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Abstract：
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.
摘要：
报道了在巴斯德毕赤酵母中来自担子菌的含铁脂氧合酶的首次异源表达。克隆了白灵菇脂氧合酶基因LOX1的五种不同表达构建体，并成功转移到巴斯德毕赤酵母SMD1168中，但仅一种pPIC9K载体构建体被功能性表达。在该构建体中,通过在信号序列和LOX1基因之间插入第二Kozak序列来替换载体提供的α因子信号序列。筛选His(+)转化体对遗传霉素(G418)的抗性水平。Lox1在不同的培养条件下表达并使用N末端His标签纯化。相对酶活性在甲醇诱导后48h显着增加，并且在2mll（-1）诱导剂中最高。重组酶在pH7和30-35°C下表现出最佳的脂氧合酶活性，并且vmax与野生型酶相似。
