Biotechnology Reports 5 (2015) 31–39

Contents lists available at ScienceDirect

Biotechnology Reports
journal homepage: www.elsevier.com/locate/btre

Laccase production by Pleurotus ostreatus and its application in

synthesis of gold nanoparticles$
Ahmed I. El-Batal a , Nora M. ElKenawy a , Aymen S. Yassin b, * , Magdy A. Amin b
a
Drug Radiation Research Department, National Center for Radiation Research & Technology, Cairo 11787, Egypt
b
Department of Microbiology and Immunology, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt

A R T I C L E I N F O A B S T R A C T

Article history: In this work, the production of fungal laccase was optimized from local isolate of Pleurotus ostreatus using
Received 7 July 2014 solid state fermentation. Factorial design was used to study the effect of several nutrients on enzyme
Received in revised form 3 November 2014 production. Purification and characterization of the enzyme and the effect of temperature, pH and
Accepted 6 November 2014
gamma radiation on fungal growth and enzyme production was investigated.
Available online 11 November 2014
Optimization of production conditions yielded an enzyme with activity over 32,450 IU/g of fermented
substrate. Factorial design was capable of establishing the conditions that multiplied the activity of the
Keywords:
enzyme several folds, consequently, reducing the cost of production. The enzyme was capable of
Laccase
Pleurotus ostreatus
decolorizing several dyes with over 80% reduction in color confirming the aromatic degrading capability
Gold nanoparticles (GNPs) of laccase. The enzyme was also used in the synthesis of gold nanoparticles, proving that laccase from
Pleurotus ostreatus has a strong potential in several industrial applications.
ã 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

1. Introduction and soil treatments and also biosensor developments [8,9]. In

particular, the ability to biodegrade various types of dyes by white-
Enzyme production is an expanding field of biotechnology. rot fungi has proven to be effective, with their elimination being
Laccase (E.C. 1.10.3.2, p-benzenedial: oxygen oxidoreductase) is mediated through oxidoreduction reactions catalyzed by the lignin
able to catalyze the oxidation of various aromatic compounds degrading enzymes they produce, such as lignin peroxidase,
(particularly phenol) with the concomitant reduction of oxygen to manganese peroxidase and laccase [10].
water [1]. Although the enzyme is present in plants, insects and Most studies, dealing with ligninolytic enzyme production by
bacteria, the most important source are fungi and particularly white-rot fungi, have been carried out using the liquid culture
basidiomycetes [1,2]. The white-rot fungi are the most efficient conditions, in spite of the fact that these organisms grow in nature
microorganisms capable of extensive aerobic lignin degradation. in solid-state conditions. Recent reviews on solid-state fermenta-
Due to the higher redox potential of fungal laccase compared to tion (SSF) point out the enormous potential of this culture
plant or bacterial laccase, they are utilized in several biotechno- technique for the development of different bioprocesses [11].
logical applications [3]. Fungal laccase is considered a key player in A lot of reports have emerged in the last few years describing
lignin degradation and/or the removal of potentially toxic phenols the preparation and characterization of gold nanoparticles
arising during morphogenesis, sporulation, or phytopathogenesis (GNPs), due to the extraordinary physicochemical characteristics
and fungal virulence [4]. The role of laccase in lignin and phenolic and wide usages in different fields. Although preparation of
compound degradations has been evaluated in a large number of nano-gold by physical procedures (such as laser ablation)
biotechnological applications such as dye degradation [5,6], provides GNPs with narrow range of particle size, it needs
bioremediation of some toxic chemical wastes [5,7], wastewater expensive equipment and has low yield [12]. Hazardous effects of
organic solvents, reducing agents and toxic reagents applied for
chemical synthesis of GNPs on the environment, has encouraged
$
This is an open-access article distributed under the terms of the Creative the development of eco-friendly methods for preparation of gold
Commons Attribution-NonCommercial-No Derivative Works License, which nanoparticles [13].
permits non-commercial use, distribution, and reproduction in any medium, The aim of the present work is to optimize the production of
provided the original author and source are credited.
* Corresponding author. Tel.: +20 2 25353100/200/300/400;
laccase by Pleurotus ostreatus under SSF and to evaluate the
Mobile: +20 100 9610341. industrial applications of laccase in the decolorization of several
E-mail address: [email protected] (A.S. Yassin). dyes and in the synthesis of GNPs.

