François Shareck

Seven lipolytic genes were isolated and sequenced from a metagenomic library that was constructed following biomass enrichment in a fed-batch bioreactor submitted to high temperature (50–70°C) and alkaline pH (7–8.5). Among those sequences, lipIAF1-6 was chosen for further study and cloned in Streptomyces lividans 10–164. The G+C content within the sequence was 64.3%. The encoded protein, LipIAF1-6, was related to various putative lipases previously identified in different genome sequences. Homology of LipIAF-6 with the different lipases did not exceed 31%. The optimum pH (8.5) and temperature (60°C) of the purified enzyme were in agreement with the enrichment conditions. Furthermore, the enzyme was thermostable for as long as 30 min at 70°C. The maximum activity of the purified lipase was 4,287 IU/mg towards p-nitrophenyl (p-NP) butyrate (60°C; pH 8.5). LipIAF1-6 does not seem to need the presence of metal ions for its activity. The enzyme was slightly inhibited by 10 mM CoCl₂ (14%), HgCl₂ (12%), and dithiothreitol (DTT) (15%). The serine protease inhibitor phenylmethylsulphonyl fluoride (PMSF) reduced activity by 39% and 71% when incubated at concentrations of 1 and 10 mM, respectively. Finally, LipIAF1-6 was stable in different organic solvents, and against several surfactants and oxidative agents commonly found in detergent formulations. These results are quite encouraging for further use of this enzyme in different industrial processes.

The construction of a cosmid library from the biomass produced in an enriched Sequencing Fed-Batch Reactor allowed the isolation of a new lipase by functional screening. The open reading frame of 928 bp encoded a polypeptide of 308 amino acids with a molecular mass of 32.6 kDa. The amino acid sequence analysis revealed the presence of the conserved pentapeptide GXSXG essential for lipase activity. Alignment with known sequences of proteins showed no more than 52% identity with different lipases, confirming the discovery of a novel gene sequence. The lipase was cloned and expressed in Streptomyces lividans and further purified by a combination of hydrophobic interaction and size-exclusion chromatography. Spectrophotometric assays with different p-nitrophenyl esters demonstrated a preference for long-length acyl chains, especially p-nitrophenylmyristate (C14). Moreover, the enzyme presented an optimal activity at 60°C and at alkaline pH of 10.5.