Screening and Isolation of Novel Glutaminase Free L-asparaginase from Fungal
Sathyanarayana N. Gummadi
L-asparaginase (E.C.188.8.131.52) has been commonly used for the treatment of acute
lymphoblastic leukemia in adults and children. It is also used in food industry
to reduce acrylamide formation during the preparation of fried food items containing
starch at high temperatures. Several microorganisms from the diverse group of
bacteria and yeast were reported to be used for L-asparaginase production however,
many of the strains also coproduce L-glutaminase which is highly undesirable
as it results in cellular stress and neurotoxicity. Thus identification of new
sources for the production of glutaminase free L-asparaginase needs to be explored.
In this study, we screened endophytic fungi isolated from trees of moist deciduous
and semi evergreen forests of the Western Ghats and plants growing in Rono Hills,
Arunachal Pradesh, India for the production of glutaminase free L-asparaginase.
Using a simple agar plate assay, we found that 33 strains were positive for
the L-asparaginase activity among which 19 strains showed glutaminase free L-asparaginase
activity. Our results show that: Alternaria sp. endophytic in
the leaf of Withania somnifera and growing in the moist deciduous forest
of the Western Ghats showed maximum enzyme activity. Optimization of process
parameters reveal that maximum L-asparaginase production was observed at 96
h of fermentation and high concentration of glucose in the medium as the carbon
source inhibited enzyme production in Alternaria sp. This is the first
report on production of glutaminase free L-asparaginase by fungal endophyte
January 28, 2014; Accepted: April 06, 2014;
Published: June 02, 2014
L-asparaginase (E.C.184.108.40.206) is an enzyme which is present in diverse types
of microorganisms, plants and animals (Mohan Kumar et
al., 2013). L-asparaginase has been commercially used to prevent acrylamide
formation in fried food products (Pedreschi et al.,
2008; Mohan Kumar et al., 2013). L-asparaginase
from bacterial sources has been reported to be used especially as a therapeutic
agent in the treatment of acute lymphoblastic leukemia in children (Muller
and Boos, 1998; Pieters et al., 2011). This
enzyme depletes malignant cells by preventing the formation of essential growth
factors for tumor development. L-asparaginase production by various microorganisms
including, Erwinia carotovora (Maladkar et al.,
1993), Escherichia coli (Cedar and Schwartz,
1968; Wei and Liu, 1998), Aspergillus sp.
(Sarquis et al., 2004) and also from marine derived
fungal endophytes such as Fusarium sp., Phomopsis sp., Trichoderma
sp. and Sargasam wightii (Thirunavukkarasu
et al., 2011) has been studied extensively. A recent report revealed
that clinical E. coli L-asparaginase is an important drug in treatment
of patients with Acute Lymphoblastic Leukemia (ALL) (Rytting,
2012). Though it is a strong candidate for treatment of ALL, several secondary
complications hinder the use of this enzyme in tumor treatment including side
effects such as breathing problems, neural disorders, pancreatitis and also
affects reproductive fertility (Duval et al., 2002).
Pyrococcus furiosus and its mutants MTCC 5580-5582 produces L-asparaginase
which was found to be highly stable (Kundu et al.,
2013). Aspergillus tamari and Aspergillus terreus have been
used for production of L-asparaginase but the yields were very low due to the
presence of glutaminase and urease (Sarquis et al.,
2004). Aspergillus niger and Aspergillus oryzae have been
preferred because of their high yields (Laan et al.,
2008; Eisele et al., 2011). L-asparaginase
from Bacillus licheniformis (RAM 8) with low glutaminase levels was optimized
for the removal of acrylamide that is formed in baked and fried food (Mahajan
et al., 2012). Although the production of L-asparaginase has been
reported from various organisms, the major constraint in commercialization is
L-glutaminase coproduction with L-asparaginase wich leads to toxicity. Very
few reports are available on glutaminase free L-asparaginase produced by microorganisms.
