1. INTRODUCTION
Actinomycetes are omnipresent and are most valued organisms. They are branching bacteria with the fungal type of morphology. They are Gram-positive bacteria with DNA rich in G+C content (57–75%), frequently filamentous and sporulating. Most of the actinomycetes are saprophytic bacteria and thrive in a wide range of habitat. Actinomycetes are major source of important bioactive compounds having high commercial value. They are reported to produce a wide range of bioactive metabolites which include antibiotics, antitumor agents, immunosuppressive agents, and enzymes [1]. Streptomyces is the largest antibiotic-producing genus, producing both antibacterial agents (Erythromycin, Neomycin, Streptomycin, Tetracycline, Vancomycin, etc.) and antifungal agents (Nystatin, Amphotericin B, Natamycin, etc.). Other genera which are frequent producers of antibiotics include Micromonospora, Actinomadura, Streptoverticillium, Actinoplanes, Nocardia, Saccharopolyspora, and Streptosporangium [2]. An actinomycetes metabolite, Adriamycin avert DNA replication process and hence is used in the treatment of leukemia. Another metabolite, rapamycin has immune suppression property and is used in organ transplantation [3]. Actinomycetes are also known for their capacity to produce various enzymes such as amylase, protease, lipase, cellulase, pectinase, xylanase, laccase, asparaginase, and glutaminase [4].
Microbial L-asparaginases (L-asparagine amidohydrolase, E.C.3.5.1.1) are generally used as therapeutic agents in the treatment of human cancers [5]. L-asparaginase enzyme catalyzes the hydrolysis of L-asparagine to aspartic acid and ammonia [6]. L-asparagine is required by both normal and tumor cells. Under natural conditions, L-asparagine is synthesized by enzyme L-asparagine synthetase using substrates aspartic acid and glutamine. L-asparagine can also be absorbed from external sources. Cancerous cells lack the enzyme L-asparagine synthetase and hence are unable to synthesize their own L-asparagine [7]. Hence, these cancerous cells are dependent on L-asparagine present in the blood of the patient. Tumor cells require an enormous quantity of L-asparagine for their rapid growth. L-asparagine is limited in the circulating pool when the enzyme L-asparaginase is present resulting in the death of tumor cells [8]. This approach helps in the use of L-asparaginase as an anti-cancerous agent [9]. In addition to clinical applications of L-asparaginase have potential application in food industry. It can be used in eliminating acrylamide formation during frying of starchy foods. Amino acids and reducing sugars present in starchy food, when heated, give a characteristic aroma and flavor. When L-asparagine and reducing sugars react, it leads to the formation of acrylamide. Addition of L-asparaginase enzyme before baking or frying the food leads to conversion of L-asparagine to aspartic acid. Thus, the enzyme reduces the risk of acrylamide formation in food [10].
Various actinomycetes such as Streptomyces griseus [11], Nocardia sp. [12], and Actinomycete strain LA-29 [13] were explored for L-asparaginase production. L-asparaginase enzyme purification was carried out from various organisms such as actinobacteria [14], Streptomyces halstedii [15], Streptomyces tendae [16], and marine Streptomyces spp. [17]. Different purification techniques such as salt precipitation [13,17] and column chromatography [13,14,18] were used during purification steps. Enzyme was characterized with respect to various parameters such as temperature, pH, and molecular weight. Furthermore, studies were undertaken to assess the effect of metal ions and kinetic parameters [14,15,17-23]. Production and purification of extracellular L-asparaginase are easier compared to intracellular enzymes. Hence, the present study aims at isolation of actinomycetes, screening for L-asparaginase production and purification, and characterization of extracellular L-asparaginase.
