ISOLATION AND CHARACTERIZATION OF L-ASPARAGINASE EXTRACELLULAR ENZYME PRODUCING BACTERIA FROM INDUSTRIAL SOIL SAMPLES

ISOLATION AND CHARACTERIZATION OF L-ASPARAGINASE EXTRACELLULAR ENZYME PRODUCING BACTERIA FROM INDUSTRIAL SOIL SAMPLES

Rajesh Singh Tomar, Neha Sharma ^* and Shuchi Kaushik

Amity Institute of Biotechnology, Amity University, Gwalior - 474005, Madhya Pradesh, India.

ABSTRACT: Industrial soil contains a variety of L-Asparaginase producing microorganisms such as bacteria and fungi. Microorganisms can produce different types of enzymes which are frequently used in different types of industries for different purpose. In the present study, we focused on isolation of L-Asparaginase enzyme producing bacteria from industrial soil samples collected from different regions of Gwalior. These bacterial isolates were Salmonella sp., Proteus vulgaris, Bacillus subtilis which were identified based on colony morphology, Gram staining, biochemical tests and using selective and differential media. All the tested isolates were able to produce enzymes like L-Asparaginase. All the three bacteria which were isolated from soils of a different region of Gwalior able to produce L-Asparaginase enzyme. Maximum L-Asparaginase activity was shown in second (II) bacterial isolate Salmonella sp., which was 12.734 mg/ml. Salmonella sp. were not reported to show any type of allergenicity when their sequences were checked by bioinformatic tool Algpred. So, these bacterial isolates are considered as a good source of enzyme production and may be used at an industrial level. Thus from the present study, it may be concluded that microbes from soil sample can be a good source of industrially important enzymes.

Enzyme activity, Salmonella sp., Proteus vulgaris, Bacillus subtilis, L- Asparaginase, Algpred bioinformatics tool

INTRODUCTION: Enzymes have valuable medical and industrial applications. L-asparaginase (EC 3.5.1.1) belongs to family amidohydrolases, which catalyzes the hydrolysis of L-asparagine amino acid and makes L-aspartate and ammonia as byproducts ¹. Biochemistry of enzyme and their enzyme kinetics and the selection of proper substrate are required for enzyme assay ². For enzyme assays pH temperature, ionic strength, the concentration of substrate, and enzyme should be in optimum condition ³.

It is produced by a large number of microorganisms that include Pseudomonas stutzeri ⁴, Pseudomonas aerugenosa ^5, and E. coli ⁶. It is used for the treatment of acute lymphoblastic Leukemia, preliminary and advanced stages of cancer ^{7, 8} so L- Asparaginase is a critical extracellular enzyme of medical importance as an anti-cancer agent and an anti-microbial agent. Asparaginase is commonly used in place of heat treatment of starchy food in the industry to reduce the formation of the acrylamide, which is suspected human carcinogenic ^{8, 9}. Currently, L-Asparaginase has great demand in recent years at the level of food industries and pharma industries (40%) ^{10, 11}.

Databases and bioinformatics tools can be used to find allergens, haptens, epitopes, and the presence of mast cell ¹². Algpred a bioinformatics tool is used in the prediction of allergic proteins, mast cell, and IgE epitopes in enzyme producing bacteria by various methods incorporated in the software ¹³.

MATERIALS AND METHODS:

Collection of Soil Samples: Soil samples were collected from different sites of Gwalior region, Madhya Pradesh.

Isolation and Identification of Bacteria: First, the microorganisms present in soil samples were cultured on nutrient agar medium, and the morphology, colony characteristics of organisms were based on shape, size, odor, margin and surface characteristics. Gram staining was used to differentiate between gram positive and gram negative microorganism. Spore staining was also done to check spore chain morphology in all the bacterial isolates.

After phenotypical identification of bacteria, further confirmation was done based on their biochemical characterization viz., annitol salt agar test, amylase production, hydrolysis of gelatin, IMViC test, catalase test, Widal test, skim milk test, carbohydrate fermentation test, Simmon citrate test and urease test by standard methods.

Enzymatic Assay: Enzyme assay was used to identify the production of a particular enzyme by the bacteria. The enzyme assay was done at the level of both qualitative and quantitative.

Qualitative Analysis: Qualitative test was done with the help of Nessler’s reagent (Hans Bisswanger 2014). For this assay screening media was prepared. The constituents of this media (gm/ltr) include Na₂HPO₄ (6), NaCl (0.5), KH₂PO₄ (3), MgSO₄ (1 M/ltr), L-Asparaginase (5), CaCl₂ (0.1M/ltr), 20% glucose stock solution (10 ml), agar (2%), autoclaved at 15 psi for 15 min. Then, streaking of all five bacterial isolates was done on these media plates, but the concentration of phenol red (2.5%) which was prepared in ethanol was different in each plate. First media plate having 80 µl phenol red, the second plate contains 120 µl phenol red, the third plate contains 240 µl phenol red and the fourth plate contain 360 µl phenol red. Incubated the plates at 37 °C for 24 h to produce colonies with pink color zone around them ¹⁴.

Quantitative Analysis: Determination of specific activity.

Enzyme Preparation: All the bacterial isolates in nutrient broth media was cultivated at room temperature for 16 h on the shaker. After incubation 1 ml of all five bacterial culture was centrifuged at 10,000 rpm for 10 min at 4 °C. The Supernatant was taken, which is used in the enzyme assay.

Estimation of Protein: Amount of protein in the sample is estimated by Lowry’s method. This method is used for determining the concentration of protein in sample ¹⁵.

