【作者】 陈蕾；

【导师】 林英武；

【作者基本信息】 南华大学 ， 化学， 2022， 硕士

【摘要】 蛋白质工程通过修饰血红素活性位点、引入非天然氨基酸以及在天然或从头设计的蛋白支架中用非天然金属辅因子替换血红素,为设计功能性酶（如血红素酶）提供了一种强大的方法。例如,血红素蛋白中的成员之一--神经红蛋白（Ngb）,通过合理修饰血红素活性位点,被转化为高效的亚硝酸盐还原酶和多功能过氧化物酶。古老的靛蓝染料在世界范围内一直具有需求,化学合成靛蓝可能会导致环境问题,而靛蓝的生物合成是一种绿色环保的方法。在这项研究中,通过蛋白质工程和理性设计,我们表明人神经红蛋白可以转化为一种高效的酶,能够有效的催化吲哚合成靛蓝,合理设计了三个突变位点以分别稳定蛋白质（A15C）、增强H₂O₂活化（H64D）和促进电子转移（F49Y）。通过检查human Ngb的X射线晶体结构,我们注意到Phe49是一个合适的突变目标,因为它位于血红素活性位点附近,靠近蛋白质表面。因此,我们通过用Tyr49替换Phe49,构建了A15C/H64D/F49Y Ngb的三突变体,该突变体有一个羟基的差异,并且引起最小的结构扰动。氨基酸的侧链,如Tyr,也可以通过氢键和疏水相互作用来调整吲哚与血红素活性部位的结合。动力学研究和产物分析表明,三突变体A15C/H64D/F49Y Ngb的活性远高于工程化肌红蛋白突变体,而无需细胞色素P450所需的昂贵辅助因子。该酶还可以有效催化含有-Cl/-Br/-NO₂基团的吲哚衍生物的偶联氧化,产生靛蓝衍生物染料,产率和化学选择性分别高达～90%和～97%。这些合成的染料能应用于棉纺织品上染成蓝色、红色、黄色、紫色甚至绿色等均匀颜色,因此我们预计Ngb的工程酶将在纺织印染行业有实际应用更多还原

【Abstract】 Protein engineering provides a powerful method for designing functional enzymes,such as heme enzymes,by modifying the heme active site,introducing non-natural amino acids into intrinsic or novel protein scaffolds,and replacing heme with non-natural metal cofactors.For example,one of the member of the globin family,neuroglobin（Ngb）,has been converted into a highly efficient nitrite reductase and multifunctional peroxidase by rational modification of the hemoglobin active site.The old dye indigo is still demanded worldwide,whereas chemical synthesis of indigo may cause environmental problems.lternatively,the biosynthesis of indigo is an eco-friendly approach.In this study,by protein design and engineering,we showed that human neuroglobin（Ngb）can be converted into an enzyme capable of efficient synthesis of indigo from indole.Three mutations were rationally designed to stabilize the protein（A15C）,enhance H₂O₂ activation（H64D）,and facilitate electron transfer（F49Y）,respectively.By examining the X-ray crystal structure of human Ngb,we noticed that Phe49 is a suitable mutation target,which is located near the heme active site and close to the protein surface.Therefore,we constructed a triplet mutant of A15C/H64D/F49Y Ngb by replacing Phe49 with Tyr49,which has a hydroxyl difference and causes minimal structural perturbation.Sidechains of amino acids,such as Tyr,can also be used to adjust the binding of indole to the active site of hemoglobin through hydrogen bonding and hydrophobic interactions.Kinetic studies and product analysis revealed that the triple mutant A15C/H64D/F49Y Ngb exhibits an activity much higher than those of the engineered myoglobin mutants,without the need of expensive cofactors required for cytochromes P450.This enzyme can also efficiently catalyze the coupling oxidation of indole derivatives containing–Cl/–Br/–NO₂ groups,producing indigo derivative dyes,with a yield up to～90%and chemoselectivity up to～97%,respectively.Moreover,these synthesized dyes were successfully applied to dye cotton textiles with uniform colors such as blue,red,yellow,purple,and even green.Thus,we expect that the engineered enzyme of Ngb will have practical applications in the textile dyeing industry.更多还原