PARTIAL PURIFICATION AND CHARACTERISATION OF L-ASPARAGINASE FROM GORDONIA SP KM067296 ISOLATED FROM LONAR LAKE (MAHARASHTRA.INDIA)

Ganjave Snehal, Bhat Manish and Marar Thankamani*. School of Biotechnology and Bioinformatics, D.Y. Patil University, CBD Belapur, Navi Mumbai, Pin-400614, Maharashtra, India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


ISSN: 2320-5407
Int. J. Adv. Res. 4 (12), 467-474 468 administration of such an enzyme protein, in general, produces the corresponding antibody in the tissues, resulting in the anaphylactic shock or neutralization of drug effect, thus exploring novel serologically different L-asparaginase is a need of the hour (Jois et al., 2013).
L-asparaginase is an enzyme drug choice for acute lymphoblastic leukemia in children used in combination chemotherapy. Normal cells possess an enzyme L-asparagine synthase which satisfies for the requirement of the amino acid unlike in leukemic cells which have trivial or no L asparagine synthase. This deficit leads to the inhibition of DNA and RNA synthesis, blocking protein synthesis and destruction of cell function and cell death (Chande and Bhat, 2014). It is widely used in acute lymphoblastic leukemia, Hodgkin disease, acute myelocytic leukemia, acute myelomonocytic leukemia, chronic lymphocytic leukemia, lymphosarcoma treatment, and reticulosarcoma and melanosarcoma regimen. Apart from its therapeutic value, L-asparaginase also finds application in food industry. It has been found that L-asparaginase reduces the formation of carcinogenic acryl amides in deep fried potato recipes. Addition of asparaginase before baking or frying the food, results in conversion of asparagine into common amino acid, aspartic acid and ammonium. As a result, asparagine is unavailable to complete Maillard reaction, and thereby the formation of acryl amide is greatly lowered (Friedman, 2003).The present study aims to isolate, purify and characterize L-asparaginase from selected bacterial species.
Microbes residing in supreme habitat like alkaline or saline areas tend to express proteins with different characteristics than those inhabiting in the usual environment. Lonar Lake located in Buldhana district of Maharashtra appears to be one of the marvels of state and is considered to be the world's third largest crater formed due to a meteor impact. It is a saline soda lake with pH in the range of 10-12, serves as a distinctive habitat for isolation of halophiles and alkaliphilic organisms (Chande and Bhat, 2014). Studies on L-asparaginase producing isolate from Lonar Lake have not been reported very extensively and an attempt for the same is made in this study. The current study was aimed at exploring Gordonia terrae strain DSM43249 as a source of L-asparaginase.

Materials and Methods:-
Soil samples were collected in sterile containers from four side of Lonar lake (19°58′36″N 76°30′30″E, Buldhana, Dist. Maharashtra, India) in the month of December. The pH of the lake was noted immediately at the site. Luhana's method (Luhana et al., 2013) was modified for carrying out production assay. It was grown at 37°C in shaker incubator and then transferred to Erlenmeyer flask for 72h at 37°C in M-9 media containing (per 1L distilled water): Na 2 HPO 4 , 6.0g; KH 2 PO 4 , 3.0g; NaCl, 0.5g; L-asparagine, 1%;MgSO 4 .7H 2 O 0.12g; CaCl 2 .2H 2 O, 0.01g;peptone 0.6%;yeast extract,1%; pH-7.0. At the end of incubation time, the samples were centrifuged at 10,000rpm for 15min, at 4°C, and then cell free supernatant was used in further purification steps. All the assays were performed in triplicates.

Enzyme Assay:-
The enzyme was assayed by direct nesselerization method. One unit of L-asparaginase activity (IU) is defined as the amount of enzyme which liberates l μmol of ammonia per min at 30˚C and pH 7.4 (Luhana et al., 2013).

Estimation of Protein:-
The protein contents were estimated by the method of Lowry using bovine serum albumin as the standard (Lowry, 1951).

