Monica et al., J Biocatal Biotransformation 2014, 2:2 http://dx.doi.org/10.4172/2324-9099.1000108
Journal of Biocatalysis & Biotransformation
Research Article
a SciTechnol journal BSA: Bovine Serum lbumin; EDTA: Ethylenediaminetetraacetate; NBS: N-Bromosuccinimide; NEM: N-Ethylemaleimide; PMSF: Phenylmethylsulphonylfluoride; Rf: Retardation factor; SDS-PAGE: Sodium Dodecyl Sulfate-Polyacryalamide Gel Electrophoresis; SSF: Solid State Fermentation; SmF: Submerged Fermentation; TLC: Thin Layer Chromatograph; TCA: Trichloroacetic Acid;
Isolation, Purification and Characterization of Fungal Extracellular L-Asparaginase from Mucor hiemalis
Introduction
Monica Thakur , Lynette Lincoln , Francois N Niyonzima and Sunil S More1* 1
1
1
Abstract L-asparaginase is an enzyme that deaminates the free L-asparagine to yield aspartic acid and is used as an antileukemic agent. L-asparaginase producing fungus was screened from a local soil sample and identified as Mucor hiemalis based on morphological and microscopic characteristics. The selected modified CzapekDox growth medium contained 1% (w/v) L-asparagine as a sole nitrogen source and inducer. The best culture conditions noted after optimization were an initial pH of 7.0 at 30°C, 4 days of incubation period, 1.25% L-asparaginase and 0.4% glucose. A 4.2-fold increase in an extracellular L-asparaginase secretion was observed after optimization. On lectin-agarose column, L-asparaginase was purified to homogeneity with 69.43 U/mg specific activity, 4.59fold enhancement and 18.46% recovery. The apparent molecular weight as sized by sodium dodecyl sulfate-polyacryalamide gel electrophoresis and on comparison with the standard molecular weight markers was approximately 96.32 kDa. The purified L-asparaginase was a non-metalloprotein but a metal activated one and was active at an optimum pH of 7 and a temperature of 37°C. The enzyme almost remained unaltered at 37°C, and at pH 7.0 for 24 h. It was inhibited by sodium dodecyl sulfate but its activity was enhanced by the nonionic surfactants (Tween 80 and Triton X 100). The enzyme was very specific for L-asparagine and showed a low Km value of 4.3 mM and a Vmax of 625 U/ml. Thin layer chromatography confirmed the L-aspartic acid as a hydrolytic product of L-asparaginase. The purified L-asparaginase exhibited a good scavenging activity towards 2, 2’-azinobis-3ethylbenzothiazoline-6-sulfonic acid when compared to ascorbic acid. The production of the therapeutic enzyme in higher amounts at physiological temperature and good scavenging activity suggested the enzyme to be used in the formulations for the therapy of acute lymphoblastic leukaemia.
Keywords Mucor hiemalis; L- asparaginase; Non-metalloprotein; Lectinagarose column; Antioxidant
Abbreviations ABTS: 2’-azinobis-3-ethylbenzothiazoline-6-sulfonic acid; ALL: Acute Lymphoblastic Leukaemia; β-ME: β-Mercaptoethanol; *Corresponding author: Sunil S More, Department of Biochemistry, CPGS, Jain University, 18/3, 9th Main Jayanagar 3rd Block, Bangalore-560011, India, Tel: +91 9481787729; Fax: +91 080 43226501; E-mail:
[email protected] Received: September 20, 2013 Accepted: December 12, 2013 Published: December 23, 2013
International Publisher of Science, Technology and Medicine
L-asparaginase (E.C 3.5.1.1) is a tetrameric protein that hydrolyzes free L-asparagine to give aspartic acid and ammonia [1,2]. Under physiological conditions, the hydrolytic reaction is mostly irreversible [3]. L-asparaginases are among the largest group of therapeutic enzymes as they account for about 40% of the total worldwide sale of anti leukemic and antilymphoma agents [4]. As the enzyme is in a great demand in clinical applications and in food processing industries, the demand for this therapeutic enzyme is increasing several folds every year [5]. For the growth of lymphatic tumor cells, L-asparagine served as an essential amino acid since L-asparagine synthetase is present in lower amounts. However, normal cells are capable of manufacturing enough L-asparagine for their metabolic needs using L-asparagine synthetase [6]. As the lymphatic tumor cells require sufficient asparagine for their development, they utilize all the asparagine from the diet and from blood circulation or body fluid. As a result, the lymphatic tumor cells are died off [3]. L-asparaginase drug is used as chemotherapeutic agents because it depletes the L-asparagine present in blood circulation, indirectly starving tumor cells resulting in lymphatic tumor cell death [7]. One of the most important malignant disorders is acute lymphoblastic leukemia (ALL) most often observed in children. ALL is in general caused by exposure to chemical carcinogens, genetic disorders and viral infections [8]. L-asparaginase can be obtained from animals, plants and microorganisms such as filamentous fungi, yeasts, actinomycetes and bacteria. However, the organisms of choice are microorganisms as they can be cultured easily using cheap substrate, the culture conditions for enzyme production are easily optimized, easily genetically modified to increase the yield, the enzyme can be produced in bulk, extracted and purified economically, good stability, and consistency than animal and plant enzymes [2,9]. The extracellular L-asparaginase secretion depends on the medium components such as nitrogen and carbon sources and cultural parameters like initial pH of the culture medium, incubation temperature, inoculum size and fermentation time. All these factors vary from one organism to another [10]. Solid state fermentation (SSF) and submerged fermentation (SmF) are in use for extracellular production of fungal L-asparaginase. Although SSF uses cheap substrates like agricultural wastes, the SmF is preferred since the medium composition is known and the medium composition can be easily altered to get a considerable yield of good quality [11]. Although L-asparaginases of Escherichia coli and Erwinia chrysanthemi have emerged as the potent chemotherapeutic agents for the last 40 years, they were associated with minor side effects such as vomiting, leucopenia, fever, skin rash, nausea, thromboembolysis, difficulty in breathing, hyperglycaemia, weight loss, decreased blood pressure, sweating, immunosuppression, loss of consciousness, acute
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Citation: Monica T, Lincoln L, Niyonzima FN, Sunil SM (2013) Isolation, Purification and Characterization of Fungal Extracellular L-Asparaginase from Mucor Hiemalis. J Biocatal Biotransformation 2:2.
doi:http://dx.doi.org/10.4172/2324-9099.1000108 pancreatitis and neurological seizures [2,8,12-14]. The extensive research is therefore going on worldwide with eukaryotic fungal L-asparaginases which are having less adverse effects than bacterial enzymes [13]. Few reports are available with the extracellular secretion of L-asparaginases by fungi [15]. Mold of the genera Fusarium, Penicillium and Aspergillus have been reported to secrete L-asparaginase showing an anti-lymphoma activity with less adverse effects [11,13]. Few reports are available for the optimization of culture conditions for maximum L-asparaginase production by fungal species. Studies on properties of L-asparaginase of Mucor species are scarce. No even single species has been reported to produce sufficient amount of enzyme that can be commercialized. Since the search for an ideal asparaginase producing organism is a continuous exercise. The present investigation reports the screening of Mucor hiemalis which is a potent producer of an L-asparaginase, optimization of the culture parameters for maximum enzyme secretion, purification of enzyme to homogeneity, characterization of the purified enzyme and the study of its antioxidant property.
