2 Km, kcat, kcat/Km and standard error values determined from the double reciprocal plots
using DMP, ABTS and syringaldazine as the substrates.
Km (µM)
kcat (s-1)
kcat/Km (×104 M-1s-1)
DMP
ABTS
420 ±2.55
13.04 ±0.02
3.11 ±0.03
225 ±3.06
13.03 ±0.03
13.04 ±0.04
13.00 ±0.03
5.80 ±0.04
Syringaldazine
100.00 ±3.00
Page 27 of 35

Table 3 A comparative chart showing the selective oxidations of toluene and substituted
toluenes to corresponding benzaldehydes by laccases obtained from various
fungal sources.
Laccases from different fungal
sources.
Selective oxidation in
Selective oxidation in the References
the absence of mediator presence of mediator
Trametes hirsuta MTCC-1171
Trametes hirsuta MTCC-11397
Trametes versicolor
Excellent (0.5-2.0 hrs)
-
This work
Poor (>24 hrs)
Excellent (0.5-2.0 h)
Excellent (0.5-1.0 h)
Excellent (0.5-2.0 h)
Excellent (0.5-1.5 h)
Excellent (0.5-1.5 h)
Excellent (0.5-1.5 h)
*
No
No
No
No
No
*
Coriolus versicolor MTCC-138
Pleurotus ostreatus MTCC-1803
Xylaria polymorpha MTCC-1100
Phellinus linteus MTCC-1175
37
38
39, 40
41
* The selective oxidation by these species is not reported and authors have done it for the
purpose of comparing its catalytic efficiency with laccase of T. hirsuta MTCC-1171.
Thus, this is a part of the manuscript.
Page 28 of 35

Table 4 Retention times (min) for reactant, standard benzaldehydes/substituted
benzaldehydes and respective synthesized benzaldehyde/substituted benzaldehydes with
percentage yield of products.
Toluene
(Reactant)
Benzaldehyde
(Standard)
Benzaldehyde
(Product)
Yield
(%)
2.2
3.2
3.2
96
3-Nitrotoluene
(Reactant)
3-Nitrobenzaldehyde
(Standard)
3-Nitrobenzaldehyde
(Product)
3.0
3.3
3.3
100
92
4-Nitrotoluene
(Reactant)
4-Nitrobenzaldehyde
(Standard)
4-Nitrobenzaldehyde
(Product)
4.2
3.6
3.6
3-Chlorotoluene
(Reactant)
3-chlorobenzaldehyde
(Standard)
3-Chlorobenzaldehyde
(Product)
7.3
6.2
6.2
97
95
4-Chlorotoluene
(Reactant)
4-Chlobenzaldehyde
(Standard)
4-Chlorobenzaldehyde
(Product)
3.3
3.7
3.7
3,4-Dimethoxytoluene 3,4-Dimethoxybenzaldehyde 3,4-Dimethoxybenzaldehyde
(Reactant)
(Standard)
(Product)
6.6
5.9
5.9
100
Page 29 of 35