Construction and Characterization of Composite Biocatalysts

复合生物催化剂的构建和表征

基本信息

批准号：
13125203
负责人：
ESAKI Nobuyoshi
金额：
$ 51.01万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

项目摘要

We analyzed the mechanism of biosynthesis of iron-sulfur clusters, which are useful as components of composite biocatalysts. We also developed novel processes for the production of chiral compounds by the use of novel composite biocatalysts. In addition, reaction mechanisms of several composite biocatalysts such as amino acid racemases which require pyridoxal phosphate as a coenzyme were clarified.1. Sulfur atoms of iron-sulfur clusters are supplied from L-cysteine by the sulfur-elimination reaction catalyzed by cysteine desulfurases. We studied the detailed mechanisms of iron-sulfur cluster assembly involving the following cysteine desulfurases : IscS and SufS from Escherichia coli and SsCsd3 from Synechocystis sp. PCC6803. Interactions between these cysteine desulfurases and proteins functioning as scaffold proteins for the assembly of iron-sulfur clusters were analyzed.2. We isolated and characterized a novel NADPH enzyme, N-methyl-L-amino acid dehydrogenase. The gene coding for this enzyme was cloned from Pseudomonas putida ATCC12633, and its overexpression system was constructed. Using this enzyme, we developed an enzymatic process in which chiral N-methyl-L-amino acids are produced from α-keto acids and methylamine.3. We isolated and characterized a novel NADPH enzyme that ctalyzes the asymmetric reduction of a carbon-carbon double bond in halogenated unsaturated compounds. We developed an enzymatic process in which (S)-2-CPA, which is used as a building block for the synthesis of aryloxyphenoxypropionic acid herbicide, is produced by asymmetric reduction of a carbon-carbon double bond in 2-chloroacrylate.

我们分析了铁硫簇的生物合成机理，这些机制可作为复合生物催化剂的组成部分。我们还通过使用新型复合材料生物催化剂来开发用于生产手性化合物的新过程。此外，将几种复合材料生物催化剂的反应机制（例如氨基酸种族酶）塑造为磷酸吡啶多毒素为辅酶。1。铁硫簇的硫原子是由半胱氨酸反应由半胱氨酸脱硫酶催化的。我们研究了涉及以下半胱氨酸脱硫酶的铁硫簇组件的详细机制：来自Synechocystis sp的Escherichia Coli和SSCSD3的ISC和SUF。 PCC6803。这些半胱氨酸脱硫酶和蛋白质作为支架蛋白的蛋白质之间的相互作用分析了铁硫簇的组装。2。我们分离并表征了一种新型的NADPH酶N-甲基-L-氨基酸脱氢酶。该酶的基因编码是从pseudomonas putida atcc12633克隆的，并构建了其过表达系统。使用这种酶，我们开发了一种酶促过程，其中通过α-酮酸和甲胺产生手性N-甲基-L-氨基酸。3。我们分离并表征了一种新型的NADPH酶，该酶在卤化的不饱和化合物中构成了碳碳双键的不对称还原。我们开发了一种酶促过程，在该过程中，（S）-2-CPA被用作合成芳氧苯氧基丙酸除草剂的基础，是通过在2-氯丙烯酸酯中不对称碳碳双键的不对称还原而产生的。