L-asparaginase Production by a new isolate Bacillus aryabhattai strain ITBHU02 in solid
Yogendra Singh, S. K. Srivastava*
School of Biochemical Engineering, Institute of Technology, Banaras Hindu University,
Varanasi 221005, India
Corresponding author. Address: School of Biochemical Engineering, Institute of Technology,
Banaras Hindu University, Varanasi 221005, India
Tel: +91 542 6702886; Fax: +91 542 2368428
E-mail addresses: firstname.lastname@example.org (S. K. Srivastava),
1st International Conference on Biosciences and Bioengineering: A collaborative Approach
The enzyme, L-asparaginase (L-asparagine amino hydrolase, EC 220.127.116.11), catalyzes L-
asparagine breakdown into aspartic acid and ammonia. In recent years, asparaginases have been
receiving meticulous attention because of their potential medicinal uses and application in food
industries. This study presents production of L-asparaginase by a new isolate Bacillus
aryabhattai Strain ITBHU02 on the solid surface of rice husk, wheat bran, rice bran, cotton seed
powder, coconut oil cake, and groundnut oil cake as substrates. Optimization of the solid state
fermentation media and parameters resulted in a 14% increase in the L-asparaginase activity.
Optimum L-asparaginase production 16.1 U/gds was observed on wheat bran supplemented with
1%, (w/w) yeast extract, 0.25%, (w/w) L-asparagine at pH 7.5, 100%, (v/w) initial moisture and
30°C after incubation 36 hrs.
KEYWORDS: L-asparaginase production; Bacillus aryabhattai strain ITBHU02; solid state
fermentation; wheat bran
L-asparaginase (L-asparagine amino hydrolase, EC 18.104.22.168) is a high value enzyme with
intensive chemotherapeutical uses against a wide variety of tumors especially acute
lymphoblastic leukemia (ALL) (Story et al. 1993; Verma et al., 2007). Recently it is also gaining
much importance in food industries for producing acrylamide free food products (Pedreschi et
al., 2008). On the basis of numerous studies, International Agency for Research on Cancer has
classified acrylamide as “probably carcinogenic to human” (IARC report, 1994). The enzyme L-
asparaginase is widely distributed in nature from bacteria to mammals and plays a vital role in
amino acid metabolism and utilization. L-asparaginase catalyses the hydrolysis of amide group
of L-asparagine to form L-aspartic acid and ammonia.
L-asparaginase production from microbial sources through the fermentation has been a
promising method, owing to its cost-effectiveness and eco-friendly nature. Although submerged
fermentation is a cost-intensive method, it is still a commonly used technique for L-asparaginase
production throughout the world because of its apparent advantages in consistent enzyme
production with defined medium and process condition, and advantage in downstream
processing. However, the major shortcomings of L-asparaginase production by submerged
fermentation are low concentration production, and consequent handling, reduction, and disposal
of large volumes of water during the downstream processing (Datar, 1986). In this context, solid
state fermentation (SSF) has gained fresh and plentiful attention of researchers to overcome the
drawbacks of submerged fermentation. SSF has several advantages over submerged fermentation
such as lesser energy requirements, very low risk of bacterial contamination, lower need of water
and less environmental concerns regarding the disposal of solid waste (Doelle et al., 1992).
Additionally, the utilization of agro-waste solid as a substrate for carbon and energy requirement
under SSF makes this approach environmental friendly. Usually substrates used for solid state
fermentation are water insoluble polymer of starch or cellulosic materials.
Presently, L-asparaginase purified from two microbial sources viz. E. coli and Erwinia
carotovora is extensively utilized in chemotherapy of leukemia. However, their prolonged
administration induces immunogenic side effects like allergic reactions, anaphylaxis, pancreatitis
and neurological seizures. The anti-asparaginase antibodies so formed, abolish the enzyme
activity from circulating plasma (Heinemann and Howard, 1996; Savitri and Azmi 2003; Verma
et al. 2007). To overcome the cytotoxicity associated with the clinical preparations of
asparaginases from two aforesaid microorganisms, an alternative source of L-asparaginase is
required, which is attributed to produce serologically different enzyme but having same
antineoplastic activity. The increasing importance of L-asparaginase in recent years for its
therapeutic applications as well as extensive uses in food industries promoted us to utilize newer
microbial sources for L-asparaginase production.
In the present study, an L-asparaginase producing soil isolate Bacillus aryabhattai strain
ITBHU02 (accession no. JQ673559) was utilized in solid state fermentation for L-asparaginase
production. Selection of most appropriate agro-residual substrate, with optimal media
composition and fermentation conditions to maximize the yield of L-asparaginase enzyme, was
MATERIALS AND METHODS
Bacillus aryabhattai strain ITBHU02 (Accession No. JQ673559) was isolated from a soil
site containing hospital waste and screened as potent L-asparaginase producer according to the
method described by Gulati et al. (1997). The culture was maintained in nutrient agar (NA) slant.
