L-Asparaginase (L-asparagine amino hydrolase, E.C. 18.104.22.168, LA) catalyzes the
hydrolysis of L-asparagine into L-aspartic acid and ammonia. As the several
types of tumour cells require L-asparagine is an essential amino acid for protein
synthesis; they are deprived of an essential growth factor in the presence of
LA. Effective depletion of L -asparagine results in cytotoxicity for leukemic
cells (Savitri and Azmi, 2003). The LA is present in
many animal tissues, bacteria, plants and in the serum of certain rodents but
not in mankind. Whereas microbial LA has attracted considerable attention since,
the demonstration that LA from E. coli has antitumor activity (Mashburn
and Wriston, 1964; Broome, 1963). The most economical
and the most commonly used microorganisms to produce LA are Erwinia caratovora,
Bacillus sp. (Mohapatra et al., 1995) Corynebacterium
glutamicum and Escherichia coli (Netrval, 1977).
The LA is the first enzyme with antitumor activity is intensively studied
in human beings. It is the enzyme drug of choice for acute lymphoblastic leukemic
in children used in combination therapy.
Wide range of bacteria, fungi, yeast, actinomycetes and algae are very efficient
producer of LA. Among the bacteria, Coliform bacteria (are defined as rod-shaped,
Gram -ve, oxidase negative and lactose fermenting) have a diverse range of enzymatic
activity and are capable of catalyzing various biochemical reactions. Thus,
there is enormous scope for investigation to explore the possibilities of deriving
new products of economic importance. The reported work deals with isolation
of coliform bacteria and optimization of various parameters to get the maximum
yield of L-asparaginase.
MATERIALS AND METHODS
Collection of Water Samples
Water samples were collected from different places of Belgaum, Karnataka,
India in a month of July-August. The reported work was carried out in Department
of Biotechnology, K.L.E.S.s College of Pharmacy, Belgaum, Karanataka,
India. For collection, heat-sterilized bottles (100 mL capacity) were used containing
0.1 mL of a fresh 1.8% (w/v) aqueous solution of sodium thiosulphate to neutralize
the bactericidal effect of any chlorine or chloramines in the water (Senior,
Screening of Coliform Bacteria
Serial Tube Method (Multiple Tube Lactose Fermentation)
MacConkey broth was sterilized (autoclaved at 120°C for 20 min), distributed
equally (5 mL) in each test tubes. All the tubes were inoculated with 1mL of
water sample and kept at 37°C for overnight incubation. Presence of coliform
was further characterized by streaking from positive MacConkey broth tubes to
Eosin-Methylene Blue agar (Dubey and Maheshwari, 2005).
Modified ISP-5 Medium (Rapid Screening Method)
Modified ISP-5 medium (L-asparagine 1 g, Dipotassium phosphate 1 g, trace
salts solution 1 mL, lactose 20 g, phenol red 1 mL, agar 20 g, pH 7.0) was sterilized
(autoclaved at 120°C for 20 min), inoculated with 1 mL of collected water
samples and poured to the petri plates. All the plates were incubated at room
temperature including the control plates, which were without carbon source (lactose).
Identification of Isolates
Isolated colonies were identified through the lactose fermentation test,
Gram staining and biochemical tests such as H2S production, indole
production, MR-VP test, citrate utilization test etc. (Cappuccino
and Shearman, 2006; Sleigh and Duguid, 2005). The
isolated were screened for asparaginase activity as per the direct nesslerization
method. The TLC was performed to check the conversion of L-asparagine into L-aspartic
acid. Mobile phase: n-butanol: acetic acid: water (5:4:1) and ninhydrine as
Optimization of Fermentation Parameters
Both the environmental and nutritional parameters were optimized by using
TGY (Tryptone glucose yeast extract) broth as a Basal medium; Glucose 1 g, Dipotassium
hydrogen phosphate 1 g, Yeast extracts 5 g, Tryptone 5 g, pH 7.0 (Peterson
and Ciegler, 1969) in lab scale fermentor (Sartorius B-lite, Bangalore).
Enzyme activities were determined at regular intervals of 12 upto 48 h.