2. MATERIALS AND METHODS
2.1. Isolation, Screening, and Characterization of Actinomycetes
Soil samples from different farms, in and around Hubballi, Karnataka, were collected. 22 isolates of actinomycetes were obtained from soil by dilution plate technique using starch casein (SC) agar plates (Composition: Soluble starch-1%; K2HPO4-0.2%; KNO3-0.2%; NaCl-0.2%; casein-0.03%; MgSO4.7H2O-0.005%; CaCO3-0.002%; FeSO4.7H2O-0.001%; and agar-1.5%) [24]. The plates were incubated for 7 days at 30°C. Single discrete isolates were screened for L-asparaginase production. The isolates were maintained on SC agar and sub-cultured periodically. Isolates were screened for production of L-asparaginase using the method as described by Mahajan et al. [25]. Modified M-9 medium (composition: Na2HPO4-0.6%; KH2PO4-0.3%, NaCl-0.05%, L-asparagine-0.5%, MgSO4.7H2O-2ml (1 M), CaCl2.2H2O-1ml, (0.1 M), glucose-5%, agar-2%, and pH-7) was incorporated with a pH indicator (0.007 % Bromo Thymol Blue) [26]. L-asparaginase activity was identified by a change in the color of medium from yellow to green or blue. Modified M-9 medium without dye served as one control. Another control was Modified M-9 medium without L-Asparagine but supplemented with NaNO3. The isolate was identified according to the standard methods as described by Shirling and Gottileb [27]. Various properties of the isolates were studied as reported earlier [4].
2.2. Production of L-Asparaginase and Enzyme Assay
Production of L-asparaginase was undertaken by the method as described by Peterson and Ciegler [28]. The isolate was inoculated in 100 ml of Tryptone glucose yeast extract medium (composition: Glucose-0.l%; K2HPO4-0.l%; yeast extract-0.5%; and tryptone-0.5%, pH-7.0) and incubated in rotary incubator shaker at 30°C, 120 rpm for 120 h. At a fixed interval, known volume of samples was withdrawn and centrifuged in a cooling centrifuge (Eppendorf) at 8000 × g for 15 min at 4°C. The supernatant served as the crude enzyme preparation. Enzyme was assayed using the Berthelot reaction which estimates the amount of ammonia liberated [29,30]. To 0.5 ml of crude enzyme, 0.5 ml of 1 mM L-asparagine and 1 ml of 0.5 M Tris-HCl buffer (pH 6) were added and incubated at 37°C for 10 min. The reaction was stopped by adding 1 ml of phenol reagent, 1 ml of 1N sodium hydroxide, and 1 ml of alkaline hypochlorite. The mixture was kept for 15 min. Absorbance was recorded at 600 nm using ultraviolet-visible spectrophotometer (Make: LABINDIA) using appropriate blank.
Quantity of enzyme required to liberate 1 μM of ammonia per min is defined as one international unit (IU) of L-asparaginase enzyme. Lowry’s method was used to determine protein content [31].
2.3. Enzyme Purification
L-asparaginase was purified using following steps: Ammonium sulfate salt precipitation and column chromatography (diethylaminoethyl [DEAE]-Cellulose and Sephadex G-50). The culture medium was centrifuged in a cooling centrifuge (Eppendorf) at 6000 × g for 8 min. The supernatant obtained was precipitated using ammonium sulfate at a salt concentration ranging from 40% to 80%. It was left overnight at 4°C and then centrifuged at 6000 × g for 20 min at 4°C. The precipitate was dissolved in 0.5 M Tris-HCl buffer (pH 6), and enzyme activity was determined. The precipitate with the highest activity was dialyzed overnight against the same buffer at 4°C and assayed. This was used for further purification. The crude enzyme was applied to a column of DEAE-Cellulose column (15 cm × 2 cm), and protein was loaded. The column was equilibrated with 0.5 M Tris-HCl buffer (pH 6). The elution was carried out using gradient of NaCl (0.1 M–0.5 M) with a flow rate of 0.5 ml/min. Fractions of 1 ml were collected. It was followed by monitoring the A280 of each fraction to determine protein content. The active fractions were assayed for enzyme activity. L-asparaginase active fractions were pooled and desalted by dialysis against 0.5 M Tris-HCl buffer (pH 6). Purification of L-asparaginase was continued with Sephadex G-50 at room temperature. The column (15 cm × 2 cm) was equilibrated with 0.5 M Tris-HCl buffer, and then enzyme was loaded onto the column. It was eluted with the same buffer in the beginning and then using a linear gradient of KCl (0–200 mM) in 0.5 M Tris-HCl, buffer. Flow rate of 0.5 ml/min was maintained. 1 ml fractions were collected and estimated for protein content and assayed for enzymatic activity. Absorbance measurement (A280) method was used to estimate protein content. L-asparaginase active fractions were pooled and used for further analysis [13,17,21,23].
