本文選題：脫氧核糖醛縮酶 + 固定化��； 參考：《浙江大學(xué)》2016年博士論文

【摘要】：2-脫氧-D-核糖醛縮酶(EC 4.1.2.4, DERA)催化的羥醛縮合反應(yīng)可以生成兩個(gè)手性中心,特別是它能夠以三分子乙醛為底物,經(jīng)過(guò)兩步連續(xù)羥醛縮合反應(yīng)得到他汀類藥物手性側(cè)鏈。他汀類藥物能夠有效降低人體內(nèi)膽固醇含量,是一種重要的的降血脂藥物。盡管如此,DERA在應(yīng)用上仍存在一些問(wèn)題：底物譜窄,偏好磷酸化底物,對(duì)2-脫氧-D-核糖(DR)等非磷酸化底物催化活力低,對(duì)底物乙醛的耐受性差等。因此本課題在改進(jìn)現(xiàn)有熒光高通量篩選方法的基礎(chǔ)上,對(duì)DERA進(jìn)行同源模建和分子動(dòng)力學(xué)計(jì)算,綜合運(yùn)用定向進(jìn)化和理性設(shè)計(jì)改造DERA,以提高其對(duì)非磷酸化底物的催化活力和對(duì)高濃度乙醛的耐受性,并用于手性藥物前體的合成。(1)熒光高通量篩選方法的建立：為了快速有效的篩選新酶和測(cè)定改造后DERA的催化活性,在現(xiàn)有熒光高通量篩選方法的基礎(chǔ)上,研究香豆素類衍生物熒光發(fā)光機(jī)制,發(fā)現(xiàn)母環(huán)分子中取代基的種類及其位置的變化能夠?qū)晒鈴?qiáng)度產(chǎn)生作用。通過(guò)對(duì)熒光底物的結(jié)構(gòu)進(jìn)行重新設(shè)計(jì),在香豆素母環(huán)3號(hào)位引入苯并咪唑基,在6號(hào)位引入甲氧基,使底物在可見(jiàn)光下散發(fā)強(qiáng)烈的綠色熒光,相比于現(xiàn)有方法,其檢測(cè)靈敏度提高了58.2倍。(2)DERA的定向進(jìn)化改造：從實(shí)驗(yàn)室前期重組表達(dá)的8種DERA中,選擇對(duì)非磷酸化底物DR催化活力乙醛耐受性最高的DERAGth和DERASep作為研究對(duì)象。這兩種酶均可在E. coli BL21(DE3)中實(shí)現(xiàn)過(guò)量表達(dá),純酶的比活分別為22.5U/mg和16.5U/mg。通過(guò)易錯(cuò)PCR對(duì)DERAGth和DERASep進(jìn)行定向進(jìn)化后,利用構(gòu)建的高通量篩選方法獲得突變株DERAGth (F159I,S209G),其比活為105.8U/mg,相比原始酶提高了3.7倍。(3)理性設(shè)計(jì)提高DERA對(duì)N-丙烯醛鄰苯二甲酰亞胺的催化活力：考察了DERAGth, DERASep催化受體底物N-丙烯醛鄰苯二甲酰亞胺(N-AMP)和乙醛縮合這一模型反應(yīng)的活力,DERAGth和DERAGth (F159I, S209G)比活分別為0.52U/mg和0.71 U/mg,而DERASep比活為6.2U/mg,因此選擇DERASep作為目標(biāo)酶進(jìn)行改造。以DERATma(PDB1o0y)為模板,采用同源建模法構(gòu)建了DERASep的結(jié)構(gòu)模型,并對(duì)模型進(jìn)行評(píng)價(jià)。通過(guò)分子對(duì)接,發(fā)現(xiàn)殘基Thr10,Ser205,Ala206的側(cè)鏈可能阻礙N-APM進(jìn)入催化口袋。根據(jù)對(duì)接能的大小選擇Thr10,Ser205,Ala206進(jìn)行模擬突變,突變體結(jié)合N-AMP的分子動(dòng)力學(xué)計(jì)算,得到DERASep(A206G)和DERASep(S205E)的結(jié)合自由能分別為-12.39kcal/mol和-7.11kcal/mol(DERASep為-3.17kcal/mol),推測(cè)這兩個(gè)位點(diǎn)的突變可能會(huì)引起酶活力的變化。定點(diǎn)突變后考察突變體對(duì)底物催化活力的變化,結(jié)果顯示,DERASep突變體(A206G)比活為22.8U/mg,比DERASep提高了2.7倍。(4)理性設(shè)計(jì)提高脫氧核糖醛縮酶乙醛耐受性：以DERAsep和DERASep(A206G)為研究對(duì)象,根據(jù)研究報(bào)道發(fā)現(xiàn)酶的熱穩(wěn)定性和其乙醛等有機(jī)溶劑的耐受性有正相關(guān)性,因此提出通過(guò)增強(qiáng)DERASep的穩(wěn)定性提高其乙醛耐受性的思路。利用分子模擬工作站Discovery Studio對(duì)DERASep的結(jié)構(gòu)模型進(jìn)行虛擬突變掃描計(jì)算,得到單個(gè)氨基酸的突變能�；谲浖倪\(yùn)算規(guī)則,突變能越低,結(jié)構(gòu)越穩(wěn)定,選取七個(gè)突變能最低的單點(diǎn)位點(diǎn)D15F:S16I,T41C,T120I,G174Y,G174,G213C,在此基礎(chǔ)上進(jìn)行雙點(diǎn)突變和多點(diǎn)虛擬突變,得到的組合突變能最低的突變體DERASep Variant10(T120C, G174I, G213C)和DERASep Variant11(T120C, G174I, A206G,G213C)�？疾炱湟胰┠褪苄�,結(jié)果顯示DERASep Variant10在300mM乙醛濃度下,靜置2小時(shí)后,活力殘余70.5%,DERASep Variant11在相同條件下靜置2小時(shí)后,活力殘余66.7%,而被廣泛研究的DERAEco在同樣條件下,靜置2小時(shí)后活力殘余幾乎為O。在全細(xì)胞催化N-AMP (46.2mM)和乙醛(166.7mM)的縮合反應(yīng)中,在24h內(nèi)DERASep Variant11催化反應(yīng)了43.6%的底物,比DERASep和DERAEco分別提高了1.32倍和3.1倍,是文獻(xiàn)中報(bào)道的DERA催化活力的1.55倍。(5) DERAEco的固定化改造：通過(guò)交聯(lián)將DERA結(jié)合在納米Fe304磁性顆粒上,確定最適的交聯(lián)條件為：酶量與載體量的比為1：10,交聯(lián)pH為6.0,交聯(lián)時(shí)間為5h。制備后的交聯(lián)體中,76.8%的酶交聯(lián)在載體上,酶活回收率最高為65.04%同時(shí)顯著的提高了酶的熱穩(wěn)定性和乙醛耐受性,使其在300mM乙醛濃度下,25℃,靜置10h后殘余61.4%的2-脫氧-D-核糖(DR)裂解活力。
