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Comparative structure modelling and mutational analysis of the low-temperature-active metagenomically derived lipase h1Lip1
Södertörn University, School of Life Sciences. Karolinska Institutet.
University of Dublin, Dublin, Ireland.
Södertörn University, School of Life Sciences.ORCID iD: 0000-0002-5802-5126
(English)Manuscript (preprint) (Other academic)
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:sh:diva-31554OAI: oai:DiVA.org:sh-31554DiVA: diva2:1060758
Note

Som manuskript i avhandling. As manuscript in dissertation.

Available from: 2016-12-29 Created: 2016-12-29 Last updated: 2016-12-29Bibliographically approved
In thesis
1. Exploring the metagenome of the Baltic Sea sediment
Open this publication in new window or tab >>Exploring the metagenome of the Baltic Sea sediment
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
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.

Place, publisher, year, edition, pages
Stockholm: Karolinska institutet, 2008. 47 p.
Keyword
Maringeologi, Bakterier
National Category
Biological Sciences
Identifiers
urn:nbn:se:sh:diva-31555 (URN)978-91-7357-386-3 (ISBN)
Public defence
2008-05-30, MA624, Huddinge, 10:00 (English)
Opponent
Supervisors
Available from: 2016-12-29 Created: 2016-12-29 Last updated: 2016-12-29Bibliographically approved

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