The slant was incubated at 30°C for 24 h and stored at 4°C±1°C. Stock culture was transferred to
fresh NA medium every 3-4 weeks.
Preparation of inoculum
For inoculum preparation, strain ITBHU02 was cultivated in nutrient broth media
containing beef extract 10.0 g, peptone 10.0 g, and sodium chloride 5.0 g per liter of distilled
water (pH 7.0). The cells were cultivated in this medium at 30°C on a rotary shaker at 160 rpm
for 24 h.
Different agro-waste based products like rice husk, wheat bran, rice bran, cotton seed
powder, coconut oil cake, and groundnut oil cake were used as substrate. Ten grams of each
substrate was measured into 250 ml Erlenmeyer flasks into which a supplemental salt solution
was added properly to get the desired moisture level. The salt solution composed of 6.0 g/L
Na2HPO4.2H2O, 3.0 g/L KH2PO4, 0.5 g/L MgSO4.7H2O, and 0.5 g/L NaCl. L-asparagine was
supplemented as inducer for synthesis of enzyme L-asparaginase (Gulati et al. 1997). Content
was mixed properly and autoclaved at temperature 121°C (pressure 15 psi) for 15 min.
Solid state fermentation and crude enzyme production
The sterilized fermentation media was inoculated with 2.0 ml of inoculum, mixed
thoroughly and incubated at 30°C for 4 days in a stationary condition. Each experiment was done
in triplicate. The recovery of crude L-asparaginase from the fermented material was done by
simple extraction method. For this, the fermented substrate was mixed thoroughly with 50 ml of
50 mM phosphate buffer (pH 7.0) and the contents were agitated for 1 h at room temperature in a
rotary shaker at 150 rpm. At the end of extraction, the liquid was filtered o ff through Whatman
No.1 filter paper and the resulting clear filtrate was used for L-asparaginase assay.
L-asparaginase activity assay
L-asparaginase activities were assayed at 37°C with using L-aspartic acid β-hydroxamate (AHA)
as the substrate (Derst et al. 1992). Reaction mixture containing 0.3 mL AHA solution (0.01 M
solution in 0.05 M HEPES buffer, pH 7.0) and 0.1 mL culture filtrate was incubated for exactly
30 min at 37°C. The reaction was stopped by addition of 2.4 mL stopping reagent (1 M Na2CO3
solution: 1% (w/v) 8-hydroxyquinoline in ethanol: 1% (w/v) NaIO4 solution; 8:1:0.2). The green
color, developed after keeping the mixture in boiling water bath for 1-2 min, was measured at
705 nm. One unit (U) of asparaginase activity is defined as the amount of enzyme that liberates
1.0 µmol of NH2OH from AHA per min at 37°C.
L-asparaginase activity was also measured by direct nesslerization method (Imada et al.,
1973). Wherein, 1 unit (U) of L-asparaginase was the amount of enzyme that liberated 1 µmol of
ammonia in 1 min at 370C and pH 8.6. The enzyme activity was expressed in terms of units per
gram dry fermented substrate (U/gds).
Optimization of solid state fermentation condition
Different process parameters influencing yield of the enzyme during SSF were optimized.
Different incubation periods (1–5 days) were studied for their effect on enzyme production.
Initial moisture contents (40, 60, 80, 100 and 120%) of solid substrates (before autoclaving) were
adjusted by means of aforesaid supplemental salt solution. For initial pH optimization, substrates
were moistened with supplemental salt solution prepared in different buffers viz. 20 mM citrate-
phosphate, Tris–HCl or glycine-NaOH buffers at pHs ranging from 4 to 10, respectively.
Fermentation at different incubation temperatures (20, 25, 30, 35, 40, 45 and 55°C) were carried
out to observe their effect on enzyme production. Effect of different inoculum sizes (1.0 to 5.0
ml of 24 h old culture containing 4.0 x 108 CFU/ml) on the yield of L-asparaginase was also
scrutinized. For each experimental variable all other parameters were kept at their optimum
level. All the experiments were conducted in triplicate and data obtained were average of the
Effect of additional nutrients
The outcome of additional nutrients on L-asparaginase production was tested by adding
carbon sources (1.0%, w/w) such as maltose, glucose, fructose, sucrose, galactose or soluble
starch, and nitrogen sources (1.0%, w/w) such as yeast extract, malt extract, peptone, casein,
sodium nitrate, ammonium nitrate or ammonium sulphate. Optimum concentration level of L-
asparagine as an inducer for maximum yield of L-asparaginase was also scrutinized.