[Abstract]:The aldol condensation catalyzed by 2- deoxy -D- ribose aldolase (EC 4.1.2.4, DERA) can produce two chiral centers. In particular, it can be used as a substrate with three molecular acetaldehyde. After two steps of continuous aldol condensation, a statin chiral side chain is obtained. Statins can effectively reduce the content of cholesterol in the human body. It is an important kind of drug. In spite of this, there are still some problems in the application of DERA: narrow substrate spectrum, preference for phosphorylation of substrates, low catalytic activity for 2- deoxy -D- ribose (DR), and poor tolerance to substrate acetaldehyde. Therefore, on the basis of improving the existing high throughput screening method, this topic is based on the homologous modeling of DERA. Molecular dynamics calculation, integrated use of directional evolution and rational design to transform DERA to improve its catalytic activity on non phosphorylated substrates and tolerance to high concentration acetaldehyde, and to synthesize chiral precursors. (1) the establishment of a high throughput screening method: a rapid and effective screening of new enzymes and the determination of the catalytic DERA catalysis On the basis of current high throughput screening method, the fluorescence luminescence mechanism of coumarin derivatives is studied. It is found that the variety of the substituents and their positions in the mother ring molecules can affect the fluorescence intensity. By redesigning the structure of the fluorescent substrates, the benzimidazole group is introduced at No. 3 of the coumarin mother ring, in No. 6 By introducing methoxy to emit a strong green fluorescence in visible light, the sensitivity of the substrate was increased by 58.2 times compared with the existing methods. (2) directional evolution of DERA: from the 8 kinds of DERA expressed in the earlier stage of the laboratory, the DERAGth and DERASep with the highest tolerance to the active acetaldehyde, which catalyze the non phosphorylated substrate DR, were selected as the study These two enzymes can be overexpressed in E. coli BL21 (DE3). The specific activity of pure enzymes is 22.5U/mg and 16.5U/mg., respectively, after the directed evolution of DERAGth and DERASep through the wrong PCR. The mutant DERAGth (F159I, S209G) is obtained by the high throughput screening method constructed, which is 3.7 times higher than that of the original enzyme. (3) Rational design improves the catalytic activity of DERA to N- acrolein two methyl imide: the activity of DERAGth, DERASep catalytic receptor substrate N- acrolein phthalimide (N-AMP) and acetaldehyde condensation, DERAGth and DERAGth (F159I, S209G) are different from the 0.52U/mg and 0.71 U/mg. This selection of DERASep as a target enzyme is transformed. Using DERATma (PDB1o0y) as a template, the structure model of DERASep is constructed by homologous modeling method and the model is evaluated. Through molecular docking, it is