Materials and Methods Isolation and identification of L-asparaginase producing fungi The soil sample was collected from Kengeri (Karnataka, India), serially diluted and spread plated on modified Czapek-Dox agar plates (pH 6.2) containing L-asparagine as a sole nitrogen source. The constituents of the medium were (g/l) L-asparagine (10.0), KCl (0.52), KH2PO4 (1.52), MgSO4∙7H2O (0.52), glucose (2.0), CuNO3∙3H2O (trace) FeSO4 (trace), ZnSO4∙7H2O (trace) and agar (20.0). It was supplemented with phenol red of 0.009% (v/v) strength. 0.02% (w/v) tetracycline was also added to the medium before being poured on Petri plates. The incubation was carried at room temperature (30 ± 2°C) for 72 h. The formation of a pink zone around the organism was an indication of the L-asparaginase-producing fungi [16]. The fungus with bigger zone was purified by streaking on the same medium and maintained at room temperature and at 4°C. The isolated fungus was identified on the basis of morphological and microscopic characteristics [17]. It was sent to a National Fungal Culture Collection of India (Agharkar Research Institute, Pune) for further identification.
Inoculum preparation and submerged fermentation The isolate was grown on the modified Czapek-Dox agar plates at room temperature for 72 h. A 5 mm disc of inoculum was taken from the culture plate edge with a cork borer [7] and inoculated into 100 ml of the fermentation medium contained in 250-ml Erlenmeyer flask. The fermentation medium was the same as the one used for isolation without agar. After 72 h of incubation at 30 °C, the broth was centrifuged with cooling centrifuge (C-30 BL Remi, India) at 10000 rpm for 10 min at 4 °C. The clear supernatant served as crude enzyme and was used in L-asparaginase activity determination.
L-asparaginase assay and protein estimation Extracellular L-asparaginase activity was assayed according to the modified method of Imada et al. [18] using L-asparagine as substrate. 0.5 ml of 0.5 M phosphate buffer (pH 7), 0.5 ml of 0.04 M L-asparagine, 0.5 ml of distilled water and 0.5 ml of crude enzyme were all mixed together, shaken well and incubated at 37°C for 30 min. The reaction was terminated by the addition of 0.5 ml of 1.5 M trichloroacetic acid (TCA). The ammonia liberated was quantified
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calorimetrically by adding 0.2 ml Nessler’s reagent to the mixture of 0.1 ml filtrate and 3.75 ml distilled water. The absorbance was recorded at 450 nm with a UV-spectrophotometer (SL 159 Elico, India) after a final incubation for 10 min. The blank was prepared as above except that the enzyme was added after the TCA addition. The amount of ammonia liberated by the test sample was calculated using the ammonium sulfate standard curve. One unit of L-asparaginase is defined as the amount of enzyme which liberated 1 µmol of ammonia per min per ml under the assay conditions. Protein content was estimated using BSA as standard [19].
Optimization of culture conditions production of L-asparaginase
for
maximum
The important factors influencing L-asparaginase production was evaluated, optimizing one parameter at a time and keeping other factors unaltered. To observe the effect of incubation temperature on enzyme production, the production medium of pH 6.2 was inoculated with 2 culture discs and incubated at different temperatures, viz. 30, 37 and 45°C. The effect of the initial pH of the medium on the enzyme production was studied by inoculating the 2 culture discs into the 100 ml production medium adjusted to different pHs ranging from 4.0 to 9.0 and by incubating for 3 days at room temperature (30 ± 2°C). The influence of substrate concentration was investigated using different concentration of L-asparagine ranging from 0.25 to 1.5% (w/v) in the medium of pH 7.0 and growing the organism at 37°C for 3 days. The effect of glucose concentration on enzyme secretion was evaluated using different concentrations in the 0.2 - 1.0 % range at an initial pH 7.0 and by incubating at 37°C for 72 h. The time course study for optimal enzyme production was carried out by inoculating the 2 culture discs into the production medium of pH 7.0 at 37°C and by checking the enzyme activity every day for a period of 7 days. The L-asparaginase activity in all cases was quantified as described earlier.
L-asparaginase purification Acetone precipitation: The bulk enzyme production was done under optimized condition and culture broth was centrifuged as before. The enzyme in the supernatant was subjected to partial purification by acetone precipitation as per the modified method of Obi and Odibo [20]. 3 volumes of chilled acetone were slowly added to one volume of the supernatant and the mixture incubated for 2 h at -20°C. After the incubation period, the chilled mixture was subjected to centrifugation at 10000 rpm for 10 min. The supernatant was discarded carefully and pellet served as partially purified enzyme. The pellet was re-dissolved in a small amount of 0.5 M phosphate buffer (pH 7.0) and subjected to the lyophilization (Freeze dryer, Model LY3TTE, Snijders Scientific, Tilburg Holland). Affinity chromatography: The partially purified lyzophilized sample was subjected to glycoprotein test using sulfuric acid (99%) and ethanol (100%). The formation of brown color was an indication of a glycoprotein. The acetone sample was then purified by affinity chromatography as per the method of Spivak et al. [21] with minor changes. A lectin (concalvin A) - agarose column was used to totally purify the protein. The column was first equilibrated with 0.5 M phosphate buffer (pH 7.0). The sample was loaded and the unbound fraction collected. A sucrose solution of 1 M strength was applied to the column and bound fraction collected. The protein content and enzyme activity of bound and unbound samples were determined as earlier. The bound fraction showing higher protein content and higher enzyme activity was lyophilized. The resulted sample was used for the properties studies. The protein content and L-asparaginase • Page 2 of 9 •
Citation: Monica T, Lincoln L, Niyonzima FN, Sunil SM (2013) Isolation, Purification and Characterization of Fungal Extracellular L-Asparaginase from Mucor Hiemalis. J Biocatal Biotransformation 2:2.
