Biochemical and Molecular Characterization of a New Aspartase Producer Aeromonas media NFB-5 from Effluent of a Fertilizer Factory

Ram      Sarup Singh; Mukesh      Yadav

Abstract

Aspartase (L-aspartate ammonia-lyase; EC 4.3.1.1) catalyzes the reversible amination of fumaric acid to produce L-aspartic acid. Aspartase producing bacterial strains were isolated from the effluent of a fertilizer industry. The effluent was alkaline in nature and rich in nitrogenous contents. Aspartase producing bacterial strains were isolated by enrichment technique. Screening of isolated strains for aspartase activity has shown isolate NFB-5 to be the most efficient aspartase producer. This isolate showed higher aspartase activity than reference strains of Escherichia coli and Bacillus cereus. The genus of strain was identified by morphology, physiological and biochemical tests. Species specific identification was determined by 16S rDNA analysis. The phylogenetic analysis identified the isolate as Aeromonas media NFB-5, a motile member of aeromonads of family Aeromonadaceae which are ubiquitous inhabitants of fresh water and estuarine environments. The strain was capable of expressing maximum aspartase (48.22±0.324 U/g fresh biomass) within 24 h of growth at 37ºC. Aspartase gene (aspA) from A. media has been successfully cloned and sequenced. The gene consisted of an ORF of 1,424 bp encoding a protein of 474 amino acid residues. The amino acid sequence of the enzyme showed a high degree of identity (91%) to aspartase from E. coli but differed from aspartases from other microorganisms. The results suggest that A. media NFB-5 is a new potent aspartase producer and cloning of its aspartase gene (aspA) having potential applications in food industry.

Keywords: Aeromonas media, aspartase, biochemical characterization, cloning, degenerate primers, nucleotide sequence, effluent, 16S rDNA sequence, phylogenetic analysis, A. media NFB-5, aspartase gene (aspA), fumaric aminase catalyzes, aspartame, L-aspartic acid, NFB-5