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

我们对以下实验进行了研究，以阐明1)醛肟脱水酶(Oxd)的反应机理，2)醛肟脱水酶的多样性，以及3)“醛-腈途径”的多样性和生理功能。我们构建了芽孢杆菌Nit连接oxD基因的高效表达系统。结果：1.将OXB-1在重组大肠杆菌中表达，获得了大量高纯度、均一的Oxd蛋白(OxdB)。对OxDB蛋白进行分光光度分析，以确定其在酶催化过程中所处的血红素环境和功能残基。从原代培养物中筛选出oxd基因，并克隆了其全长oxd基因。我们在大肠杆菌中过度表达了新的OXD，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-腈途径”的菌株进行了丁腈降解酶的遗传筛选，并克隆了它们的基因。我们比较了丁腈降解酶的性质和一级结构，并从酶学和遗传学的角度研究了它们之间的关系。并对该途径的生理和进化进行了讨论。

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.