found that the side chain of residues Thr10, Ser205, Ala206 may block N-APM into the catalytic pocket. According to the size of docking energy, Thr10, Ser205, Ala206 advance is selected. The mutation combined with the molecular dynamics calculation of N-AMP, the binding free energy of DERASep (A206G) and DERASep (S205E) is -12.39kcal/mol and -7.11kcal/mol (DERASep -3.17kcal/mol) respectively. It is speculated that the mutation of these two loci may cause the change of enzyme activity. The results showed that the DERASep mutant (A206G) was 22.8U/mg and 2.7 times higher than that of DERASep. (4) rational design improved the tolerance of deoxyribose aldolase acetaldehyde: DERAsep and DERASep (A206G) as the research object. According to the study, it was found that the thermal stability of the enzyme was positively correlated with the tolerance of its acetaldehyde and other organic solvents. By strengthening the stability of DERASep to improve its acetaldehyde tolerance, using the molecular simulation workstation Discovery Studio to calculate the structural model of DERASep, the mutation energy of the single amino acid is obtained. Based on the software operation rules, the lower the mutation energy, the more stable the structure, and the selection of the seven single points with the lowest mutation ability D15F:S16I, T41C, T120I, G174Y, G174, G213C, on the basis of double point mutation and multi point virtual mutation, the lowest mutants of the mutant DERASep Variant10 (T120C, G174I, G213C) and DERASep tolerance are obtained. Under the same condition, 2 hours after statics, the residual vitality is 70.5%, and DERASep Variant11 is retained for 2 hours under the same condition, and the residual vitality is 66.7%. While the widely studied DERAEco is under the same condition, the residual vitality is almost O. in the condensation reaction of N-AMP (46.2mM) and acetaldehyde (166.7mM) catalyzed by the whole cell for 2 hours, and the DERASep Variant11 is catalyzed in 24h. 43.6% of the substrate, 1.32 times and 3.1 times higher than that of DERASep and DERAEco, is 1.55 times of the catalytic activity of DERA reported in the literature. (5) the immobilized transformation of DERAEco: the optimum crosslinking condition is determined by crosslinking DERA on the nano Fe304 magnetic particles: the ratio of the amount of enzyme to the carrier amount is 1:10, the crosslinked pH is 6, and the crosslinking is crosslinked. In the crosslinking body after 5h. preparation, 76.8% of the enzyme was crosslinked on the carrier, the activity recovery of the enzyme was 65.04% and the enzyme's thermal stability and acetaldehyde tolerance were increased significantly, and at the concentration of 300mM acetaldehyde at 25, the residual 2- deoxidization -D- ribose (DR) decomposition activity after the static 10h was placed.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q55

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