http://dx.doi.org/10.1016/j.btre.2014.11.001
2215-017X/ã 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
32 A.I. El-Batal et al. / Biotechnology Reports 5 (2015) 31–39

2. Materials and methods fungi is highly regulated by nutrients [16]. The main effects of
parameters on laccase production were estimated by subtracting
2.1. Fungal strains the mean responses of parameters at their lower levels from their
corresponding higher levels and divided by the total number of
Seven locally isolated fungal strains (Gliocladuim virens, experimental runs. The adequacy of the model was tested and the
Sclerotiam rolfsii, Penicilluim chrysogenum, Pleurotus ostreatus, parameters with statistically significant effects were identified
Gliocladuim deliquescence, Rhizoctania solani and Penicilluim using Fisher’s test for the analysis of variance (ANOVA).
citrinum) were used in the study obtained from the culture
collection in the Pharmaceutical Microbiology Laboratory Drug 2.7. Radiation of fungus
Radiation Research Department (NCRRT, Egypt). All strains were
microscopically identified and kept on potato dextrose agar (PDA) The process of irradiation was carried out using 60Co Gamma
at 4  C and periodically sub-cultured to maintain viability. All Chamber (4000-A-India) at a dose rate 10.28 kGy/h at the time of
strains were tested for production of laccase enzyme. experiment. Seven days old Pleurotus ostreatus slant about
(8  106 spores/ml) was irradiated at different doses (0.1, 0.25,
2.2. Fermentation medium 0.5, 0.75, 1, 1.5 and 2 kGy) then cultivated at optimized conditions
for laccase production. Non-irradiated culture was used as control.
Fermentation was done in 250 ml Erlenmeyer flasks, where 8 ml
of distilled water were added to 5 gm carbon source (66% moisture 2.8. Laccase partial purification and characterization
content) [14]. The chosen concentrations of inducers were then
added (according to the experiment design) and autoclaved at Ammonium sulphate was added to the cell free filtrate obtained
121  C for 20 min. The fungus was added to the medium as a 2 ml from Pleurotus ostreatus to attain 80% saturation and the flask was
spore suspension (8  106 spores/ml) and incubated at 29  C kept at 4  C for 48 h. Content was centrifuged at 2415 g for 15 min at
statically in complete darkness. 4  C and the supernatant was discarded. The pellet was dissolved in
a 50 ml, 1 mM citrate phosphate buffer pH 5. The precipitate was
2.3. Extraction of enzyme desalted by dialysis bag to remove low molecular weight
substances and other ions that interfere with the enzyme activity
After seven days, the whole contents of the flask were soaked in as previously described [17]. Protein concentration was quantified
100 ml, 1 mM citrate phosphate buffer (pH 5) for 2 h and put in a using the Bradford assay with bovine serum albumin as standard
shaker at 200 rpm (LAB-Line R Orbit Environ, U.S.A) and then [18].
extracted in tincture press, centrifuged in cooling centrifuge The effect of pH on the activity of partially purified enzyme was
(Hettich Universal 16 R, Germany) for 10 min at 6  C and 2415  g to studied by incubating it with the following buffers for 7 min:
remove particulate. citrate phosphate buffer for pH (3–5) and sodium phosphate for pH
(6–8). The effect of temperature on activity was determined by
2.4. Enzyme assay incubating the enzyme in water bath in the range from 30  C to
90  C with 10  C increments for (15 min). The effect of 5 doses of
