Functions, diversity, and physiological functions of novel heme-containing enzyme "aldoxime dehydratase"

为了阐明1)醛肟脱水酶（Oxd）的反应机理，2)醛肟脱水酶的多样性，3)“醛肟-腈途径”的多样性和生理功能，我们进行了以下实验研究。我们在重组大肠杆菌中构建了nitrilase (Nit)-linked oxd基因OxB-1的过表达系统，获得了大量高纯度和均质形式的oxd蛋白（OxdB）。我们对OxdB蛋白进行了分光光度分析，以确定其在酶催化过程中的血红素环境和功能残基。从家畜培养物中筛选了oxd基因，并克隆了oxd全长基因。我们在大肠杆菌中过表达了新的氧化酶，并检测了它们的性质。在此基础上，对Oxds的生理功能进行了研究。我们对具有“醛肟-腈途径”的菌株进行了基因筛选，并克隆了它们的基因。比较了两种氮化物降解酶的性状和主要结构，研究了两种氮化物降解酶的酶学和遗传关系。生理学和进化的途径也进行了讨论。

We examined the following experiments in order to clarify 1) reaction mechanism of aldoxime dehydratase (Oxd), 2) diversity of aldoxime dehydratase, and 3) diversity and physiological function of "aldoxime-nitrile pathway".1. We constructed an overexpression system of nitrilase (Nit)-linked oxd gene from Bacillus sp. OxB-1 in a recombinant E. coli and obtained large quantities of Oxd protein (OxdB) in highly pure and homogenous form.2. We analyzed OxdB protein spectrophotometrically to identify its heme environment and functional residue during enzyme catalysis.3. We screened oxd genes genetically from stock cultures and cloned whole lengths oxd genes.4. We overexpressed new oxds in E.coli and examined their characters. Based on the findings, physiological functions of Oxds are studied.5. We genetically screened nitrile-degrading enzymes in strains having "aldoxime-nitrile pathway" and cloned their genes.6. We compared the characters and primary structures of the nitrile-degrading enzymes and studied the relationship between Oxds enzymologically and genetically. Physiology and evolutions of the pathway are also discussed.