植物乳杆菌P-8亚油酸异构酶酶学性质及必需基团的研究

发布时间：2018-05-24 23:44

  本文选题：亚油酸异构酶 + 共轭亚油酸　； 参考：《内蒙古农业大学》2017年硕士论文

【摘要】：共辄亚油酸(Conjugated linoleic acid,CLA)是一系列具有抗肿瘤、提高免疫力、降血脂、降低胆固醇等功效的含有顺式或反式共轭双键亚油酸的总称。亚油酸异构酶(Linoleic acid isomerase,LAI)是亚油酸(Linoleic acid,LA)生物转化为共辄亚油酸的一个重要酶。植物乳杆菌P-8也有LAI,但其结构和功能及其催化机制不甚清楚。本研究为了探究植物乳杆菌P-8亚油酸异构酶的酶学性质和一些必需基团,构建了植物乳杆菌P-8LAI的原核表达重组菌BL21-pET28a-LAI。对目的基因IPTG诱导表达条件进行优化,结果表明重组LAI的最佳表达条件是:IPTG终浓度为.0.1mM,130r/min,14h摇床诱导培养。诱导表达的野生型重组LAI经镍柱子纯化后用气相色谱法进行酶学性质分析,结果表明该酶的最适底物浓度是0.3mg/mL,最适温度为37℃,最适酶液体积为100μL,最适 pH 值为 7.0。Km=211.368 g/L,Vmax=75.188 mg/L×h。利用 Specia lized BLAST、http://smart.embl-heidelberg.de/和 vecterNTI 对亚油酸异构酶进行结构分析,锁定可能的6个必需位点,用QuickChange法成功构建了六个点突变体的重组质粒 pET28a-T563C-LAI、pET28a-G320T-LAI、pET28a-A515C-LAI、pET28a-G203C-LAI、pET28a-A389T-LAI和 pET28a-G458T-LAI。诱导表达的6个重组突变LAI,经镍柱子纯化后,用气相色谱法分析其活力并与重组野生型LAI相比较,六个位点的改变均引起酶活力发生大的改变。因此,可以确定LAI的188位、107位、172位、68位、130位和153位的氨基酸残基全为必需基团。
[Abstract]:Linoleic acid (CLA) is a series of conjugated linoleic acid containing cis-or trans-conjugated linoleic acid, which has the functions of anti-tumor, improving immunity, lowering blood lipid and lowering cholesterol. Linoleic acid isomerase (Lai) is an important enzyme for the bioconversion of linoleic acid to linoleic acid. Lactobacillus plantarum P-8 also has LAI, but its structure, function and catalytic mechanism are not very clear. In order to investigate the enzymatic properties and some essential groups of Lactobacillus plantarum P-8 linoleic isomerase, a prokaryotic expression recombinant strain BL21-pET28a-LAI was constructed for Lactobacillus plantarum P-8LAI. The expression conditions of target gene IPTG were optimized. The results showed that the optimal expression condition of recombinant LAI was induced culture by shaking bed for 14 h. The final concentration of LAI was. 0.1 mm MMT 130r / min ~ (-1) 路min ~ (-1) ~ (-1). The enzyme properties were analyzed by gas chromatography after purified by nickel column. The results showed that the optimal substrate concentration was 0.3 mg / mL, the optimum temperature was 37 鈩，

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