导　　师： 冯雁

学科专业： 071010

授予学位： 博士

作　　者： ;

机构地区： 吉林大学

摘　　要： 来源于超嗜热古菌Aeropyrum pernix K1酰基氨酰肽酶/(APE1547/)是具有酰基肽酶//酯酶等活性的多功能/(promiscuous/)酶,具有超常的生物学稳定性。本课题组在前期研究中,通过定向进化的方法,获得了酯酶活性提高的突变体P01/(R526V/),对系列对硝基苯酚酯底物表现良好的酯酶水解活性,最适酰基链长底物为对硝基苯酚辛酸酯/(pNP‐C8/)。本文结合定向进化与半理性设计的蛋白质工程方法和溶剂工程技术,增加该酶对长链酰基底物的活性和选择性,并对酶活性和底物选择性与酶细微结构之间的关系进行了深入的分析。采用的研究方法和取得的结果如下: 1、以P01为出发酶,应用非理性的定向进化方法,对其催化结构域基因进行易错PCR后与推进器结构域连接,转化BL21菌,在多重选择压力下/(高温及不同长度碳链的底物/),应用优化的高通量筛选方法,筛选了万余株突变体,发现1个远离活性中心18.2/(?/)的关键位点T560,得到突变体E01/(R526V//T560W/)。催化动力学分析发现:E01对pNP‐C12的亲和力/(1//K/_m值/)是P01的9.7倍,转化数/(kcat值/)是P01的1.5倍,因此催化效率/(k/_/(cat/)//K/_m值/)是P01的14倍,表明对底物特异性的提高主要的原因是提高了对pNP‐C12的亲和力。将P01分子与不同链长底物作模拟通道对接/(SLITHER/),发现T560位于次要底物通道的入口处,突变为W后,通道变得狭窄,阻碍了pNP‐C12的进入。结构分析表明,酶的推进器结构域中以及推进器结构域与催化结构域之间存在两条通道,推测由于改善了pNP‐C12在酶分子内部的交通,而使催化效率明显提高。此外,T560W突变使酶的80℃半衰期由123小时下降为77小时/(酶浓度0.8mg//ml/)。结构分析表明,Thr560的侧链O与563的主链NH之间形成氢键对局部构象的稳定性有重要作用,突变破坏了这一氢键,可能是热稳定性下降的原因之一。 2、以E01为出发酶,根据底物与E01活性中心的分子对接结果,应用半合理设计的方法,选择11个底物结合部位的残基/(3个活性位点附近残基Y444、Y446、Y449和8个底物口袋处的残基P370、E419、W474、M477、F485、F488、I489、T527/)进行饱和突变,在多重选择压力下/(高温及不同长度碳链的底物/)的高通量筛选,发现了2个关键位点W474和F488,对酶的酰基链长选择性和活性有显著影响。并获得3个突变体:S01/(F488G//R526V//T560W/)、S02/(F488P//R526V//T560W/)和S03/(W474V//R526V//T560W/)。它们对pNPC12的k/_/(cat/)//K/_m值分别是P01的4.5、4.6和21倍,k/_/(cat/)值分别是P01的3、1.5和4倍。 应用同源模建和底物对接,对F488和W474位点影响底物选择性和活性的机制进行了分析。F488位点位于底物结合口袋的侧壁,突变为G后,底物结合口袋体积扩大为577/(?/)~3,推测更利于容纳长链酰基;W474位点位于底物口袋的底部,突变为V后,底物结合口袋的深度由6.07/(?/)延长为11.4/(?/)左右,同时该口袋的疏水性增强,有利于长链酯底物的进入与定位,因而提高了活性。为证实结构分析的合理性,通过理性设计,分别获得了E01基础上的突变F488→S、A、Y、W和突变W474→A、Q和双点突变W474V//F488G连同饱和突变筛选得到的F488G、F488P和W474V共12种突变体酶,分别测定催化动力学参数,与相应氨基酸侧链理化性质作二维构效分析,其结果支持结构分析的结论。理性设计得到的D01/(W474//F488G// R526V//T560W/)性质最优,对pNPC12的k/_/(cat/)//K/_m值是P01的24倍,k/_/(cat/)值是P01的5倍。 3、溶剂工程是通过改变介质环境来达到调节酶活性和底物选择性的重要技术。选择乙腈、二甲基亚砜和二甲基甲酰胺作为研究对象,分别建立了反应体系,研究了野生型酶和各种突变体在水//有机共溶反应体系中对不同底物的催化反应,发现三个带有F488G和W474V的突变体在低浓度有机溶剂中的催化特征与WT、P01和E01存在显著的不同。随着有机助溶剂浓度的提高,S01、S03和D01的活性有明显的提高,而WT、P01和E01的活性却受到强烈的抑制。在10/%乙腈、30/%二甲基亚砜和20/%二甲基甲酰胺中,最佳突变体D01与WT相比,活性分别提高240倍、99倍和280倍。有机溶剂对S01、S03和D01的底物选择性也有明显增强,其中最佳突变体D01在10/%乙腈、30/%二甲基亚砜和20/%二甲基甲酰胺中,对pNPC12// pNPC4的选择性增加了12、18、和19倍。 分析发现酶在乙腈溶剂中的C/_/(50/)与F488残基的疏水性参数呈正相关性/(R~2=0.946/)。在10/%乙腈中的模拟分子动力学也发现乙腈分子可以渗透进入活性中心,而且突变后的G488附近有乙腈分子留滞的现象。推测该激活现象是由于有机溶剂分子渗入酶活性中心,进入了F488G和W474V突变产生的空穴,使催化部位的微环境发生改变,因而改变了酶的催化效率。 本论文对酶本身及酶反应溶剂体系的优化进行了系统的探索。研究表明:1、APE1547对酯底物的选择性与底物通道、底物结合口袋关键氨基酸理化性质、催化中心的微环境密切相关。2、酶的分子改造与溶剂工程相结合,是改变酶底物选择性的有效手段,对酶分子改造和应用具有很好的借鉴意义。 An acylaminoacyl-peptidase /(apAAP/) from hyperthermophilic archaea Aeropyrum pernix K1 is a promiscuous acyl peptidase and esterase. Our previous studies showed that saturation mutation of conservative residues R526 near the active site can effectively improve the esterase activity, but does not change the selectivity to pNP-ester. In order to fulfill the urgent requirement of thermostable lipolytic enzymes, the substrate preference of apAAP was further improved from p-nitrophenyl caprylate /(pNP-C8/) to p-nitrophenyl laurate /(pNP-C12/) by protein and solvent engineering. We start from the best mutant P01 /(R526V/), directed evolute its esterase activity toward long-chain acyl ester under the multiple selection pressure /(high temperature and different carbon chain length of the substrate/), applied of optimized high-throughput screening methods, found a key site: T560 among about 10000 mutants, which is 18.2 ? away from the active center. A mutant E01 /(R526V//T560W/) showed high catalytic efficiency /(k/_/(cat/)//K/_m/) for pNP-C12, which is about 14-fold higher than P01.The study found that was mainly due to improve the affinity for pNP-C12. We use SLITHER, a web server to generate contiguous conformations of a molecule along