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）醛肟还原酶（Oxd）的反应机理和2）微生物中"醛肟-腈途径"的生理功能，我们检查了以下实验：1.我们构建了腈水解酶（Nit）连接的axd基因在重组大肠杆菌中的过表达系统.大肠杆菌中获得了大量高纯度、均一的Oxd蛋白（OxdB）.通过对酶的详细性质分析和分光光度分析，推测了OxdB的反应机理.我们从重组E.大肠杆菌菌株在优化的条件下生长（1），并应用于初步的结晶研究.我们从我们的储备培养物和土壤样品中筛选了几个具有腈水合酶（NHase）的Oxd喜欢的菌株.我们从球形红球菌A-4（已在（4）中分离）中高度纯化了NH酶连接的Oxd（OxdRG），并将其性质与先前表征的Oxds进行了比较。我们从黑曲霉基因组文库中克隆了oxd基因。根据OxdRG的N端氨基酸序列信息，阐明oxd基因与编码腈水合酶、酰胺酶及其调控蛋白和激活因子的基因共存于该菌株基因组中，形成基因簇.我们克隆了存在于Rhodococcus erythropolis N-771酰胺酶基因上游的oxd基因同源物，该菌株已被分离为NH ase生产菌。我们构建了Oxd基因在重组大肠杆菌中的过表达系统。coli，并优化了其表达体系。在优化的条件下，重组E.大肠杆菌中培养，并从菌株中纯化OxdRE，表征，并将其性质与已知的Oxds进行比较。

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.