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An Optimized Medium for Screening of L-Asparaginase production by Escherichia coli

Purified L-asparaginase II from Escherichia coli has been supplied and employed in the acute leukemia and other malignant neoplasms chemotherapy. L-asparaginase II gene (ansB) in E. coli is under regulation and certain conditions is needed for expression of this gene. In this investigation ,the various concentrations of modified M9 medium ingredients and various carbon source were tested to optimize the medium for expression and identification of L-asparaginase in E. coli. Finally a semiquantitative plate assay for L-asparaginase producing Escherichia coli is reported.


INTRODUCTION
In 1953 Kidd found that guinea pig serum inhibited a number of transplantable lymphomas in mice and rats as well as certain spontaneous and radiation-induced leukemias in mice. Broome has presented some evidence that the antitumor principle in guinea pig serum is L-asparaginase [1] . Deamidation of Lasparagine by extracts of E. coli was first reported in 1957 [2] . Later, Mashburn and Wriston observed that Lasparaginase (L-asparagine amidohydrolase, Enzyme Commission 3.5.1.1) purified from cell extract of E. coli has an antitumor activity similar to that of guinea pig serum [3] . Although other microorganisms such as Aerobacter, Bacillus, Erwinia, Pseudomonas, Serratia, Xanthomonas, Photobacterium [4] , Streptomyces [5] , Proteus [3] , Vibrio [6] and Aspergillus [7] have a potential for asparaginase production, purified enzyme from E. coli has been supplied and employed in the clinical application for acute leukemia and other malignant neoplasms in human. L-asparaginase belongs to an amidase group that hydrolyses the amide bond in Lasparagine to aspartic acid and ammonia. Unlike the normal cells, neoplastic cells cannot synthesize Lasparagine due the absence of L-asparagine synthetase. Therefore, they obtain the required asparagine from circulating pools. For this reason, intravenous injection of free enzyme results in selective neoplastic cell death, directly by the depletion of circulating asparagine levels or indirectly from some other metabolite of the asparaginase reaction [7][8][9] .
E. coli has two isozymes of L-asparaginase [10] , Lasparaginase I (AnsA) which is found in the cytoplasm and has a low affinity for L-asparagine (EC1; Km = 3.5 mM) and L-asparaginase II (AnsB) that is a periplasmic enzyme used in the treatment of acute lymphoblastic leukemia and in contrast, it is a high-affinity enzyme (EC2; Km = 10 pM). AnsA is thought to be constitutively produced but ansB gene expression is changed by aeration, carbon source and variation of available amino acids [1,2,11] . In this investigation, an optimized medium for semi-quantitative plate assay and screening of L-asparaginase producing E. coli is reported based on modified M-9 medium [12] . In this medium, L-asparagine is used as the sole nitrogen source. The production of L-asparaginase by E. coli leads to ammonia formation and increase in pH of the medium. A pH indicator (phenol red) makes medium pink around the colonies producing L-asparaginase.

Isolation and identification of E. coli:
Environmental samples were collected from waste water and the Khoshk River, Shiraz, Iran (in February 2008). Clinical  Table 1). The pH of the media was adjusted at 7.0. Stock solution of the phenol red was prepared (2.5% in ethanol, pH 7.0), filtered by 0.2 m cellulose filter and added to autoclaved media. E. coli isolates were inoculated on the prepared plates and incubated at 37 o C for 48h. The Modified M9 medium was used as blank. After incubation, L-asparaginase activity were reported as (-), trace or (+) based upon intensity of produced pink color and pink zone diameter.

RESULTS
Studies with different concentrations of ingredients revealed that the most appropriate concentrations in 1 liter of medium are KH 2 PO 4 ( 0.75 g), NaCl (0.5 g), Lasparagine ( 10 g), Maltose (1 g), agar (17 g) and phenol red (0.05 g). On optimized medium with above concentrations among the 130 isolates of E. coli, 35 isolates produced trace pink zone and 8 isolates produced (+) zone (Fig. 1), while only 4 trace zones were produced on modified M9 medium ( Table 2).

DISCUSSION
The ansB gene of E. coli is regulated by catabolite activator protein (CAP; also called cyclic AMP receptor protein or CRP) [1,2,11] . The low level of intracellular glucose results in a high level of cAMP, which means high cAMP-CAP complexes. cAMP-CAP binds to a specific site on the DNA that is located adjacent to the promoter for the genes regulated by CAP. The -10 and -35 motifs in the promoter sequence for such genes are not a perfect match to the consensus sequence for 70 promoters. cAMP-CAP bound to the CAP binding site increases the binding of 70 RNA polymerase to the promoter and increases gene expression [13] . Therefore, glucose is an inhibitor for ansB gene. We changed the carbon source of medium from glucose, to disaccharide for reduction in the level of intracellular glucose and more AnsB production; maltose was the best. Also, in contrast to modified M9 medium, Na 2 HPO 4 has been eliminated to reduce bufferic properties of medium and little ammonia identification. The optimum concentration of phenol red in the medium is 0.05 mg L −1 which keeps the colour yellow for a better identification of colour change to pink for trace amounts of L-asparaginase. High concentrations of phenol red giving the colour of medium dark red make difficult to identify the pink zone. The observed results encourage us to continue further screening for Lasparaginase production in the other microorganisms (Bacteria and Fungi) on our optimized medium.