Physiological functions of "aldoxime-nitrile pathway" in microorganisms and the use of enzymes responsible for the pathway to organic synthesis

为了阐明微生物中醛肟脱水酶（Oxd）的反应机理和“醛肟-腈途径”的生理功能，我们进行了以下实验：我们在重组大肠杆菌中构建了nitrilase （Nit）连接的axd基因过表达系统，获得了大量高纯度、高均质形式的Oxd蛋白（OxdB）。通过对该酶的详细性质分析和分光光度分析，推测了其反应机理。我们从(1)优化条件下培养的重组大肠杆菌菌株中纯化了数百mg的OxdB，并进行了初步的结晶研究。我们从我们的家畜培养物和土壤样品中筛选了几种具有腈水合酶（NHase）的Oxd菌株。我们从(4)中分离出的球状红球菌A-4中高度纯化了nase -linked Oxd (OxdRG)，并将其与先前表征的Oxd进行了比较。我们根据OxdRG的n端氨基酸序列信息，从球芽草基因组文库中克隆出oxd基因。结果表明，在该菌株的基因组中，oxd基因与编码nase、amidase及其调控蛋白和激活因子的基因共存，形成基因簇。我们克隆了红红红球菌N-771氨基酶基因上游的oxd基因同源物，该基因已被分离为nase产生菌。构建了Oxd （OxdRE）在重组大肠杆菌中的过表达体系，并对其过表达体系进行了优化。在优化的条件下，对重组大肠杆菌进行培养，从该菌株中纯化OxdRE，对其进行表征，并与已知Oxds进行性能比较。

We examined the following experiments in order to clarify 1) reaction mechanism of aldoxime dehydratase (Oxd) and 2) physiological function of "aldoxime-nitrile pathway" in microorganisms:1. We constructed an overexpression system of nitrilase (Nit)-linked axd gene in a recombinant E. coli and obtained large quantities of Oxd protein (OxdB) in highly pure and homogenous form.2. We speculated a reaction mechanism of OxdB by analyzing detailed characters and spectrophotometric analysis of the enzyme.3. We purified several hundreds mg of OxdB from the recombinant E. coli strain grown under optimized conditions at (1) and applied for a preliminary crystallization studies.4. We screened several strains having Oxd liked with nitrile hydratase (NHase) from our stock cultures and soil samples.5. We highly purified NHase-linked Oxd (OxdRG) from Rhodococcus globerulus A-4, which has been isolated at (4), and compared its properties with the previously characterized Oxds.6. We cloned oxd gene from thegenomic library of R. globerulus based on an information of N-terminal amino acid sequence of OxdRG. We clarified that the oxd gene coexisted with genes coding NHase, amidase, and their regulatory proteins and activators at the genome of the strain as to form gene clusters.7. We cloned oxd gene homologue existing at an upstream of amidase gene of Rhodococcus erythropolis N-771, which had been isolated as NHase producer. We constructed the overexpression system of the Oxd (OxdRE) in the recombinant E. coli and optimized its overexpression system. Under the optimized conditions, the recombinant E. coli was cultivated and OxdRE was purified from the strain, characterized, and compared its properties with the known Oxds.