International Conference on Global Trends in Engineering, Technology and Management (ICGTETM-2016)
L-Asparginase Production by Bacterial and Fungal Source
Gaurav D. Khodape1, Dr. I.D.Patil2, Jayant P.Parpalliwar3
Assistant Professor1, Professor2, Assistant Professor3 Department of Biotechnology, Shram Sadhana Bombay Trust
College of Engineering & Technology Bambhori, Jalgaon - 425-001, Maharashtra, India.
Abstract:
L-asparginase (L-aspargine aminohydrolase), the enzyme which converts L-aspargine to L-aspartic acid and
ammonia, has been used as a chemotherapeutic agent which has anti carcinogenic potential. The study on the
localization of any enzyme plays a vital role in the development of bioprocess. A wide range of microorganisms
such as filamentous fungi; bacteria have proved to be beneficial sources of L-asparginase enzyme. Bacterial isolates
has been screened for the production of L-asparginase and parameters of fermentation process were evaluated for
the production using E.coli for submerged fermentation(SMF) and Aspergilus niger for solid state fermentation
(SSF). Comparative study has been done for the SSF and SMF for identifying the qualitative and quantitative
analysis of the produced enzyme.
Key words: L-asparginase, E.coli, Aspergilus niger, SSF, SMF.
INTRODUCTION:
Enzyme is a biocatalyst which accelerates biological
reactions. There are various sources of enzymes and
it includes microorganisms, higher plants and
animals. Plant enzymes are papain, proteases,
amylases and soyabean lopoxygenase. Animal
enzymes includes lipase, tripsin etc. Microbial
enzymes are classified into two categories namely
extracellular and intracellular. Extracellular enzymes
are secreted out the cell. They help in establishment
in host tissue or decomposition of organic substrates,
e.g. cellulase. Intracellular enzymes remain within
the cell. These are difficult to extract and they have
high economic value, e.g.invertase, asparginase etc.
Intracellular enzymes obtained by breaking the cells
by means of a homogenizer or a bead mill and
extracting them through biochemical process. The
enzyme L-asparginase is one of the most industrially
important enzymes. The discovery of L-asparaginase
(L-asparaginase aminohydrolase), a medicinal agent
for the treatment of malignant tumors, was made in
1922. This enzyme is also a choice for acute
lymphoblastic leukemia, lymphosarcoma and in
many other clinical experiments relating to tumors
therapy in combination with chemotherapy. The
therapeutic potential of this enzyme is well
established, as it has remarkably induced remission in
most patients suffering from acute lymphoblastic
leukemia. With the development of its new functions,
a great demand for L-asparaginase is expected in the
coming years. Guinea pig serum contained a high
activity of L-asparaginase. L-asparginase production
using microbial systems has attracted considerable
attention, owing to cost effective and eco friendly
nature. A wide range of microbes has proved
beneficial sources for this enzyme. L-asparginase
production from bacterial origin can cause
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hypersensitivity in the long term use leads to allergic
reactions.
L-asparginase
production
using
microorganism such as filamentous fungi yeast and
bacteria have proved to be beneficial source. Lasparginase is produced throughout the world by
submerged fermentation technique. This technique
has many disadvantages such as low concentration
production and consequent handling reduction and
disposal of large volumes of water during
downstream processing. Therefore the SMF
technique is a cost intensive and highly problematic
and poorly understood unit operation. SSF is very
effective technique as the yield of product is many
times higher than that of SMF and it also offers many
advantages including resistance to contamination,
ease of product extraction and simpler methods for
treating the fermented residue. The objective of this
study was to isolate L-asparaginase from fungi by
solid-state fermentation using soybean oil extracts as
a substrate and submerged fermentations and partial
purification, and characterization of the crude
enzyme extract. Solid state fermentation generally
defined as the growth of microorganisms on (moist)
solid material in absence or near absence of free
water. SSF involves the growth of microorganism on
moist solid particles in situation in which the spaces
between the particles contain a continuous gas phase
and minimum of visible water. Although droplets of
water may be present between the particles and there
may thin films of water at particle surface. The
interparticle water phase is discontinuous and most of
the interparticle phase is filled by the gas phase. The
majority of water in the system is absorbed within the
moist solid particles SSF offers numerous advantages
over submerged fermentation, this include high
volume productivity, relatively higher concentration
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of products, less effluent generation and simple
fermentation equipment etc.
