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 α-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 48h after methanol induction and was highest with 2mll(-1) inducer. The recombinant enzyme showed an optimal lipoxygenase activity at pH 7 and 30-35°C and a vmax like the wild-type enzyme.
Gottfried Wilhelm Leibniz University Hannover, Institute of Food Chemistry, Callinstr. 5, D-30167 Hannover, Germany. Electronic address: sebastian.kelle@lci.uni-hannover.de.
Sebastian
S
Kelle
Gottfried Wilhelm Leibniz University Hannover, Institute of Food Chemistry, Callinstr. 5, D-30167 Hannover, Germany.
Katerina
K
Zelena
Gottfried Wilhelm Leibniz University Hannover, Institute of Food Chemistry, Callinstr. 5, D-30167 Hannover, Germany.
Ulrich
U
Krings
Gottfried Wilhelm Leibniz University Hannover, Institute of Food Chemistry, Callinstr. 5, D-30167 Hannover, Germany.
Diana
D
Linke
Gottfried Wilhelm Leibniz University Hannover, Institute of Food Chemistry, Callinstr. 5, D-30167 Hannover, Germany.
Ralf G
RG
Berger
D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008084:Lipoxygenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013058:Mass Spectrometry; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D020076:Pleurotus / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D013696:Temperature
D005656:Fungal Proteins; D011994:Recombinant Proteins; D008084:Lipoxygenase
Functional expression; Kozak sequence; Lipoxygenase; Pichia pastoris; Pleurotus sapidus