doi:http://dx.doi.org/10.4172/2324-9099.1000108 activity of the crude, partially purified sample and purified sample were estimated as described before. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE): The SDS-PAGE was carried out according to Laemmili [22] under non reducing conditions to size and to determine the enzyme purity. The gel system contained a separating gel (12.5% acrylamide) and a stacking gel (6%). After electrophoresis, the gel was silver stained using the method of Blum et al. [23] with slight modifications. In brief, the gel was placed in a tray on a gel rocker and a fixative (40 % methanol and 10% acetic acid) added over the gel and left undisturbed for 20 min. The gel was washed 3 times with a rinse solution (30% ethanol) for 20 min each. A reductant (0.02% sodium thiosulfate) was added and left for 1 min. The gel was washed trice with distilled water for 30 s each. The cold silver stain in a brown bottle (0.2% AgNO3 + 0.02% formaldehyde just added before staining) was added and the gel left in the dark for 20 min. It was again washed with distilled water for only 30 s. The gel was developed (3% sodium carbonate + 0.05% formaldehyde added before use). After the appearance of the bands, the stop solution was added (5% glacial acetic acid). After gel silver staining, the molecular mass of L-asparaginase was quantified by comparing its eletrophoretic mobility to the mobilities of the proteins in the marker (kDa): phosphorylase b (97.4), BSA (66.0), ovalbumin (43.0), carbonic anhydrase (29.0), soyabean trypsin inhibitor (20.1) and lysozyme (14.3). L-asparaginase activity staining: 1% agarose in 25 ml of 0.5 M phosphate buffer (pH 7.0) containing 1% L-asparagine was prepared by heating and poured over the glass plates (5×5 cm). The wells were made with gel puncher (5 mm). 20 µl enzyme was added to each well and incubated overnight in a moist chamber at 37°C. The control was without the substrate L-asparagine. The gel was stained with Nessler’s reagent. The formation of a brown colored zone was an indication of ammonia production.
Physicochemical characterization of the enzyme Effect of pH on purified L-asparaginase activity and stability: The effect of pH on L-asparaginase activity was studied over the pH range of 3.0 to 10.0 using different buffers of 0.05 M: citrate buffer (pH 3), acetate buffer (pH 4.0 and 5.0), phosphate buffer (pH 6.0 and 7.0), Tris-HCl buffer (pH 8.0 – 9.0) and carbonate buffer (pH 10.0). The assay was performed by pre-incubating 0.25 ml of purified enzyme with 0.75 ml of each of the buffers at room temperature for 30 min. The enzyme activity was determined as reported earlier. The pH stability was analyzed by taking 1.5 ml of purified enzyme and 4.5 ml buffer (pH 7.0) together and incubating at room temperature. The activity was quantified at regular intervals viz. 0 min, 30 min, 1 h, 2 h, 4 h and 24 h. Effect of temperature on purified L-asparaginase activity and stability: The influence of temperature on L-asparaginase activity was analysed over the temperature range of 0°C to 90°C. 0.25 ml of enzyme was pre-incubated with 0.75 ml of 0.05M phosphate buffer (pH 7.0) at the respective temperature for 30 min. 0.5 ml of asparagine was added and the activity determined as before. The temperature stability was studied by mixing 1.5 ml of purified enzyme and 4.5 ml buffer (pH 7.0) together and incubating at 37°C. 1 ml of the mixture was removed each time at different time intervals (0 min, 30 min, 1 h, 2 h, 4 h and 24 h) and assayed. The effect of various metal ions on on purified L-asparaginase activity: The effect of different metal ions on L-asparaginase enzyme
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was evaluated using the chloride of Fe2+, Fe3+, Hg2+, Ca2+, Ba2+, Mg2+, Zn2+, Cu2+, K+, Na+, Co2+, Pb2+ and Mn2+ of 2 mM strength. The assay was performed by pre-incubating 0.25 ml of enzyme with 0.75 ml of the respective metal ion at 37 °C for 30 min. The activity was determined as before. Effect of inhibitors, activators and chelators on L-asparaginase activity: The influence of inhibitors and activators was analysed using ethylenediaminetetraacetate (EDTA), N-bromosuccinimide (NBS), N-ethylemaleimide (NEM), phenylmethylsulphonylfluoride (PMSF), urea, sodium azide (NaN3), Triton-X 100, Tween-80, sodium dodecyl sulfate (SDS). and β-mercaptoethanol (β-ME). A pre-incubation was conducted for 30 min after mixing 0.25 ml of the enzyme with 0.75 ml of the concerned inhibitor/activator. The activity was determined as earlier.
Substrate specificity of L-asparaginase and kinetic constant determination The affinity of L-asparaginase towards various amino acids was checked. L-aspartic acid, L-arginine, L-histidine, L-phenylalanine and L-glutamine were used [7]. The assay was carried as by replacing 0.04 M L-asparagine (control) by the concerned amino acid and the activity determined as earlier. The kinetic parameters (Km and Vmax) of the purified L-asparaginase were determined by linear regression from Lineweaver-Burk plot [24] with L-asparagine (1 mM - 40 mM) as substrate.
Thin layer chromatography (TLC) Chromatography of L-asparaginase hydrolysed products was evaluated by TLC. 0.5 ml L-asparaginase was mixed with 0.5 ml of 0.2% L-asparagine dissolved in 1 ml phosphate buffer (pH 7.0) and incubated at 37 °C for 24 h. The hydrolysed products were spotted on the chromatographic plate (8 × 5 cm). L-aspartic acid and asparagine of 0.2% (w/v) strength were also applied as the standards. The solvent system used was butanol, acetic acid and water in the 5:1:4 ratio. The enzymatic products were detected by staining with 0.25% ninhydrin in acetone.
