Expression and characterization of a novel heterologous moderately thermostable lipase derived from metagenomics in Streptomyces lividans.
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 degrees 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 degrees 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 degrees C. The maximum activity of the purified lipase was 4,287 IU/mg towards p-nitrophenyl (p-NP) butyrate (60 degrees 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.
Côté
Amélie
A
INRS-Institut Armand-Frappier, 531 boul. des Prairies, Laval, QC H7V1B7, Canada.
Shareck
François
F
D000468:Alkalies / Q000737:chemistry; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D001482:Base Composition; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005798:Genes, Bacterial; D006358:Hot Temperature; D008049:Lipase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D056186:Metagenomics; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D048170:Streptomyces lividans / Q000201:enzymology / Q000235:genetics*; D013379:Substrate Specificity
D000468:Alkalies; D001426:Bacterial Proteins; D008049:Lipase