本文選題：亞油酸異構(gòu)酶 + 共軛亞油酸��； 參考：《內(nèi)蒙古農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：共輒亞油酸(Conjugated linoleic acid,CLA)是一系列具有抗腫瘤、提高免疫力、降血脂、降低膽固醇等功效的含有順式或反式共軛雙鍵亞油酸的總稱。亞油酸異構(gòu)酶(Linoleic acid isomerase,LAI)是亞油酸(Linoleic acid,LA)生物轉(zhuǎn)化為共輒亞油酸的一個(gè)重要酶。植物乳桿菌P-8也有LAI,但其結(jié)構(gòu)和功能及其催化機(jī)制不甚清楚。本研究為了探究植物乳桿菌P-8亞油酸異構(gòu)酶的酶學(xué)性質(zhì)和一些必需基團(tuán),構(gòu)建了植物乳桿菌P-8LAI的原核表達(dá)重組菌BL21-pET28a-LAI。對(duì)目的基因IPTG誘導(dǎo)表達(dá)條件進(jìn)行優(yōu)化,結(jié)果表明重組LAI的最佳表達(dá)條件是:IPTG終濃度為.0.1mM,130r/min,14h搖床誘導(dǎo)培養(yǎng)。誘導(dǎo)表達(dá)的野生型重組LAI經(jīng)鎳柱子純化后用氣相色譜法進(jìn)行酶學(xué)性質(zhì)分析,結(jié)果表明該酶的最適底物濃度是0.3mg/mL,最適溫度為37℃,最適酶液體積為100μL,最適 pH 值為 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 對(duì)亞油酸異構(gòu)酶進(jìn)行結(jié)構(gòu)分析,鎖定可能的6個(gè)必需位點(diǎn),用QuickChange法成功構(gòu)建了六個(gè)點(diǎn)突變體的重組質(zhì)粒 pET28a-T563C-LAI、pET28a-G320T-LAI、pET28a-A515C-LAI、pET28a-G203C-LAI、pET28a-A389T-LAI和 pET28a-G458T-LAI。誘導(dǎo)表達(dá)的6個(gè)重組突變LAI,經(jīng)鎳柱子純化后,用氣相色譜法分析其活力并與重組野生型LAI相比較,六個(gè)位點(diǎn)的改變均引起酶活力發(fā)生大的改變。因此,可以確定LAI的188位、107位、172位、68位、130位和153位的氨基酸殘基全為必需基團(tuán)。
[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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