新型转氨酶的挖掘及其应用于手性胺和邻氨基醇合成的研究

发布时间：2018-04-22 16:56

本文选题：转氨酶 + 手性胺　；参考：《太原理工大学》2017年硕士论文

【摘要】：手性胺和手性邻氨基醇类化合物在手性医药工业、有机配体、手性助剂、拆分剂以及光学显示材料等方面有广泛的应用。生物催化具有高效率、高选择性、反应条件温和和环境友好等优点,因此,比传统化学催化法更具有吸引力和竞争力。ω-转氨酶反应过程无需辅酶循环再生吸引了大批研究者的关注。本课题以手性邻氨基醇特异性转氨酶为研究对象,通过显色法构建了转氨酶高通量筛选方法;通过构建的高通量筛选方法获得了一株高活力高选择性邻氨基醇转化菌株;通过基因组挖掘技术从目的菌株中成功克隆到4个新型转氨酶,并对其在大肠杆菌中进行了高效表达、纯化及表征,同时对其底物特异性进行了分析。首先,利用模板底物2-羟基苯乙酮与无色2,3,5-三苯基-2H-四唑氯化物(2,3,5-triphenyltetrazolium chloride,TTC)反应生成的红色TPF(简称显色反应)在波长510 nm处有吸收值这一原理,建立了一种高效、快速、灵敏的高通量转氨酶筛选方法。该显色反应的适宜pH范围是6.0-10.0,适宜温度范围是20oC-30oC,显色反应时间约3-5 min。该方法在以苯甘氨醇为底物时,底物转化率水平的灵敏度范围为1.6%-32.5%,且该方法对细菌的全细胞具有很好的适用性。本实验通过构建的高通量筛选方法成功筛选得到一株对苯甘氨醇催化活性较高且选择性专一的恶臭假单胞菌(Pseudomonas putida NBRC 14164)。其次,运用基因组挖掘技术,从Pseudomonas putida NBRC 14164的基因组中挖掘到与ω-转氨酶cv2025的氨基酸序列同源性分别为37%、35%、56%和58%的pp21050、ppbaua、pp36420、ppspuc四种ω-转氨酶基因序列,构建表达载体于大肠杆菌中成功可溶性表达。对目标蛋白进行了镍柱纯化,经sds-page检测得到电泳纯的转氨酶。再次,以外消旋苯乙胺为底物对纯化后的ω-转氨酶进行了酶学性质研究。实验结果发现pp21050、ppbaua、pp36420和ppspuc四种转氨酶酶活最佳ph分别为9.0、10.0、8.0和9.0;最适温度分别为35oc、50oc、35oc和35oc;ppbaua具有相对较好的酶活温度稳定性,尤其在高温60oc下孵育2h还保留有70%左右的相对酶活,而pp21050、pp36420和ppspuc对温度敏感,热稳定性较差;pp21050、ppbaua、pp36420和ppspuc的km值分别为161.3mm、136.7mm、398mm、130.9mm;vmax分别为2.8u/mg、0.5u/mg、5.6u/mg、3.6u/mg。其中ppspuc的催化效率最高,达到0.023mm-1s~(-1),pp21050的催化效率次之,达到0.015mm-1s~(-1),pp36420的催化效率达到0.012mm-1s~(-1),ppbaua的催化效率最低仅有0.003mm-1s~(-1)。最后,本课题选择了4种外消旋胺类化合物和3种不同的外消旋邻氨基醇类化合物对ω-转氨酶的底物特异性进行了研究。实验结果发现四种转氨酶对邻氨基醇类化合物和胺类化合物表现出不同的催化活性与立体选择性。在动力学拆分外消旋胺过程中,四种转氨酶均只对s构型胺有催化活性,其中pp21050、pp36420和ppspuc表现出了良好的催化能力,底物转化率大部分能达到48-50%,ee值也达到98-99%。在动力学拆分外消旋邻氨基醇过程中,四种转氨酶均只对r构型的邻氨基醇有催化活性,其中Pp21050、Pp36420和PpspuC对外消旋苯甘氨醇的催化活性较好,转化率分别为50.2%,49.4%和49.3%,ee值达到99%,97%和99%;PpbauA对底物外消旋2-氨基-1-丁醇和缬氨醇有较高的催化能力,转化率和ee值分别达到50.0%和99.0%。本实验还通过Ppspu C动力学拆分外消旋苯甘氨醇成功制备得到S构型的苯甘氨醇,收率为45.0%,纯度大于99.0%。综上所述,四种新型转氨酶具有高活力、高立体选择性和广泛底物谱的特征,使其在生物法制备手性胺和手性邻氨醇中具有潜在的应用价值。
[Abstract]:Chiral amines and chiral aminophenol compounds have extensive applications in the chiral pharmaceutical industry, organic ligands, chiral auxiliaries, resolution agents and optical display materials. Biocatalysis has the advantages of high efficiency, high selectivity, mild reaction conditions and friendly environment. Therefore, it is more attractive and competitive than traditional chemical catalysis. A high throughput screening method for aminotransferase was constructed with chiral aminoalcohol specific aminotransferase, and a high activity and high selective aminoalcohol transformation strain was obtained by high throughput screening method. 4 new aminotransferase was cloned from the target strain by over genome mining technique, and its expression in Escherichia coli was highly expressed, purified and characterized. At the same time, its substrate specificity was analyzed. First, the template substrate 2- hydroxyacetophenone and the colorless 2,3,5- three phenyl -2H- four azolides (2,3,5-triphenyltetrazolium C) were used. Hloride, TTC) the reaction generated red TPF (abbreviated color reaction) has the absorption value at the wavelength of 510 nm. A high efficient, rapid and sensitive high throughput transaminase screening method is established. The suitable pH range of the color reaction is 6.0-10.0, the suitable temperature range is 20oC-30oC, the color reaction time is about 3-5 min., and the method is with benzoglyol. The sensitivity range of