高山被孢霉中Δ9脂肪酸脱饱和酶的表达、纯化和其细胞色素b_5功能域的鉴定  

Expression,Purification of Mortierella alpina Δ9 Desaturase and Characterization of Its Cytochrome b_5 Domain

作　　者：王明轩[1] 陈海琴[1] 顾震南[1] 陈卫[1] 陈永泉[1] 

机构地区：[1]江南大学食品学院食品科学与技术国家重点实验室,无锡214122

出　　处：《中国生物工程杂志》2017年第3期43-50,共8页China Biotechnology

基　　金：国家自然科学基金资助项目(21276108)

摘　　要：在毕赤酵母中表达和纯化源自高山被孢霉ATCC 32222的膜结合Δ9-I脂肪酸脱饱和酶,测定其活性,并探究其细胞色素b_5功能域的性质。构建含有高效纯化标签ZZ-tag的表达载体;用Western blotting和SDS-PAGE筛选Δ9-I脂肪酸脱饱和酶高表达量转化子;通过梯度离心和去垢剂筛选确定膜蛋白质提取条件;采用IgG亲和纯化色谱和阴离子交换色谱对Δ9-I脂肪酸脱饱和酶进行纯化;利用酿酒酵母细胞破碎物为底物考察Δ9-I脂肪酸脱饱和酶活性;通过波长扫描和Na_2S_2O_4还原实验对Δ9-I脂肪酸脱饱和酶细胞色素b_5功能域进行表征。结果显示,目的蛋白质被成功表达并筛选出高表达量转化子;20 000g离心1h为最佳膜分离条件,Fos-Choline-16为最佳去垢剂;纯化后的Δ9-I脂肪酸脱饱和酶结构完整,具有细胞色素b_5功能域;在酿酒酵母提取物中Δ9-I脂肪酸脱饱和酶对C16:0和C18:0底物的转化效率分别为(16.88±9.32)%和(20.61±7.55)%;波长扫描显示Δ9-I脂肪酸脱饱和酶在411nm处有强吸收,并且在Na_2S_2O_4作用下被还原至422nm,说明细胞色素b_5功能域在体外能够被还原。因此,含有细胞色素b_5功能域的脂肪酸脱饱和酶的首次成功表达、纯化和鉴定为亚铁血红素脂肪酸脱饱和酶脱饱和反应机制的研究奠定了基础。The integral Δ9-I desaturase gene from Mortierella alpina ATCC 32222 was expressed and purified from Pichia pastoris. The codon-optimized gene for the Δ9-I desaturase was appended to a cassette containing the human rhinovirus 3C protease cleavage site,the Ig G-specific ZZ-tag,and an RGS-His10-tag. The highest expressing clone was determined by Western blotting and SDS-PAGE analysis. A membrane fraction was isolated by gradient centrifugation and detergents were used for efficient extraction of desaturase. Affinity purification of desaturases was conducted using the C-terminally fused Ig G-binding ZZ-tag. The enzymatic activity was determined using cell lysate of Saccharomyces cerevisiae as substrate. The cytochrome b_5 domain was characterized by wavelength scan and Na_2S_2O_4 reduction experiment. Results showed that the Mortierella alpina Δ9-I desaturase was expressed successfully and transformant with high expression level was selected. The membrane fraction was collected at a centrifugation of 20 000 g for 1h and only Fos-Choline-16 effectively extracted the desaturases from the membrane,when compared to the efficiency of SDS. The purified desaturase was intact and the cytochrome b_5 domain was present. In the yeast lysate,the conversion rate of C16 ： 0 and C18： 0 substrate were（ 16. 88 ± 9. 32） % and（ 20. 61 ± 7. 55） %,respectively. Wavelength scan of the purified protein revealed an absorbance feature at 411 nm and it shifted to 422 nm in the presence of Na_2S_2O_4. This result indicated that the cytochrome b_5 domain can be reduced in vitro. Thus,the first successful purification of an integral desaturase that contained the fusion cytochrome b_5 domain lays the foundation for the mechanism study of heme integral desaturase.

关 键 词：脂肪酸脱饱和酶 膜结合蛋白质 蛋白质纯化 高山被孢霉 细胞色素b5功能域 

分 类 号：Q819[生物学—生物工程]