Vantamuri and Kaliwal, IJPSR, 2016; Vol. 7(12): 4978-4987.

E-ISSN: 0975-8232; P-ISSN: 2320-5148

IJPSR (2016), Vol. 7, Issue 12 (Research Article)

Received on 22 June, 2016; received in revised form, 07 September, 2016; accepted, 13 September, 2016; published 01 December, 2016

PRODUCTION OF LACCASE BY NEWLY ISOLATED MARASMIUS SP. BBKAV79 IN SOLID

STATE FERMENTATION AND ITS ANTIPROLIFERATIVE ACTIVITY
A. B. Vantamuri 1 and B. B. Kaliwal *2
Department of Studies and Research in Biotechnology and Microbiology 1, Karnatak University, Dharwad-
580003, Karnataka, India.
Department of Studies and Research in Biotechnology and Microbiology 2, Davangere University,
Davangere-577 002, Karnataka, India.
Keywords: ABSTRACT: The aim of the present study was to investigate the
Anti-cancer, Laccase, Marasmius sp. production of laccase by Marasmius sp. BBKAV79 in Solid State
BBKAV79, Solid state fermentation Fermentation (SSF) and its antiproliferative activity. Experiments were
Correspondence to Author:
also conducted to determine the variables that could affect activity of
Prof. B. B. Kaliwal laccase. In particular, the effects of the initial moisture content, pH,
temperature, and inoculum volume, incubation period in addition to
Post Graduate Department of Studies
carbon and nitrogen sources were evaluated. Rice bran was found to be
and Research in Biotechnology and
Microbiology, Davangere University, the best supported lignocellulosic substrate for extracellular laccase
Davangere-577 002, India. production under SSF. Highest activity of laccase was achieved at pH 6.0
and temperature at 40°C. Inoculum size and incubation period for laccase
Email: [email protected] production at 2000µl culture broth and 10 days respectively. The
presence of starch and ammonium sulphate in the growth medium was
found to be favourable for the production of the laccase. Such a high
activity was obtained without any addition of inducers. Thus, the
indigenous isolate is found to be a potential producer of laccase using
SSF and can be exploited for further biotechnological applications. The
process also promises economical utilization and value addition of agro-
residues and it potently suppressed the proliferation of tumor cell lines
KB, MCF7 and VERO with an IC50 value of 88.61 µl, 49.5 µl, and
~67603 µl respectively, signifying that it is an anticancer protein.
INTRODUCTION: A number of anthropological The increasing exploitation has resulted in need of
activities has resulted in extensive pollution 1 technology for remediation of toxic chemicals and
aggravating the need to understand the toxic dyes 7. Enzymatic degradation of these stains and
impacts of xenobiotics in the environment. The dyes through microbial approach has become an
deteriorating conditions of water and soil quality integrated and preferred method of addressing the
has affected a wide range of organisms including issues of environmental pollution globally and
fishes 2, 3, 4, 5 and amphibians 6. hence enzyme production is noted to be an
QUICK RESPONSE CODE emerging field of biotechnology 8.
DOI:
10.13040/IJPSR.0975-8232.7(12).4978-87 Annual world sales figures are close to billion
dollars 9 with increasing number of patents and
Article can be accessed online on: research articles related to this field. Since the
www.ijpsr.com biotechnological applications require large amounts
of low cost enzymes, one of the appropriate
DOI link: http://dx.doi.org/10.13040/IJPSR.0975-8232.7 (12).4978-87

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Vantamuri and Kaliwal, IJPSR, 2016; Vol. 7(12): 4978-4987. E-ISSN: 0975-8232; P-ISSN: 2320-5148

