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Metagenomic approach for the isolation of a novel low-temperature-active lipase from uncultured bacteria of marine sediment
Södertörns högskola, Institutionen för livsvetenskaper. Karolinska Institute.
Södertörns högskola, Institutionen för livsvetenskaper.ORCID-id: 0000-0002-5802-5126
2007 (Engelska)Ingår i: FEMS Microbiology Ecology, ISSN 0168-6496, E-ISSN 1574-6941, Vol. 59, nr 2, s. 524-534Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

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 C-14. 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 t(1/2) < 5 min.

Ort, förlag, år, upplaga, sidor
United Kingdom: Wiley-Blackwell Publishing Ltd. , 2007. Vol. 59, nr 2, s. 524-534
Nyckelord [en]
metagenomic cloning, lipase, bacteria, sediment, marine, environmental genome
Nationell ämneskategori
Mikrobiologi
Forskningsämne
Miljövetenskapliga studier
Identifikatorer
URN: urn:nbn:se:sh:diva-6183DOI: 10.1111/j.1574-6941.2006.00206.xISI: 000243506800029PubMedID: 17328767Scopus ID: 2-s2.0-33846247798OAI: oai:DiVA.org:sh-6183DiVA, id: diva2:397055
Tillgänglig från: 2011-02-11 Skapad: 2011-02-11 Senast uppdaterad: 2017-12-11Bibliografiskt granskad
Ingår i avhandling
1. Exploring the metagenome of the Baltic Sea sediment
Öppna denna publikation i ny flik eller fönster >>Exploring the metagenome of the Baltic Sea sediment
2008 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Environmental microorganisms are fundamental to ecosystem function, acting as drivers in processes such as primary production, organic matter remineralisation, pollution remediation and global biogeochemical cycling. However, the study of the bacterial communities requires the application of advanced culture-independent methods considering that only a small fraction of the community is otherwise accessed. The goal of this thesis was to investigate the bacterial community structures and functions of Baltic Sea coastal sediments. To assess the distribution and identity of metabolically active bacteria along a vertical redox gradient, a polyphasic method was applied including: reverse transcriptase-PCR (transcription) and bromodeoxyuridine immunocapture (replication) for 16S rRNA gene analyses through both clone library sequence analysis and terminal restriction fragment length polymorphism (T-RFLP). It was demonstrated that the bacterial communities were highly diverse and significantly different at different redox layers. Phylogenetic analysis identified several novel bacterial groups, some with potentially important ecological roles, notably the first genetic evidence of active anammox bacteria, demonstrating that the bacterial community of the Baltic Sea sediment includes several largely unexplored groups. A metagenomic approach was used to access the bacterial diversity. Considering that the Baltic Sea sediment contained a diverse and largely unexplored bacterial community and also represent a permanently cold environment. This community is likely to harbor bacteria with enzymes adapted to low temperatures that would have a potential biotechnological value. The capacity of functional metagenomics for bioprospecting was demonstrated though the construction of a fosmid library of the prokaryotic genomic pool and expression screening, which enabled the identification of several novel lipolytical enzymes. A novel lipase, h1Lip1 (DQ118648) was isolated, overexpressed, purified and characterized for catalytic activity, substrate specificity, apparent temperature optimum and thermo-stability, demonstrating that the enzyme was low temperature active. 3D protein structure modelling of the lipase supported the presence of an alpha/beta-hydrolase fold, a catalytic triad and a lid structure, covering the active site. Comparative structure analyses and site directed-mutagenesis further showed the importance of a region within the N-terminal and lid for substrate affinity and thermal stability. In conclusion, these targeted molecular strategies demonstrate that the Baltic Sea sediments contain a highly diverse and unique bacterial community that also represents a useful source of biotechnologically interesting molecules.

Ort, förlag, år, upplaga, sidor
Stockholm: Karolinska institutet, 2008. s. 47
Nyckelord
Maringeologi, Bakterier
Nationell ämneskategori
Biologiska vetenskaper
Identifikatorer
urn:nbn:se:sh:diva-31555 (URN)978-91-7357-386-3 (ISBN)
Disputation
2008-05-30, MA624, Huddinge, 10:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2016-12-29 Skapad: 2016-12-29 Senast uppdaterad: 2016-12-29Bibliografiskt granskad

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Hårdeman, FredrikSjöling, Sara

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