Purification of L-Asparaginase:-
The activity of extracellular and intracellular crude enzyme was assayed. The extracellular concentration of the enzyme was significantly higher and hence it was used for further purification and characterization. The supernatant from the production media (crude enzyme) was subjected to serial ammonium sulfate precipitation 0%-40%, 40%-60% and 60%-80% and 80%-100% and enzyme assay for each fraction was carried out. Maximum activity was found in 60% saturation and hence this fraction was dialyzed against Tris buffer (50mM, pH 8.4) with three buffer changes at 4°C overnight. Gel filtration chromatography was done for dialyzed sample using Sephadex G-75 column (1×25m) equilibrated and eluted with 150ml Tris-HCl (50mM, pH 8.4).The separated proteins were visualized by PAGE (Laemmli,1970) using Coomassie brilliant blue R-250 staining solution and the molecular weight of Lasparaginase was determined using standard molecular weight marker(medium range 97-14KDa;Sigma-Aldrich).

Characterization of L-Asparaginase:-
The optimum conditions for the enzyme activity were determined by assaying at different temperatures (10-45°C)pH (4-10) (Sabbagh et al., 2013) and incubation time (10-60 minutes). The enzyme kinetics as measured by the Michaelis constant (Km) is defined as the substrate concentration at half the maximum velocity, the rate of enzymatic reactions, by relating reaction rate to the concentration of a substrate. The Michaelis constant (Km) value of the purified enzyme was estimated in a range of L-asparagine concentrations of 0.02M-0.08 M. Line Weaver-Burk plot analysis was carried out to calculate the apparent Km and Vmax value of purified L-asparaginase. The activity was determined in different concentrations of metal ion MgCl 2 and metal ion chelator ethylenediamine tetra acetic acid (EDTA) in the concentration of 0.2-0.8mg/ml in the reaction mixture (Mohapatra et al., 1995).

Identification of Isolate:-
The potential isolate was identified by 16S rRNA sequencing method (Chromus Biotech Pvt.Ltd., Bangalore). The sequence was aligned with representative sequence. The isolate was found to be Gordonia sp KM067296. Purification of L-asparaginase:-The sequential multi steps purification procedures has been summarized in Table 1.  Figure 1 shows the elution profile of the partially purified L-asparaginase from Sephadex G-75 (Sigma-Aldrich) column. A sharp distinctive peak of L-asparaginase activity, which fits with only one protein peak, was noticed. The most active fractions with specific activity 38.61IU/ mg and about 14-fold purification were pooled together, concentrated and stored at -20°C. Characterization of Partially Purified L-asparaginase Enzyme Effect of pH on enzyme activity:-Results revealed that the pH 7.4 was optimal for L-asparaginase from Gordonia terrae. These results coincide with that of Dhevagi and Poorani (2006) who reported the maximal L-asparaginase activity in Streptomyces spp. which was between pH 7.0-8.0, whereas pH 5.0-9.0 were reported earlier to be optimum for amidase activity (Ohshima et al., 1976).

Effect of metal ions on enzyme activity:-
Among the salts tested, considerable loss of activity was observed only with EDTA. The highest inhibition value was recorded with a concentration of 0.8 mg/ml. There are reports that L-asparaginase extracted from Bacillus spp. was strongly inhibited by EDTA (Mohapatra et al., 1995). Addition of MgCl 2 enhanced the enzyme activity to

Effect of substrate concentration on enzyme activity:-
A Line weaver Burk analysis showed a Km value of 0.061mM and Vmax of 0.57 IU. Higher Km values 6.6 and 7.0 mM for L-asparaginase from Lupinus arboreusand Lupinus angustifolius, respectively, has been reported (Chang and Farnden, 1981

Conclusion:-
The present study focused on production, partial purification and characterization of L-asparaginase from Gordonia terrae strainKM067296.The isolated Gordonia terrae has the ability to produce a significant amount of extracellular L-asparaginase.The present study indicates the isolated Gordonia terrae strain KM067296 used in this study will be a potential source for extracellular L-asparaginase enzyme. The kinetic parameters for optimal production of biologically active L-asparaginase have been ascertained. The high catalytic activity of the enzyme at physiological pH and temperature and its considerable stability over a wide range of pH and temperature makes it highly favorable to be exploited as a potent anticancer agent. Studies on the enzyme relating to purification and characterization would open new avenues in the application of the enzyme in the healthcare industry and for pharmaceutical, therapeutic, commercial purposes. Enzyme Activity(IU/ml)