MATERIALS AND METHOD:
Chemicals, Media and Reagents used are Glucose
(C6H12O6), Luria Bertani broth (LB), Tryptone,
Yeast Extract, Sodium Chloride (NaCl), Eosin
Methylene Blue (EMB), McConkey’s Agar, Lactose
Broth, Crystal Violet, Gram’s Iodine, Saffranin ,
Peptone, Kovac’s reagent, Nutrient Agar, Nutrient
Broth, Sodium Carbonate (Na2Co3), Sodium
hydroxide (NaOH), Copper Sulphate (CuSO4),
Sodium Tartarate, Bovine Serum Albumin (BSA),
Potassium Dihydrogen Phosphate (KH2PO4), LAsparginase, Magnesium sulphate (MgSO4),
Calcium Chloride (CaCl2), Agar agar, Phenol
Reagent (Folin And Ciocalteu’s), Tris HCl
(Hydroxymethyl methylamine), Trichloroacetic Acid,
Nessler’s Reagent, Sodium dihydrogen Phosphate
(NaH2PO4),
Sodium
hydrogen
Phosphate
(NaHPO4), Sodium Cholride (NaCl), Potassium
Chloride (KCl), Dipotassium Hydrogen phosphate
(K2HPO4), Ferrous sulphate(FeSO4), Citric Acid,
Hydrochloric Acid (HCl), Manganese sulphate
(MnSO4), Zinc Sulphate (ZnSO4), Czapek Dox,
Ethanol.
METHODOLOGY:
COLLECTION OF SAMPLE:
10gm of soil sample was collected from premises of
Shram Sadhana Bombay Trust College of
Engineering and Technology, Bambhori, Jalgaon
(MS) (Coordinates: 21° 0' 54.5436'' N 75° 30'
10.3896''E) for the isolation of desired bacteria for
production of L-Asparginase enzyme by submerged
fermentation.
SUBSTRATE PREPARATION:
The Groundnut oil Cake that was needed as a
substrate for production Enzyme by Solid State
Fermentation (SSF) was collected from Balaji Oil
Mills, Erandol, Jalgaon dst. (MS), (Coordinates: 20°
55' 35.6844'' N75° 19' 56.8992'' E) which produces
oil from groundnut seed, cotton seed, and sunflower.
COLLECTION OF MICROORGANISM:
Fungi were collected from National Center for
Industrial Microorganism (NCIM) a division at
National Chemical Laboratory (NCL) Pune,
Maharashtra, India.
SUBMERGED FERMENTATION PROCESS:
Bacterial species was isolated in departmental
laboratory from soil sample collected form Shram
Sadhana Bombay Trust COET, Bambhori, Jalgaon.
By serial dilution method strain was allowed to grow
on nutrient agar media which was then sub cultured.
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The sub cultured strain was then cultured on sterile
McConkeys Agar plate to check for pink colonies of
E.coli which confirmed the E.coli growth. The pink
Colonies were isolated and grown on sterile Nutrient
media (KH2PO4 0.2%, MgSO4 0.1%, CaCl2 0.1%,
Glucose 3%, Agar 2.5%, L-Aspargine 0.6%, pH 6.2
and phenol red indicator 2 drops) and incubated for
48 hours at 370C, there was change in pH from 6.2 to
8.3 which noted using a pH strip which indicated the
production of L-asparginase and also resulted in
formation of pink zone around the colonies. Also a
control was prepared on sterile Petri dish having
composition of (KH2PO4 0.2%, MgSO4 0.1%,
CaCl2 0.1%, Glucose 3%, NaNO3 0.6, pH 6.2 and
phenol red indicator 2 drops) which after incubation
of 48hours at 370C showed no pink zone formation
and there was no change in pH. The isolated bacteria
from pink zone was isolated and inoculated on
nutrient agar slant which was used as starter culture
for submerged fermentation. The sterilized nutrient
medium containing (KH2PO4 0.2%, MgSO4 0.1%,
CaCl2 0.1%, Glucose 3%, peptone 0.5%, yeast
extract 0.3%, L-Aspargine 0.6% was prepared &
sterilized. This sterilized nutrient media was cooled
at room temperature and inoculated with the isolated
strain of E.coli in flask. Inoculated broth was
incubated at 37 0C for 48 hrs at 120 rpm in an
incubator shaker for the fermentation. At the end of
fermentation, 10 ml broth was collected and
centrifuged at 10000 rpm for 10 min at 40C and the
supernatant was carefully collected and used as a
crude enzyme for protein estimation and enzyme
assay which was maintained at 40C.