Enzyme Activity: Enzymatic activity means the rate at which substrate molecule converts into a chemically modified product with the help of an enzyme. Activity is defined in unit/ml.

Calculation of Enzyme Activity: Activity of all five enzymes in all five bacterial isolates was calculated by the equation of standard graph of 4 mM ammonium chloride.

Calculation of Specific Activity of Enzyme: Specific enzyme activity is the number of enzyme units/ ml divided by the protein concentration in mg/ml. It is a ratio of enzyme activity to protein concentration. Its unit is µmol/min/mg or units/mg. It is used to measure the purity of an enzyme. Specific activities of each enzyme of each bacterial isolates were calculated by the following formula ¹⁶.

Specific activity = Enzyme concentration / Protein concentration

RESULTS AND DISCUSSION:

Qualitative Analysis of Asparaginase Enzyme: Primary screening of L-asparaginase producing isolates from the soil is done by the qualitative method.  All the screened isolates were found to be positive for L-asparaginase production by Nessler’s reagent. Upon primary screening, all the five isolates were found to be positive as they showed growth on modified M9 medium. When the concentration of phenol red dye was increased from 80 µl to 360 µl, the first bacterial isolate showed growth and the pink zone around the colonies on the medium containing the dye (240 µl). Second, fourth, fifth bacterial isolates, which showed very good L-Asparaginase enzymatic activity were not able to grow on M9 media plate when a high concentration of phenol red. The results are similar to the work done by Bhat et al. (2015) ¹⁷.

FIG. 1: L-ASPARAGINASE PRODUCING BACTERIA

Estimation of Concentration of Protein: It was done by Folin Lowry Method. Estimation of protein in each bacterial isolates was calculated by the following equation of standard graph Table 1.

Estimation of Enzyme Activity: All the five bacteria which were isolated from soils of a different region of Gwalior able to produce L-Asparaginase enzyme.

TABLE 1: ENZYME ACTIVITY, PROTEIN CONCENTRATION AND SPECIFIC ACTIVITY OF L-ASPARAGINASE

Maximum L-Asparaginase activity was shown in the second (II) bacterial isolate, which was 12.734 mg/ml. Since the results are similar to the work done by Pradhan et al., (2013) ¹⁸. Hence, it can be concluded that the soil of the plant growing field and agricultural field are a good source of asparaginase producing bacteria. These fields are a good source of minerals like nitrogen which are required for the production of asparaginase producing enzyme Table 1.

Algpred Method for Test of Allergy: It plays an important role in the pharmaceutical industry to check the allergenicity of any drug that the particular drug can be used in the treatment of diseases. The presently available L- Asparaginase is not sufficient to meet industrial demands. Hence, there is a continuous search for new L-Asparaginase with novel characteristics for industrial application obtained from diverse bacteria isolates Table 2.

So, their allergenicity nature can be identified with the help of bioinformatics tool Algpred. In it, Allergenicity is identified based on reactivity with the mast cell, Mapping of IgE epitopes, Amino acid composition, BLAST and presence of dipeptides. The sequences from different organisms belonging to the same group as studied in the present work were analyzed for their antigenicity. Since the results are similar to the work done by Bucholska et al., 2018. He used bioinformatics tools to find allergens, haptens, epitopes, and the presence of mast cell, which has an advantage in the field of medicine.

Mahboobi et al., in 2017, ¹⁹ focused on the application of bioinformatics tools to study about E.coli model because it is a good source of L-asparaginase enzyme which has an anticancer ability.

Hence, it can be concluded that Databases and Associated bioinformatics tools are very useful to study Allergens, Epitopes, and Haptens present in the test organisms. Although the use of such in-silico tools provide us rapid and reliable results and minimize the experimental work, cost, and time but these results further need to be validated by in-vitro and in-vivo studies.

TABLE 2: ALGPRED TABLE FOR L-ASPARAGINASE PRODUCING SALMONELLA SP.

CONCLUSION: This study indicates that soil can provide a good source of asparaginase producing bacteria. This study aimed to isolate and characterize the enzyme producing bacteria from soil samples collected from different regions of Gwalior. The environment where we live is the habitat for various microorganisms, mostly bacteria. They have various industrial applications like enzymes production, fabric manufacturing, bioremediation, pharmaceutics, etc.

Microorganisms have become increasingly important as a producer of industrial enzymes. From the results obtained in this research, it could be concluded that organism with the potential to produce enzyme can be isolated from the soil of different environmental condition. This isolates could be exploited for the commercial production of the enzyme in the industry like baking, brewery, and detergent, pharmaceutical especially L- Asparaginase can be used in the treatment of different types of cancer and textile industry. Microorganism from a soil sample is a good source of L- Asparaginase producing bacteria. These bacterial isolates were pathogenic and also showed resistance towards antibiotics might be due to the environmental condition where they present, but they were not showing any type of allergenicity when it was checked by bioinformatic tool Algpred.  So, these bacterial isolates are considered as a good source of enzyme production at industrial level.

ACKNOWLEDGEMENT: Nil

CONFLICT OF INTEREST: The authors declare no competing or conflicts of interest.

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    How to cite this article:

    Tomar RS, Sharma N and Kaushik S: Isolation and characterization of L-Asparginase extracellular enzyme producing bacteria from industrial soil samples. Int J Pharm Sci & Res 2019; 10(11): 4937-41. doi: 10.13040/IJPSR.0975-8232.10(11).4937-41.

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