approaches for this purpose is to utilize the laccase by Marasmius sp. BBKAV79 and
potential of lignocellulosic wastes, some of which antiproliferative assay was performed.
may contain significant concentrations of soluble
carbohydrates and inducers of enzyme synthesis MATERIALS AND METHODS:
ensuring efficient production of ligninolytic Microorganism: Organism screening for laccase-
enzymes 10, 11. SSF is defined as any fermentation producing microbes on potato dextrose agar plates
process performed on a non-soluble material that (PDA) containing indicators namely, guaiacol,
acts both as physical support and source of ABTS, syrindalzine and tannic acid resulted in
nutrients in absence of free flowing liquid 12. The isolation of 8 fungal strains. Isolates showing
low moisture content means that fermentation can positive reaction were maintained on PDA plates at
only be carried out by a limited number of 30°C and stored at 4°C. The best laccase producing
microorganisms, mainly yeasts and fungi, although isolate was identified by molecular characterization
some bacteria have also been used 13. technique at Agharkar Research Institute, Pune,
India and identified as Marasmius sp. BBKAV79
Laccase (E.C. 1.10.3.2, p-benzenedial: oxygen (NCBI Gen Bank accession number KP455496,
oxidoreductases) belongs to oxidoreductase class KP455497).
and is able to catalyse the oxidation of various
aromatic compounds (particularly phenol) with the Screening of different lignocellulosic substrates:
concomitant reduction of oxygen to water 14. The following agro wastes were used for the initial
Laccase was first discovered in the sap of the screening: rice bran, rice straw, sugarcane bagasse,
Japanese lacquer tree Rhusvernicifera, and its saw dust and pigeon pea waste. All of them were
characteristic as a metal containing oxidase was locally procured and were sterilized at 121 °C and
discovered by Bertrand in 1985 15. Since then, 15lb pressure for 20 mins and were used for the
laccases have also been found in various study.
basidiomycetous and ascomycetous fungi and thus
far fungal laccases have accounted for the most Media preparation: The media was prepared by
important group of multicopper oxidases (MCOs) adding 2% of each of the agro wastes to the
with respect to number and extent of Mineral Salt (MS) medium. The media was then
characterization 15. The potential use of laccases in sterilizing at 121ºC and 15lb pressure for 20
biotechnology has stimulated the need to discover minutes. This agro wastes mineral salt (AWMS)
suitable enzymes in large quantities. Production of media was used for the study. The 250 ml conical
any enzyme including that of lacasses is known to flasks with 100 ml of the above AWMS media and
be affected by fermentation factors such as, were inoculated with well grown fungal discs from
medium composition, pH, temperature and PDA plates and the flasks were adjusted at a pH of
aeration. There have been reports describing 6 and incubated at 30 ºC in shaking incubator with
increased production of extracellular laccases in 150 rpm for 5 days and the enzyme extraction and
many species of white rot fungi when grown on assay were done as guaiacol assay method 21. The
natural substrates, such as cotton stalk 16, molasses best agro waste which supports the maximal
wastewater 17, wheat bran 18 and barley bran 19. laccase assay were selected and used for the
optimization study.
However, the literature support towards
optimization is found to be very limited. Utilization Laccase Harvesting: After specified days of
of industrial and agricultural wastes for laccase incubation, laccase was extracted by a simple
production is an effective way to reduce production contact method. For this purpose 100mL of Sodium
costs and also simultaneously utilise these acetate buffer (pH 5.5) was added in the flasks. The
substrates efficiently 20. On other hand, strategies flasks were placed on incubator shaker at 150 rpm
for effective large scale production of laccases for 1 hour. Mixture was then filtered with filter
through SSF is found to be limited as well. Hence, paper and the filtrate was centrifuged at 10,000 rpm
the present work is planned to study various for 10 minutes at -10 ºC to remove all spores and
physicochemical parameters for optimization of other impurities. The supernatant was collected and
subjected to laccase assay 22.