Fermentation was carried out for 5 days and all other parameters were kept at their optimal level.
RESULT AND DISCUSSION
Evaluation of different agro-residual substrates for L-asparaginase production
The screening of an ideal agro-residual based substrate for maximum enzyme production
in a solid-state fermentation process mainly depends upon its easier degradation into nutrients
and uptake by the microorganism to synthesize the targeted metabolite, its cost effectiveness and
availability in the nature. The present study revealed that L-asparaginase production pattern
varied with the type of agro-residual substrates. This could be accredited to solid materials dual
role-supply of nutrients to the microbial culture for its growth and anchorage for the growing
cells. Maximum enzyme production (11.5 U/gds) was observed with wheat bran, while minimum
L-asparaginase production (7.2 U/gds) was noticed with rice husk as substrate/support material.
Wheat bran contains approximately 18% protein, 5% fat and 62% carbohydrate (Madruga et al.,
2000) and is rather complete source of nutrients for microorganisms (Ellaiah et al., 2004; Beg et
al., 2000). Evaluation of L-asparaginase production values of this strain with different substrates
tare shown in (Fig. 1).
Fig.1 Effect of various agro-residual wastes on L-asparaginase production by B. aryabhattai ITBHU02 in
SSF (initial pH 7.0; temperature 30°C; initial moisture 100%; 1.0 mL inoculum with spore count 4 x 108 CFU/mL)
Effect of incubation time
Fig. 2 depicts that production of L-asparaginase by B. aryabhattai ITBHU02 in wheat
bran was gradually increased with incubation time under solid state cultivation until 36 h and
reached to a maximum yield of 11.8 U/gds. Further incubation after 36 h showed slight
declination in enzyme activity till 120 h reaching 7.6 U/gds.
Fig. 2 Effect of incubation time on L-asparaginase production by B. aryabhattai ITBHU02 in
SSF (substrate: wheat bran; initial pH 7.0; temperature 30°C; initial moisture 100%; 1.0 mL inoculum with spore
count 4 x 108 CFU/mL)
Effect of moisture content
Moisture content is one of the most significant factors under solid state fermentation
using a specific substrate as it plays a key role in microbial growth and enzyme production.
Growth of microbial population and synthesis of metabolite under solid state fermentation is
totally different as that in submerged fermentation culturing, because these occur alongside the
surface of solid substrate particle having low water content. Water behaves like a vehicle for
substrate transport and so the activity of water greatly affects the synthesis of product (Fogarty
and Kelly, 1980). Therefore, optimization of the available moisture content within fermenting
substrate to get maximum product is quite essential. A maximum L-asparaginase production,
13.7 U/gds was achieved at 100% moisture content (Fig. 3). A linear correlation between
moisture content and L-asparaginase yield was observed until 100% and maximum activity of
the enzyme sharply declined at further increase in moisture content.
Fig. 3 Effect of moisture content (%) on L-asparaginase production by B. aryabhattai ITBHU02 in SSF
(substrate: wheat bran; initial pH 7.0; temperature 30°C; 1.0 mL inoculum with spore count 4 x 108 CFU/mL;
incubation time: 36 hours)
Effect of pH
The production of L-asparaginase by microorganisms is strongly dependent upon
medium pH as it plays a pivotal role in transportation of various components across the cell
membrane and in managing the metabolic activities of the cell (C. Krishna, 2005). Results
showed that enzyme production was increased with pH achieving an optimum level at pH 7.5
(13.9 U/gds) as Fig. 4. Further production of the enzyme decreased subsequently at all higher pH
values attaining activity 6.8 U/gds at pH 10.0. Preceding studies dealing with the effect of initial
pH on L-asparaginase production by other Bacillus strains such as B. circulans and Bacillus sp.
(isolated from marine alga) indicated pH values 7.0 and 8.0 respectively for optimal L-
asparaginase activity (Prakasham et al., 2010; Mohapatra et al., 1995).