Antioxidant activity of L-asparaginase The ABTS assay was carried out as per Re et al. [25] to evaluate the antioxidant activity of L-asparaginase. The ABTS•+ radical cation was produced by mixing 10 ml potassium persulfate of 2.45 mM with 10 ml aqueous ABTS of 7 mM and incubating in the dark for 12 h at room temperature. The cation was then diluted 60 times with 0.01 M phosphate buffered saline (PBS) (pH 7.4) to an optical density of ~0.7 at 734 nm. The L-asparaginase was prepared in the range of 200 to 1000 µg/ml using PBS. 1 ml of each L-asparaginase concentration was mixed with 1 ml of diluted ABTS•+. After mixing, the absorbance was recorded with a spectrophotometer (SL 159 Elico, India) at 734 nm after exactly 1 min. The L-ascorbic acid was used as standard and the blank was without ABTS.
Statistical Analysis The experiments to determine L-asparaginase activities were performed in triplicates. The differences between mean values were recorded by one way analysis of variance (ANOVA) and Duncan’s multiple range test (DMRT) at the 5% significance level using the SPSS statistical package.
Results and Discussion • Page 3 of 9 •
Citation: Monica T, Lincoln L, Niyonzima FN, Sunil SM (2013) Isolation, Purification and Characterization of Fungal Extracellular L-Asparaginase from Mucor Hiemalis. J Biocatal Biotransformation 2:2.
doi:http://dx.doi.org/10.4172/2324-9099.1000108
Isolation and identification of L-asparaginase producing fungi In the present study, L-asparaginase producing fungus was isolated from soil fields of Kengeri (Karnataka, India). The modified Czapek-Dox agar contained L-asparagine as a sole nitrogen source and phenol red as an indicator. L-asparagine also served as an enzyme inducer. The formation of a pink zone around the microorganism was an indication of the L-asparaginase production (Figure 1). The change in color (from yellow to pink) of the indicator resulted from the increase in pH due to ammonia release. The ammonia along with L-aspartic acid is formed by the deamidation reaction of the substrate L-asparagine by L-asparaginase [16,18]. The positive L-asparaginase producing fungus was identified morphologically and microscopically as Mucor sp. The identity was further confirmed as Mucor hiemalis by a National Fungal Culture Collection of India (Agharkar Research Institute, Pune). A Mucor sp. secreting extracellular L-asparaginase was also isolated from the marine sponge Spirastrella sp. [26]. Aspergillus, Penicillum, Fusarium, Helminthosporium, Scophulariopsis, Paecilomyces and Pestalotiopsis screened from Bhitarkanika mangrove forest soil of Orissa coast (India) were found to be a good source of L-asparaginase [27]. Many fungal species producing L-asparaginase were also isolated
from soil. For example, Emericella nidulans from different soils of Tumkur university campus (Karnataka, India) [28], Aspergillus flavus (KUFS20) from garden soil of Coimbatore (Tamilnadu, India) [29] and Penicillium sp. was screened from soil samples of Bangalore (Karnataka, India) [30]. Hosamani and Kaliwal [31] reported the screening of Fusarium equiseti from rhizosphere soil of various plants around Dharwad campus (Karnataka, India) and suggested that the presence of the fungus might be due to the presence of natural source of amino acid present in root exudates of the plants in the rhizophere soil. The microorganisms from the rhizosphere may have thus adapted the uptake and utilization of available nutrients like free amino acids.
Optimization of culture parameters for maximum L-asparaginase production The various parameters influencing L-asparaginase secretion were optimized. The L-asparaginase-producing fungi must be provided with optimum growth conditions in order to improve and increase the enzyme production without increasing the cost. A balance between various medium components is maintained, reducing the amount of unused nutrients after fermentation completion. Incubation temperature, initial pH, nitrogen and carbon source, and incubation temperature were optimized.
Table 1: Effect of incubation temperature, initial pH, L-asparagine concentration, glucose concentration and incubation time on L-asparaginase production by Mucor hiemalis. Means and standard deviation for each factor are shown. A significant difference at P0.05 is shown by different letters in rows Incubation temperature (°C) 30
37
Enzyme activity (U/ml)
212.5±15.7a
190.9±12.2a
45 142.0±16.8b
Initial pH.
4
5
6.2
7
8
Enzyme activity (U/ml)
130.9±10.7d
163.6±11.2d
220.1±31.3c
320.0±26.8a
275.9±25.0b
234.6±10.0c
L-Asn [ ] (%, w/v)
0.50
0.75
1.00
1.25
1.50
1.75
Enzyme activity (U/ml)
259.2±15.5e
285.0±41.6c,d
325.4±13.7c
518.4±23.2a
426.2±22.7b
231.7±9.2e
1.0
9
L-Asn [ ] (%, w/v)
0.2
0.4
0.6
0.8
Enzyme activity (U/ml)
522.1±10.3c
815.6±34.9a
701.2±22.7b
534.3±35.1c
200.6±16.9d
Incubation time (days)
1
2
3
4
5
6
7
Enzyme activity (U/ml)
499.7±28.1d
693.6±30.6c
833.2±27.4a
888.1±28.5a
666.0±39.9c
425.5±24.6e
314.7±14.1f
Effect of incubation temperature on enzyme production by Mucor hiemalis The incubation temperature is a critical environmental factor for L-asparaginase production by microbes because it regulates microbial growth and consequently enzyme secretion. The fungus was able to grow and produce the enzyme on all the temperatures evaluated with maximum production at 30 °C although statistically at par with 37 °C. However a noticeable decrease in enzyme yield was seen at 45 °C (Table 1). The optimum temperature of 30 or 37 °C was reported in most of the L-asparaginase producing fungal species [5,13,15,16,28,29,31]. The low enzyme activity value recorded at 45 °C may be attributed to partial enzyme denaturation resulted from a change in metabolic activities. Any increase or decrease from optimum incubation temperature slows down the metabolic activities of microorganisms [28,41].
Effect of initial pH of the medium on L-asparaginase production Figure 1: Fungal isolate showing a pink colour around the organism. The fungus was grown at room temperature for 3 days on modified Czapek-Dox agar plate.
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The initial pH of the production medium is an important parameter affecting the enzyme production since it can indirectly act on the fungal growth by affecting the availability of medium nutrients. The maximum L-asparaginase production was noted at
• Page 4 of 9 •
Citation: Monica T, Lincoln L, Niyonzima FN, Sunil SM (2013) Isolation, Purification and Characterization of Fungal Extracellular L-Asparaginase from Mucor Hiemalis. J Biocatal Biotransformation 2:2.
doi:http://dx.doi.org/10.4172/2324-9099.1000108 an initial pH of 7.0; thereafter a decline in enzyme production was seen (Table 1). Similarly, an initial pH of 7.0 led to maximum enzyme yield by Fusarium equiseti [31] and Aspergillus terreus [32]. The initial pH reported for maximum L-asparaginase production is in the pH 6.0 - 9.0 range [5,15,28,33,34]. A decline in enzyme activity seen after optimum pH may due to partial denaturation of the enzyme resulted from dissociation of the ionizable groups of enzymes. The change in pH prevents the binding of a substrate to the enzyme owing to change of shape and properties of an enzyme and/or the substrate [35].
