耐热转酮酶的定向进化及其在手性羟基酮合成中的应用

发布时间：2018-01-30 13:01

本文关键词： 耐热转酮酶定向进化手性羟基酮　出处：《华东理工大学》2016年硕士论文　论文类型：学位论文

【摘要】：本课题从Geobacillus属芽孢杆菌中克隆得到三个耐热性转酮酶基因,并将其导入大肠杆菌中进行可溶性表达验证。选取α位无羟基、a位羟基构形分别为R和S构形的三类转酮酶典型的受体底物,借助于高通量的转酮酶酶活测定方法对这三个耐热转酮酶进行底物谱测定,测定结果证实这三个耐热转酮酶同其它来源的转酮酶一样对受体底物的α位羟基具有选择性,即对α位羟基为R构形的受体底物的催化活力比α位羟基为S构形或α位无羟基的受体底物的催化活力高10至40倍。本课题通过比较三个耐热转酮酶的活力,最终选定来自于Geobaicillus stearothermophilus的转酮酶TK456作为研究对象,借助于计算机Pymol软件和PDB及Swiss-mod el网络数据库对其晶体结构进行了模拟。在晶体结构的催化口袋中Asp470与α位羟基为R构形的受体底物的αα位羟基形成氢键,与氢键距离较近的有Leu382、Leu191、Phe435三个氨基酸残基位点。为了改变此耐热转酮酶对a位羟基的选择性,本课题建立了L191、F435、D470/L191、D470/F435的饱和突变库,并通过测序的方法对建立的突变库质量进行了评估。借助于以pH为基础建立的转酮酶高通量酶活测定和筛选方法对饱和突变库用α位羟基为5构形的模式受体底物L-甘油醛和a位无羟基的模式受体底物丙醛同时筛选,共筛选得到了45个阳性突变体。选取a位无羟基的丙醛、丁醛和甲氧基乙醇醛、α位构形分别为R、S构形的D-甘油醛、L-甘油醛、乙醇醛等几种受体底物对筛选得到的45个阳性转酮酶突变体和野生型转酮酶的催化活力和立体选择性进行了测定,其中对a位羟基为S构形的受体底物L-甘油醛和a位没有羟基的模式受体底物丙醛的催化活力最大分别提高约8.5倍和10倍。利用衍生化试剂对45种阳性突变体催化丙醛形成的产物进行衍生化,并借助于手性气相色谱柱对其光学纯度进行测定,筛选出立体选择性较好的两种突变体,Val191/Arg470 f口Leu435/Glu470,二者催化丙醛形成产物的ee值分别为0.81(R)和0.79(S)。最后利用这两种立体选择性较好的突变体催化α位没有羟基的两种底物丙醛、丁醛,进行了生物催化合成,并对其产物结构进行验证,结果表明突变体Val191/Arg470催化这两种底物的得率分别为：34%、53%；突变体Leu435/Glu470催化这两种底物的得率分别为52%、50%。
[Abstract]:In this study, three thermostable transketozyme genes were cloned from Bacillus Geobacillus, and then introduced into Escherichia coli for soluble expression. Three types of transketozyme typical receptor substrates with R and S configurations were obtained. The three thermostable transketonases were determined by high-throughput transketozyme activity method. The results showed that the three thermostable transketonases were as selective to the 伪 -hydroxyl groups of the receptor substrates as the other transketonases. That is to say, the catalytic activity of the receptor substrate with 伪 -hydroxyl group is 10 to 40 times higher than that of 伪 -hydroxyl group or 伪 -hydroxyl free receptor substrate. In this study, the activity of three thermostable transketonases was compared. . Finally, the transketonase TK456 from Geobaicillus stearothermophilus was selected as the research object. With the help of computer Pymol software and PDB and Swiss-mod. The crystal structure was simulated by el network database. In the catalytic pocket of the crystal structure, Asp470 formed a hydrogen bond with the 伪伪 -hydroxyl group of the receptor substrate with 伪 -hydroxyl group as R configuration. In order to change the selectivity of the thermostable transketonase to the hydroxyl group at position a, L191 was established in order to change the selectivity of the thermostable transketonase to the hydroxyl group at position a, and the three amino acid residues of Leu382Phe435 were close to the hydrogen bond. The saturated mutation library of F435 / D470 / L191 / D470 / F435. The quality of the mutant library was evaluated by sequencing. The model receptor with 伪 -hydroxyl group of 5 configuration was used to determine and screen the high throughput enzyme activity of transketonase based on pH. L- glyceraldehyde and propionaldehyde, the model receptor substrates without a hydroxyl group, were screened simultaneously. A total of 45 positive mutants were obtained, including propionaldehyde, butyraldehyde and methoxyglycolic aldehyde without hydroxyl at position a, and D- glyceraldehyde and L-glyceraldehyde with r-S configuration, respectively. The catalytic activity and stereoselectivity of 45 positive transketonase mutants and wild type transketonases were determined by glycolaldehyde and other receptor substrates. The catalytic activity of L- glyceraldehyde, a receptor substrate with S configuration at position a, and propionaldehyde, a model receptor substrate without hydroxyl group, was increased by about 8.5 times and 10 times respectively. The products of propionaldehyde formation catalyzed by sexual mutants were derivatized. The optical purity was determined by chiral gas chromatography. Two mutants, Val191- Arg470f Leu435/Glu470 with good stereoselectivity were selected. The ee values of propanal products catalyzed by these two mutants were 0.81g R) and 0.79N Sol, respectively. Finally, two kinds of substrates, propionaldehyde and butyraldehyde, which had no hydroxyl group at 伪 site, were catalyzed by these two mutants with good stereoselectivity. The biocatalysis synthesis was carried out, and the structure of the product was verified. The results showed that the yield of the two substrates catalyzed by the mutant Val191/Arg470 was: 34 / 34 / 53, respectively. The yield of the two substrates catalyzed by the mutant Leu435/Glu470 was 52% and 50%, respectively.
【学位授予单位】：华东理工大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q814;O622.4

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