Optimization of Environmental Parameters
Optimization of pH
The effect of pH on L-asparaginase production was studied by growing AS-2
strain in basal medium of different pH (5, 6, 7, 8, 9 and 10). The pH was maintained
by using phosphate buffer.
Optimization of Temperature
Optimal temperature for the productivity of AS-2 strain was determined by
keeping the inoculated basal media at 27, 30, 34 and 37°C separately.
Optimization of Nutritional Parameters
Optimization of Carbon Source
The basal medium contains 0.1% of glucose as carbon source. In this study,
optimization was done using Glucose and Sucrose with concentrations of 0.1,
0.5, 1.0 and 1.5%, separately. Basal medium with these different carbon sources
with their individual concentrations were sterilized and inoculated with AS-2
strain; incubated at 37°C for 48 h.
Optimization of Nitrogen Source
The basal medium contains 0.5% of yeast extract as nitrogen source. In this
study, optimization was done using Peptone and yeast extract in individual concentrations
of 0.1, 0.5, 1.0 and 1.5%. Basal medium with these nitrogen sources were sterilized
and inoculated with AS-2 strain; incubated at 37°C for 48 h.
Determination of Biomass Production and Optimal Duration
Optimal duration for the enzyme production was measured by using optimized
fermentation parameters. The AS-2 strain was inoculated in optimized TGY broth
and incubated for 40 h. Samples were withdrawn at every hour. The optimal duration,
the time at which the enzymatic activity high was determined by measuring the
liberated ammonia through nesslerization method. Biomass production was measured
by dry weight method.
Partial Purification of L-asparaginase
L-asparaginase was partially purified by the method described by Bilimoria
(1969). Solid (NH4)2SO4 was added to the
supernatant fluid to achieve 20% saturation. The suspension was further centrifuged
and solid (NH4)2SO4 was added to supernatant
to reach 40 and 60% saturation. The precipitates (0-20, 20-40 and 40-60%) were
collected by centrifugation. The collected precipitates were dissolved in Tris-HCL
buffer, pH 8.0 and dialyzed overnight against the same buffer. After the exclusion
of ammonium sulphate, enzymatic activity in each dialyzed solution was determined
by nesslerization method. The fraction showing the highest enzyme activity was
designated as Partially Purified Extract (PPE).
RESULTS AND DISCUSSION
Coliform bacteria are commonly used indicator of sanitary quality of foods
and water. Here, two different methods used for the isolation of coliform mentioned.
In multiple tubes lactose fermentation after the incubation period, we observed
the change in color of MacConkey broth initially red to yellow due to the fermentation
of lactose into the acidic metabolites by coliform bacteria which were differentiated
from non-coliform as well as other gram negative bacteria by streaking EMB agar
from previously positive MacConkey broth tubes. By this method we have screened
two bacteria coded as AS-1 and AS-2.
|| ISP-5 Medium with lactose after 7 days of incubation
|| ISP-5 Medium without lactose after 7 days of incubation
In another method developed in our laboratory in which we modified the original
ISP-5 medium. Which contains L-asparagine, a substrate for the L-asparaginase
and original carbon source i.e., glycerol was replaced with lactose. Phenol
red was incorporated in the medium as pH indicator. Modified ISP-5 medium without
lactose was served as control to observe whether the medium color changes from
yellow to pink due to the lactose fermentation or the conversion of L-asparagine
into the L-aspartic acid and ammonia. We observed high intensity pinkish colour
after 2 days incubation and low intensity color after the 7 days incubation
in modified ISP-5 medium with lactose and without lactose, respectively (Fig.
1, 2). From this we concluded that pinkish color surrounding
the bacterial colonies was due to the liberation of ammonia which makes the
pH of the medium alkaline. Colonies with the pinkish surrounding were coded
as AS-3, AS-4, AS-5, AS-6, AS-7, AS-8, AS-9, AS-10, AS-11 and AS-12 (Fig.
