迭代取代结构柔区位点增强胆固醇氧化酶稳定性的研究

Enzyme stability does have great impacts on its application and industrial value. This project aims on enhancing the stability of microbial cholesterol oxidase (COD), by using of database and software to analysize the previous result from site directed mutagenesis, scan the atom and electron thermal parameters of the amino acid residues along the peptide, find the free energy high points distanced from the enzymatic activity center, and work on simulation of amino acid substitution to those residues in order to enhancing the stability of COD. From the simulated substitution results, the base iterative substitution of COD gene would be implemented precisely on purpose. Thus a systematic method combining of in situ selection and pured enzyme screening would be established based on the measurement of both thermal and kinetic properties of this enzyme, which ensures the positive mutants with better enzyme stability could be bred and screened out. Finally, the related mechanism of the enzyme stability change would be illustrated. Depended on this methodological system of "Iterative substitution on structural flexible site to enhance the enzyme stability", the blind variation of base substitution would be weakened and the possibility of positive mutation would be obviously increased.It will provide a novel proposol on improving microbial enzyme stability through rational design. Meanwhile,as one of the biochemical diagnostic enzyme, a much stable cholesterol oxidase has great industrial value, which help rectify the situation of diagnostic enzymes mainly relied on imports and promote the domestic production of those enzymes.

酶的稳定性直接影响到酶的实际应用及工业化价值。本研究以微生物胆固醇氧化酶（COD）为具体对象，利用数据库软件对前期的点诱变成果进行理性分析，再扫描肽链氨基酸残基的原子与电子热运动参数，探明远离活性区域的结构柔性高点，在虚拟取代的基础上，开展理性的碱基迭代取代，后通过原位筛选法初选正变体，并兼顾酶的热力学稳定性和动力学稳定性特点，利用亲和纯化制取纯酶,精确甄别出酶稳定性能得到显著提高的正突变株，并诠释有关的分子机理。 本研究拟建立的"迭代取代改造结构柔区位点提高酶稳定性" 方法体系，减少基因体外诱变的盲目性，提高获得正突变株的几率，为理性设计改善微生物酶的稳定性研究提供新思路。同时，本研究对于打破发达国家在诊断用酶领域的知识产权垄断，扭转大多数诊断用酶依赖进口的局面，促进诊断用酶的国产化方面，具有重要的意义。

本研究以黄素氧化酶类中的胆固醇氧化酶（ChOAb酶）为对象，借助数据库软件分析，探讨理性诱变提高酶分子稳定性的方法，并对稳定性提高的变异机理开展分子结构水平的探究。.研究思路是，基于酶蛋白解折叠自由能分析，搜索可能引起解折叠自由能变化的氨基酸残基作为诱变位点。方法设计应用生物信息学软件Rosetta software，对ChOAb酶蛋白的506个氨基酸位点分别进行解折叠自由能以及总能量的计算，输出可能引起正向突变的计算结果，进行酶分子突变实证操作，得到了六株热稳定性正向提高突变株(D71G, N86H, P149D, P354D, N459G and P464E)，六个突变氨基酸位点均位于酶的表面，其中N86H获得较理想的正突变效应，热稳定性提高2.6倍。虚拟酶蛋白折叠研究表明，N86H位于酶表面的lid-loop上。再以N86为基点，进行了饱和突变和新一轮的组合诱变，研究发现稳定性正向提高的变异位点皆出现在ChOAb酶蛋白分子结构的表面。虚拟折叠演绎还表明，上述氨基酸位点变换后致使酶稳定性出现正向效应的主要机理是，酶结构相应部位的分子内键力得以加强。有关现象和机理的解释为深入开展酶稳定性改造，提供有价值的理论依据和使用方法。