高山被孢霉中Δ6脱饱和酶关键位点及其对活性的影响  

Key sites of Δ6 desaturase from Mortierella alpina and its effect on catalytic activities

作　　者：史海粟[1] 陈海琴[1] 顾震南[1] 张灏[1] 陈永泉[1] 陈卫[1] 

机构地区：[1]江南大学食品学院

出　　处：《食品与发酵工业》2016年第9期1-7,共7页Food and Fermentation Industries

基　　金：国家自然科学基金(No.21276108)

摘　　要：高山被孢霉中Δ6脱饱和酶(Ma FADS6)是决定ω3/ω6多不饱和脂肪酸代谢流的关键酶。利用定点突变技术研究Ma FADS6-Ι一级结构与功能的关系,为高山被孢霉多不饱和脂肪酸的合成提供了理论依据。利用非链取代式质粒扩增技术对已构建质粒p YES2/NT C-Ma FADS6-Ι内的表达单元(即Ma FADS6-Ι)中细胞色素b5区(HPGG)下游的位点进行定点突变,将所有突变体分别转化酿酒酵母进行诱导表达,进一步通过在培养基中添加Δ6脱饱和酶的底物来考察重组菌对各底物的催化作用。实验结果表明,所有突变体催化α-亚麻酸(ALA)的活性没有改变;K61和D68两个位点对催化亚油酸(LA)起关键作用,其对应的4个突变体对LA的转化率分别为(6.2±0.5)%(K61T)、(0.3±0.0)%(K61F)、(8.2±0.7)%(D68N)和(6.6±0.8)%(D68L),相比原始转化率各降低50%以上;G63和T69两个位点对LA的催化无直接影响,但G63T突变体由于突变位点极性改变,其对LA的转化率为(8.5±0.9)%,比原始转化率降低了49.9%;而V66位点的突变体对LA的转化率分别为(22.3±2.6)%(V66T)和(20.7±2.5)%(V66A),比原始转化率分别降低了36.1%和37.8%。确定了Ma FADS6-Ι氨基酸序列中K61和D68两个关键氨基酸位点经突变后均会使其酶活明显降低,这为研究Ma FADS6-Ι一级结构与功能的关系提供了理论依据。Δ6 desaturase from oleaginous Mortierella alpina（ Ma FADS6） is a key enzyme in biosynthesis of ω3 /ω6 polyunsaturated fatty acids（ PUFAs）. This study focused on the relationship between the primary structure and function for Ma FADS6-Ι based on site-directed mutagenesis. Non-chain plasmid amplification method was used in this study to target downstream of cytochrome b5 region（ HPGG） in the expression unit（ Ma FADS6-Ι） of p YES2 / NT CMa FADS6-Ιby site-directed mutagenesis. Each recombinant plasmid was transformed and expressed in Saccharomyces cerevisiae. The results showed that the catalytic activity of all mutant using α-linolenic acid（ ALA） as substrate has not changed. Two catalytic sites,K61 and D68,played a key role for its catalysis on linoleic acid（ LA）. The LA conversion rate of four corresponding mutants was（ 6. 2 ± 0. 5） %（ K61T）,（ 0. 3 ± 0. 0） %（ K61F）,（ 8. 2 ±0. 7） %（ D68N） and（ 6. 6 ± 0. 8） %（ D68L）,respectively,which reduced by more than 50% compared to the original conversion rate. G63 and T69 were two sites which not directly affect the catalysis of LA,but LA conversion of G63 T mutant was（ 8. 5 ± 0. 9） % which decreased 49. 9% compared the original conversion rate due to polarity change of mutation site. LA conversion rates of V66 mutants were（ 22. 3 ± 2. 6） %（ V66T） and（ 20. 7 ± 2. 5） %（ V66A）,which decreased by 36. 1% and 37. 8% compared to the original conversion rate. Enzymatic activities were significantly reduced after mutation of two key site of amino acid sequence of Ma FADS6-Ι,K61 and D68. It provided a theoretical basis for the study on relationship between primary structure of Ma FADS6-Ιand its function.

关 键 词：Δ6脱饱和酶 高山被孢霉 定点突变 

分 类 号：TS201.3[轻工技术与工程—食品科学]