Laccase activity measurement was performed spectrophoto- gamma radiation (2, 3, 4, 5 and 6 kGy) on the activity of laccase was
metrically (JASCO V/560 UV/Vis, Japan) at wavelength of 525 nm in studied. Also, the effect of several activators and inhibitors such as
a reaction medium containing 1 mM syringaldazine (e = 65 mM 1 Cu2+, Zn2+ and Mg2+, used as sulphate salts and Ca2+, Cd2+, Co2+ and
cm 1), 50 mM phosphate buffer pH 5 and culture filtrate. Oxidation Ba2+ used as chloride salts and EDTA with the concentration of
of syringaldazine was monitored by measuring the increase in 1 mM. Laccase activity was monitored under standard assaying
absorbance for 4 min. Enzyme activity was expressed in units (U); conditions.
one unit was defined as 1 mmol of syringaldazine oxidized per min The reaction assay mixture of laccase was incubated with
[15]. activators or inhibitors, optimized buffer and syringaldazine and at
respective optimum temperature. The change in absorbance was
2.5. Screening of carbon source and nitrogen source measured spectrophotometrically to evaluate the influence of
these activators and inhibitors on enzyme activity. Results were
Four agricultural wastes were screened as carbon sources expressed as percentage of the control (non-treated laccase).
for production of laccase. Banana peelings (dried in oven at 55  C
for 36 h), spent coffee ground (brought from local coffee factory), 2.9. Decolorization of dyes
rice straw and wheat bran flakes (brought from local market) were
all tested. Six nitrogen sources of natural and synthetic origin were Five dyes namely methyl orange, trypan blue, ramazol brilliant
screened which are yeast extract, tryptone, malt extract, ammoni- red, ramazol brilliant blue and ramazol brilliant yellow (Dye Star
um sulphate, urea and ammonium chloride. company, Germany) were chosen to test the enzyme’s ability to
remove their color. A volume of 0.1 ml of the stock solution
2.6. Experimental factorial design (20 ppm) was added to 2 ml distilled water and 2 ml of the partially
purified enzyme extract with activity 417 U/ml respectively, the
The statistical software package (Minitab 16, U.S.A) was used for percentage reduction of color was monitored for 3 h and was
designing the experiment, regression analysis of experimental data determined spectrophotometrically (JASCO V/560 UV/Vis, Japan)
and in plotting the relation between variables. The effects of the six by monitoring the absorbance at the characteristic wavelength of
variables in two level form namely: malt extract (1% nitrogen each dye. The decolorization efficiency (R%) was calculated as
content or 2% nitrogen content), Tween-80 (0.01%(v/v) or 0.02%(v/ follows:Dye decolorization percentage = [(Initial absorbance fi-
v)), CuSO4 (0.625 mM or 1.25 mM), resorcinol (10 mg or 20 mg), nal absorbance)/(initial absorbance)]  100
DL-Methionine (5 mg or 10 mg) and tannic acid (2.5 mg or 5 mg) Initial absorbance indicated absorbance of the untreated dye at
were assessed. The possible interactions between them were the characteristic peak and the final absorbance indicated
investigated using 32 experiments; the choice of the variables was absorbance of dye after treatment with laccase at the same peak
based on the fact that the production of ligninolytic enzymes by after 3 hours.
 A.I. El-Batal et al. / Biotechnology Reports 5 (2015) 31–39 33
[(Fig._1)TD$IG]
Table 1
Results of screening carbon sources.

