NEW APPROACH TO SUSTAINABLE CHEMICAL RESOURCES BASED ON SEARCH, PRECISE ANALYSIS, AND APPLICATION OF BACTERIAL SUGAR-CARBOCYCLIZATION ENZYMES

以二氧化碳固定为基础制造可持续化工资源的新模式对节约化石资源和保护我们的生存环境是非常理想的。本文提出了一种利用碳水化合物环化酶- 2-脱氧-基-肌糖（DOI）合成酶（DOIS）的方法，并对DOIS进行了详细的分析和替代应用。(1)纯化了圆芽孢杆菌的DOIS，鉴定了其致病基因（btrC）并在大肠杆菌中表达。(2)确定了转化大肠杆菌的发酵条件和DOI的制备方案。(3)通过简单的化学处理，证实了DOI可转化为邻苯二酚或1,2,4 -三乙酰氧基苯。(4)利用底物类似物分析了酶与底物之间的详细相互作用，以阐明特定氢键的重要性。(3)利用酶切法和LC/ESI/MS/MS分析对活性位点进行定位。(4)为了利用代谢工程技术制备替代性DOI，广泛分析了丁香的生物合成基因簇。(5)鉴定出一种新的转氨酶（BtrS）负责DOIS后的步骤，并在大肠杆菌中过表达以证实其功能。(6)环虫btrS基因因内框缺失而被破坏。目前正在调查这种干扰物生产DOI的可能性。(7)利用过表达的BtrS进行制备。2-脱氧-三基肌糖胺是开发药物的潜在资源。(8) DOI被成功转化为葡萄糖，这是一种很有前途的无膳食负荷的葡萄糖替代品。(8)从Streptomyces sp.中鉴定出一个新的DOIS并过表达。其功能得到了证实。所有这些工作似乎构成了对dois的精确理解及其在有机资源制备中的应用的重要基础。少

New paradigm for manufacturing sustainable chemical resources based on the CO_2 fixation is highly desirable to conserve fossil resources and to protect our living environments. An approach using carbohydrate-cyclizing enzyme, 2-deoxy-scyllo-inosose (DOI) synthase (DOIS), being originally involved in the biosynthesis of butirosins in Bacillus circulans, has been developed, and detailed analysis and alternative application of DOIS have been exploited. (1) DOIS of B. circulans was purified, and the responsible gene (btrC) was identified and expressed in E. coli. (2) Fermentation conditions of the transformed E. coli and preparative protocol of DOI were established. (3) Conversion of DOI into catechol or 1, 2, 4-triacetoxybenzene was confirmed by simple chemical treatment. (4) Detailed interaction between the enzyme and substrate was analyzed using substrates analogues to elucidate importance of specific hydrogen-bondings. (3) Active site mapping was pursued with mechanism-based inhibitor … More with aids of enzymatic digestion and LC/ESI/MS/MS analysis. (4) For alternative preparation of DOI by metabolic engineering, the biosynthetic gene cluster of butirosin was extensively analyzed. (5) A new transamination enzyme (BtrS) was identified to be responsible for the step next to DOIS, and was over-expressed in E. coli to confirm the function. (6) The btrS gene in B. circulans was disrupted by in frame deletion. Potential of the disruptant for DOI production is currently under investigation. (7) The over-expressed BtrS was utilized to prepare.2-deoxy-scyllo-inosamine as a potential resource for the development of pharmaceuticals. (8) DOI was successfully converted into carbaglucose which is a promising substitute for glucose without dietary load. (8) A new DOIS was identified from a Streptomyces sp. and overexpressed. Its function was confirmed. All these works appear to constitute significant fundamentals for the precise understanding of DOISs and for its application to the preparation of organic resources. Less