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Effect of initial moisture content on laccase bran broth on laccase production was studied by
production: To investigate the influence of the inoculating the Marasmius sp. BBKAV79 and the
initial total moisture content (before autoclaving) of flasks were incubated at room temperature and
the substrate was carried out under various initial assay was done as guaiacol assay method.
moisture content adjusted with salt solution.
Samples containing 5 moisture levels (30%, 45%, Effect of nitrogen sources for laccase
50%, 65% and 70%) were prepared by moistening production: Nitrogen sources as tryptone, peptone,
5 g of studied substrates with salt solution 23. The sodium nitrate, ammonium sulphate and sodium
optimum initial moisture content of solid substrate carbonate were added to rice bran broth on laccase
achieved by this step was fixed in subsequent production was studied by inoculating the
experiment. After soaking, the sample was again Marasmiussp.BBKAV79 and the flasks were
dried as described above and percent moisture incubated at room temperature and assay was done
content was calculated as follows, Percent of as guaiacol assay method.
moisture content (initial) of solid medium = (wt. of
Extracellular laccase assay: The Laccase activity
the rice bran - dry wt.) x 100 / dry wt. 24, 25.
was assayed at room temperature by using
Effect of pH for laccase production: The effect of 10mMGuaiacol in 100 mM sodium acetate buffer
various pH viz., 3, 4, 5, 6, 7, 8 and 9 on laccase (pH 5.0).The reaction mixture contained 3.0 ml
production in rice bran broth was done by acetate buffer, 1.0 ml Guaiacol and 1.0 ml enzyme
inoculating the flasks (with the above pH) with source. The change in the absorbance of the
Marasmius sp. BBKAV79 and the flasks were reaction mixture containing guaiacol was
incubated and assay was done as guaiacol assay monitored at 470 nm for 10 mins of incubation
method. using UV Spectrophotometer. Enzyme activity is
measured in U/ml which is defined as the amount
Effect of temperature for laccase production: of enzyme catalysing the production of one
The effect of various temperature ranges on laccase micromole of coloured product per min per ml 21, 26,
27, 28
production in rice bran broth was studied by .
inoculating the Marasmius sp. BBKAV79 and the
incubating the flasks at different temperatures viz., Calculation:
37°C, 40°C, 45°C , 50°C and 55°C. The flasks Volume activity (U/ml) =
were incubated for 5 days and assay was done as
guaiacol assay method. ΔA470nm/min × 4.0 × Vt × dilution factor
€ × Vs
Effect of inoculums volume for laccase Where,
production: The effect of inoculum volume was Vt = final volume of reaction mixture (ml) = 5.0
studied by adding different levels of inoculum Vs = sample volume (ml) = 1.0
(250, 500, 1000 and 2000) from 5 days old fungal €=extinction co-efficient of guaiacol = 6,740/M/cm
broth to the rice bran broth and incubated at room 4 = derived from unit definition & principle
temperature and assay was done as guaiacol assay
method. Assay of Antiproliferative Activity:
Antiproliferative activity has been reported for
Effect of incubation period for laccase many mushroom proteins. Performed cytotoxicity
production: The effect of incubation period on by the 3-(4,5-dimethylthiazol-2-yl)-2,5-
laccase production in rice bran broth was studied diphenyltetrazolium bromide (MTT) assay. The
by inoculating with Marasmius sp. BBKAV79 and cell lines were cultured in DMEM medium
incubated at room temperature for various time (Invitrogen) which was supplemented with 10%
intervals. The enzyme was extracted and its activity FBS (Gibco, Invitrogen) and 1% Antibiotic–
was determined. Antimycotic 100X solution (Thermo fisher
Scientific).
Effect of carbon sources for laccase production:
Carbon sources as maltose, sucrose, starch,
fructose, glucose and arabinose were added to rice