Carbon source Laccase activity (U/gfs)

Wheat bran 4610
Banana peelings 1280
Rice straw 569
Spent coffee ground 135

2.10. Preparation and characterization of GNPs

GNPs were prepared as previously described [19], briefly,

to 3 ml of laccase enzyme, containing 417 IU/mg, 0.1 ml of
tetrachloroauric acid with concentration of (10 mg/1 ml) was
added, (49% purity). The reaction mixture was stirred properly
using magnetic stirrer, within 90 min the yellow colored solution Fig. 1. Main effects of variables showing the positive effect of nitrogen source,
started changing to pink then violet, detected visually and by UV/ Tween-80, CuSO4 and methionine.
Visible spectrophotometer indicating the formation of GNPs.
Average particle size and size distribution were determined by
(PSS-NICOMP 380-ZLS) particle sizing system (St. Barbara,
California, USA). UV/Visible Spectra of GNPs were recorded using 3.2. Screening of carbon source and nitrogen source
a spectrophotometer (JASCO V-560UV/Vis, Japan) operated at a
resolution of 1 nm from range of 200–700 nm and observed The screening of the four agricultural wastes showed that
absorption peak at 550 nm due to excitation of surface plasmon wheat bran which is an abundant byproduct formed during wheat
vibration in GNPs solution or the SPR band. FT-IR measurements flour preparation was the highest in laccase activity 4610 (U/gfs)
were carried out using a spectrophotometer (JASCO FT/IR-6300 compared to other sources (Table 1). When screening different
infra-red) by employing KBr pellet technique. The size and nitrogen sources, malt extract gave the highest laccase activity
morphology of synthesized GNPs were recorded using transmis- (8460 U/gfs) compared to yeast extract (7205 U/gfs) and tryptone
sion electron microscope, TEM (JEM-100CX.TEM JEOL, Japan). TEM (6500 U/gfs) (Table 2). Wheat bran and malt extract were both used
studies were carried by drop coating GNPs onto carbon-coated to support growth of Pleurotus ostreatus.
TEM grids. The film on the TEM grids were allowed to dry, the extra
solution was removed using blotting paper.
3.3. Experimental factorial design

