PCR detection of ansA from marine bacteria and its sequence characteristics from Bacillus tequilensis NIOS4

As many as 71 marine bacterial DNA extracts were PCR screened for L-asparaginase (ansA), a key gene in anti-cancer molecular-searches. Over 62% (44) of them were positive for ansA gene. The positive cultures were from genera Bacillus and Staphylococcus. The ansA gene cloned from isolate NIOS4 belonging to recently described Bacillus tequilensis is 1099bp in length with a 990bp ORF coding for 329 amino acids. BLASTx analysis revealed this sequence to be 98% similar to earlier reported ansA sequence from B. subtilis (Accession no. NP390239.1). By comparing its deduced amino acid sequence with other bacterial asparaginase sequences six substitutions at positions 305(Thr), 313(Lys), 314(Leu), 315(Asp), 318(Arg), and 320(Gln) are observed. Key residues like Thr(12), Thr(85), Asp(86), Lys(156), and Phe(165) taking part in active-site formation and imparting catalytic properties are conserved. The phylogenetic tree based of the ansA amino acid sequences revealed close relatedness of the NIOS4 ansA sequence with B. subtilis (Accession no. NP 390239.1). It's very close genetic resemblance to B. subtilis and conservation of certain key amino acid residues suggest it as a prospective candidate for evaluation and, production of L-asparaginases.