|748-1||INDUCTON OF EXPRESSION AND CHARACTERIZATION OF LIPOXYGENASE KINETICS OF Shewanella woodyi.|
|Autores:||Jhoanne Hansen (UB - University of Barcelona, Faculty of Pharmacy / UNESP - State University of Sao Paulo) ; Albert Garreta (UB - University of Barcelona, Faculty of Pharmacy) ; Karla Stropa (UNESP - State University of Sao Paulo) ; Montserrat Busquets (UB - University of Barcelona, Faculty of Biology) ; Angeles Manresa (UB - University of Barcelona, Faculty of Pharmacy) |
Lipoxygenases (LOX) are a class of nonheme iron oxygenases that catalyze the conversion of arachidonic acid (AA) and other polyunsaturated fatty acids to their hydroperoxy derivatives. Occur ubiquitously in plants and mammals, and only recently they have been detected in coral, moss, fungi and a number of bacteria as well.
In this work, we cloned a fragment of a gene SW.LOX.ARAC encoding a lipoxygenase from Shewanella woodyi and characterized the enzyme biochemically.
In order to characterize the biochemical properties of SW.LOX.ARAC, the corresponding coding region was placed under the control of an IPTG-inducible promoter of an Escherichia coli expression vector. Bacterial cells containing the SW.LOX.ARAC construct and grown at 18ºC for 18 h, expressed the corresponding His-tagged protein, which was purified by affinity chromatography. SDS–PAGE analysis revealed a protein with a molecular mass of around 85 kDa. Crude extracts and purified preparations of these bacteria exhibited LOX activity, using the latter for biochemical characterization.
The lipoxygenase activity was determined by spectrophotometry at 234 nm
in the presence of arachidonic acid. One unit of activity (UA) was defined as the increase of one unit of absorbance at 234 nm in a minute. All measurements were performed in quadruplicate.
The highest enzymatic activity was observed in at a concentration of 100 mg / ml of LOX and 0,4mM of the arachidonic acid. The optimum temperature for activity was 27º C, and its thermal stability between 22°C and 36°C. We observed maximal activity at pH 7,0.
Acknoledgements:The financial support of the Comissió Interdepartamental de Recerca I Tecnologia CIRIT project 2009SGR819 and the Comisión Interministerial de Ciencia y Tecnologia (CICYT), project CTQ2010-21183-C02-01/PPQ is gratefully acknowledged.
Palavras-chave: lipoxygenase, protein expression, Shewanella woodyi