8 Egypt. J. Bot., Vol. 55, No.1 pp. 127 - 147 (2015) ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ ــــــــــــــ ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ* E-mail: [email protected]tel.: +201003643976 Biosynthesis of laccase by Aspergillus flavus NG85 Isolated from Saint Catherine protectorate M.I. A. Ali, S. A. Ouf, N.M. Khalil * and M. N. Abd El-Ghany Department of Botany and Microbiology, Faculty of Science, Cairo University, Giza 12613, Egypt. HE MICROFLORAL pictures of Saint Catherine protectorate, Giza Zoo Garden and Cairo University soil were studied. The obtained 31 microbial isolates were qualitatively and quantitatively screened for laccase production. Aspergillus flavus from Saint Catherine protectorate achieved highest laccase production on both solid and liquid media. Identification of this fungal species was further confirmed at the molecular level based on nuclear ribosomal DNA internal transcribed spacer (ITS) identities and was found to be A. flavus strain NG85. The fungus produced statistically highest amounts of laccase after 10 days of growth at 36.7 o C and when growth medium was adjusted at pH 5. D-glucose at a concentration of 24 g/l was the best carbon source. The leading nitrogen source was peptone used at 2.51 g/l. Supplementation of copper sulfate at concentration 10 μM to the optimized growth medium caused an increase of 122% in enzyme yield. The crude laccase preparation of A. flavus NG85 from Saint Catherine protectorate showed antiproliferative activity against colon carcinoma cells (HCT-116) andbreast carcinoma cells (MCF-7) with IC 50 values of 24.3 and 41.3 μg/ml, respectively, and a less inhibitory effect against hepatocellular carcinoma cells (HepG-2). Keywords: Laccase, Aspergillus flavus NG85, Biosynthesis, Optimization, Cytotoxic activity. Laccase (benzenediol oxygen oxidoreductase, EC 1.10.3.2) is a highly unspecific enzyme containing up to 4 copper atoms within their catalytic sites (Duran and Esposito, 2000).It catalyses the oxidation of various phenolic compounds and aromatic amines with molecular oxygen as an electron acceptor (Palmeri et al., 1993). Laccase production has been described in fungi, plants and bacteria (Mayer and Staples, 2002). Yoshida first described laccase in 1883 from the exudates of the Japanese lacquer tree, Rhusvernicifera. However in 1896, for the first time, both Bertrand and Laborde demonstrated laccase to be a fungal enzyme (Levine, 1965 and Thurston, 1994). Laccase production occurs in various fungi over a wide range of taxa. Fungi from the deuteromycetes, ascomycetes (Aisemberg et al., 1989) as well as basidiomycetes (Sadhasivam et al., 2008) are the known producers of laccase. Some soil bacteria were also reported to produce extracellular laccase (Martins et al., 2002). T
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8 Egypt. J. Bot., Vol. 55, No.1 pp. 127 - 147 (2015)
7 cell line) and c-hepatocellular carcinoma cells (HepG-2 cell line).
M.I. A. ALI et al .
Egypt. J. Bot., 55, No. 1 (2015)
142
Discussion
Laccase is most widely distributed in a wide range of higher plants and fungi
(Benfield et al., 1964) as well as in bacteria (Diamantidis et al., 2000). Most of
the laccases described in literature were isolated from higher fungi. Laccases have
been isolated from ascomycetes, deuteromycetes and basidiomycetes (Assavanig
et al., 1992).The present study firstly aimed to isolate fungi and bacteria from
different localities. The microfloral pictures in Saint Catherine protectorate, Cairo
Zoo garden and Cairo University soil were studied. Despite the fact that the Saint
Catherine protectorate soil showed the least species diversity (H= 0.4), compared
with the other tested soils, Aspergillus flavus isolate obtained from this soil
exhibited the highest laccase production on solid as well as on liquid media.
Accordingly, this isolate was chosen to optimize its cultural conditions for
maximum laccase production and activity. It was thought advisable to confirm its
identification at the molecular level. The obtained 545bp-long nucleotide
sequence was deposited in NCBI GenBank and was given a strain identifier, A.
flavus NG85, with accession number: KJ855143. It was found that maximum laccase production by A. flavus NG85 was
reached after10 days of growth. Various incubation periods were achieved by
different microorganisms. The white rot fungus Pycnoporus cinnabarinus produced
maximum level of laccase on day 5 and accounted for about 70% of the total
extracellular protein (Eggert et al., 1996). Laccase production from
Cyathusbulleri was detectable after 2 days and reached maximum on day 7
(Vasdev et al., 1994). The onset oflaccase activity in Trichoderma harzianum
WL1occurred on day 2 and reached its maximum on day 4 and then the rate of
enzyme production declined gradually. The difference in time course of laccase
production by the various fungal systems mainly dependson the source, media
composition and type of inducers (Sadhasivam et al., 2008). Pointing et al. (2000) stated that the optimum temperature range for laccase
production is between 25 and 30oC. However, A. flavus NG85 in this work
showed maximum production of laccase at 36. 7oC.