2.11. Effect of temperature, gamma radiation and different volumes of

After carrying out 32 experiments, reflecting different combi-
HAuCl4 on GNPs synthesis
nations of the variables (Table shown in supplementary data), the
results revealed that run number 28 gave the highest enzyme
The effects of temperature (40 –100 with 10  C increments),
activity of (32,450 U/gfs) with all of the variables in their higher
radiation (2, 2.5, 3, 3.5, 4, 5 and 6 kGy) and different concentrations
level except for tannic acid that is in its lower level.
of tetrachloroauric acid (0.1, 0.2, 0.3, 0.4 and 0.5 ml of 10 mg/ml
Main effects plotting showed the positive effect of nitrogen
solution) were assessed to study their effect on the formation of
source, Tween-80, CuSO4 and methionine. In case of nitrogen
GNPs. Blank sample was done in case of radiation before mixing
source effect, a major difference between means was noticed.
with HAuCl4.
However, tannic acid had negative effect in its higher level as
shown in Fig. 1.
3. Results
The significance of the effect of the six variables was assessed
using one way (ANOVA). Both the T-value and P-value statistical
3.1. Screening for laccase producing fungi
parameters were used to confirm the significance of factors studied
as shown in Table 3. The results shown proved that nitrogen source,
After screening of the seven fungal strains, Pleurotus ostreatus
Tween-80 and tannic acid all had a significant effect with
was chosen due to its relatively high laccase activity (4610 U/gfs),
(P = <0.05).
(where gfs is defined as the number of units of enzyme produced
The model determination coefficient (R = 0.81) suggested that
from 5 g fermented substrate), compared to other fungi and
the fitted model could explain 81% of the total variation which
consequently, Pleurotus ostreatus was used to optimize laccase
implies a satisfactory representation of the process by the model.
production throughout the whole study.
The coeffecient of determination (R-value) always lies between
0 and 1. As the closer the value of R is to 1.0, the stronger the
model and the better it predicts the response. The analysis of
variance for the selected factorial model showed that the model
Table 2 was significant with a model F-value of 17.75 (P = <0.05) as shown
Screening of nitrogen sources. in Table 4, as the larger the magnitude of the T-value and smaller
Nitrogen source Laccase activity (U/gfs) the P-value, the more significant is the corresponding coefficient
[20].
Malt extract 8460
Yeast extract 7205 The mathematical expression for the relationship between the
Tryptone 6500 six variables for laccase production is given below in equation
Ammonium sulphate 5420 shown. The equation allows the prdection of the response in
Urea 1789 further future experiments where a relationship between tested
Ammonuim chloride 819
factors is expressed based upon the experiments we did.The
34 A.I. El-Batal et al. / Biotechnology Reports 5 (2015) 31–39
[(Fig._2)TD$IG]
Table 3
Test of significance of variables.

Term T P
Nitrogen source 8.94 0.000*
Tween-80 2.61 0.015*
CuSO4 1.63 0.115
Methionine 1.99 0.057
Resorcinol 1.26 0.218
Tannic acid 2.59 0.016*
*
Significant.

mathematical formula is originally: [Y = a + bx], where Y is

the enzyme activity, a is the constant (slope of the line) and x
is the concentration of the variable (we have six variables). The
numbers before every factor are constants generated by the
program based upon our results. The variables are as folows: malt
extract (1% nitrogen content or 2% nitrogen content), Tween-80
(0.01%(v/v) or 0.02%(v/v)), CuSO4 (0.625 mM or 1.25 mM), resor-
cinol (10 mg or 20 mg), DL-methionine (5 mg or 10 mg) and tannic Fig. 2. Actual enzyme activity versus predicted activity.

acid (2.5 mg or 5 mg). Substitution in the equation by the

concentarions of the six variables will give the predicted enzyme
activity.Predicted enzyme activity = 8511 + 7460 [nitrogen Ca2+ had an enhancing effect on activity of laccase with different
Source] + 2207 [Tween 80] + 1397 [CuSO4] + 1590 [methionine] + extents where Cu2+, and Mg2+ gave the highest activation (15%) at
1054 [resorcinol] 2197 [tannic Acid] the concentration used (Fig. 7). Whereas, inhibitors as Cd2+, Co2+
Fig. 2 shows the actual enzyme activity and the predicted and Ba2+, caused decrease in the activity of laccase, with Co2+
activity with an equation describing the relationship between giving highest inhibition reaching 70% (Fig. 8). However, EDTA did
them. not inhibit laccase activity at the concentration used.