Fig. 4 Effect of medium pH on L-asparaginase production by B. aryabhattai ITBHU02 in SSF (substrate:
wheat bran; temperature 30°C; moisture content 100%; 1.0 mL inoculum with spore count 4 x 108 CFU/mL;
incubation time: 36 hours)
Effect of incubation temperature
Effect of different incubation temperature was shown in Fig. 5. The maximum enzyme
production was accomplished at 30°C (14.5 U/gds). Although, the physiological changes
persuaded by high temperatures during enzyme production are not entirely understood, it has
been recommended that at high temperatures, microorganisms may synthesize only a reduced
number of proteins essential for growth and other physiological processes (Gawande and Kamat,
1999). Further increase in incubation temperature resulted in reduction of the enzyme
Fig. 5 Influence of incubation temperature on L-asparaginase production by B. aryabhattai ITBHU02 in
SSF (substrate: wheat bran; pH 7.5; moisture content 100%; 1.0 mL inoculum with spore count 4 x 108 CFU/mL;
incubation time: 36 hours)
Effect of inoculum level
Enzyme production varied with inoculum level and showed parabolic nature in the
studied range. Maximum L-asparaginase synthesis (14.7 U/gds) was observed with inoculum
size of 2.5 ml (containing 4.0 x 108 CFU/ml) (Fig. 6). Increase of inoculum level from 2.5 ml to
3.5 ml or 4.5 ml negatively affected the enzyme production and caused 16% and 27% reduction,
respectively. Nevertheless, a higher inoculum level increased the moisture content to a
significant point and free excess liquid present in an unabsorbed form will therefore give rise to
an additional diffusional barrier together with that imposed by the solid nature of the substrate
and lead to a decrease in growth and enzyme production (Krishna and Chandrasekaran, 1996).
Fig. 6 Effect of size of inoculum on L-asparaginase production by B. aryabhattai ITBHU02 in SSF
(substrate: wheat bran; moisture content 100%; pH 7.5; temperature 35°C; incubation time: 36 hours)
Effect of different carbon source
In order to scrutinize the optimum carbon source, various sources such as maltose,
glucose, fructose, sucrose, galactose or soluble starch were used for the study. It is clear from the
Fig. 7 that glucose was best carbon source for L-asparaginase production by B. aryabhattai
ITBHU02. The glucose was reported as a catabolite repressor for production of L-asparaginase
in bacteria (Abdel-Fattah et al., 2002). But these experimental data showed that glucose was not
repressor for L-asparaginase production by the strain at lower concentrations. Higher
concentrations (beyond 0.5%, w/w) suppress the synthesis of the enzyme. Glucose was shown to
be the best carbon source with combination of L-asparagine for production of L-asparaginase by
Pectobacterium carotovorum also (S. Kumar et al., 2010). Further to facilitate the optimum
concentration of carbon source, L-asparaginase production was explored by supplementation of
different glucose concentration (0-2.0%, w/w). Experimental data revealed that the maximum
activity of 15.0 U/gds was obtained for 0.5% and the least activity of 11.3 U/gds for 1.5%
Fig. 7 Effect of various carbon source on L-asparaginase production by B. aryabhattai ITBHU02 in SSF
(substrate: wheat bran; pH 7.5; temperature 35°C; moisture content 100%; 2.5 mL inoculum with spore count 4 x
108 CFU/mL; incubation time: 36 hours)
Effect of different Nitrogen source
The impact of supplementation of various nitrogen sources (1% w/w) such as yeast
extract, malt extract, peptone, casein, sodium nitrate, ammonium nitrate or ammonium sulphate
on L-asparaginase production is shown in Table 1. Each source was supplied additionally with
whean bran in solid state fermentation. Thereafter, with the optimal nitrogen source, L-
asparagine (0.25%, 0.50%, 0.75 % and 1.0 %, w/w) was supplemented to get optimum induction
of enzyme L-asparaginase. Yeast extract followed with ammonium sulphate were observed to
improve the enzyme activity having yield of 15.6 and 14.8 U/gds, respectively in comparison to
14.2 U/gds for the control. Malt extract, peptone, ammonium nitrate, sodium nitrate and casein
greatly reduced enzyme production. It was observed that yeast extract along with L-asparagine
(0.25%, w/w) synergistically showed maximum induction effect for enzyme synthesis and
improved the production 16.1 U/gds.
Table 1 Effect of various nitrogen sources on L-asparaginase production by B. aryabhattai ITBHU02 in a solid-
state fermentation (substrate: wheat bran; pH 7.5; temperature 35°C; moisture content 100%; 2.5 mL inoculum with
spore count 4 x 108 CFU/mL; incubation time: 36 hours)
Nitrogen source %
Yeast extract (YE)
+ L-asparagine (%)
Production of an anti leukemic drug, L-asparaginase by a soil isolate B. aryabhattai
ITBHU02 under solid-state fermentation was influence d by physiological and chemical nature of
the wheat bran and associated with growth of the microbial strain. The use of agro-waste raw
materials is cheaper and more advantageous than conventional substrates for L-asparaginase
production. Moisture content of the solid medium was found to be important to achieve
maximum enzyme production yields. Wheat bran, as nutrient source, was insufficient medium
for production of enzyme. Enhanced L-asparaginase production yields could be obtained by
supplementation of yeast extract and L-asparagine to the solid medium.