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

(1)羟基腈裂解酶（HNL）的研究进展及其在光活性氰醇中的应用：为了构建羟基腈裂解酶文库，我们在异种宿主中表达了几个HNL基因。我们成功克隆了甲醇酵母毕赤酵母（Pichia pastoris）中P. mume (Plum) HNL的cDNA，并对其进行了初步结构解析和cDNA表达。克隆了植物巴利ospermum montanum S-HNL的全长cDNA和基因组DNA，并对其进行了测序。基因组DNA包含两个内含子和一个ORF，编码263个残基蛋白（亚基：29.5 kDa）。c端短截短被证明在大肠杆菌培养基中获得了更高的产量，最高可达3倍。在大肠杆菌中表达了hnl基因，并对该酶进行了表征，包括对合成(S)-氰醇的20种底物的动力学研究。拟南芥中的R- hnl接受硝基甲烷（MeNO2）作为供体，与芳香醛反应生成(R)-b-硝基醇（Henry反应）。这是第一个用R- hnl催化合成(R)-b-硝基醇的例子。在水-有机双相体系中，以枇杷叶（EjHNL）为原料，以苯甲醛和丙酮氰醇为催化剂，通过酶促转氰化反应成功地合成了(R)- mandelonnitrile。首次从西番莲（Passiflora edulis）叶中分离纯化了一种高纯度分子链（HNL）。采用PeHNL在双相体系中不对称合成了(R)-丙二腈。(2)马尼奥特大肠杆菌HNL的功能表达机制：在大肠杆菌中实现了马尼奥特大肠杆菌HNL的功能表达。高可溶性突变体MeHNL-His103Leu在毕赤酵母、链状利什曼原虫等系统以及大肠杆菌PURE系统和小麦胚芽翻译系统两种无细胞翻译系统中均有表达。结果表明，该功能表达现象是大肠杆菌系统特有的。(3)氨基酸酰胺的动力学解析：求解了折叠型I型外消旋酶的天然形式和e-氨基-e-己内酰胺络合形式的第一个晶体结构。成功地进行了突变以影响酶的底物特异性。进一步筛选了新的氨基酸酰胺消旋酶。少

(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