發(fā)布時(shí)間：2018-01-30 13:01

  本文關(guān)鍵詞： 耐熱轉(zhuǎn)酮酶 定向進(jìn)化 手性羥基酮　出處：《華東理工大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：本課題從Geobacillus屬芽孢桿菌中克隆得到三個(gè)耐熱性轉(zhuǎn)酮酶基因,并將其導(dǎo)入大腸桿菌中進(jìn)行可溶性表達(dá)驗(yàn)證。選取α位無(wú)羥基、a位羥基構(gòu)形分別為R和S構(gòu)形的三類轉(zhuǎn)酮酶典型的受體底物,借助于高通量的轉(zhuǎn)酮酶酶活測(cè)定方法對(duì)這三個(gè)耐熱轉(zhuǎn)酮酶進(jìn)行底物譜測(cè)定,測(cè)定結(jié)果證實(shí)這三個(gè)耐熱轉(zhuǎn)酮酶同其它來(lái)源的轉(zhuǎn)酮酶一樣對(duì)受體底物的α位羥基具有選擇性,即對(duì)α位羥基為R構(gòu)形的受體底物的催化活力比α位羥基為S構(gòu)形或α位無(wú)羥基的受體底物的催化活力高10至40倍。本課題通過(guò)比較三個(gè)耐熱轉(zhuǎn)酮酶的活力,最終選定來(lái)自于Geobaicillus stearothermophilus的轉(zhuǎn)酮酶TK456作為研究對(duì)象,借助于計(jì)算機(jī)Pymol軟件和PDB及Swiss-mod el網(wǎng)絡(luò)數(shù)據(jù)庫(kù)對(duì)其晶體結(jié)構(gòu)進(jìn)行了模擬。在晶體結(jié)構(gòu)的催化口袋中Asp470與α位羥基為R構(gòu)形的受體底物的αα位羥基形成氫鍵,與氫鍵距離較近的有Leu382、Leu191、Phe435三個(gè)氨基酸殘基位點(diǎn)。為了改變此耐熱轉(zhuǎn)酮酶對(duì)a位羥基的選擇性,本課題建立了L191、F435、D470/L191、D470/F435的飽和突變庫(kù),并通過(guò)測(cè)序的方法對(duì)建立的突變庫(kù)質(zhì)量進(jìn)行了評(píng)估。借助于以pH為基礎(chǔ)建立的轉(zhuǎn)酮酶高通量酶活測(cè)定和篩選方法對(duì)飽和突變庫(kù)用α位羥基為5構(gòu)形的模式受體底物L(fēng)-甘油醛和a位無(wú)羥基的模式受體底物丙醛同時(shí)篩選,共篩選得到了45個(gè)陽(yáng)性突變體。選取a位無(wú)羥基的丙醛、丁醛和甲氧基乙醇醛、α位構(gòu)形分別為R、S構(gòu)形的D-甘油醛、L-甘油醛、乙醇醛等幾種受體底物對(duì)篩選得到的45個(gè)陽(yáng)性轉(zhuǎn)酮酶突變體和野生型轉(zhuǎn)酮酶的催化活力和立體選擇性進(jìn)行了測(cè)定,其中對(duì)a位羥基為S構(gòu)形的受體底物L(fēng)-甘油醛和a位沒(méi)有羥基的模式受體底物丙醛的催化活力最大分別提高約8.5倍和10倍。利用衍生化試劑對(duì)45種陽(yáng)性突變體催化丙醛形成的產(chǎn)物進(jìn)行衍生化,并借助于手性氣相色譜柱對(duì)其光學(xué)純度進(jìn)行測(cè)定,篩選出立體選擇性較好的兩種突變體,Val191/Arg470 f口Leu435/Glu470,二者催化丙醛形成產(chǎn)物的ee值分別為0.81(R)和0.79(S)。最后利用這兩種立體選擇性較好的突變體催化α位沒(méi)有羥基的兩種底物丙醛、丁醛,進(jìn)行了生物催化合成,并對(duì)其產(chǎn)物結(jié)構(gòu)進(jìn)行驗(yàn)證,結(jié)果表明突變體Val191/Arg470催化這兩種底物的得率分別為：34%、53%；突變體Leu435/Glu470催化這兩種底物的得率分別為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 偽 -hydroxyl groups of the receptor substrates as the other transketonases. That is to say, the catalytic activity of the receptor substrate with 偽 -hydroxyl group is 10 to 40 times higher than that of 偽 -hydroxyl group or 偽 -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 偽 偽 -hydroxyl group of the receptor substrate with 偽 -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 偽 -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 偽 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.
【學(xué)位授予單位】：華東理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q814;O622.4

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