Tripathi_2004_Enzyme.Microb.Technol_34_437

An expression library generated from genomic DNA of Streptococcus sp. N1 resulted in C1N1 Escherichia coli recombinants forming haloes on lipase indicator tributyrin/Tween-80 agar plates. The excised insert in the recombinant C1N1 was of 3,489 kb size. Analysis of the nucleotide sequence of the insert showed two significant operons of 1032 bp (ORF1) and 729 bp (ORF3). ORF1 on the (+)-strand is the structural lipase gene codes for a protein of 343 amino acid residues with a theoretical mass of 38.6 kDa, which is close to the value of the lipase expressed in E. coli (39.36 kDa). The primary structure of the lipase deduced from the nucleotide sequence shows a consensus sequence containing the active serine [VAGHSIGG], a conserved H-G dipeptide in the N-terminal part of the enzyme and a potential site for N-linked glycosylation at amino acid residues [129-131]. The sequence, AAGGA at -6 upstream of the start codon ATG seems to be putative ribosomal binding site (rbs) and is similar to the rbs of lip3 of Moraxella sp. TA 144. The putative-10[TTTAT] and -35[AATG] promoter sequences showed similarity to lip3 of Moraxella sp. TA 144. The lipase shared no significant homology with the members of different lipases families except with group V comprising of Psychrobacter immobilis B-10 [28% identical and 56% positive similarity] and Moraxella sp. TA 144, lip3 [28% identical and 54% similarities]. The 729bp operon present on (-) strand of 3.489 kb C1N1 insert was essential for the secretion of the enzyme. The putative protein on BLAST/FASTA search showed 57% identities and 72% positives with ATP-binding component of ABC-transporter. The recombinant lipase was resistant to organic solvents, non-ionic surfactants, metal ions and showed alkaline pH optima (pH 8.4).