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

为了阐明1)乙醛肟脱水酶(Oxd)的反应机理和2)“乙醛-腈途径”在微生物中的生理功能，我们进行了以下实验：1.在重组大肠杆菌中构建了与腈酶(Nit)连接的axd基因的高效表达系统，并获得了大量高纯度、均一的Oxd蛋白(OxdB)。通过对该酶的详细性质分析和分光光度分析，推测了该酶的反应机理。我们从(1)优化条件下生长的重组大肠杆菌中提纯了数百mg OxDB，并应用于初步的结晶研究。我们从我们的原种培养和土壤样品中筛选出了几株具有氰基水合酶(NHase)活性的OXD菌株。我们从(4)处分离到的Globerulus A-4中高度纯化了NHase连锁Oxd(OxdRG)，并将其性质与以前鉴定的Oxd进行了比较。根据OxdRG的N-端氨基酸序列信息，我们从大黄鱼基因组文库中克隆了oxD基因。我们明确了oxd基因与编码氨酶、酰胺酶及其调控蛋白和激活子的基因在菌株基因组中共存，形成了基因簇。我们克隆了已分离的产氨酶红球菌N-771的酰胺酶基因上游的oxd基因同源物。构建了Oxd(OxdRE)在重组大肠杆菌中的过表达系统，并对其过表达系统进行了优化。在优化的条件下，培养重组大肠杆菌，从菌株中纯化OxdRE，对其进行了鉴定，并与已知Oxds进行了性质比较。

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.