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GDS(L) lipase from Streptomyces coelicolor: cloning, purification and biochemical characterization (CROSBI ID 538503)

Neobjavljeno sudjelovanje sa skupa | neobjavljeni prilog sa skupa | međunarodna recenzija

Bielen, A, Abramić, M, Pigac, J, Franekić, J, Vujaklija, D GDS(L) lipase from Streptomyces coelicolor: cloning, purification and biochemical characterization // Applied Molecular Microbiology“ MedILS, Split, Hrvatska, 2007 Split, Hrvatska, 23.06.2007-01.07.2007

Podaci o odgovornosti

Bielen, A, Abramić, M, Pigac, J, Franekić, J, Vujaklija, D

engleski

GDS(L) lipase from Streptomyces coelicolor: cloning, purification and biochemical characterization

GDS(L) lipases are interesting group of hydrolytic enzymes, often with multifunctional properties and potential for use in biotechnology. A putative lipase gene from Streptomyces coelicolor showed high amino acid sequence homology (66%) with cloned and characterised GDS(L) lipase from Streptomyces rimosus. The gene with corresponding RBS, was PCR-amplified with the addition of restriction sites and 6 histidine codons at the 3’ terminus. Amplifed DNA fragment was ligated into the pET-15b vector, resequenced, and subsequently ligated into the Streptomyces expression plasmid pANT849. This construct was transformed into heterologous, lipase defficient host Streptomyces lividans. The time-course of lipase production was monitored spectrofotometrically following degradation of p-nitrophenyl palmitate and the cultivation was stopped after the increase of lipase activity ceased. A simple three step protein purification procedure was developed. Firstly ammonium sulphate precipitation followed by Ni-NTA metal-affinity chromatography were applied. Obtained sample contained lipase (approximately 80%) and a contaminant, lipase-activity deficient protein of higher molecular mass. Therefore, gel-filtration was applied as the final purification step gaining > 95% purified lipase. Basic biochemical features of the enzyme were determined and will be disscused.

S.coelicolor; GDSL lipase; cloning; biochemical characterization

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Podaci o prilogu

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Podaci o skupu

Applied Molecular Microbiology“ MedILS, Split, Hrvatska, 2007

poster

23.06.2007-01.07.2007

Split, Hrvatska

Povezanost rada

Kemija, Biotehnologija, Biologija