分子改造提高脱氧核糖醛缩酶的催化活性和底物耐受性

发布时间：2018-04-20 04:41

本文选题：脱氧核糖醛缩酶 + 固定化　；参考：《浙江大学》2016年博士论文

【摘要】：2-脱氧-D-核糖醛缩酶(EC 4.1.2.4, DERA)催化的羟醛缩合反应可以生成两个手性中心,特别是它能够以三分子乙醛为底物,经过两步连续羟醛缩合反应得到他汀类药物手性侧链。他汀类药物能够有效降低人体内胆固醇含量,是一种重要的的降血脂药物。尽管如此,DERA在应用上仍存在一些问题：底物谱窄,偏好磷酸化底物,对2-脱氧-D-核糖(DR)等非磷酸化底物催化活力低,对底物乙醛的耐受性差等。因此本课题在改进现有荧光高通量筛选方法的基础上,对DERA进行同源模建和分子动力学计算,综合运用定向进化和理性设计改造DERA,以提高其对非磷酸化底物的催化活力和对高浓度乙醛的耐受性,并用于手性药物前体的合成。(1)荧光高通量筛选方法的建立：为了快速有效的筛选新酶和测定改造后DERA的催化活性,在现有荧光高通量筛选方法的基础上,研究香豆素类衍生物荧光发光机制,发现母环分子中取代基的种类及其位置的变化能够对荧光强度产生作用。通过对荧光底物的结构进行重新设计,在香豆素母环3号位引入苯并咪唑基,在6号位引入甲氧基,使底物在可见光下散发强烈的绿色荧光,相比于现有方法,其检测灵敏度提高了58.2倍。(2)DERA的定向进化改造：从实验室前期重组表达的8种DERA中,选择对非磷酸化底物DR催化活力乙醛耐受性最高的DERAGth和DERASep作为研究对象。这两种酶均可在E. coli BL21(DE3)中实现过量表达,纯酶的比活分别为22.5U/mg和16.5U/mg。通过易错PCR对DERAGth和DERASep进行定向进化后,利用构建的高通量筛选方法获得突变株DERAGth (F159I,S209G),其比活为105.8U/mg,相比原始酶提高了3.7倍。(3)理性设计提高DERA对N-丙烯醛邻苯二甲酰亚胺的催化活力：考察了DERAGth, DERASep催化受体底物N-丙烯醛邻苯二甲酰亚胺(N-AMP)和乙醛缩合这一模型反应的活力,DERAGth和DERAGth (F159I, S209G)比活分别为0.52U/mg和0.71 U/mg,而DERASep比活为6.2U/mg,因此选择DERASep作为目标酶进行改造。以DERATma(PDB1o0y)为模板,采用同源建模法构建了DERASep的结构模型,并对模型进行评价。通过分子对接,发现残基Thr10,Ser205,Ala206的侧链可能阻碍N-APM进入催化口袋。根据对接能的大小选择Thr10,Ser205,Ala206进行模拟突变,突变体结合N-AMP的分子动力学计算,得到DERASep(A206G)和DERASep(S205E)的结合自由能分别为-12.39kcal/mol和-7.11kcal/mol(DERASep为-3.17kcal/mol),推测这两个位点的突变可能会引起酶活力的变化。定点突变后考察突变体对底物催化活力的变化,结果显示,DERASep突变体(A206G)比活为22.8U/mg,比DERASep提高了2.7倍。(4)理性设计提高脱氧核糖醛缩酶乙醛耐受性：以DERAsep和DERASep(A206G)为研究对象,根据研究报道发现酶的热稳定性和其乙醛等有机溶剂的耐受性有正相关性,因此提出通过增强DERASep的稳定性提高其乙醛耐受性的思路。利用分子模拟工作站Discovery Studio对DERASep的结构模型进行虚拟突变扫描计算,得到单个氨基酸的突变能。基于软件的运算规则,突变能越低,结构越稳定,选取七个突变能最低的单点位点D15F:S16I,T41C,T120I,G174Y,G174,G213C,在此基础上进行双点突变和多点虚拟突变,得到的组合突变能最低的突变体DERASep Variant10(T120C, G174I, G213C)和DERASep Variant11(T120C, G174I, A206G,G213C)。考察其乙醛耐受性,结果显示DERASep Variant10在300mM乙醛浓度下,静置2小时后,活力残余70.5%,DERASep Variant11在相同条件下静置2小时后,活力残余66.7%,而被广泛研究的DERAEco在同样条件下,静置2小时后活力残余几乎为O。在全细胞催化N-AMP (46.2mM)和乙醛(166.7mM)的缩合反应中,在24h内DERASep Variant11催化反应了43.6%的底物,比DERASep和DERAEco分别提高了1.32倍和3.1倍,是文献中报道的DERA催化活力的1.55倍。(5) DERAEco的固定化改造：通过交联将DERA结合在纳米Fe304磁性颗粒上,确定最适的交联条件为：酶量与载体量的比为1：10,交联pH为6.0,交联时间为5h。制备后的交联体中,76.8%的酶交联在载体上,酶活回收率最高为65.04%同时显著的提高了酶的热稳定性和乙醛耐受性,使其在300mM乙醛浓度下,25℃,静置10h后残余61.4%的2-脱氧-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.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q55

【相似文献】