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The cells were seeded at a density of approximately The selection of a substrate for SSF processes
5×103 cells/well in a 96-well flat-bottom micro depends upon several factors mainly related with
plate (NEST-Biotechnology) and maintained at cost and availability and thus may involve
37ºC in 95% humidity and 5% CO2 for overnight. screening of several agro-industrial residues.
Different concentration (100, 50, 25, 12.5, 6.5, Moreover, the utilization of this type of supports
3.125 µg/300 mL) of purified laccase were treated. helps to solve the pollution problems caused by
The cells were incubated for another 48 hours. The their disposal. According to these results, rice bran
cells in well were washed twice with phosphate was selected to be used for improving laccase
buffer solution, and 20 µL of the MTT staining production as solid substrate. Rice bran was the
solution (5mg/ml in phosphate buffer solution) was best for laccase productions in our result, which
added to each well and plate was incubated at 37ºC. may be due to it contain protein and carbohydrates
After 4h, 100 µL of di- methyl sulfoxide (DMSO) enhance fungal growth and increase its metabolic
was added to each well to dissolve the formazan activity. Laccase synthesis was encouraged by
crystals, and absorbance was recorded with a 570 phenolic compounds containing in rice bran,
nm using micro plate reader. leading to increasing of laccase production. This
induction mechanism may help to humiliate lignin
Formula: or aromatic compounds in rice bran to supply
Surviving cells (%) = Mean OD of test compound further nutrients especially carbon and nitrogen 29.
/Mean OD of Negative control ×100 As previously reported similar results that the rice
bran was best substrate for maximum laccase
Inhibiting cells (%) =100- Surviving cells production from Streptomyces chartreusis and
Pleurotusostreatus, respectively 30, 31 and Hendro et
Statistical analysis: Data were analysed by one- al.,32 have evaluated rice straw and rice husk for the
way analysis of variance (ANOVA) followed by production of laccase by white rot fungi.
Tukey’s Highest Significant Difference (HSD) post
hoc test. Readings were considered significant Influence of initial moisture content on laccase
when P was ≤0.05. production: In the present study an initial moisture
content of 65%was found optimum for laccase
RESULTS AND DISCUSSION production. The laccase production varied
Screening of lignocellulosic substrates: Different significantly between 65% and all other
lignocellulosic substrates like rice bran, rice straw, percentages of moisture content with an exception
sugarcane bagasse, saw dust and pigeon pea waste being at 70% of moisture content (Graph 2).
were screened for laccase production. But these
substrates showed marked variation with respect to
the laccase production. Rice bran was supported
maximum laccase production followed by other
substrates. The laccase production varied
significantly between rice bran and all other
substrates (Graph 1).

GRAPH 2: EFFECT OF INITIAL MOISTURE CONTENT

FOR LACCASE PRODUCTION FROM MARASMIUS SP.
BBKAV79

Moisture is another key parameter to control the

growth of microorganisms and metabolites
production in solid state fermentation 23.
GRAPH 1: SCREENING OF LIGNOCELLULOSIC
SUBSTRATES FOR LACCASE PRODUCTION BY
MARASMIUS SP. BBKAV79

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The optimum moisture level in SSF is governed by Most reports indicated that the initial pH between
the water holding capacity of the substrate, the type 4.5 and 6.0 was suitable for enzyme production 14,
46
of the end product, and water requirements of the . Sodenet al.,47 have reported that the laccase
fungus 33, 34. Higher and lower water contents showed maximum activity at pH 6.5 by
adversely affect the primary metabolic activities of Pleurotussajor-cajuLac4.Ronak and Modi,30 have
microbes, leading to secretion of lower activities of suggested that the Streptomyces chartreusis
ligninases in secondary growth 35, 36. Low moisture showed maximum laccase production at pH 8.
contents in SSF have also been reported to decrease
the enzyme formation and metabolic activities of pH is important parameters that determine the
fungi due to reduced solubility of nutrients, low growth rate of fungi and significantly affect the
substrate swelling, and higher water tension 37. level of laccase produced. The production of the
enzyme strongly depends upon the initial pH of the
This result is consistent with previously reported culture medium as it may influences many
moisture content 65%, 60%, 66%, 60% and 60 % enzymatic processes and transport of nutrients
by Streptomyces psammoticus, Schyzophylum across the cell membrane 42. At higher alkaline pH,
commune, Pleurotusostreatus, Pleurotusostreatus the enzyme activity decreased gradually, due to the
HP-1 and Pleurotusostreatus PVCRSP-7, difference in redox potential between a reducing
23, 38, 39, 40, 41
respectively . The moisture level in SSF substrate and the type 1 copper in the active site of
has a great impact on the physical properties of the the enzyme and the inhibition of type 3 copper by
substrate. Solid substrates used in SSF are insoluble the hydroxyl ion at higher pH 48. Laccase was
in water therefore; water will have to be absorbed proved to be active pH in the range of 3.3 to 6.0
on to the substrate particles, which could be used and in consensus with date reported by others 49, 50,
51, 52, 53
by the microorganisms for growth and metabolic .
activity. Thus the degree of hydration of the
substrate plays an important role on the growth of Influence of temperature for laccase production:
the fungi and subsequently the enzyme production The results indicate that the Marasmius sp.
12
. BBKAV79 strain was able to produce high laccase
production at temperature 40ºC. The laccase
Influence of pH for laccase production: The production was found to be significantly high at
results showed that the Marasmius sp. BBKAV79 40ºC as compared with other temperatures except
was able to produce maximum laccase production for 45ºC which was found to be insignificant when
at pH 6. The laccase production was found to be compared with 40 ºC (Graph 4).
significantly high at pH 6 as compared with all
other pH conditions (Graph 3).

