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Extracellular lipase from bacterium Streptomyces rimosus: cloning, expression and purification for crystallization (CROSBI ID 483153)

Prilog sa skupa u zborniku | sažetak izlaganja sa skupa | međunarodna recenzija

Leščić, Ivana ; Vujaklija, Dušica ; Pigac, Jasenka ; Abramić, Marija Extracellular lipase from bacterium Streptomyces rimosus: cloning, expression and purification for crystallization // 9th International Conference on the Crystallization of Biological Macromolecules - Book of Abstracts. Jena, 2002

Podaci o odgovornosti

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

engleski

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

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.

lipase; cloning; expression; purification

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Podaci o prilogu

2002.

objavljeno

Podaci o matičnoj publikaciji

9th International Conference on the Crystallization of Biological Macromolecules - Book of Abstracts

Jena:

Podaci o skupu

9th International Conference on the Crystallization of Biological Macromolecules

poster

23.03.2002-28.03.2002

Jena, Njemačka

Povezanost rada

Kemija