substrate conversion rate is 1.6%-32.5% when the substrate is used as substrate, and the method has good applicability for all cells of bacteria. This experiment successfully screened a strain of Pseudomonas aeruginosa (Pseudomonas putida NBRC 14164) with high catalytic activity and selectivity. Secondly, using genomic mining technology, the homology of amino acid sequences from Pseudomonas putida NBRC 14164 was extracted from the amino acid sequence of Omega transaminase cv2025, which were 37%, 35%, 56% and 58% of pp21050, ppbaua, pp36420, ppspuc and four Omega transaminase gene sequences, which were constructed to achieve the successful soluble expression of the vector in Escherichia coli. The purification of the nickel column was carried out, and the electrophoretic transaminase was obtained by SDS-PAGE. Again, the enzymatic properties of the purified Omega transaminase were studied by the racemic benzoethylamine as the substrate. The experimental results showed that the best pH for the four transaminase activities of pp21050, ppbaua, pp36420 and ppspuc were 9.0,10.0,8.0 and 9, respectively, and the optimum temperature was 35oc, 50oc, respectively. 35oc and 35oc, ppbaua had relatively good enzyme activity temperature stability, especially at high temperature 60oC, 2h still retained about 70% relative enzyme activity, while pp21050, pp36420 and ppspuc were sensitive to temperature and poor thermal stability; pp21050, ppbaua, pp36420 and ppspuc, respectively. In 6u/mg, 3.6u/mg., ppspuc has the highest catalytic efficiency, reaching 0.023mm-1s~ (-1). The catalytic efficiency of pp21050 is second to 0.015mm-1s~ (-1). The catalytic efficiency of pp36420 is reached to 0.012mm-1s~ (-1). The catalytic efficiency of ppbaua is the lowest. Finally, 4 kinds of racemate amines and 3 different racemate adjacent ammonia are selected. The substrate specificity of Omega aminotransferase was studied by the basic alcohols. The experimental results showed that the four aminoalcohols showed different catalytic activity and stereoselectivity for the aminoalcohols and amines. In the kinetic resolution of racemines, the four transaminase had a catalytic activity to the S structure amine, of which pp21050, P P36420 and ppspuc showed good catalytic ability, the conversion rate of substrate could reach 48-50%, and the EE value also reached 98-99%. in the process of kinetic resolution of ortho amino alcohols. The four aminotransferase had a catalytic activity to the ortho amino alcohols of R configuration, of which the catalytic activity of Pp21050, Pp36420 and PpspuC was better. The conversion rates were 50.2%, 49.4% and 49.3% respectively, EE values reached 99%, 97% and 99%, and PpbauA had high catalytic ability for substrate racemization of 2- amino -1- butanol and valerine. The conversion rate and EE value reached 50% and 99.0%. respectively. The S configuration of benzglyl was successfully prepared by the kinetic resolution of Ppspu C, and the yield was 45. The four new aminotransferase has the characteristics of high vitality, high stereoselectivity and broad substrate spectrum, which has the potential application value in the preparation of chiral amines and chiral anaminol in biological process.

【学位授予单位】：太原理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q55;O621.3

【相似文献】