GRAPH 4: EFFECT OF TEMPERATURE ON LACCASE

PRODUCTION BY MARASMIUS SP. BBKAV79
GRAPH 3: EFFECT OF pH ON LACCASE PRODUCTION BY
MARASMIUS SP. BBKAV79 Temperature is influencing the growth in SSF, the
As previously reported similar results that the production enzyme and their metabolites 54.
highest laccase production observed at pH 6 from Temperature is much significance in the SSF
Pleurotuseous, Rigidoporus sp., Pleurotusostreatus systems because during fermentation there is a
POXA1 and Rigidoporuslignosus, respectively 42, general increase in the temperature of the
43, 44, 45
. fermenting mass due to respiration 55.

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Even though the impact of temperature is more Pleurotuspulmonarius mycelial plugs measuring 10
prominent in the scale up processes it remains an mm in diameter were used for inoculation of 5 g of
inevitable factor in all fermentation systems due to wheat bran 10. Moleds et al., 64 have observed that
its impact on microbial growth and metabolite the highest specific activity for laccase was
production 46. Earlier reported similar results that obtained when 20 Trametes hirsute agar plugs
the highest laccase production was observed at measuring 3 mm in diameter for inoculation 2.5 gm
40ºC from Trametes hirsute 56. Narayanan et al.,57 barley bran or 5 g grape seed was used. Ronak and
have reported that the temperature profile showed Modi, 31 have reported that the maximum
differences in the enzyme activity between rice production of laccase was observed when using 1.5
bran and wheat bran the maximum laccase activity x 104Colony formation unit (CFU). Patel et al., 40
was observed at 30ºC for rice bran when compared have observed that the highest activity of laccase
to wheat bran at 40ºC by Bacillus subtilis. A was obtained when five Plerotusostreatus agar
significant influence of incubation temperature on plugs measuring 8 mm in diameter for inoculation.
ligninolytic enzymes of Pleurotus sp. and Hafiz et al., 65 have showed the maximum ligninase
Dichomitussqualen and other white rot fungi has activity by Trametesversicolor at 15 ml of
been reported, and temperatures ranging from 25 to inoculum. The maximum production of laccase was
30ºC were found optimum for laccase production observed by Streptomyces psammoticus when using
58, 59, 61, 62
. Shanmugam et al., 42 have reported the 1.5 x 107CFU 23.
Pleurotuseous showed highest laccase production
at temperature 50 ºC. Abrahão et al., 62 have Influence of incubation time for laccase
reported a class of isolated Basidiomycetes with an production: The results indicate that the 13 days of
optimum laccase activity at 70ºC. incubation showed excellent laccase production.
The laccase production varied significantly
Influence of inoculum volume for laccase between 13 day of incubation as compared with
production: The results showed that the highest other durations with exceptions being 12, 14 and 15
laccase production was obtained when using 2000 day of incubation which were found to
µl of culture broth. The laccase production was insignificantly vary as compared to 13 day (Graph
found to be significantly high at 2000 µl as 6).
compared with all other volumes of inoculum
considered for present study (Graph 5).