Maximum production of laccase by A. flavus NG85 occurred in the acidic pH (5). Earlier reports suggested that pH between 4.5 and 6.0 is suitable for laccase
production (Thurston, 1994).
Laccase production by fungi has been found to be largely affected by
nutritional conditions, such as carbon and nitrogen source and related
concentrations and microelements. Laccases are generally produced in low
concentrations by fungi, but higher concentrations could be obtained by adding
various supplements to liquid growth media (Lee et al., 1999 and Vasconcelos
et al., 2000). In this work, the monosaccharide D-glucose was the most potent carbon
source inducing maximum laccase production at an optimum concentration of
24g/l.This is in accordance with Leifa et al. (2007) who reported that utilization
BIOSYNTHESIS OF LACCASE BY ASPERGILLUS FLAVUS …
Egypt. J. Bot., 55, No. 1 (2015)
143
of monosaccharides for laccase production is better compared to sugar alcohol
and complex sugar due to their simple nature.
A. flavus NG85 in this study achieved highest laccase production when using
peptone as a nitrogen source with optimum concentration of 2.51g/l. It was
proven that nitrogen sources and their concentrations were as important
nutritional factors as carbon sources in regulating laccase production (Minussi
et al., 2002). Vahidi et al. (2004) reported that when yeast extract was used as
nitrogen source it increased laccase production. A decrease in enzyme production
was seen when inorganic nitrogen sources were used alone in the growthmedium.
Oppositely, Elisashvili et al. (2001) reported high laccase production in C.
unicolor IBB 62, grown in a medium with ammonium sulfate as the only nitrogen
source.
The data reached in this study showed that supplementation of copper sulfate
at a concentration of 10 μM caused significant enhancement in laccase
production. These findings are in agreement with previous reports showing that
the addition of 2 mM CuSO4 during the exponential growth phase of fungal
growth led to a remarkably increased laccas eproduction (Galhaup et al., 2002;
Couto and Sanroman, 2005).Almost a similar effect was observed in the cultures
of Trametes multicolor MB 49 and Trametes trogii BAFC 463 with copper
concentrations ranging from 0.5 to 2.0 mM. Sadhasivam et al. (2008) found that
copper sulfate supplementation at 1 mM concentration yielded high amounts of
laccase. The formation of laccase by a Bacillus sp. was considerably increased by
addition of 1 mM copper sulfate (Kaushikand Thakur, 2014). Addition of
inducers enhanced the production of laccase at the level of gene transcription.
The promoter regions of the genes encoding for laccase contain various recognition
sites that are specific for xenobiotics and heavy metals. It has been demonstrated that
the Pleurotuso streatus laccase genes poxc and poxa1b are transcriptionally induced
by copper, and several putative metal responsive elements (MREs) were found in
the promoter regions of thes egenes (Faraco et al., 2003).
Urgent need for novel anticancer drugs has paved way for the usage of fungi
and their products withanti-cancer properties. In this work, the crude enzyme
preparation produced by A. flavus NG85 showed high cytotoxic activities against
colon carcinoma cells or breast carcinoma cells and a less inhibitory effect against
hepatocellular carcinoma cells was detected. This effect could be due to laccase
and/or other proteins produced by A. flavus NG85, since the cultivation medium
was oriented to high laccase production. However, it is recommended for future
work to purify and characterize laccase produced by A.flavus NG85. Hu et al.
(2011) purified alaccase from fresh fruiting bodies of the edible white common
Agrocybe cylindracea mushroom. It caused HIV-1 reverse transcriptase
inhibitory activity (IC50=12.7 μM) and antiproliferative activity against HepG2
cells (IC50=5.6 μM) and MCF7 cells (IC50=6.5 μM).
M.I. A. ALI et al .
Egypt. J. Bot., 55, No. 1 (2015)
144
In conclusion, A. flavus NG85 strain isolated from Saint Catherine
protectorate achieved highest amounts of extracellular laccase. Its growth
medium was directed toward maximum biosynthesis of the enzyme. The crude
enzyme extract of the fungal filtrate proved high antitumor potency against colon
carcinoma cells and breast carcinoma cells and a less cell toxicity against
hepatocellular carcinoma cells.
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