Table 2: Summary of purification procedure of L-asparaginase of Mucor hiemalis. Fraction
Total activity (U)
Total protein (mg)
Specific Activity (U/mg)
Purification Recovery fold (%)
Crude enzyme
1203.55 79.64
15.11
1
100
Acetone precipitate
481.38
21.0
22.92
1.51
40
3.2
69.43
4.59
18.46
Affinity 222.18 chromatography
Effect of L-asparaginase concentration on the enzyme production by Mucor hiemalis The yield of L-asparaginase can be increased by varying the nitrogen sources or the concentration of a good nitrogen source since L-asparaginase is nitrogen regulated enzyme [13]. Maximum enzyme secretion was achieved at 1.25% (Table 1). A similar observation was reported in the 1 to 2% range for L-asparaginase of Bipolaris sp. [15], Aspergillus terreus [34] and Fusarium sp. [36]. However, maximum yield was obtained at 0.5% L-asparagine in Aspergillus oryzae [16]. The amino acid proline (0.5-2%) was also the best nitrogen source for L-asparaginase production in many filamentous fungal species [7,13,33]. A decrease in L-asparaginase production seen at elevated L-asparagine concentration (Table 1) may be ascribed to nitrogen catabilite repression. The same repression was also reported by Savitri et al. [9]. However, the decrease in L-asparaginase production in Aspergillus terreus IOC 217 was attributed to the presence of a protease by Sarquis et al. [13] since the protease levels enhanced when L-asparaginase levels declined.
Effect of different concentrations of the glucose on L-asparaginase production An extracellular L-asparaginase is produced during the postexponential and stationary growth phases by fungi under most culture conditions. It is therefore regulated by carbon and nitrogen sources. An important L-asparaginase production was recorded at 0.4% glucose concentration beyond which the enzyme activity gradually declined (Table 1). Likewise 0.4% (w/v) was the best glucose concentration for the L-asparaginase production in Aspergillus terreus MTCC 1782 [5] and Bipolaris sp. BR438 [15]. A decline in L-asparaginase synthesis observed after optimum glucose concentration may be attributable to glucose catabolite repression. A stimulatory effect is thus seen at the low glucose concentrations whereas higher glucose concentrations are inhibitory. The similar glucose effect was observed for the L-asparaginase of Aspergillus terreus MTCC 1782 [5] and Bipolaris sp. BR438 [15].
Time course analysis of L-asparaginase production by Mucor hiemalis The effect of fermentation time was evaluated to note the optimal time for harvesting L-asparaginase. The optimal time found was 96 h although statistically at par 72 h (Table 1). The same incubation period was also noted for L-asparaginase of Aspergillus species [3,5,16,32,34], Bipolaris sp. BR438 [15] and Penicillium sp. [29]. The shorter incubation time makes the present submerged fermentation costeffective and reduces the chance of L-asparaginase decomposition by proteolytic enzymes. Contrastingly, a higher incubation time of 120 h was seen in the secretion of L-asparaginase by Fusarium sp. [36]. In the present study, the prolonged incubation time led to a decrease in L-asparaginase secretion and this may be due to the exhaustion of some medium constituents or the production of inhibitory compounds. Volume 2 • Issue 2 • 1000108
Figure 2: Silver stained SDS-PAGE of L-asparaginase of Mucorhiemalis. Protein molecular weight marker (Lane 1), affinity chromatography purified enzyme (Lane 2), Acetone purified enzyme (Lane 3). Proteins in the marker (kDa): Phosphorylase b (97.4), BSA (66.0), ovalbumin (43.0), carbonic anhydrase (29.0), soybean trypsin inhibitor (20.1) and lysozyme (14.3).
Enzyme purification The ammonium sulphate and acetone precipitation methods were used to partially purify the enzyme; however an important yield was seen with acetone precipitation. The L-asparaginase of Candida utilis was also partially purified by acetone precipitation [37]. A lectin-agarose column was used to totally purify the enzyme because the L-asparaginase from Mucor hiemalis was glycosylated. The L-asparaginase of Candida utilis was also a glycoprotein. Similarly, the L-asparaginase purified from the mesophilic fungus Cylindrocarpon obtusisporum MB-10 was a glycoprotein with 37.3% (w/w) carbohydrate [38]. The purification of the L-asparaginase to homogeneity was efficient, rapid and cost-effective since two purification steps were used. Bora and Bora [39] reported that few purification steps are preferable since a loss of about 10 % enzyme yield is seen at each purification step. A recovery of 18.46% with a specific activity of 69.43 U/mg was achieved (Table 2). Different specific activities and yields have been reported for various fungal species. For example a recovery of 36.2% and 1.9-fold with 13.97 U/ mg was recorded for L-asparaginase of Penicillium sp. [7]. A yield of 3.6% with 7.6-purification fold was seen for L-asparaginase of Aspergillus terreus [32]. A specific activity of 207 U/mg with 0.54% recovery and 267.75-purification fold was noted for the enzyme of Aspergillus aculeatus [40].
SDS-PAGE and Molecular weight estimation The acetone purified sample and the affinity purified sample were • Page 5 of 9 •
Citation: Monica T, Lincoln L, Niyonzima FN, Sunil SM (2013) Isolation, Purification and Characterization of Fungal Extracellular L-Asparaginase from Mucor Hiemalis. J Biocatal Biotransformation 2:2.
doi:http://dx.doi.org/10.4172/2324-9099.1000108 demonstrate the activity of the enzyme through L-asparagine-agarose gel. The gel was sprayed with Nessler’s reagent and a clear brown coloured zone was seen in the gel where the enzyme had degraded L-asparagine (Figure 3). The enzyme was therefore very active and degraded the co-polymerized L-asparagine to L-aspartic acid and ammonia.
Characterization of L-asparaginase
Figure 3: Activity staining of L-asparaginase. Left: clear brown coloured zone indicating L-asparagine degradation. Right: Control.
1000
Enzyme activity (U/ml)
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Figure 4: Effect of pH on the activity of L-asparaginase of Mucorhiemalis.The pre-incubation was done at room temperature (30 ± 2°C) in various buffers of 0.05 M strength for 30 min. A significant difference at P0.05 is shown by different letters on the error bars.