SOLID STATE FERMENTATION:
Substrate preparation:
Oil industry waste (Groundnut oil cake) was
collected from the Balaji Oil Mills, Erandol, and
Jalgaon. Collected substrate dried but 2% oil and
some percentage of water were maintained. Collected
substrate was exposed to the sunlight for drying for
48 hrs. After sun drying the substrate again dried in
oven at 80 0C for 24 hrs. Oven dried substrate were
made in powdered form in an Electric grinder. The
above grinded substrate was sieved for studying the
effect of particle size and other parameter. The fine
grinded 10 gm substrate was collected in Petri dish
and moistened with salt solution containing
gm/100ml (yeast extract 0.3, peptone 0.15, NaCl 1.5,
NaH2PO4.2H2O 0.61, KCl 0.3, MgSO4.7H2O 0.01,
MnSO4 0.1 and FeSO4 0.1). The substrate was
moisturized to 100% (w/v) by salt solution.
Moisturized substrate was taken in to autoclave and
sterilized for 15 minute at 121 0 C for proper cooking
of the substrate and to increase its amenability for
microorganisms.
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Inoculum preparation:
A microorganism used in the project was Aspergillus
niger (NCIM No.1056, L-Asparginase producer).
The spores of these microorganisms were transferred
aseptically to 100 ml conical flask containing 25 ml
of sterilized inoculum medium (sterilized at 1210C
for 15 minutes) containing g/100ml:(glucose 2, yeast
extract 0.3, peptone 0.5, NaCl 0.5, NaH2PO4 0.61,
KCl 0.3 MgSO4.7H2O 0.01, FeSO4 0.1, KH2PO4
0.75 and K2HPO4 0.75)in laminar air flow. The flask
was then kept in incubator at 37 o C for 72 hrs.
SOLID STATE FERMENTATION:
The enzyme was extracted by a simple contact
method.
After incubation, the fermented oil cake waste
sample was added to conical flask containing 100ml
distilled water (ratio of 1:10 (w:v )) in the laminar air
flow. The flasks were shaken at 150 rpm for 120
minute and material was filtered through muslin cloth
or whatmans filter paper 1. Filtrate collected was
centrifuged at 10000 rpm for 10 minutes at room
temperature. Supernatant was carefully collected and
used as crude enzyme for protein estimation and
assay.
by colorimeter to determine the protein content of
crude enzyme extract followed by specific activity
determination of enzyme.
Fig. 1. Standard Protein Graph
Ammonia standard graph:
Ammonia standard graph was prepared using liquid
ammonia (25%). 24.7 μg /1ml ammonia was taken
and standard graph was plotted by measuring the
absorbance at 470nm using calorimeter.
PROTEIN ESTIMATION:
Estimation of protein was carried out according to the
method of Lowry. 2ml of crude enzyme with 5ml of
alkaline copper reagent and 0.5ml FC reagent dilute
with water in ratio(1:2). The Optical density was
measured at 660nm using Calorimeter.
ENZYME ASSAY:
L-asparaginase enzyme assay was performed by a
colorimetric method at room temperature using a
calorimeter at 470nm by estimating the ammonia
produced during L-asparaginase catalysis using
Nessler’s reagent. A reaction mixture consisting of 0.5
ml of crude enzyme, 0.01 M L-aspargine and 0.05 M
Tris-HCl buffer (pH 8.6) was incubated for 30 min at
37°C. The reaction was stopped by the addition of 0.5
ml of 15% Trichloroacetic acid solution. The liberated
ammonia was coupled with Nessler’s reagent and was
quantitatively determined using an ammonium
(0.247gm/10ml DW) reference standard. 1 unit of the
L-asparginase (IU) is defined as the amount of enzyme
capable of producing 1 μmol of ammonia per minute
at 37°C.
