Development of novel cyclodextrin glucanotransferase (CGTases) for applications in biotechnology

环糊精葡聚糖转移酶（cyclodextrin glucanotransferases, CGTases）是一种来自细菌和古细菌的多功能酶，被广泛应用于生物技术中，用于从淀粉中生产环糊精。CGTases催化四种机制相关的转化(1)环化- - -1,4-葡聚糖（麦芽寡糖，淀粉）转化为环糊精；（ii）偶联，线性麦芽寡糖作为线性化环糊精的受体；（iii）歧化，线性低聚麦芽糖向线性受体的转移和（iv）水解。从机理上讲，这些反应都是相似的，因为它们都涉及到立体选择性α -葡萄糖转移到醇受体上，要么是糖苷（i-iii），要么是水（iv）。这些酶在生物技术方面的一个特别吸引人的特点是糖基转移酶反应（i-iii），因为它们允许从廉价和丰富的起始材料（即天然存在的糖苷，如淀粉）形成新的糖苷键，而不需要昂贵的激活。通过利用CGTases的混杂活动，可以很好地扩展CGTases的应用程序。研究表明，烷基糖苷等“非天然”受体可以作为反应（ii）中的受体，以可持续的方式生成新的表面活性剂。该项目旨在解决以下挑战：选择性-生成只介导（i-iv）之间所需反应的生物催化剂，例如偶联（ii）而没有副反应（i， iii和iv）b。酶生产-找到可以大规模生产的酶（类似于当前的CGTases）酶的稳定性-催化剂的寿命由最终用户定义，包括固定和溶液。酶活性-产品的时空产量和最终用户定义的起始材料的经济使用。项目目标和描述本研究项目将重点研究酶的稳定性和活性。目的是评估一组非天然基质及其类似物，以确定理想的基质，如果需要进一步发展。该学生将领导在批量和流动条件下评估酶的稳定性和活性的大规模工作的发展。生物催化：评估新发现的酶对化学合成的一组底物的生物催化活性，包括标记的底物。这将使在复杂生物样品/粗表达系统中直接进行质谱分析成为可能。在实验设计的指导下，最佳候选物质将用于放大研究，以优化反应参数。

BackgroundCyclodextrin glucanotransferases (CGTases) are multifunctional enzymes from bacteria and archaea that are widely used in biotechnology for the production of cyclodextrins from starch. CGTases catalyze four mechanistically related transformations (i) cyclization - the conversion of alpha-1,4-glucans (malto-oligosaccharides, starch) into cyclodextrins; (ii) coupling, where linear malto-oligosaccharides acts as acceptors for linearized cyclodextrins; (iii) disproportionation, transfer of linear maltooligosaccharides to linear acceptors and (iv) hydrolysis. Mechanistically, these reactions are all similar in that they involve stereoselective alpha-glucosyl transfer on to an alcohol acceptor, either a glucoside (i-iii) or water (iv). A particularly attractive feature of these enzymes for biotechnology are the glycosyltransferase reactions (i-iii), because they allow new glycosidic bond formation from cheap and abundant starting materials, i.e. naturally occurring glycosides such as starch, without the need for costly activation.There is a great opportunity to expand the applications of CGTases by exploiting their promiscuous activities. It has been shown that 'unnatural' acceptors such as alkyl glucosides can be used as acceptors in reaction (ii) generating new surfactants in a sustainable manner. The project will aim to address the following challenges:a. Selectivity - to generate biocatalysts that only mediate the desirable reaction among (i-iv), such as coupling (ii) without side reactions (i, iii and iv)b. Enzyme production - find enzymes that can be produced on scale (similar to current CGTases)c. Enzyme stability - lifetime of catalyst as defined by end-user, both immobilized and in solutiond. Enzyme activity - space-time yields of products and economic use of starting material as defined by end user.Project Aim and DescriptionThis research project will have an emphasis on Enzyme stability and activity. The aim will be to evaluate a panel of non-natural substrate and their analogues to determine ideal substrate and if required further evolution. The student will lead the development of scale-up work evaluating both enzyme stability and activity under batch and flow conditions. Biocatalysis: To evaluate the biocatalytic activity of the newly discovered enzymes against a panel of substrates that will be chemically synthesized, including labelled substrates. This will enable direct MS analysis in complex biological samples/crude expression system. Best candidates will be used in scale-up studies guided by design of experiments to optimize reaction parameters.