王珏;胡丽丽;张育敏;吴娜;李睿;

• 1:山西中医药大学基础医学院

摘要(Abstract)：

研究结合理性设计和定向进化技术,通过2轮易错PCR、2次定点突变和4轮定点饱和突变提高米黑霉脂肪酶(Rhizomucor miehei Lipase,RML)的热稳定性,共得到9株理想的脂肪酶突变体,在经70℃热处理2 h后,均能保留对甘油三酯的水解活性。以M9(D48V/V67A/S168P/S240A/S311C/D313H)的热稳定性最佳,热处理后保留活性可达(1 316±31)U/mg,这一数据是野生型RML常温水解活性的5.2倍(野生型RML经热处理后完全失活)。经测序分析,突变位点D48V和V67A位于目的基因前导肽上,S168P、S240A、S311C和D313H位于目的基因成熟区,且对前导肽上的两个突变点进行饱和突变后,突变体的热稳定性会发生改变,说明前导肽的突变会影响RML全基因的热稳定性,因此,RML热稳定性得以提高是源于其前导肽和成熟区的共进化作用。

关键词(KeyWords)： 前导肽;基因突变;米黑霉脂肪酶;热稳定性

Abstract:

Keywords:

基金项目(Foundation): 山西中医药大学科技创新能力培育计划(2018PY-20);; 山西省自然基金青年项目(201801D221278)

作者(Author): 王珏;胡丽丽;张育敏;吴娜;李睿;

Email:

DOI:

参考文献(References)：

• [1]CHEN K,ARNOLD F H.Tuning the activity of an enzyme for unusual environment:sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide[J].Proceedings of the National academy of Sciences,1993,90(4):5618-5622.
• [2]曲戈,张锟,蒋迎迎,等.2018诺贝尔化学奖:酶定向进化与噬菌体展示技术[J].生物学杂志,2019,36(1):1-6.
• [3]ROBERTO A C,NICOLAS D,JOELLE N P.Semi-rational approaches to engineering enzyme activity:combining the benefits of directed evolution and rational design[J].Current Opinion in Biotechnology,2005,16(2):378-384.
• [4]DEREWENDA Z S,DEREWENDA U.Relationships among serine hydrolases:evidence for a common motif in triacylglyceride lipases and esterases[J].Biochemistry and Cell Biology,1991,69(2):842-851.
• [5]李婷婷,张天喜,武俊明,等.嗜热酯酶的研究进展[J].生物学杂志,2019,36(5):78-81.
• [6]易华伟,唐晓峰.基于氨基酸序列和模拟结构预测蛋白质稳定性的研究进展[J].生物技术通报,2017,33(4):83-89.
• [7]LI G L,FANG X R,SU F,et al.Enhancing the thermostability of Rhizomucor miehei lipase with a limited screening library by rational-design point mutations and disulfide bonds[J].Applied and Environmental Microbioloy,2018,84 (2):1-16.
• [8]WANG J,WANG D,YU H W.Enhanced activity of Rhizomucor miehei lipase by directed evolution with simultaneous evolution of the propeptide[J].Applied Microbiology and Biotechnology,2012,96(2):443-450.
• [9]MARK B,CORRINE M L,UWE T B.A microtitre plate-based assay method to determine fat quality[J].European Journal of Lipid Science and Technology,2007,109(3):180-185.
• [10]WANG D,WANG J,YU H W.A new and efficient colorimetric high-throughput screening method for triacylglycerol lipase directed evolution[J].Journal of Molecular Catalysis B:Enzymatic,2012,82(1):18-23.
• [11]王珏,王丹,张兵,等.米黑霉脂肪酶基因的高效表达及活性测定[J].食品与生物技术学报,2012,31(12):1262-1268.
• [12]朱国萍,滕脉坤,王玉珍.脯氨酸对蛋白质热稳定性的贡献[J].生物工程进展,2000,20(4):48-51.
• [13]NOBUHIKO T,FRANCOIS S,LUIS S,et al.How protein stability and new functions trade off[J].PLoS Computational Biology,2008,4(2):1-7.
• [14]吴厚军,喻晓蔚,沙冲,等.D190V点突变提高华根霉Rhizopus chinensis CCTCC M201021 脂肪酶的最适温度和热稳定性[J].微生物学通报,2013,40(11):1955-1961.
• [15]ZHOU C,XUE Y F,MA Y H.Enhancing the thermostability of α-glucosidase from Thermoanaerobacter tengcongensis MB4 by single proline substitution[J].Journal of Bioscience and Bioengineering,2010,110(1):12-17.
• [16]TAKANO K,HIGASHII R,OKADA J,et al.Proline effect on the thermostability and slow unfolding of a hyperthermophilic protein[J].Journal of Biochemistry,2009,145(1):79-85.
• [17]谭中标,李剑芳,邬敏辰,等.脂肪酶热稳定性改造研究进展[J].食品与生物技术学报,2014,33(7):673-681.
• [18]COSTANTINI S,COLONNA G,FACCHIANO A M.ESBRI:a web server for evaluating salt bridges in proteins[J].Bioinformation,2008,3(3):137-138.