3.4. Effect of gamma irradiation on growth of Pleurotus ostreatus and 3.6. Dyes decolorization
laccase activity
Decolorization of four of the used dyes exceeded 50% within 3 h
The results showed that as the radiation dose increased, the and was confirmed by the decrease in absorbance in the
growth of Pleurotus ostreatus decreased gradually (Fig. 3), conse- characteristic wavelength of every dye. The highest decolorization
quently, the production decreased. As for the activity of the value was obtained in case of methyl orange and trypan blue,
produced laccase, it was highly affected by irradiating the fungus as almost no decolorization was detected in case of ramazol yellow.
it decreased to almost half (17,200 U/gfs), compared to the non-
irradiated enzyme (32,450 U/gfs). The decrease in activity was 3.7. Preparation and characterization of GNPs
directly proportional to the increase in the dose until complete loss
in enzyme activity at 1.5 and 2 kGy. Formation of GNPs was confirmed by the formation of violet
color after 90 min at room temperature that gave a significant peak
3.5. Laccase partial purification and characterization at 550 nm. Size distribution of the formed GNPs using DLS and TEM
imaging of GNPs showed highly mono dispersed GNPs with size
After precipitation of the enzyme using ammonium sulphate, range of 22–39 nm. The FTIR spectrum of laccase before and after
total activity decreased from 675,000 to 622,000 U/1500 ml but the formation of GNPs (Figs. 9 and 10), showed the change in the
specific activity increased from 112.5 to 204 U/mg. The enzyme corresponding peaks of functional groups before and after
kept 90% of activity at pH 6 with abrupt decrease before and formation of GNPs, expressing change in intensity of the major
beyond that value (Fig. 4). Thermal stability of purified laccase peak at 3016 cm 1 that corresponds to OH and/or NH functional
showed that at temperature 40  C, laccase exhibited the highest groups and the peak of 1631 cm 1 corresponds to carbonyl group,
activity while above 60  C laccase activity decreased sharply and at both could be ascribed to secondary amide structure.
80  C only 10% of initial activity remained after 15 min incubation
(Fig. 5). Gamma irradiation of the enzyme at 2 kGy decreased its 3.8. Effect of temperature, gamma radiation and different volumes of
activity to half, whereas up to 5 and 6 kGy, where 20% of the HAuCl4 on GNPs synthesis
activity remained (Fig. 6). Activators including Cu2+, Zn2+, Mg2+ and
Incubation of laccase enzyme in the presence of HAuCl4 at
different temperatures showed that as temperature increased,
Table 4 absorbance increased which indicated higher concentration of
Analysis of variance (ANOVA) results for experiments. formed GNPs. Testing the effect of gamma radiation on the
production of GNPs showed that increasing the dose of radiation
Source DFa Sum of squares Mean squares Fb Pc
increased the production of GNPs; maximum GNPs production was
Regression 6 2138109479 356351580 17.75 0.000
noticed at 5 kGy. No color was detected in blank sample (radiation
Residual error 25 501854395 20074176
Total 31 2639963874 before mixing with HAuCl4). In case of effect of different
concentrations of HAuCl4 on GNPs synthesis, the best volume of
R2 = 81.0%, R2 (adjusted) = 76.4%.
a
DF: degree of freedom.
HAuCl4 was 0.3 ml as it gave the highest concentration of
b
F: F-value. GNPs; further increase in gold volumes caused decrease in GNPs
c
P: probability. concentration
 A.I. El-Batal et al. / Biotechnology Reports 5 (2015) 31–39 35
[(Fig._3)TD$IG]

Fig. 3. Effect of Gamma radiation on the growth of the fungus and consequently the production of the enzyme. As radiation dose increased, production decreased.

