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

背景环糊精葡聚糖转移酶（Cyclodextrin glucanotransferases，CGTases）是来源于细菌和古细菌的多功能酶，广泛应用于淀粉制备环糊精的生物技术中。CGTase催化四种机械相关的转化：（i）环化-α-1，4-葡聚糖（麦芽寡糖，淀粉）转化为环糊精;（ii）偶联，其中直链麦芽寡糖充当直链环糊精的受体;（iii）缩合，直链麦芽寡糖转移至直链受体;和（iv）水解。从机理上讲，这些反应都是相似的，因为它们涉及立体选择性α-葡糖基转移到醇受体上，葡糖苷（i-iii）或水（iv）。这些酶用于生物技术的一个特别有吸引力的特征是糖基转移酶反应（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，而不需要昂贵的活化。已经表明，“非天然”受体如烷基葡糖苷可以用作反应（ii）中的受体，以可持续的方式产生新的表面活性剂。该项目旨在应对以下挑战：a.选择性-产生仅介导（i-iv）之间的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-发现可以大规模生产的酶（类似于目前的CGTase）酶的稳定性-催化剂的寿命由最终用户定义，包括固定化和溶液。酶的活性-时空产率的产品和经济使用的起始材料所定义的最终用户。项目目标和描述本研究项目将有一个重点酶的稳定性和活性。目的是评估一组非天然底物及其类似物，以确定理想的底物，并在需要时进一步进化。学生将领导规模扩大工作的发展，评估酶的稳定性和活性在批量和流动条件下。生物催化：评价新发现的酶对一组化学合成底物（包括标记底物）的生物催化活性。这将使得能够在复杂的生物样品/粗表达系统中进行直接MS分析。最佳候选物将用于以实验设计为指导的放大研究，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.