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Use of the enzymes in the microbial and plant "aldoxime-nitrile pathway"for chiral synthesis

利用微生物和植物“醛肟-腈途径”中的酶进行手性合成

基本信息

批准号：
20380053
负责人：
ASANO Yasuhisa
金额：
$ 12.4万
依托单位：
Toyama Prefectural University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2009
资助国家：
日本
起止时间：
2009 至 2011
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-20380053/
关键词：
酵素応用微生物植物遺伝子有機工業化学

项目摘要

(1) Development of hydroxynitrile lyase (HNL) and its use in optically active cyanohydrin : To construct a library of HNLs, several HNL genes were expressed in heterogonous hosts. We succeeded in cDNA cloning, primary structure elucidation and expression of cDNA of P. mume (Plum) HNL in methanol yeast Pichia pastoris. Full-length cDNA and genomic DNA of a novel S-HNL from a plant, Baliospermum montanum were cloned and sequenced. The genomic DNA contained two introns and one ORF encoding a 263-residue protein (subunit : 29.5 kDa). C-terminal short truncations proved to be successful to access higher productivity in culture medium of E. coli up to 3-fold. The hnl gene was expressed in E. coli and the enzyme was characterized including kinetic studies of 20 substrates for (S)-cyanohydrin synthesis. An R-HNL from Arabidopsis thaliana accepts nitromethane (MeNO2) as a donor in a reaction with aromatic aldehydes to yield (R)-b-nitro alcohols (Henry reaction). This is the first example of the … More R-HNL-catalyzed synthesis of (R)-b-nitro alcohols. (R)-Mandelonitrile was successfully synthesized by an enzymatic transcyanation reaction of benzaldehyde and acetone cyanohydrin catalyzed by a HNL from Eriobotrya japonica (EjHNL) in an aqueous-organic biphasic system. An HNL from leaves of Passiflora edulis (PeHNL) was purified and characterized for the first time. Asymmetric synthesis of (R)-mandelonitrile in a biphasic system employing PeHNL as carried out.(2) Mechanism of functional expression of HNL from Manihot esculenta : Functional expression of HNL from M. esculenta, MeHNL, was achieved in E. coli. The highly soluble mutant MeHNL-His103Leu were expressed in the systems such as Pichia pastoris, Leishmania tarentolae and two cell-free translation systems, such as E. coli PURE system and Wheat germ translation system. The phenomenon of the functional expression was proven to be specific to the E. coli system.(3) Dynamic kinetic resolution of amino acid amide : The first crystal structures of a fold-type I racemase are solved for the native form and e-caprolactam-complexed form of a-amino-e-caprolactam racemase. Mutation was successfully done to affect the substrate specificity of the enzyme. Further screening for new amino acid amide racemase was carried out. Less

(1)羟基腈裂解酶（HNL）的开发及其在光学活性氰醇中的用途：为了构建HNL文库，在异源宿主中表达几种HNL基因。本研究成功地克隆了乌梅HNL基因的cDNA，并对其进行了一级结构鉴定，同时在甲醇酵母Pichia pastoris中进行了表达。克隆了一种新的植物Baliospermummontanum的S-HNL的全长cDNA和基因组DNA，并进行了序列测定。基因组DNA含有两个内含子和一个ORF，编码一个263个残基的蛋白质（亚基：29.5 kDa）。C端截短法在大肠杆菌培养基中获得了较高的产量。大肠杆菌中，最高可达3倍。hnl基因在E.大肠杆菌和酶的特点，包括动力学研究的20个底物的（S）-氰醇合成。来自拟南芥的R-HNL在与芳香醛的反应中接受硝基甲烷（MeNO 2）作为供体，以产生（R）-b-硝基醇（亨利反应）。这是第一个例子 ...更多信息 R-HNL催化合成（R）-b-硝基醇。在水-有机两相体系中，以枇杷叶提取物HNL（EjHNL）为催化剂，催化苯甲醛和丙酮氰醇的转氰反应合成了（R）-扁桃腈。首次从西番莲叶中分离纯化了一种HNL（PeHNL）。采用PeHNL在两相体系中进行（R）-扁桃腈的不对称合成。(2)木薯HNL功能性表达的机制：木薯HNL的功能性表达。Esculenta，MeHNL，在E.杆菌在巴斯德毕赤酵母（Pichia pastoris）、塔伦妥利什曼原虫（Leishmania tarentolae）和大肠杆菌（E. coliPURE系统和麦胚翻译系统。这种功能表达现象是大肠杆菌特有的。coli系统。(3)氨基酸酰胺的动态动力学拆分：对α-氨基-ε-己内酰胺消旋酶的天然形式和ε-己内酰胺络合形式解析折叠型I消旋酶的第一晶体结构。成功地进行了突变以影响酶的底物特异性。进一步筛选新的氨基酸酰胺消旋酶。少