4. Discussion environment similar to its natural habitat, therefore encouraging

growth of the fungus, also it is an abundant source for
The most efficient lignolytic fungi are the basidiomycetes. They hydroxycinnamic acids, particularly ferulic and p-coumaric acids,
could be either white or brown-rot fungi, both of which are which are known to stimulate laccase production and it was
taxonomically so close to each other that they sometimes appear in successfully used in laccase production by Trametes versicolor
the same genus. Almost all species of white-rot fungi were UAMH 8272 providing several fold increase in laccase activity
reported to produce laccase to varying degree [21]. After screening [24,25].
seven fungal strains, Pleurotus ostreatus (a well-known white-rot For the nitrogen source to be used, malt extract was selected as
fungus) was chosen due to its relatively high laccase activity it gave significantly higher yield of laccase compared to other
compared to other laccase producing fungi. Pleurotus ostreatus is a synthetic nitrogen sources. Malt extract served as nitrogen and also
common edible mushroom also known as Oyster mushroom. It as carbon source in the growth of Cyathus bulleri where it resulted
was first cultivated in Germany as a subsistence measure during in a much higher yield of laccase than in mineral medium [26].
the World War I [22]. It is now grown commercially around the For laccase application in industrial processes, large amounts of
world for food. enzyme are required. The major aim of the study was to find the
Increasing the production of lignolytic enzymes may be optimized conditions for maximum laccase production. Reaching
achieved by modifying the source of carbon and nitrogen in the the optimized production conditions using the conventional one
medium. Since the high cost of the enzyme is a major limitation in factor at a time technique would be quite laborious and time
using laccase in an industrial scale; using agricultural wastes not consuming. One of the currently available statistical designs to
only decreases the cost but also solves an environmental problem predict the behavior of a reaction is the factorial design. Such
[23]. Wheat bran is an abundant byproduct formed during wheat design of experiments completely explains the reaction and brings
flour preparation; it has been selected to perform the present study out the finer details by carrying out selected experiments. The
for its high yield of laccase. Wheat bran’s physical integrity serves variables chosen to assess their effects on laccase production were
as a supporting material and it provides the fungus an nutrients, surface active agents or possible inducers for enzyme
[(Fig._5)TD$IG]
[(Fig._4)TD$IG]

Fig. 4. Effect of pH on enzyme activity. The highest activity was observed at around Fig. 5. Effect of temperature on enzyme activity. The highest activity is observed at
pH 6. 40  C.
 36 A.I. El-Batal et al. / Biotechnology Reports 5 (2015) 31–39
[(Fig._6)TD$IG]
laccase, consequently, resorcinol and tannic acid were chosen. The
effect of resorcinol was not significant but the optimized laccase
production was observed at high concentration of resorcinol.
Attempts were made to increase laccase production by the
addition of the reported laccase inducer tannic acid to enhance
the expression of laccase gene at the transcription level in the
growth medium [31]. However, the optimized production condi-
tion required low concentration of tannic acid with significance of
(p = 0.016) which might be due to the reaction between the
produced laccase and tannic acid, which resulted in making laccase
in an undetectable state by syringaldazine since tannic acid is one
of the traditional screening reagents for laccase [32].
The effect of copper on laccase synthesis was studied in
Trametes versicolor and Pleurotus ostreatus among several other
white-rot fungi [33,34]. As laccase is a multi-copper oxidase in its
structure, the availability of copper in the medium might allow the
synthesis of the enzyme. In addition, the presence of copper in
Fig. 6. Effect of radiation on enzyme activity. Radiation decreased the activity of the Pleurotus ostreatus cultures decreases the activity of extracellular
laccase enzyme.
proteases which might degrade laccase [35]. However, copper
present in high concentration was extremely toxic to microbial
production or activity. Their choice depended on previous studies cells [36]. In the present study, copper was not a significant
done, in addition to the nature of the enzyme and its chemical variable indicting that copper was not a critical component in both
structure. concentrations which was quite unexpected.
Nitrogen source had always been an important nutrient for the Gamma radiation was used in many cases to induce general
growth of fungi and the production of enzymes. However, several metabolic processes and consequently increases enzymes produc-
fungi require the concentration of nitrogen to be in excess to tion due to the well-known phenomena of “Hormesis”; which is
produce laccase, while other fungi produce laccase only when the stimulation of any system by low doses of environmental,
induced by nitrogen starvation. Lentinula edodes and Phanerochaete biotic and abiotic stress factors including pathogens, physical
chrysosporium provide examples of improved laccase production in and chemical agents [37]. However, the reduction of growth and
nitrogen sufficient media [27,28]. A nitrogen deficient medium was decrease of enzymes production by gamma radiation had also been
however required for high production of laccase in Pycnoporus recorded by other studies. The results obtained showed that, as the
sanguineus (cinnabarinus) [29]. Our results supported the first radiation dose increased, Pleurotus ostreatus growth decreased
finding showing that laccase production was in excess with the which was in agreement with other studies as in case of the strain
higher concentration of malt extract (2% nitrogen content) as it was Pleurotus sajor-caju [38]. The decrease in growth accompanying the
a significant variable (p = 0.000). This is probably due to the fact increase in dose (up to 1.5 kGy) and subsequent decrease in laccase
that fungi require nitrogen for their growth and their general production, might be due to reduction in the viable count of fungi
metabolic processes and so providing nitrogen in excess as a result of the over accumulation of free radicals that usually
subsequently increases enzyme production. accompany the gamma irradiation process, when these rays
For the surfactant Tween-80, it was a significant variable interact with water molecules in an organism, they generate
(p = 0.015), as high concentration of the enzyme was usually transient free radicals that can cause additional indirect damage to
accompanied by high concentration of Tween-80. The addition DNA and so causes injury in the microbial cells resulting in
of the surfactant Tween-80 has resulted in higher yields of incomplete inhibition [39]. Complete inhibition of fungal growth
ligninolytic enzymes in certain fungi because there is evidence that and subsequent loss of enzyme activity were detected with 2 kGy,
these surface acting agents result in higher permeability of oxygen which might be due to break down of DNA structure of cells by that
and extracellular enzyme transport through the cell membranes of dose of gamma irradiation resulting in complete death [40].
fungi [29,30]. For a variety of industrial applications characterization of
Most common inducers used for laccase production included produced enzyme would be a crucial step in order to know the
phenolic compounds, which are structurally related to lignin or conditions (pH, temperature, gamma radiation and activating and
lignin derivatives, considered to be the natural substrate for inhibiting ions) that the enzyme could function. Optimum pH for
[(Fig._7)TD$IG] [(Fig._8)TD$IG]