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Enzyme activity (U/ml)
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Figure 5: Effect of temperature on activity of L-asparaginase of Mucorhiemalis. The pre-incubation was done at required temperature for 30 min in phosphate buffer (pH 7.0). A significant difference at P0.05 is shown by different letters on the error bars.
subjected to SDS-PAGE under non reducing conditions. A single band was observed next to the phosphorylase b. L-asparaginase was therefore a homogenous monomeric protein. Since L-asparaginase is a tetrameric protein [1], the purified enzyme may be a homotetramer because a single band was seen. On comparison with the standard molecular weight markers, the apparent molecular weight of L-asparaginase was approximately 96.32 kDa (Figure 2). The molecular weight was close to 94 kDa of L-asparaginase purified from Aspergillus terreus [32,34]. 48 and 66 kDa were also recorded for L-asparaginase of Rhizopus sp. [7] and Aspergillusniger [33], respectively. Higher molecular weight of 216 kDa was also reported for the L-asparaginase of mesophilic fungus Cylindrocarpon obtusisporum [38]. The variation of L-asparaginase molecular weight may be ascribed to genetic differences.
Activity staining of L-asparaginase A simple enzymatic diffusion reaction was performed to
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Effect of pH on enzyme activity and stability: The effect of pH on the enzyme activity was evaluated. The optimum pH was 7.0 although statistically at par with 6.0 and 8.0 (Figure 4). Likewise, pH 7.0 was the optimum for the L-asparaginase of Penicillium sp. [7] and Aspergillus terreus [32]. The optimum pH 9.0 was also reported for L-asparaginase purified from Aspergillus terreus KLS2 [3] and Aspergillus aculeatus [40]. Low activity values were seen at higher acidic or alkaline pH because the purified enzyme was a neutral enzyme. Indeed, enzymes contain on their surface a large number of basic and acidic groups which get ionized depending on the pH environment. Since the enzyme is neutral, any important deviation affects the active site of L-asparagine resulting in an activity decline. The enzyme was checked for its stability in phosphate buffer of pH 7.0. It was observed that the enzyme maintained its stability for 4 h at 30 °C. However, a marginal reduction of 1.2 % in relative enzyme activity was noted after 24 h (data not shown). The L-asparaginase of Aspergillus terreus KLS2 was 100% stable for only 1 h at pH 8.0 [3] while the enzyme from Aspergillus aculeatus was stable up to 8 h at pH 9.0 [40]. The purified enzyme in the present study appeared to be stable than other reported for fungal species. Effect of temperature on enzyme activity and stability: The effect of temperature on L-asparaginase activity was investigated and the maximum enzyme activity was recorded at 37 °C although statistically at par with 30 °C (Figure 5). The L-asparaginase of Aspergillus terreus KLS2 [3] and Penicillium sp. [7] also exhibited an optimum temperature of 37 °C. L-asparaginase of Aspergillus aculeatus [40] showed an optimum activity at 30 °C while 35 °C was optimum for the enzyme of Aspergillus terreus [32]. Low L-asparaginase activity observed at higher temperature may be attributable to partial denaturation. Comparable denaturation was also reported [37,40]. The enzyme was 100% stable for 4 h. However, after 4 h the enzyme started losing its activity and an overnight incubation resulted in a slight drop of 2.4% in the activity of the enzyme (data not shown). Dange and Peshwe [40] purified an L-asparaginase of Aspergillus aculeatus that was stable for 2 h at 30 °C. Sakamoto et al. [37] reported L-asparaginase that was stable at 55 °C for 10 min at pH 7.0. The enzyme of the present study displayed a higher stability as compared with other fungal enzymes as no other reports about L-asparaginase to retain stability after overnight incubation. The stability of an enzyme at physiological pH and temperature is a desirable characteristic for a medical enzyme. Effect of metal ions on L-asparaginase activity: The effect of various cations on L-asparaginase was studied. It was found that Ba2+, K+, Cu2+, Mn2+ and Hg2+ enhanced the enzyme activity, specifically in the presence of Mn2+ the enzyme activity doubled. A slight decrease in the activity was observed with Fe2+ and Na+. However, no inhibition or activation seen with Ca2+, Mg2+, Na+, Fe3+, Zn2+ and Co2+ (Figure 6). From the results it can be concluded that L-asparaginase is in general a metaL-activated enzyme. Similar results were reported by Sakamoto et al. [37] where the enzyme of Candida utilis was not inhibited by Ca2+, Mg2+, Mn2+, Fe2+, Zn2+, and Cu2+. Contrastingly,
• Page 6 of 9 •
Citation: Monica T, Lincoln L, Niyonzima FN, Sunil SM (2013) Isolation, Purification and Characterization of Fungal Extracellular L-Asparaginase from Mucor Hiemalis. J Biocatal Biotransformation 2:2.
doi:http://dx.doi.org/10.4172/2324-9099.1000108
Residual activity (%)
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Figure 6: Effect of various cations on L-asparaginase activity. The enzyme was pre-incubated with the respective metal ion of 2 mM strength for 30 min at 37 °C. The relative activity was 100% when no metal ion was added. A significant difference at P0.05 is shown by different letters on the error bars.
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Substrate specificity and determination of kinetics of L-asparaginase
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Figure 7: Effect of various inhibitors and activators (2 mM) on L-asparaginase activity.The control was pre-incubated at 37 °C for 30 min without any inhibitor and the residual protease activity was considered as 100%. A significant difference at P0.05 is shown by different letters on the error bars.
0.0090
y = 0.006x + 0.001 R² = 0.997
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to know the identity and the type of the enzyme. The effect of inhibitors and activators was studied. SDS was only the reagent which showed a reduction in relative enzyme activity and retained 17% of activity (Figure 7). EDTA displayed no effect on L-asparaginase activity confirming the non metalloproteinic nature of the enzyme. L-asparaginase is therefore a metaL-activated enzyme but not a metalloenzyme. Similarly, EDTA did not show any effect on L-asparaginase of Candida utilis [37]. In contrast to SDS, the nonionic surfactants (Tween 80 and Triton X 100) enhanced the enzyme activity (Figure 7) as they may provide a good environment for enzyme action. 2-mercaptoethanol enhanced the enzyme activity. A similar result was reported by Raha et al. [38] with L-asparaginase of Cylindrocarpon obtusisporum. However, it did not enhance or inhibit the enzyme purified from Candida utilis [37]. NEM, urea, NBS and PMSF did not have any effect on the enzyme of Mucor hiemalis. Cysteine, tryptophan and serine may thus be absent in the L-asparaginase active site.