An intercellular glutaminase free L-asparaginase from few microorganisms such
as Pectobacterium carotovorurm MTCC 1428 (Kumar
et al., 2011), Vibrio succinogenes (Distasio
et al., 1982), Pseudomonous stutzeri (Manna
et al., 1995), Pyrococcus furiosus and its mutants MTCC 5580-5582
(Kundu et al., 2013) has been reported. Endophytic
fungi reside inside living tissues of all groups of plants without causing any
disease in them. These fungi are known to produce novel bioactive compounds
(Gunatilaka, 2006; Weber, 2009;
Suryanarayanan et al., 2009; Debbab
et al., 2011). These fungi have also been studied for their use in
biological control of plant diseases (Vega, 2008; Rocha
et al., 2011). Our recent studies and those by others indicate that
endophytic fungi are also a source of novel industrial enzymes (Nagaraju
et al., 2009; Rajulu et al., 2011;
Suryanarayanan et al., 2012; Robl
et al., 2013). In an earlier study, it was reported that many of
the endophytic fungi associated with marine algae produce L-asparaginase (Thirunavukkarasu
et al., 2011). In this study, we screened endophytic fungi isolated
from trees of moist deciduous and semi evergreen forests of the Western Ghats
and plants growing in Rono Hills, Arunachal Pradesh, India for the production
of glutaminase free L-asparaginase.
MATERIALS AND METHODS
L-asparagine and L-glutamine was procured from Sigma-Aldrich. All chemicals used in the production medium and protein analysis were of analytical grade and of the highest purity from SRL, Hi-Media and Merck, India.
Isolation of endophytes: From each plant species, mature healthy leaves
or outer bark were collected, washed thoroughly in running tap water and cut
into segments (0.5 cm2) and surface sterilized by serial washing
in 70% ethanol and sodium hypochlorite (Suryanarayanan
et al., 1998). They were then plated on antibiotic-amended Potato
Dextrose Agar (PDA) medium in petri dishes, sealed using ParafilmTM
and incubated in a light chamber with 12 h light: 12 h dark cycle for 28 days
at 26±1°C (Bills and Polishook, 1992). The
tissue segments were observed periodically and the endophytic fungi growing
out of them were cultured in PDA slants and were identified using standard taxonomic
keys (Ellis, 1971, 1976; Subramanian,
1971; Barnett and Hunter, 1972; Von
Arx, 1974; Sutton, 1980; Onions
et al., 1981; Ellis and Ellis, 1987).
Plate assay for L-asparaginase: A modified Czapek Dox (CD) medium (glucose
2 g L-1, L-asparagine 10 g L-1 or L-glutamine 10 g L-1,
KH2PO4 1.52 g L-1, KCl 0.52 g L-1,
MgSO4.7H2O 0.52 g L-1, FeSO4.7H2O
0.01 g L-1 and agar 20 g L-1) was used for plate assay
(Thirunavukkarasu et al., 2011). A 2.5% stock solution of phenol red
was prepared in ethanol (pH 6.2) and 3 mL of this was added to 1000 mL of Czapek
Dox medium. A mycelial plug (5 mm diameter) cut from the growing margin of the
colony of an endophyte or phellophyte was placed in a petri dish containing
20 mL of this medium. After 72 h of incubation at 26±1°C, the appearance
of a pink zone around the fungal colony in an otherwise yellow medium indicated
L-asparaginase activity (Gulati et al., 1997).
In order to study the effect of glucose on growth and enzyme production, fungal
endophytes were inoculated on modified CD media with and without 2% (w/v) glucose.
Fungal growth and the enzyme zone were measured as described earlier.
Shake flask experiments for enzyme production: Fungal endophyte strains
producing glutaminase free L-asparaginase were maintained and sub-cultured every
7 days in PDA medium and incubated at room temperature. The production of L-asparaginase
was optimized in modified CD medium (Kumar et al.,
2010). Modified CD medium contained glucose 2.0 g L-1, L-asparagine
10.0 g L-1or L-glutamine 10.0 g L-1, KH2PO4
1.52 g L-1, KCl 0.52 g L-1, MgSO4.7H2O
0.52 g L-1, (NH4)2SO4 5.0 g L-1,
FeSO4.7H2O 0.01 g L-1 and the initial pH was
set to 6.2 (Thirunavukkarasu et al., 2011; Kumar
et al., 2011). Seven days old fungal endophyte mycelium plug was
cut (~5 mm diameter) using cork borer and inoculated in 50 mL modified CD medium
in a 250 mL Erlenmeyer flask. The culture flask was incubated at 30°C and
180 rpm in an orbital shaker for 120 h. Samples were withdrawn at regular intervals
of 24 h and used for the measurement of L-asparaginase production. In order
to study the effect of carbon and nitrogen source, varying concentrations of
glucose (1.0, 2.0 and 5.0 g L-1) and L-asaparagine (5.0, 10.0 and
20.0 g L-1) was incorporated in CD medium and growth was carried
out for five days. The samples were collected for every 24 h and assayed as
Enzyme assays: The culture filtrates were centrifuged at 10,000xg for
10 min at 4°C and their protein contents were determined by Lowry method
(Lowry et al., 1951). The enzyme assay reaction
mixture contained 0.5 mL of 0.1 M potassium phosphate buffer pH 8.0, 0.1 mL
of 40 mM L-asparagine and 100 μg of extracellular protein (culture filtrate
of each endophyte) and final volume was made upto 2.0 mL with distilled water.