Fig. 7. Activators and their effect on activity of the enzyme. Cu+2, and Mg+2 gave the Fig. 8. Inhibitors and their effect on the activity of the enzyme. Co+2 gave the
highest activation. highest inhibition.
 A.I. El-Batal et al. / Biotechnology Reports 5 (2015) 31–39 37
[(Fig._9)TD$IG]
cleavage of azo dyes [48]. The laccase oxidative transformation of
dyes depends on their chemical structure. The presence of ortho-
hydroxy groups with respect to the azo link was found to enhance
the decolorization rates of azo-dyes with laccase whereas nitro
groups stabilized the dye molecules against laccase action [49].
Green synthesis of nanoparticles using microorganisms or
enzymes provides advancement over chemical and physical
method as it is cost effective, environment friendly, easily scaled
up for large scale synthesis and in this method there is no need to
use high pressure, energy, temperature and toxic chemicals [50].
Studies have shown that the secreted proteins/enzymes and
reducing agents such as amino acids, peptides and organic acids in
biological entities, are found to be responsible for nanoparticle
production. Similarly, in this study, laccase from Pleurotus ostreatus
Fig. 9. FTIR spectrum for laccase before GNPs formation, arrows point to peak served as a rich source for the proteins and free amino groups
numbers that corresponds to OH and/or NH functional group at 3089 cm 1 and the reducing gold into GNPs. These compounds contain functional
carbonyl group at 1635 cm 1. groups that play a role in the reduction and hence the synthesis as
well as the stabilization of nanoparticles [51,52]. These proteins
laccase exhibited variation which may be due to changes in create electrostatic attraction of negatively charged carboxylic
the reaction caused by the substrate (syringaldazine), oxygen or groups, therefore stabilizing these nanoparticles by “capping” to
the enzyme itself. The highest activity of the produced laccase was prevent their aggregation through the creation of repulsive forces
at pH 5 with syringaldazine as a substrate in agreement with the [53]. Laccase was performing the reduction process as a protein
previous work [41]. Relative high thermostability is an attractive and not as an active enzyme as laccase in its active form was
and desirable characteristic of an enzyme. In general, the optimum actually catalyzing oxidation and upon exposure to increasing
temperature for laccase activities can differ from one strain to temperature or gamma radiation, laccase lost its activity as
another, with a range for most fungal laccases being 50–70  C [42], breaking down the integrity of its protein structure and exposing of
in our case, laccase had optimum temperature at 30–50  C and various amino acids began.
rapidly lost activity at temperatures above 60  C which might be FTIR measurements (Figs. 9 and 10) were carried out to identify
due to breaking down the integrity of laccase protein structure and the possible interactions between gold ions and enzyme protein
so losing much of its activity [43,44]. which acted as reducing agent to synthesize and stabilize gold
In general, laccase responds similarly to several inhibitors of nanoparticles. Enzyme protein contains three main functional
enzyme activity. Many ions such as azide and halides can bind to groups, including the amino, carboxylic, and thiol group, which are
the type 2 and type 3 copper atoms, resulting in the interruption of easily used as active sites to modify the other molecules or
internal electron transfer with the subsequent inhibition of activity nanomaterials. FTIR spectrum confirmed the presence of the
[45]. EDTA did not inhibit laccase activity as was observed with the functional groups, 3016 cm 1 peak corresponded to OH and/or NH
laccase obtained from an unidentified basidiomycete [46]. functional groups and presence of carbonyl group could be
Some of the most toxic dyes are amino-substituted azo dyes, ascribed to the peak of 1631 cm 1 [54].
which are often mutagenic and carcinogenic. Current methods for Our finding was in agreement with previous studies [55], which
dye-decolorization are chemically derived and include adsorption, characterized the GNPs produced by marine microalgal strain of
chemical transformation, and incineration [47]. It has been Tetraselmis suesica and according to that study, these functional
suggested that enhanced microbial decolorization of dyes may groups could be used in bioconjugation and/or immobilization of
provide a less expensive and more environmentally acceptable various compounds.
alternative to chemical treatment. An advantage of using fungal The broad band contour which appears in the range of
oxidative mechanisms to degrade azo dyes over other micro- 3000–3400 cm 1 is the summation of associated intermolecular
organisms is that it is possible to avoid the formation of hazardous hydrogen bonds arising from —NH2 and —OH groups in protein
breakdown products such as anilines formed by the reductive molecules which becomes much broader and more intense after
the reaction with gold ions, indicating that the N—H vibration is
[(Fig._10)TD$IG] affected due to the gold attachment and revealing that nitrogen
atoms are the binding sites for gold on protein [56]. The peaks at
1637 cm 1 and 1151 cm 1 arise from a carbonyl stretching
vibration and phenolic groups which shows the carbonyl stretch-
ing vibration from the carboxylate ions and the hydroxyl stretching
vibration from the phenolic ions in the protein [57]. This spectrum
indicates that the secondary structure of the protein of laccase is
affected as a consequence of reaction with the gold ions or binding
with the GNPs.
Based on previous studies [12], the key role of exposing thiol
groups of a-amylase for GNPs formation is high temperature
(70  C) that destructs the appropriate folding of a-amylase
and exposes hydrophobic and hidden groups with reductive
ability and makes it possible to form nanometallic structures. The
effect of temperature was determined by carrying out the reaction
Fig. 10. FTIR spectrum for laccase after GNPs formation, arrows point to peak using (0.3 ml of 10 mg/ml) of HAuCl4 at different temperatures. It
numbers showing the change in intensity of the major peak at 3016 cm 1 that
corresponds to OH and/or NH functional groups and the peak of 1631 cm 1 that
was found that as temperature increases, the GNPs synthesis rate
corresponds to carbonyl group, both could be ascribed to secondary amide increases and the time taken for color conversion was much
structure. reduced. At room temperature, there was an initial lag period for
38 A.I. El-Batal et al. / Biotechnology Reports 5 (2015) 31–39

the formation of GNPs and the synthesis time was longer, reaching Radiation Research Department and the financial support was
90 min. At 100  C, only 10 min were required to get the highest provided by NCRRT.
absorbance (3.2) indicating the highest productivity of GNPs, this
result was in agreement with previous studies [13]. The radiation-
Appendix A. Supplementary data
induced synthesis is one of the most promising strategies because
it is simple, clean and has harmless feature [58]. During radiation,
Supplementary data associated with this article can be found, in
when aqueous solution is exposed to gamma radiation, it creates
the online version, at http://dx.doi.org/10.1016/j.btre.2014.11.001.
solvated electrons which are able to reduce metal ions forming
nano metals. Exposure of the extract to different doses of radiation
was performed after addition of HAuCl4, surface plasmon References
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