2.4. Characterization of the Purified L-Asparaginase
2.4.1. Effect of pH, temperature and substrate concentration on enzyme activity
Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was used to evaluate the molecular weight of the purified L-asparaginase enzyme [32]. The gel was stained with Coomassie Brilliant Blue R-250. Standard molecular weight marker (Bangalore Genei, India) was used to compare the molecular weight of L-asparaginase [21]. The effect of temperature on L-asparaginase activity was studied using Berthelot reaction [18,30]. The temperature of incubation was varied between 20°C and 50°C with step increase of 5°C, and the optimum temperature was evaluated. The pH was varied between 3 and 9 with a step increase of 1 unit. Acetate buffer (pH 3.0–5.0), phosphate buffer (pH 6.0–7.0), and Tris-HCl buffer (pH 8.0–9.0) at a concentration of 100 mM were used during the study [14,18,21]. Commercial L-asparagine of concentration between 0.2 mM and 2 mM was used as the standard substrate. The enzyme assay was performed by Berthelot reaction. The evaluation of Michaelis–Menten and Lineweaver–Burk plots yielded the kinetic parameters for the L-asparaginase activity.
2.4.2. Effect of metal ions and additives
Different salt solutions - sodium chloride (Na+), potassium chloride (K+), magnesium sulfate (Mg2+), calcium chloride (Ca2+), ferrous sulfate (Fe2+), copper sulfate (Cu2+), mercury chloride (Hg2+), and chemicals, namely, dithiothreitol (DTT), SDS, ethylene diamine tetra acetic acid (EDTA), and mercaptoethanol (ME) were used to determine their effect on enzymatic activity. This mixture was incubated at room temperature for 30 min, and then enzyme assay was performed [17-19,21].
3. RESULTS AND DISCUSSION
3.1. Isolation and Characterization of Actinomycetes and Screening for L-Asparaginase
Twenty-two actinomycetes were isolated from different soil samples. All the isolates were screened for L-asparaginase production using modified M-9 media with bromothymol blue (BTB) as an indicator. L-asparaginase catalyzes the breakdown of asparagine to aspartic acid and ammonia. Due to the production of ammonia, pH of the media increased. Hence, addition of the pH indicator changed its color and this forms the basis of assay. BTB was used as pH indicator in the medium. Plates/broth containing BTB were yellow at lower pH value and changed to dark blue at higher pH value. Hence, a dense dark blue color is produced if microorganism produces L-asparaginase [25]. In control plate without dye, organism growth was observed with no change in media color. In another control plate, wherein L-asparagine is substituted with NaNO3, growth was not observed indicating, organism was unable to utilize NaNO3. Of all the isolates, only eight actinomycetes were able to produce L-asparaginase as indicated by a change in the color of the media from yellow to blue. Among these isolates, one isolate (A-164) had the ability to change the media color in short duration [Figure 1], and enzyme production was more (9.8 IU/ml) and hence was selected for further studies. Based on the phenotypic, microscopic and biochemical properties of the organism [Figure 2 and Table 1], the isolate was identified as genus Streptomyces. Figure 2a shows typical colony morphology for the isolate Streptomyces sp. Figure 2b and c shows microscopic view of the organism highlighting the presence of hyphae and asexual spores. The color of mature aerial mycelium was white, and that of substrate mycelium was light yellow without any melanoid pigmentation. The spores were observed to be knob-shaped. The isolate could able to degrade casein, tyrosine, and xanthine, typical properties of genus Streptomyces [33,34]. Various researchers have reported production of L-asparaginase by Streptomyces spp. such as S. halstedii [15], S. tendae [16], and Streptomyces ginsengisoli [35].