Reaction mixture was incubated at 37°C in stirred water bath for 30 min
and enzymatic reaction was stopped by adding 0.5 mL of 1.5 M Trichloro Acetic
Acid (TCA). The reaction mixture was centrifuged at 10,000xg for 5 min at 4°C
to remove precipitates. The ammonia released was determined using colorimetric
method by adding 200 μL Nesslers reagent into a tube containing 100
μL supernatant and 1700 μL distilled water. This mixture was vortexed
and incubated at room temperature for 20 min and absorbance was measured at
436 nm (Kumar et al., 2010). Ammonium sulphate
was used to plot the standard curve to determine ammonia released in the reaction
mixture (Mahajan et al., 2012). One unit (IU)
of L-asparaginase activity is determined as the amount of enzyme needed to liberate
1 μmol of ammonia per min at 37°C when L-asparagine is used as substrate
(Imada et al., 1973; Kumar
et al., 2010). Glutaminase activity was measured by direct nesslerization
as described by Mashburn and Wriston (1964), using
L-glutamine as the substrate. One Unit (IU) of L-glutaminase activity is calculated
as the amount of enzyme needed to liberate 1 μmol of ammonia in presence
of L-glutamine per min at 37°C. Specific activity of protein is defined
as enzyme acitivity per milligram of protein used in the assay.
Statistical analysis: All the experiments are performed at least four times and one way analysis of variance (ANOVA) was performed in Minitab 16. A p<0.05 was considered significant.
RESULTS AND DISCUSSION
Screening of fungal endophytes for glutaminase free L-asparaginase:
Thirty three different fungal endophyte strains were collected from plant hosts
in Western Ghats and Rono Hills in India. The samples were critically scrutinized
for the production of L-asparaginase by plate assay method. The plate assay
method involves the use of Czapeck Dox medium with L-asparagine as a nitrogen
source and phenol red as a pH indicator to monitor the changes in pH. If the
organism produces L-asparaginase, it could grow on a normal plate containing
L-asparagine and degrades it into aspartic acid and ammonia. Release of ammonia
in the medium changes the pH towards alkaline which is indicated by a visible
change in medium colour to pink. This pink zone was initially used to screen
the fungal endophytes (Gulati et al., 1997; Thirunavukkarasu
et al., 2011). For example C. lunata showed pink zone only
when grown in plate containing L-asparaginase and not in plate containing L-glutamine
(Fig. 1a), suggesting that the strain produces glutaminase
free L-asparaginase. While another strain, L. theobromae showed pink
zone when grown both on L-asparagine as well as in L-glutamine plate implying
that this strain produces both the enzymes (Fig. 1b).
In order to screen the endophytes that produces glutaminase free L-asparaginase, all 33 fungal endophytes were allowed to grow on the modified CD medium containing L-glutamine as a nitrogen source and the colour change was monitored. Endophytes that produced pink colour zone on L-glutamine medium containing plates (Fig. 1b) were eliminated. Of the fungi studied, only a species of Periconia and S. intermedia lacked asparaginase activity (Table 1). Twelve fungi, viz., Acremonium sp., Alternaria sp. 2, Aspergillus sp., Botrytis sp., C. cladosporioides, Corynespora sp., Cylindrocladium sp., Fusarium sp., L. theobromae, Pestalotiopsis sp. 1 and 2 and Sordaria sp., were positive for both the enzymes (Table 1). Some strains producing only glutaminase free L-asparaginase (Fig. 1a) were selected and plate assay was performed. Nineteen fungal endophytes were shown to be producing glutaminase free L-asparaginase which were further examined for the highest activity of L-asparaginase production by measuring the colony diameter (mm) and diameter of the pink colour zone (mm) which indicates the enzyme production quantitatively (Fig. 2).
