近平滑假丝酵母ATCC7330羰基还原酶的定点突变及其生物催化性能研究

Carbonyl reductase from Candida parapsilosis ATCC7330 (wild-type CpCR) has been concerned by people, but still faces some challeges. The study on the wt-CpCR site-directed mutagenesis and catalysis properties will start from the catalytic active sites, the binding domain of the substrates and co-enzyme NADPH. The wt-CpCR catalytic active sites will be investigated by site-directed mutagenesis to reveal the catalytic mechanism. The twelve kinds of typic substrates with different large groups and small groups will be designed. The screening of the positive mutant sites will be obtained from the binding domain of the typical substrates and the enzymes, The catalytic dynamic parameters of kinds of one site or many sites mutants through expressing and preparing by Escherichia coli with pBAD plasmid induced by arabinose will be investigated in order to reveal the controlling laws of amino acid residues on the pocket domain of the enzyme in the course of the enzyme accepting the large groups and small groups of the substrates. Followed by the mutant enzymes, the second turns of site-directed mutagenesis will be studied from the flexibility regions of the enzyme, the mutant enzymes will be prepared, the enzymatic activity changes and the stability will be determined under the medium of aqueous-organic diphase. The relations between the constructions and properties of the amino residues distributed in the pocket binding domain and flexibility binding domain with the typical substrates and coenzyme, respectively. This project will provide scientific evidence for the research on the molecular reconstruction of carbonyl reductase CpCR and produce the excellent biocatalysts for the preparation of chiral intermediates of Betastine, relaxing peptide antagonist.

近平滑假丝酵母ATCC7330羰基还原酶wt-CpCR因其底物适应性广、催化效率高备受关注，但仍面临一些挑战。本项目拟从该酶的催化区域、与底物和辅酶NADPH结合区域的主要氨基酸入手，对该酶催化区域的关键位点进行突变，研究其催化特性，阐明该酶的催化作用机制；设计12种带有不同大、小基团的酮类特征底物，采用虚拟氨基酸饱和突变，筛选与这些特征底物结合的口袋区域氨基酸正突变位点，在阿拉伯糖为诱导剂的大肠杆菌pBAD系统中进行表达，构建突变文库和突变酶，研究单点、多点突变酶的催化动力学参数；然后，在上述优势突变酶基础上再次进行定点突变，筛选与辅酶结合的柔性区域氨基酸突变位点，制备突变酶，研究它们在双相体系的酶活变化和稳定性，揭示该酶在口袋区域和柔性区域氨基酸残基突变前后的构效关系，为CpCR酶的分子改造提供科学依据，为Betastine、舒缓肽拮抗剂B1等合成提供优异生物催化剂。

近平滑假丝酵母ATCC7330羰基还原酶wt-CpCR因其底物适应性广、催化效率高备受关注，但仍面临一些挑战。本项目在重组菌E.coliBL21（DE3）/ pACYC Duet-1-cpcr中表达带有组氨酸标签的羰基还原酶wt-CpCR基因，经过分离纯化后开展了该酶的动力学参数测定和酶学性质研究，为该酶的分子改造研究奠定基础。从该酶的催化区域的主要氨基酸入手，对该酶催化区域的4个主要氨基酸位点H71、C48、C167和S50进行突变，研究其催化特性，阐明该酶是通过H71、C48、C167的活性官能团锚定二价锌离子，然后锌离子与羰基上的氧配位，保证还原型辅酶NADPH的负氢离子的有效进攻的催化作用机制，通过蛋白质模拟可以看到H71，C48，C167上的氨基和巯基与Zn离子配位距离分别为2.02 Å、2.76 Å、2.21Å，进一步确认该催化机制；设计12种带有不同大、小基团的酮类特征底物，采用虚拟氨基酸饱和突变，筛选与大基团相关的特征底物结合的大口袋区域氨基酸正突变位点Thr171、Thr54、Ser50，构建突变文库和突变酶，研究各种突变酶催化动力学参数，重点考察了突变酶Thr171Phe对不同取代基乙酮类底物催化能力的影响，该突变酶通过将疏水性苏氨酸分子改造为芳香族氨基酸苯丙氨酸后，CpCR酶的大口袋区域接纳具有苯环取代的底物亲和力增强；通过蛋白质模拟筛选与小基团相关的特征化合物结合的小口袋区域氨基酸正突变点Ala121、Ala97、Ala98、L308、L309，只发现Ala121 Asn突变酶酶活提升37%，其他三个点的突变酶酶活增加不到10%，突变体酶Ala121Asn与对氯苯基甲酮类底物的亲和力有较大提升；最后，在上述优势突变酶基础上，通过理性设计确定影响wt-CpCR与辅酶NADPH结合的稳定性的氨基酸突变点Ala98、Ser258、Ser 216、Ser307、Gly262，再进行虚拟饱和突变，筛选正突变位点，制备突变酶，研究表明A98N和G262N 在PB/甲基叔丁基醚的双相体系中界面稳定性比原始酶 wtCpCR 分别提高 1.7倍和1.4倍，在耐剪切力方面四个突变酶更加稳定，耐氧方面G262N较强，这4个突变酶对耐热稳定性有轻微降低。本研究揭示了该酶在口袋区域和柔性区域氨基酸残基突变前后的构效关系，为Betastine等合成提供科学依据。