 | Figure 1: Qualitative screening for L-asparaginase production by Streptomyces sp. The broth shows change in color of modified M9 media from yellow to blue due to the production of L-asparaginase. (a) M-9 media containing both L-asparagine and bromothymol blue (BTB) pH indicator without inoculum, (b) control tube containing M-9 media without BTB pH indicator, (c) control tube containing M-9 media without L-asparagine, (d) M-9 media containing L-asparagine, BTB pH indicator, and inoculum. [Click here to view] |
 | Figure 2: Photographic images of Streptomyces sp. (a) Colonies of isolate after 5 days of incubation on glucose asparagine medium, (b) and (c) microscopic view of the isolate (×100). [Click here to view] |
 | Table 1: Biochemical characteristics of Streptomyces sp. [Click here to view] |
The isolated Streptomyces sp. was inoculated in production media and incubated for 120 h. Samples were drawn and assayed for enzyme activity. Enzyme activity was carried out using Berthelot reaction, wherein ammonia is estimated by phenol nitroprusside method. This reaction is based on the principle that the ammonia released reacts with Berthelot’s reagent to form blue colored product and absorbance is measured [36]. Here, ammonia reacts with hypochlorite and two molecules of phenol to form indophenols which produce characteristic blue color. The absorbance of indophenols can be measured at 600 nm. It was observed that Streptomyces sp. had a highest enzyme activity of 26.67 IU/ml at 72 h [Figure 3] and then the activity decreased. This decrease may be due to substrate limitation or product inhibition. Thaer and Ellaiah [37] also observed that L-asparaginase activity from Streptomyces strain A2 was highest after 72 h of incubation. L-asparaginase enzyme from culture media of Streptomyces sp. was purified by salt precipitation, ion exchange chromatography, and size exclusion chromatography. Enzyme was precipitated at 85% salt saturation. The precipitate was dissolved and dialyzed against buffer overnight at 4°C and assayed for enzyme activity. This was used for further purification steps. The elution profile of the chromatography on DEAE-cellulose column [Figure 4] showed one peak with L-asparaginase activity. This was applied onto a Sephadex G-50 column [Figure 5]. In the elution profile, L-asparaginase enzyme was eluted at the initial stage of chromatography, indicating that the enzyme was of high molecular weight. The purified enzyme exhibited the specific activity of 390 IU/mg with 60-fold purification and yield of 42% [Table 2]. Various researchers have used different techniques for purification of L-asparaginase enzyme from different actinomycetes and obtained specific activity in the range of 660–5035 IU/mg and yield in the range of 25–60% [14-17,21].
 | Table 2: Purification table of L-asparaginase from Streptomyces sp. [Click here to view] |
 | Figure 3: Production profile of L-asparaginase from Streptomyces sp. [Click here to view] |
 | Figure 4: Elution profile of L-asparaginase on diethylaminoethyl-cellulose. [Click here to view] |
 | Figure 5: Elution profile of L-asparaginase on Sephadex G-50. [Click here to view] |
The molecular weight of purified enzyme was found to be 34 kDa [Figure 6]. Prista and Kyridio [38] reported 33 kDa as the apparent molecular weight of L-Asparaginase from Thermus thermophilus. Kumar and Selvam [21] observed that the subunits of L-asparaginase had a molecular mass of approximately 33.3 kDa. L-asparaginase was characterized for various parameters. At pH 6, enzyme had a maximum activity of 32.8 IU/ml [Figure 7]. Above or below this pH, activity decreased indicating optimum pH for L-asparaginase is 6.0.
 | Figure 6: Determination of molecular weight of L-asparaginase by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (M: Molecular weight markers; A: L-asparaginase). [Click here to view] |
 | Figure 7: Effect of pH on L-asparaginase enzyme activity from Streptomyces sp. [Click here to view] |
 | Figure 8: Effect of temperature on L-asparaginase activity from Streptomyces sp. [Click here to view] |
 | Figure 9: Lineweaver–Burk plot for L-asparaginase from Streptomyces sp. [Click here to view] |
 | Figure 10: Effect of additives on L-Asparaginase from Streptomyces sp. [Click here to view] |
IU: International unit, DEAE: Diethylaminoethyl.
4. CONCLUSION
In the present investigation, isolates of actinomycetes were screened for L-asparaginase production. Based on enzymatic activity, a potent actinomycete was selected and was identified as Streptomyces sp., further, L-asparaginase enzyme was purified by salt precipitation, anion exchange chromatography, and size exclusion chromatography. L-asparaginase from Streptomyces sp. was purified to apparent homogeneity having a molecular size of 34 kDa. The enzyme was characterized for its various properties. This L-Asparaginase is active at a broad range of temperature and hence can be used in processes involving high temperatures. The kinetic parameters of L-asparaginase indicated that enzyme has high affinity to the substrate. Effect of various metal ions and additives indicated that the enzyme may not be a metalloenzyme and may contain free sulfhydryl groups near the catalytic site. The enzyme L-asparaginase could be of potent use in various biotechnological and industrial applications.
5. ACKNOWLEDGMENT
The authors would like to thank Dr. Ashok Shettar, Honorable Vice Chancellor, KLE Technological University, Hubballi, Karnataka, India, for his continuous support and encouragement.
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