Expression of soluble recombinant lipoxygenase from Pleurotus sapidus in Pichia pastoris

The first heterologous expression of an iron-containing lipoxygenase from a basidiomycete in Pichia pastoris is reported. Five different expression constructs of the lipoxygenase gene LOX1 from Pleurotus sapidus were cloned and successfully transferred into P. pastoris SMD1168, but only one pPIC9K vector construct was functionally expressed. In this construct the vector-provided alpha-factor signal sequence was replaced by insertion of a second Kozak sequence between the signal sequence and the LOX1 gene. His(+) transformants were screened for their level of resistance to geneticin (G418). Lox1 was expressed under different culture conditions and purified using the N-terminal His-tag. Relative enzyme activity increased significantly 48 h after methanol induction and was highest with 2 ml l(-1) inducer. The recombinant enzyme showed an optimal lipoxygenase activity at pH 7 and 30-35 degrees C and a v(max) like the wild-type enzyme. (C) 2014 Elsevier Inc. All rights reserved.