Effect of glucose on colony growth and enzyme activity by plate assay:
Previous reports showed that concomitant addition of L-asparagine and glucose
inhibits growth and activity (Geckil et al., 2006).
Few reports showed that presence of glucose in media marginally favours activity
and is a good choice compared to other substrates because of its low cost, making
it an economically viable process (Kumar et al.,
2010). The existing reports are contradictory regarding the effect of glucose
and nitrogen. Hence, to check the effect of glucose on growth and L-asparaginase
production, plate assay was performed in the presence and absence of glucose.
Plate assay for screening fungal endophytes
producing L-asparaginase, (a) Curvularia lunata grown on plates
containing L-asparagine and L-glutamine showing pink zone only when L-asparagine
was used as the substrate, (b) Lasiodiplodia theobromae showing
pink zone on both plates containing L-asparagine and L-glutamine as substrate
and (c) Glutaminase free L-asparaginase showing pink colour zones around
the fungal endophyte colony
Among the 19 fungal endophytes screened and isolated that exhibited glutaminase
free L-asparaginase activity, 10 strains which showed relatively higher enzyme
activity zone were taken and grown in the presence and absence of media containing
2 g L-1 glucose. Results showed that the presence of glucose inhibited
colony growth and enzyme activity in most of the endophytes (Fig.
3a and b). Curvularia lunata, Phomopsis sp.
and Dreschlera sp. showed an increase in both colony growth and enzyme
activity in the presence of glucose. Presence of glucose for Corynespora
sp. did not have much effect on the colony growth but the enzyme activity
zone was drastically reduced (Fig. 3b) (Narayana
et al., 2008). As a widely used carbon source, effect of glucose
on L- asparaginase activity has been well studied over the years.
|| Isolated novel fungal endophytes screened for glutaminase
free L-asparaginase production (plate assay)
Glucose is reported to induce the production of L-asparaginase in several microbes
including certain strains of Serratia marcescens (Sukumaran
et al., 1979) and Bacillus circulans (Hymavathi
et al., 2010). Glucose acts as a repressor and inhibits L-asparaginase
production in a few organisms such as Streptomyces albidoflavus (Narayana
et al., 2008) and Serratia marcescens (Heinemann
and Howard, 1969). It has been reported that when 0.4% of glucose was used,
L-asparaginase production was not inhibited but by further increase in the glucose
concentration upto 1%, pH of the medium changed from alkaline (pH 7.5-8.0) to
acidic (pH 5.2-6.9) and L-asparaginase production was inhibited (Heinemann
and Howard, 1969).
L-asparaginase production by shake flask: L-asparaginase producing microorganisms
either produce this enzyme constitutively or upon induction. The physico-chemical
conditions for L-asparaginase production differ among various microorganisms
(Savitri and Azmi, 2003). In this study, endophytes
produced L-asparaginase when grown only in the presence of L-asparagine. L-asparaginase
produced was determined by colorimetric estimation of released ammonia at 436
nm. Ten fungal endophytes, which exhibited highest activity in plate assay were
taken for production studies in shake flask. Among the 10 fungal endophytes,
WS/Alternaria sp. 2 exhibited the highest activity of 1.17±0.04
U mg-1 at 96 h, followed by TPM/Alternaria sp. 0.5±0.06
U mg-1 (Fig. 4).
Colony growth and enzyme activity for
fungal endophytes. Plate assay method was used to calculate the colony
growth and enzyme activity by measuring the colony zone diameter (mm)
and enzyme activity zone diameter (mm) in the plates. Experiment was repeated
thrice and the error bars represent standard error of mean
Effect of glucose on (a) Colony growth
and (b) Enzyme activity by plate assay in L-asparaginase producing fungal
endophytes. There is a significant decline in L-asparaginase activity
of DIOM/Phomopsis sp., WS/Alternaria sp., 2 and EU/Corynespora
sp. in the presence of glucose (p<0.05, by ANOVA). In WS/Alternaria
sp. 1, Alternaria alternata and WS/Alternaria brassicicola,
the mean colony growth decreases significantly (p<0.05, by ANOVA) in
the presence of glucose. Experiment was repeated thrice and the error
bars represent standard error of mean
Though other class of microorganisms has been reported to produce L-asparaginase
activity with higher and comparable values, this is the first report to show
glutaminase free L-asparaginase activity by fungal endophytes.