1
1/[Asparagine] (mM)
L-aspartic acid, L-arginine, L-histidine, L-phenylalanine and L-glutamine were used to investigate the substrate specificity of the L-asparaginase. The enzyme was only active against its substrate and no activity was seen with other amino acids (data not shown). Similarly, L-asparaginase of Penicillium sp. was unable to use L-aspartic acid, L-phenylalanine, L-glutamine and L-histidine as substrate [7]. Likewise, L-asparaginase of Candida utilis had specificity towards D-and L-asparagine, but it was incapable to hydrolyze L- or D-glutamine and L-aspartic acid [37]. Siddalingeshwara and Lingappa [3] purified L-asparaginase from Aspergillus terreus KLS2 that was 100% active towards the L-asparagine and 5% and 4% active towards the D-asparagine and L-glutamine, respectively. The absence of enzyme activity towards L-glutamine may be advantageous for its use as a therapeutic agent since some of the side effects of L-asparaginase are due to the specificity of enzyme towards L-glutamine. A double reciprocal plot was used to determine kinetic constants. The values found for Km and Vmax were 4.3 mM and 625 U/ml, respectively (Figure 8). Similarly, a Km value of 4 mM was found for L-asparaginase of Penicillium sp. [7]. A slightly higher Km of 12.5 mM (and a Vmax of 104.16 U/ml) was seen for the L-asparaginase of Aspergillus aculeatus [40]. A low Km value of 1 mM was recorded by Raha et al. [38] for L-asparaginase of Cylindrocarponobtusisporum. The low Km noted for L-asparaginase of Mucorhiemalis suggested the high affinity to the substrate and thus its effectiveness towards tumor cells.
Thin layer chromatography Figure 8: Lineweaver-Burk plot for the determination Km and Vmax of L-asparaginase of Mucorhiemalis. The enzyme was incubated with different concentrations of L-asparagine at 37 °C for 30 min.
the L-asparaginase of Cylindrocarpon obtusisporum was inhibited by Zn2+, Fe2+, Cu2+, Hg2+ and Ni2+ [38]. Fe2+, Co2+ and Zn2+ also enhanced enzyme activity of L-asparaginase obtained from Aspergillus aculeatus while Hg 2+, Cu 2+ and Mg 2+ had an inhibitory effect [40]. Therefore, L-asparaginases behaved differently in the presence of metal ions and this may be ascribed to the tetrameric nature of the enzyme. Effect of specific inhibitors and activators on the enzyme: The group specific reagents for amino acids in the active site help
Volume 2 • Issue 2 • 1000108
For the analysis and confirmation of the hydrolysed products from the enzymatic reaction of L-asparaginase, a TLC was conducted which confirmed the production of L-aspartic acid upon the degradation of L-asparagine by L-asparaginase. Aspartate and residual L-asparagine was observed on the TLC plate (Figure 9). Hence, it was confirmed that the enzymatic hydrolysis of L-asparagine produces aspartic acid. The comparable result was observed for the L-asparaginase of Penicillium sp. [30].
Antioxidant activity of L-asparaginase L-asparaginase and ascorbic acid showed antioxidant activities as they were able to scavenge the ABTS•+ radical cation. The concentration of L-asparaginase almost increased linearly with the increase in % inhibition (Figure 10). The purified enzyme • Page 7 of 9 •
Citation: Monica T, Lincoln L, Niyonzima FN, Sunil SM (2013) Isolation, Purification and Characterization of Fungal Extracellular L-Asparaginase from Mucor Hiemalis. J Biocatal Biotransformation 2:2.
doi:http://dx.doi.org/10.4172/2324-9099.1000108 23: 710-711. 2. Verma N, Kumar K, Kaur G, Anand S (2007) L-asparaginase: a promising chemotherapeutic agent. Crit Rev Biotechnol 27: 45-62. 3. Siddalingeshwara KG, Lingappa K (2011) Production and characterization of L-asparaginase A tumor inhibitor. Int J PharmTech Res 3: 314-319. 4. Warangkar SC, Khobragade CN, Dawane BS, Bhosale RB (2009) Effect of dihydropyrimidine derivatives on kinetic parameters of E. carotovora L-asparaginase. Int J Biotechnol Appl 1: 5-13. 5. Gurunathan B, Sahadevan R (2011) Optimization of media components and operating conditions for exogenous production of fungal L-asparaginase. Chiang Mai J Sci 38: 270-279. 6. McCredie KB, Ho DH, Freireich EJ (1973) L-asparaginase for the treatment of cancer. CA Cancer J Clin 23: 220-227. 7. Patro KR, Gupta N (2012) Extraction, purification and characterization of L-asparaginase from Penicillium sp. by submerged fermentation. Int J Biotechnol Mol Biol Res 3: 30-34. 8. Ramya LN, Doble M, Rekha VP, Pulicherla KK (2012) L-Asparaginase as potent anti-leukemic agent and its significance of having reduced glutaminase side activity for better treatment of acute lymphoblastic leukaemia. Appl Biochem Biotechnol 167: 2144-2159. 9. Savitri, Asthana N, Azmi W (2003) Microbial L-asparaginase-A potent antitumor enzyme. Ind J Biotechnol 2: 184-194. 10. Bascomb S, Banks GT, Skarstedt MT, Fleming A, Bettelheim KA (1975) The properties and large-scale production of L-asparaginase from citrobacter. J Gen Microbiol 91: 1-16.
Figure 9: Chromatogram showing hydrolyzed products. Lane 1: standard L-aspartic acid, lane 2: standard L-asparagine and lane 3: enzyme hydrolysate (aspartate and residual L-asparagine).
12. Kieslich M, Porto L, Lanfermann H, Jacobi G, Schwabe D, et al. (2003) Cerebrovascular complications of L-asparaginase in the therapy of acute lymphoblastic leukemia. J Pediatr Hematol Oncol 25: 484-487. 13. Sarquis MI, Oliveira EM, Santos AS, Costa GL (2004) Production of L-asparaginase by filamentous fungi. Mem Inst Oswaldo Cruz 99: 489-492.
100 90
14. Rossi F, Incorvaia C, Mauro M (2004) Hypersensitivity reactions to chemotherapeutic antineoplastic agents. Recenti Prog Med 95: 476-481.