RESULTS AND DISCUSSION:
Protein standard graph:
Protein standard graph was plotted by Lowry’s
method. Bovine serum albumin (BSA) was taken 100
μg / ml. Standard concentration and standard graph
was plotted by recording the absorbance at 660 nm
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Fig.2. Standard graph for Enzyme Assay
Submerged fermentation:
Effect of temperature:
The experiments were carried out at 250C, 300C,
350C, 400C, 450C, 500C by incubating the substrate at
different temperature mentioned above for 2 days.
Crude enzymes were extracted and activity was
measured. The graph of mean of enzyme activity
(shown in percentage) against incubation temperature
is shown in Fig No.3. Bacterial cell have various
mechanisms that allow them strictly to control
enzyme excretion. Change in the nature of cell
envelope can affect the release of extracellular
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enzymes to the culture medium. For L-asparginase
activity, increase in temperature from 250C to 500C,
decreases activity, whereas increase in temperature
from 350C to 400C increased the enzyme activity.
The optimum enzyme activity obtained was 79.83
IU/ min at 400C. Siddalingeshwara Kalingapplar et
al, 2010 reported optimum activity of L-asparginase
by Aspergillus sp. using carob pod medium at 350C
[39].
Effect of Incubation Time:
Submerged fermentation was performed by keeping
the culture at different incubation time from 24, 48,
72, 96, 120, 144 hrs. The graph of mean reading of
enzyme activity (shown in percentage) against
different media is shown in Fig No.4.6. The Lasparginase activity, increase in incubation time from
24 to 120 hrs increases, whereas further increase in
incubation period from 144 hrs. showed decrease in
enzyme activity. Optimum activity of 719.84 IU/ min
was observed at 120 hrs. Gradual decrease in enzyme
activity was observed after 144 hrs due decrease in
moisture and nutrient content of the medium. A.R
Soniyamby et al, 2011 reported optimum activity of
L-asparginase by Penicillium sp. using Modified
Czapek Dox medium at 370C for 96 hrs [4]. The
optimum activity was 9.8U/ml.
Fig.
3. Enzyme Estimation at various temperatures for SMF.
Effect of pH:
Among the physiochemical parameters, pH of the
growth medium plays an important role by inducing
morphological changes in the organism and in
enzyme secretion. The experiments were carried out
at different pH range 3, 4, 5, 6, 7, 8, 9 under optimum
conditions. The graph of mean reading of enzyme
activity (shown in percentage) against initial pH is
shown in Fig No.4. For L-asparginase activity,
increase in pH from 5 to 7 increases enzyme activity,
further increase in pH upto 9 decreases activity.
Optimal activity of 143.96 IU/ min was observed at
pH 7. Maysa E- Moharam et al, 2010 reported
optimum activity at pH 7 using Bacillus sp R36 [8].
Fig. 4. Enzyme Estimation at various pH range for
SMF.
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Fig. 5. Enzyme Estimation at various incubation time
for SMF.
Solid State Fermentation:
Effect of extraction pH on enzyme activity:
Solid state fermentation was performed to check the
effect of extraction pH of enzyme activity. Crude
enzyme was extracted by using buffers of different
pH from 3 to 9. Crude enzyme was extracted by
using buffers of different pH from 3 to 9. Sodium
Phosphate buffer of 0.2M was used for pH 3 to pH 8,
Tris HCl buffer of 0.2M was used for pH 9. The
enzyme activity was recorded to study the effect of
pH of extracting buffer and also to optimize the
condition for pH. The graph of mean reading of
enzyme activity (shown in percentage) against
extraction pH is shown in Fig No 4.7. For Lasparginase Activity, increase in pH from 3 to 4
resulted in increase in enzyme activity, further
increase in pH showed decrease in enzyme activity.
