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

环糊精葡聚糖转移酶（CGTases）是来自细菌和古细菌的多功能酶，其广泛用于从淀粉生产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，其中线性麦芽寡糖充当线性环糊精的受体;（iii）纯化，将线性麦芽寡糖转移至线性受体;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。抗坏血酸作为一种受体被用于生成2-O-α-D-吡喃葡萄糖基-L-抗坏血酸（AA-2G），AA-2G具有抗还原和抗氧化的特性，并且容易被α-葡萄糖苷酶降解，释放出L-抗坏血酸和葡萄糖，因此在食品、医药和化妆品中具有商业用途。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。酶活性-最终用户定义的产品时空产率和起始物料的经济使用

Cyclodextrin 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 malto-oligosaccharides 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 glycosyltransfer 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. Ascorbic acid has been used as an acceptor for the generation of 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G), which has commercial uses in food, medicine and cosmetics because of its resistance to reduction and oxidation and its easy degradation by alpha-glucosidase to release L-ascorbic acid and glucose .To take advantage of these opportunities, the following challenges will be addressed 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