Naslov
Extracellular lipase from bacterium Streptomyces rimosus: cloning, expression and purification for crystallization

Autori
Leščić, Ivana ; Vujaklija, Dušica ; Pigac, Jasenka ; Abramić, Marija

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
9th International Conference on the Crystallization of Biological Macromolecules - Book of Abstracts / - Jena, 2002

Skup
9th International Conference on the Crystallization of Biological Macromolecules

Mjesto i datum
Jena, Njemačka, 23.03.2002. - 28.03.2002

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
lipase; cloning; expression; purification

Sažetak
Lipases (triacylglycerol acylhydrolases) catalyse hydrolysis and synthesis of lipids, depending on the reaction conditions. Their natural substrates are insoluble in water, therefore hydrolysis takes place on lipid-water interface. This property distinguishes lipases from classical esterases. The ability of these enzymes to stereospecifically catalyse various reactions on a broad range of substrates gives them significant biotechnological potential. Streptomycetes are Gram-positive bacteria that exhibit remarkable capacity for synthesis of secondary metabolites. They are the most widespread producers of antibiotics. However, not much is known about their lipolytic enzymes. We previously reported purification and partial biochemical characterisation of the native extracellular lipase from Streptomyces rimosus. Now we have undertaken the effort to overexpress and purify this enzyme in larger quantities, in order to prepare sample for crystallisation studies, with final goal to solve its three-dimensional (3D) structure. A novel lipase gene from S. rimosus was cloned by a reverse genetic strategy. The gene was identified by hybridisation to a probe obtained by PCR with a mixture of degenerated oligonucleotides that correspond to the amino-terminal and an internal amino acid sequence determined for the secreted form of the lipase, and a chromosomal DNA as a template. Homologous expression of lipase gene was achieved by subcloning of S. rimosus chromosomal DNA fragment carrying the lipase gene into a high-copy bifunctional vector and then transforming S. rimosus lipase-deficient strain with the vector. A twenty-fold higher lipase activity was obtained in the culture filtrate of transformed bacterium, in comparison with the original strain. Purification of overexpressed lipase from the bacterial culture filtrate was performed by a combination of batch and column chromatography on CM-cellulose, followed by a re-chromatography on the same carrier. Final step, which gave a single protein band on SDS-PAGE was the FPLC (Mono Q) chromatography. Crystallisation experiments with purified extracellular Streptomyces rimosus lipase are under way.

Izvorni jezik
Engleski

Znanstvena područja
Kemija

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