Effect of carbon source on L-asparaginase production: Glucose is commonly
used as the primary carbon source for most of the microorganisms producing primary
and secondary metabolites. Since we have already confirmed that glucose play
a key role in enzyme activity by plate assay method, it is essential to optimize
the glucose concentration for maximizing the enzyme production in shake flasks.
WS/Alternaria sp. 2, was grown in CD media containing 1, 2 and 5 g L-1
of glucose, respectively and samples were collected for every 24 till 120 h
(Fig. 5a). Our results showed that culture grown in 1 g L-1
of glucose exhibited a maximum activity of 2.1 U mg-1 at 96h, whereas
culture grown in 2 and 5 g L-1 for 96 h showed 0.34 and 0.31 U mg-1,
respectively (Fig. 5b). Thus it is confirmed that 1 g L-1
was optimal for L-asparaginase production in WS/Alternaria sp. 2. The
enzyme production beyond 96 h decreases drastically which could be due to several
Specific activity of glutaminase free
L-asparaginase in shake flask experiments. WS/Alternaria sp. 2
shows maximum specific activity. Experiment was repeated thrice
and the error bars represent standard error of mean
Effect of glucose on specific activity
of glutaminase free L-asparaginase produced in shake flask method, (a)
WS/Alternaria sp. was inoculated in the modified CD medium containing
various concentration of glucose and (b) Specific activity at varying
Effect of L-asparagine on specific activity
of glutaminase free L- aspariginase in shake flask at varying concentrations
of L-asparagine 5, 10 and 20 g L-1. Experiment was repeated
thrice and the error bars represent standard error of mean
It was observed that the production of the L-asparaginase was significantly
reduced in the presence of glucose at higher concentrations, where it act as
repressor for L-asparaginase in Enterobacter aerogenes (Mukherjee
et al., 2000) and similar trend was observed in Fusarium sp.
(Thirunavukkarasu et al., 2011), Serrtia
marcescenss (Heinemann et al., 1970) and
Erwinia aroideae (Liu and Zajic, 1972).
Effect of nitrogen concentration on L-asparaginase production: To study
the effect of nitrogen source concentration on L-asparaginase production by
WS/Alternaria sp. 2, different concentrations of L-asparagine (5, 10
and 20 g L-1) was supplemented in the media. Culture grown in 10
g L-1 of L-asparagine showed maximum activity of 1.8 U mg-1
at 120 h, followed by 1.3 and 0.9 U mg-1 for cultures grown on 5
and 20 g L-1, respectively (Fig. 6). The time dependent
increase in the enzyme activity shows that L-asparaginase expression increases
in the presence of L-asparagine, suggesting that it is nitrogen regulated and
inducible as observed in other microbes (Sarquis et al.,
2004). Effect of varying concentration (0.5-2.0%) of L-asparagine as a sole
nitrogen source on L-asparaginase production was studied in Streptomyces
ABR2 and the optimum concentration was determined to be 1.0% (Mostafa
and Salama, 1979; Sudhir et al., 2012).
Another report showed that 1% (w/v) of L-asparagine in the media exhibited maximum
production of L-asparaginase by Aspergillus terreus MTCC 1782. The activity
of L-asparaginase decreases with increase in concentration of L-asparagine above
1% which might be due to substrate inhibition (Shaffer
et al., 1988; Baskar and Renganathan, 2011).
In this study, several endophytes have been identified that are capable of producing glutaminase free L-asparaginase. Among all the screened endophytes, WS/Alternaria sp. 2, produced maximum glutaminase free L-asparaginase. The optimal concentrations of glucose and L-asparagine were found to be 10 and 1 g L-1, respectively. Under these conditions, the strain produced a maximum specific activity of 1.65 U mg-1. This is the first report on the production of glutaminase free L-asparaginase by endophytic fungi Alternaria sp. Although, preliminary, the study gains importance as it identifies a novel eukaryotic (and hence possibly more human-compatible than a bacterial source of the enzyme) source of such a desirable enzyme for therapeutic uses. Further media components screening and optimization in shake flasks and bioreactors are needed to enhance the productivity.
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