Editor: Max Häggblom
Metagenomic approach for the isolation of a novel low-temperature-active lipase from uncultured bacteria of marine sediment
Version of Record online: 27 SEP 2006
FEMS Microbiology Ecology
Volume 59, Issue 2, pages 524–534, February 2007
How to Cite
Hårdeman, F. and Sjöling, S. (2007), Metagenomic approach for the isolation of a novel low-temperature-active lipase from uncultured bacteria of marine sediment. FEMS Microbiology Ecology, 59: 524–534. doi: 10.1111/j.1574-6941.2006.00206.x
- Issue online: 27 SEP 2006
- Version of Record online: 27 SEP 2006
- Received 1 May 2006; revised 13 July 2006; accepted 14 July 2006.First published online 27 September 2006.
- metagenomic cloning;
- environmental genome
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 Pseudomomas 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°C. Thermal stability analysis showed that h1Lip1 was unstable at 25°C and inactivated at 40°C with t1/2 <5 min.