Metagenomic approach for the isolation of a novel low-temperature-active lipase from uncultured bacteria of marine sediment.
A novel lipase was isolated from a metagenomic library of Baltic Sea sediment bacteria. Prokaryotic DNA was extracted and cloned into a copy control fosmid vector (pCC1FOS) generating a library of >7000 clones with inserts of 24-39 kb. Screening for clones expressing lipolytic activity based on the hydrolysis of tributyrin and p-nitrophenyl esters, identified 1% of the fosmids as positive. An insert of 29 kb was fragmented and subcloned. Subclones with lipolytic activity were sequenced and an open reading frame of 978 bp encoding a 35.4-kDa putative lipase/esterase h1Lip1 (DQ118648) with 54% amino acid similarity to a Pseudomonas putida esterase (BAD07370) was identified. Conserved regions, including the putative active site, GDSAG, a catalytic triad (Ser148, Glu242 and His272) and a HGG motif, were identified. The h1Lip1 lipase was over expressed, (pGEX-6P-3 vector), purified and shown to hydrolyse p-nitrophenyl esters of fatty acids with chain lengths up to C14. Hydrolysis of the triglyceride derivative 1,2-di-O-lauryl-rac-glycero-3-glutaric acid 6'-methylresorufin ester (DGGR) confirmed that h1Lip1 was a lipase. The apparent optimal temperature for h1Lip1, by hydrolysis of p-nitrophenyl butyrate, was 35 degrees C. Thermal stability analysis showed that h1Lip1 was unstable at 25 degrees C and inactivated at 40 degrees C with t1/2 <5 min.
Hårdeman
Fredrik
F
School of Life Sciences, Södertörn University College, Huddinge, Sweden.
Sjöling
Sara
S
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D003080:Cold Temperature; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D004795:Enzyme Stability; D015698:Genomic Library; D019015:Geologic Sediments / Q000382:microbiology*; D008049:Lipase / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D004269:DNA, Bacterial; D008049:Lipase