利用定点突变法对微生物精氨酸脱亚胺酶进行定向改造研究

发布时间：2018-06-11 22:35

  本文选题：精氨酸脱亚胺酶 + 定点突变　； 参考：《江南大学》2012年硕士论文

【摘要】：精氨酸脱亚胺酶(ADI)是一种针对精氨酸缺陷型癌症(如:肝癌、黑素瘤)的新药,目前处于临床三期试验。为了进一步探明M314中关键位点对精氨酸脱亚胺酶的作用,本研究通过定点突变技术分析了精氨酸脱亚胺酶的特定氨基酸位点对酶活力的影响机制。 针对已报道的关键氨基酸残基A128,H404,I410,采用QuikChange法进行定点突变,获得ADI突变株M1 (A128T)、M2 (H404R)、M3 (I410L)和M4 (A128T/H404R)。将突变株在大肠杆菌BL21 (DE3)中进行重组表达,并对纯化获得的突变蛋白进行酶学性质研究。结果表明,突变位点A128T和H404R对ADI最适pH的提高,生理中性(pH 7.4)条件下的酶活力和稳定性的提高,以及Km值的降低均具有显著的作用。 为了进一步改善ADI的酶学性质,结合序列分析以及相关文献报道,选定V10F、R18L、D38H、D44E、A128R、E296K作为突变位点,以WT-ADI为出发菌株进行单点突变,获得突变株后并对各个突变酶的酶学性质进行了考察,结果表明,D38H和E296K对WT-ADI的酶活力的提高最具有显著的促进作用。将突变热点D38H和E296K引入到突变株M314中,获得了突变株M11 (D38H/A128T/H404R/I410L)、M12 (A128T/E296K/H404R/I410L)和M13 (D38H/A128T/E296K/H404R/I410L),其中M13 (D38H/A128T/E296K/H404R/I410L)在生理中性条件下(pH 7.4)酶活力较M314(10.08 U/mg)提高了58%,比活力为17.02 U/mg,最适pH为6.5。M13的Km(0.57 mmol/L)较M314(0.67 mmol/L)有所降低,其kcat/Km较M314有显著提高。结果表明,突变株M13与底物的亲和力以及对底物的催化效率方面均有较大提高。 以来源于Pseudomonas aeruginosa的ADI蛋白质结构为模型,采用Swiss-Model对来源于P. plecoglossicida的ADI突变株M314进行同源结构建模,并对各突变位点V10F、R18L、D38H、D44E、A128R、A128T、E296K、H404R和I410L的立体结构进行分析。本研究为阐明ADI的酶活力影响机制和蛋白质的理性改造提供了一定的依据。
[Abstract]:Arginine deiminase (ADI) is a new drug for arginine deficient cancer (such as liver cancer, melanoma). In order to further investigate the effect of key sites in M314 on arginine deiminase, In this study, the effects of specific amino acid sites of arginine deiminase on the activity of arginine deiminase were analyzed by site-directed mutagenesis. Based on the reported key amino acid residues A128T404H40410, the directed mutagenesis was carried out by QuikChange method. The mutant was expressed in Escherichia coli BL21 (DE3) and the purified mutant protein was studied by enzymatic properties. The results showed that the mutation sites A128T and H404R had significant effects on the increase of the optimum pH of ADI, the increase of enzyme activity and stability under physiological neutral pH 7.4) and the decrease of km value. In order to further improve the enzymatic properties of ADI, A128T and H404R had significant effects on ADI. Combined with sequence analysis and related literature reports, we selected V10FN R18LN D38HN D44EA128RN E296K as the mutation site and WT-ADI as the starting strain for single point mutation. After obtaining the mutant strain, the enzymatic properties of each mutant enzyme were investigated. The results showed that D38H and E296K enhanced the activity of WT-ADI most significantly. 灏嗙獊鍙樼儹鐐笵38H鍜孍296K寮曞叆鍒扮獊鍙樻牚M314涓，

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