GRAPH 6: EFFECT OF INCUBATION PERIOD ON

LACCASE PRODUCTION BY MARASMIUS SP. BBKAV79
GRAPH 5: EFFECT OF INOCULUM SIZE ON LACCASE
PRODUCTION BY MARASMIUS SP. BBKAV79
In SSF process, the inoculum density is of great
importance. Too low a density may give
Inoculum size plays a significant role in enzyme insufficient biomass and permit the growth of
production in solid state fermentation. A lower undesirable contaminants, while for some processes
level of inoculum may not be sufficient to initiate high inoculum densities may produce too much
the growth, whereas a higher level may cause biomass and deplete the carbohydrates and
competitive inhibition 63. Thus, determination of nutrients necessary for product formation 54.
optimum inoculum size becomes a crucial step in Previously reported the optimum laccase
SSF. In previous studies different inoculums size production by Pleurotusostreatus was observed
have been used for laccase production, four after 7 days of incubation in solid state

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fermentation medium contain wheat straw as

substrate 39. Masutti et al., 66 have reported the
highest level of laccase production from
Pleurotusostreatus was occurred after 28 days by
solid state fermentation. Marasmius sp. and a solid
substrate of rice straw demonstrated the highest
laccase activity on day 10. Abdulkareem, 46 has
reported that the maximum level of laccase
production was observed at 15 days of incubation
period by Pleurotusostreatus.
GRAPH 8: EFFECT OF NITROGEN SOURCES ON
LACCASE PRODUCTION BY MARASMIUS SP. BBKAV79
Influence of carbon sources for laccase
production: The results showed that the starch The ligninolytic enzymes have been seen to be
supported the maximum laccase activity. The regulated by the usable concentration nitrogen in
laccase production varied significantly between the medium. The low nitrogen level can stimulate
Starch and all other carbon sources conditions the ligninoyltic enzyme production 40. Previously
(Graph 7). reported similar results that the peptone as a
nitrogen source supported maximum laccase
production from Lentinusedodes and
Pleurotusostreatus, Pleurotusostreatus,
Streptomyces psammoticus, Bacillus subtilis
MTCC 2414, Pleurotusostreatus PVCRSP-7,
Trametes hirsute, respectively 70, 71, 23, 57, 41, 56.
Ronak and Modi, 30 have showed that the yeast
extract supported maximum laccase production by
Streptomyces chartreusis. Elisashvili et al.,72 have
shown that medium with ammonium sulphate has
GRAPH 7: EFFECT OF CARBON SOURCES ON LACCASE given highest levels of laccase activity in Cerrena
PRODUCTION BY MARASMIUS SP. BBKAV79 unicolor. Zahida et al.,68 have reported that the
greatest production of laccase, chick pea powder
The carbon source is powerful nutrition regulation was used as nitrogen source in solid state
factors for producing the ligninolytic enzymes. fermentation.
Nature and type of carbon sources are among the
most important factors for any fermentation process Antiproliferative Activity: It is remarkable that
55
. The effect of different carbon sources on laccase some of mushroom components including laccases,
production has been established in the case of lectins, polysaccharopeptides, and antifungal
fungal strains 67. Earlier reported the glucose as a proteins exhibit inhibitory activities towards tumor
carbon source supported the extreme laccase cells. In the present study, the purified laccase
production from Streptomyces psammoticus, demonstrates antiproliferative activity towards
Pleurotusostreatus PVCRSP-7, Coriolusversicolor, tumor cell lines KB and MCF7 and normal cell line
Trameteshirsuta and Pleurotusostreatus LIG 19, VERO IC50 value of 88.61 µl, 49.5 µl, and ~67603
respectively 23, 41, 55, 68, 69. µl respectively (Fig.1) and this compound was not
toxic to the normal cell line. It concludes that the
Influence of nitrogen sources for laccase laccaseis acting as anticancer protein. On the other
production: The results showed that the hand, the present laccase is purified from Solid
ammonium sulphate supported the maximum State Fermentation, which means that the protein is
laccase activity. The laccase production varied very easy to obtain. It is noteworthy that it
significantly between Ammonium sulphate and all possesses further applications of agents for cancer
other nitrogen sources (Graph 8). therapy.

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How to cite this article:

Vantamuri AB and Kaliwal BB: Production of laccase by newly isolated Marasmius sp. Bbkav79 in solid state fermentation and its
antiproliferative activity. Int J Pharm Sci Res 2016; 7(12): 4978-87.doi: 10.13040/IJPSR.0975-8232.7(12).4978-87.

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