80
% inhibtion
11. Nagarethinam S, Anantha NN, Udupa N, Venkata RJ, Meenashi VB (2012) Microbial L-asparaginase and its future prospects. Asian J Med Res 1: 159168.
70 60
15. Lapmak K, Lumyong S, Thongkuntha S, Wongputtisin P, Sarsud U (2010) L-asparaginase production by Bipolaris sp. BR438 isolated from brown rice in Thailand. Chiang Mai J Sci 37: 160-164.
50 40 30
16. Gulati R, Saxena RK, Gupta R (1997) A rapid plate assay for screening L-asparaginase producing micro-organisms. Lett Appl Microbiol 24: 23-26.
20 10 0 0
2
4
6
8
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12
Concentration (mg/ml)
Figure 10: The effect of L-asparaginase (■) and L-ascorbic acid (▲) on the inhibition of the ABTS•+ radical cation. The absorbance was noted at 734 nm after 1 min at 30°C.
displayed a very good scavenging activity than L-ascorbic acid indicating its suitability as antileukemic agent. The L-asparaginase of Aspergillusflavus (KUFS20) also displayed a good scavenging activity [29].
Conclusion In the present study, a potent L-asparaginase producing Mucor hiemalis was isolated from soil. The desirable traits like stability at physiological pH and temperature, high substrate specificity and good scavenging activity suggested the purified enzyme to be exploited as an antitumor agent. References 1. Goodsell DS (2005) The molecular perspective: L-asparaginase. Stem Cells
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17. Koneman EW, Allen SD, Janda WM, Schreckenberger PC, Winn WC (1977) Colour Atlas and Textbook of Diagnostic Microbiology. (5th edn), J.B. Lippincott company, Philadelphia, USA. 18. Imada A, Igarasi S, Nakahama K, Isono M (1973) Asparaginase and glutaminase activities of micro-organisms. J Gen Microbiol 76: 85-99. 19. Lowry OH, RosebroughNJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-275. 20. Obi SK, Odibo FJ (1984) Partial Purification and Characterization of a Thermostable Actinomycete beta-Amylase. Appl Environ Microbiol 47: 571-575. 21. Spivak JL, Small D, Hollenberg MD (1977) Erythropoietin: isolation by affinity chromatography with lectin-agarose derivatives. Proc Natl Acad Sci U S A 74: 4633-4635. 22. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685. 23. Blum H, Beier H, Gross HJ (1987) Improved silver staining of plant-proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8: 93-99. 24. Lineweaver H, Burk D (1934) The determination of enzyme dissociation constants. J Am Chem Soc 56: 658-666. 25. Re R, Pellegrini N, Pannala A, Yang M, Rice-Evans C (1999) Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol Med 26: 1231-1237.
• Page 8 of 9 •
Citation: Monica T, Lincoln L, Niyonzima FN, Sunil SM (2013) Isolation, Purification and Characterization of Fungal Extracellular L-Asparaginase from Mucor Hiemalis. J Biocatal Biotransformation 2:2.
doi:http://dx.doi.org/10.4172/2324-9099.1000108 26. Mohapatra BR, Sani RK, Banerjee UC (1995) Characterization of L-asparaginase from Bacillus sp. isolated from an intertidal marine alga (Sargassum sp.). Lett Appl Microbiol 21: 380-383.
34. Balasubramanian K, Ambikapathy V, Panneerselvam A (2012) Production, isolation and purification of L-asparaginase from Aspergillus terreus using submerged fermentation. Int J Adv Pharm Res 3: 778-783.
27. Gupta N, Dash J S, Basak CU (2009) L- asparaginases from fungi of Bhitarkanika mangrove ecosystem. AsPac J Mol Biol Biotechnol 17: 27-30.
35. Khalaf ZA, AL-Ani NK, Jasim HM (2012) Optimum conditions for asparaginase extraction from Pisum sativum subspp. J Iranian J Plant Physiol 2: 517-521.
28. Jayaramu M, Hemalatha NB, Rajeshwari CP, Siddalingeshwara KG, Mohsin SM, et al. (2010) A novel approach for detection, confirmation and optimization of L-asparaginase from Emericella nidulans. Curr Pharm Res 1: 20-24.
36. Thirunavukkarasu N, Suryanarayanan TS, Murali TS, Ravishankar JP, Gummadi SN (2011) L-asparaginase from marine derived fungal endophytes of seaweeds. Mycosphere 2: 147-155.
29. Rani SA, Lalitha S, Praveesh BV (2011) In vitro antioxidant and anticancer activity of L-asparaginase from Aspergillusflavus (KUFS20). Asian J Pharm Clin Res 4: 174-177. 30. Mushtaq M S, Siddalingeshwara K G, Karthic J, Sunil D P L N S N, Naveen M, Prathiba KS (2012) Rapid screening and confirmation of L-asparaginase from Penicillium sp. Int J Res Pharmacol Pharm 1: 147-150. 31. Hosamani R, Kaliwal BB (2011) Isolation, molecular identification and optimization of fermentation parameters for the production of L-asparaginase, an anticancer agent by Fusarium equiseti. Int J Microbiol Res 3: 108-119. 32. Chandrasekhar AP (2012) Isolation, purification and characterization of Asparaginase from Aspergillusspecies. Int J Res Chem Environ 2: 38-43.
37. Sakamoto T, Araki C, Beppu T, Arima K (1977) Partial purification and some properties of extracellular Asparaginase from Candida utilis. Agric Biol Chem 41: 1359-1364. 38. Raha SK, Roy SK, Dey SK, Chakrabarty SL (1990) Purification and properties of an L-Asparaginase from Cylindrocarpon obtusisporum MB-10. Biochem Int 21: 987-1000. 39. Bora L, Bora M (2012) Optimization of extracellular thermophilic highly alkaline lipase from thermophilic bacillus sp isolated from hotspring of Arunachal Pradesh, India. Braz J Microbiol 43: 30-42. 40. Dange VU, Peshwe SA (2011) Production purification and characterization of fungal L-asparaginase. Bionano Frontier 4: 162-167.
33. Akilandeswari K, Kavitha K, Vijayalakshmi M (2012) Production of bioactive enzyme L-asparaginase from fungal isolates of water sample through submerged fermentation. Int J Pharm Pharm Sci 4: 363-366.
Author Affiliations
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Department of Biochemistry, Center for Post Graduate Studies, Jain University, Bangalore, India
1
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Volume 2 • Issue 2 • 1000108
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