Cloning and characterization of a lipoxygenase from phaeodactylum tricornutum

摘要

From the diatom p. tricornutum, the sequence of one clone which, via ests was identified as a homologous of vegetable loxen, by means of 5 ''race has been successfully extended and the sequence of the complex cdna determined. The insulation of these cdna from the underlying phage - bank was not successful, for which reason the complete cdna - sequence was composed of two overlapping fragments. One fragment corresponded to the present cdna - clone and the other fragment was isolated from the cdna - library. Since, in the area of the overlap a in the sequence unique restriction site an endonuclease existed, it was possible, these two fragments for the complete cdna - sequence be assembled. Since the purification of the enzyme does not succeeded, was for the biochemical characterization of the uncleaned enzyme from the bacterial cell lysates used. In the reaction of linoleic acid by ptlox1 could be shown that, in the case of this liquid oxygen (lox) is about a 13 - lox. The ph - optimum of the enzyme was determined on the basis of the reaction of linoleic acid and is in the range of ph 8,2. in order to detect, which often unsaturated fatty acids of ptlox1 as substrates which are acceptable and regio isomers in this case preferably, seven different fatty acids were used: linoleic acid (la), α - linolenic acid (α - lea), γ - linolenic acid (γ - lea), arachidonic acid (cg), eicosapentaenoic acid (epa), docosapentene acid (dpa) and docosahexaenoic acid (dha). The reaction of la, yielded, after reduction ..