a curved tunnel inside E01 and P01, and the binding free energy profile along the predicted channel pathway. The results showed T560W improved SUBSTRATE TRAFFIC of pNP-C12 in E01. After that, we adopt semi-rational design methods combining the crystal structure analysis, retain conservative catalytic and oxygen hole residues, chose for saturation mutagenesis at 11 residues /(P370, E419, Y444, Y446, Y449, W474, M477, F485, F488, I489 and T527/) near the substrate-binding pocket and the catalytic residue Ser. Then screen for mutant of high activity to pNP-C8 and//or pNP-C12 at 60℃. Three mutants F488G//R526V//T560W /(named S01/), F488P//R526V//T560W /(named S02/) and W474V//R526V//T560W /(named S03/) were selected from variants, with catalytic efficiency /(k/_/(cat/)//K/_m/) for pNP-C12 increased of 3-, 1.5- and 4-fold compared with the mutant P01. To investigate the effects of the mutations on the catalytic mechanism, additional mutants with diverse side chains at positions 474 and 488 were constructed and subjected to kinetic analysis alongside the previously selected mutants. The combined mutant W474V//F488G//R526V//T560W /(named D01/) had the highest esterase activity of all the mutants investigated in this study. The catalytic efficiency /(k/_/(cat/)//K/_m/) of D01 for pNP-C12 was 24-fold higher than P01. Analysis of the structure revealed that Trp474 and Phe488 are respectively located at the bottom and the wall of the substrate-binding pocket. S01 and S03 had a deeper /(11.4 /(?/) vs. 6.1 /(?/)/) and larger /(577/(?/)~3 vs. 227 /(?/)~3/) substrate-binding pocket compared to P01 and E01, which facilitates the binding of substrates with long acyl chains. The long-chain substrate selectivity and the Volume of substrate binding pocket are positively correlated well. Conjecture that the increasing volumes of pocket reduce the steric hindrance of the long-chain substrate entering the active site, so the long-chain ester substrate selectivity of the mutants increases. Solvent engineering is an important technology that can regulate activity and selectivity of enzymes by changing media environment. Because most of the lipase family are interface enzyme whose hydrophobicity are pretty high, we add several water-miscible polar organic solvents to the reaction system, found that the catalytic efficiency of the mutants was significantly affected by polar solvents. In the presence of organic solvents, the wild-type apAAP, P01 and E01 mutants were strongly inhibited, whereas the activities of the mutants S01, S02, and D01 were simulated under the same conditions. In particular, the activity of the mutant D01 increased 240-fold compared with the wild type in the presence of 10/% acetonitrile, 99-fold in 20/% DMF and more than 280-fold in 30/% DMSO. The ratio of the activities for pNP-C12 and pNP-C4 increased 12-, 18- and 19-fold for the mutant D01 in the presence of 10/% acetonitrile, 30/% DMSO and 20/% DMF, respectively. MD simulations were run for the mutants S01, S02 and their parental enzyme P01, mimicking the condition when 10/% acetonitrile was present. Detailed analysis revealed that several acetonitrile molecules penetrated the enzymes. The acetonitrile penetration for mutants S01 and S02 was more pronounced than for P01. Interestingly, the penetration of one acetonitrile molecule into mutant S01 was just occurred at the region where residue 488 was located. It means that the increase of substrate binding pocket is more favorable for polar solvent to permeate into the active site, change the catalytic site microenvironment, thus changing the enzyme catalysis efficiency.

关 键 词： 嗜热酯酶 底物特异性 半理性设计 定向进化 溶剂效应

分 类 号： [Q55]

领　　域： [生物学]