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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.

我们分析了铁硫团簇的生物合成机理，这些铁硫团簇可作为复合生物催化剂的组分。我们还开发了使用新型复合生物催化剂生产手性化合物的新工艺。此外，还阐明了以磷酸吡哆醛为辅酶的氨基酸外消旋酶等几种复合生物催化剂的反应机理。l -半胱氨酸通过半胱氨酸脱硫酶催化的消硫反应提供铁-硫簇中的硫原子。我们研究了铁硫团簇组装的详细机制，涉及以下半胱氨酸脱硫酶：大肠杆菌中的IscS和SufS以及Synechocystis sp. PCC6803中的SsCsd3。分析了这些半胱氨酸脱硫酶与作为铁硫簇组装支架蛋白的蛋白质之间的相互作用。我们分离并鉴定了一种新的NADPH酶，n -甲基- l-氨基酸脱氢酶。从恶臭假单胞菌ATCC12633中克隆该酶的编码基因，构建其过表达体系。利用这种酶，我们开发了一种由α-酮酸和甲胺合成手性n -甲基- l-氨基酸的酶促工艺。我们分离并表征了一种新的NADPH酶，该酶催化卤化不饱和化合物中碳-碳双键的不对称还原。我们开发了一种酶促工艺，其中(S)-2-CPA通过2-氯丙烯酸酯中的碳-碳双键的不对称还原产生(S)-2-CPA，它被用作合成芳氧苯氧丙酸除草剂的构建块。