Optimal activity of 143.96 IU/ min was observed at
pH 4. Ashraf A.et al, 2003 reported production of L-
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asparginase by Pseudomonas Aeruginosa on
soyabean meal and got optimum activity at pH 9[2].
Effect of particle size:
Effect of particle size on enzyme activity was
studied by groundnut oil cake as substrate. After
grinding the substrate the substrate of different
particle size from 0.1 mm to 2 mm was taken to
study the effect of particle size on enzyme activity.
Sieve shaker was used to separate the substrate
particles of different size. Sieves of different mesh
size arranged in decreasing order of mesh size 1.31
mm, 0.96 mm, 0.46 mm, 0.32 mm, 0.23 mm were
mounted on vibrator. Substrates of different particle
size were considered based on particle size.
Substrate of different particle size was taken in
conical flask and solid state fermentation was
carried out for 72 hrs at 370C.
Effect of Incubation time:
Solid fermentation was performed at varying
incubation time from 24, 48, 72, 96, 120, 144
hrs keeping the other experimental conditions at
optimum. The graph of mean reading of enzyme
activity (shown in percentage) against different
media is shown in Fig No.4.9. The l-asparginase
activity, increase in incubation time from 24 to 72 hrs
increases, whereas further increase in incubation
period from 96 hrs showed decrease in enzyme
activity. Optimum activity of 340.28 IU/ min was
observed at 72 hrs. Gradual decrease in enzyme
activity was observed after 72 hrs due decrease in
moisture and nutrient content of the medium.
Sutthinan Khanna et al, 2009 reported activity of Lasparginase by Actinomycetes sp and got the
optimal activity at 178 hrs.
Fig. 6. Enzyme Estimation at various pH for SSF.
The graph of mean reading of enzyme activity
(shown in percentage) against particle size is shown
in Fig No 4.8. Larger provides better respiration
efficiency due to increase of inter particle space. The
decrease in particle size from 0.96 mm resulted
increase in the enzyme activity. The optimal activity
of 798.36 IU/ min was seen in 0.32 mm particle size.
Siddalingeshwara K.G et al, 2010 produced Lasparginase from Aspergillus terreus KLS2 from
carob pod and got optimum enzyme activity of 5.63
IU for 2 mm[10].
Effect of temperature:
The experiments were carried out at room
temperature, 350C, 400C keeping the other
experimental conditions at optimum.
After
incubation of substrate, it was kept in incubator at
different temperature for 3 days. Crude enzyme was
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extracted and activity was measured. The graph of
mean of enzyme activity (shown in percentage)
against incubation temperature is shown in Fig
No.4.3. Microbes have various mechanisms that
allow them strictly to control enzyme excretion.
Change in the nature of cell envelope can affect
the release of extracellular enzymes to the culture
medium. For L-asparginase activity, increase in
temperature from 350C to 400C. The optimum
enzyme activity obtained was 578.87 IU/min at room
temperature. Siddalingeshwara Kalingapplar et al,
2010 reported optimum activity of L-asparginase
by Aspergillus sp. using carob pod medium at
350C [39].
room temperature 578.87 IU/min. Effect of
moisture content L-asparginase productivity was
observed high of 340.28 IU/min at 100%
moisture content. Also, effect of temperature,
incubation time, incubation pH, and media was
considered in this project productivity by SMF.
Effect of temperature on enzyme activity, nutrient
medium gave optimum activity for L-Asparginase
production of 79.83 IU/ min at incubation
temperature 400C. Effect of incubation time showed
optimum productivity for L-asparginase production
of 719.84 IU/ min at 120 hr of incubation. Effect of
pH on enzyme activity was observed at pH 7 which
showing
optimum activity for
L-asparginase
production of 143.96 IU/ min at incubation.
Effect of media for L-asparginase productivity was
observed high for nutrient broth (C) 99.46 IU/min.
Still lot of work can be carried out to optimize the
parameter for better productivity by considering the
above substrate. Effect of incubation pH, extraction
temperature, incubation time, yeast content, nutrient
composition is still to be studied. This work can be
scaled up for commercialization of the system.
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In present study, Aspergillus niger (NCIM No.1056),
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