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Elucidation of Mechanisms and Control of Functions of Versatile Biocatalysts

多功能生物催化剂的机理阐明和功能控制

基本信息

批准号：
17550155
负责人：
EMA Tadashi
金额：
$ 2.3万
依托单位：
Okayama University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17550155/
关键词：
enzyme biocatalyst enantioselectivity control mutation alcohol evolution intermediate for drug

项目摘要

1. Rational control of enantioselectivity of lipaseWe selected I1e287 as a mutation site to control the enantioselectivity of a Burkholderia cepacia lipase on the basis of a mechanism and X-ray crystal structures. A mutation that increases the steric bulkiness as compared with the wild-type enzyme will suppress the reaction of the slower-reacting enantiomer and improve the enantioselectivity. On the other hand, a mutation that decreases the steric hindrance will facilitate the reaction of the slower-reacting enantiomer and diminish the enantioselectivity. We have succeeded in controlling (both increasing and decreasing) the enantioselectivity of the lipase rationally by mutating only one amino acid residue on the basis of the mechanism.2. Synthesis of an optically active porphyrin dimer via a lipase-catalyzed reaction and its functionAn enantiomerically pure porphyrin alcohol prepared by the lipase-catalyzed kinetic resolution was linked to isophthalic acid to give a porphyrin dimer. The zinc complex of the dimer showed chiral discrimination in NMR.3. Asymmetric reduction of ketones using recombinant E. coliWe constructed a recombinant E. coli overproducing a carbonyl reductase that shows high enantioselectivity toward a wide range of ketones, and conducted the asymmetric reduction of more than 20 ketones, and 12 alcohols had enantiomeric purities of >98% ee. The reaction conditions for 2,4-octanedione were optimized, and (S)-2-hydroxy-4-octanone was obtained with the productivity of 41 g/L. An optically active intermediate for an important chiral drug was also prepared by this biotransformation.4. Preparation of the carbonyl reductase with a His-tagCarbonyl reductases with a His-tag at the N-or C-terminal were constructed and produced by E. coli.

1.脂肪酶对映体选择性的合理调控本研究根据洋葱伯克霍尔德氏菌脂肪酶的作用机理和X射线晶体结构，选择I1e287作为突变位点，对洋葱伯克霍尔德氏菌脂肪酶的对映体选择性进行控制。与野生型酶相比，增加空间体积的突变将抑制反应较慢的对映体的反应，并提高对映体的选择性。另一方面，减少空间位阻的突变将促进反应较慢的对映体的反应，并降低对映体的选择性。根据这一机理，我们通过只突变一个氨基酸残基，成功地合理地控制了脂肪酶的对映选择性。脂肪酶催化合成光学活性的卟啉二聚体及其功能通过脂肪酶催化动力学拆分制备的手性纯的卟啉醇与间苯二甲酸偶联，得到卟啉二聚体。该二聚体的锌络合物在NMR中表现出手性识别作用。利用重组大肠杆菌进行酮的不对称还原我们构建了一株对多种酮具有较高对映体选择性的重组大肠杆菌，并对20多个酮进行了不对称还原，其中12个醇的对映体纯度为98%ee。对合成2，4-辛二酮的反应条件进行了优化，得到了产率为41g/L的(S)-2-羟基-4-辛酮，并通过该转化反应制备了一种重要手性药物的光学活性中间体。含有组氨酸标签的羰基还原酶的制备构建了N端或C端带有组氨酸标签的羰基还原酶，并在大肠杆菌中进行了生产。