All isolates were firstly subjected to the lactose fermentation and gas production
to satisfy the definition of coliform bacteria. Table 1 shows
the result of lactose fermentation and gas production.
|| Gram staining of AS-2 (100x)
|| Test for lactose fermentation and gas production
|+: Positive reaction, -: Negative reaction
|| Biochemical tests
|+: Positive reaction; -: Negative reaction; ++: Strong positive
reaction; H2S: Hydrogen sulfide; MR: Methyl red; VP: Voges-Proskauer;
Ind: Indole; Pad: Phenylalanine deaminase; Cit: Citrate utilization; Arg:
Arginine decarboxylase; Ure: Urease; -Ve: Gram negative
For the further characterization we selected isolates which were either lactose
fermenting or gas producing or both. Table 2 shows the results
of various biochemical tests carried out to identify the lactose fermenting
and gas producing isolates.
From the identification studies the AS-2 strain was found to be Gram -ve, rod
shaped and lactose fermenting, also producing H2S, utilized citrate
as carbon source and Voges-Proskauer negative. All the characters of AS-2 strain
were compared with the characters of Enterobacteriaceae family described by
Sleigh and Duguid (2005) which help us to conclude that
the AS-2 was closely related to Citrobacter sp. Before the optimization
of fermentation parameters, AS-2 strain was checked for enzyme production by
growing it into TGY broth, which showed positive result. The TLC was performed
to check the conversion of L-asparagine into L-aspartic acid. The results showed
that an Rf value of sample i.e., 0.68 almost the same to that of standard L-aspartic
acid i.e., 0.70.
|| Optimization of pH
|| Optimization of temperature
As the prime requirement of the biotechnological processes is high yielding
organisms, to satisfy this requirement preliminary optimization of various fermentation
parameters are necessary. For this purpose we optimized various environmental
and nutritional parameters. The extracellular pH has a strong influence on the
pathways of metabolism and product generation by micro-organism and optimum
temperature is also important as it affects the conversion efficiency of substrate
into cell mass which affect the product formation, particularly when product
is growth associated. So in this study, we optimized pH and temperature as environmental
parameters for increasing the L-asparaginase yield. Our studies indicated that
pH-8 is optimum extracellular pH with the enzymatic activity (810 IU mL-1)
at 24 h (Fig. 4). The 37°C found to be an optimal temperature
for the L-asparaginase production with enzymatic activity (658 IU mL-1)
at 24 h (Fig. 5).
The components of the fermentation medium should be supplied in an adequate
quantity for growth, energy and building of cellular components and synthesis
of fermented products where carbon and nitrogen sources play an important role.
For this purpose we optimized carbon and nitrogen sources as nutritional parameters.
The present studies stated that 1.0% glucose in basal medium is required for
an optimal production of L-asparaginase with a yield of 650 IU mL-1
(Fig. 6) whereas 1.0% sucrose showed less yield (361 IU mL-1)
at 24 h (Fig. 7). Different nitrogen sources such as peptone
and yeast extracts were used for the production of maximum L-asparaginase and
the results showed that yeast extracts with 1.5% concentration in basal medium
gave maximum enzyme production with a yield of 371 IU mL-1 (Fig.
8) and 1.0% peptone having a lower yield of 315 IU mL-1 at 24
h (Fig. 9).
|| Optimization of glucose
|| Optimization of sucrose
|| Optimization of yeast extract
|| Optimization of peptone
|| Growth curve of AS-2 and optimal duration of L-asparaginase
|| Partial purification of L-asparaginase
|*Protein was determined by method of Lowry
et al. (1951)
Similar kind of result was obtained with the yeast extract during the production
of L-asparaginase from E. aroideae (Liu and Zajic,
By using the optimized fermentation parameters optimal study was determined,
results showed that enzymatic activity was at lowest values in the log phase
and increasing in the exponential phase; at 26 h it reached to the maximum values
and in the early stationary phase (upto 30 h) of the growth cycle the activity
was stable and continued in decreasing at late stationary phase. The results
stated that production of L-asparaginase was growth associated (Fig.
10). As the L-asparaginase is intracellular product, in the purification
studies cells were separated by centrifugation and supernatant was discarded.
A summary of purification steps is shown in Table 3. It is
clear that the enzymatic activity in the protein fraction 20-40% represented
2.15 purification fold with 20% yields and specific activity of 82 IU mL-1.