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

环糊精葡糖基转移酶(CGTase)是一种来自细菌和古菌的多功能酶，广泛应用于生物技术中以淀粉为原料生产环糊精。CGTase催化四种与机械有关的转化：(I)环化--将α-1，4-葡聚糖(麦芽寡糖，淀粉)转化为环糊精；(Ii)偶联，其中线性麦芽寡糖作为线性化环糊精的受体；(Iii)歧化，将线性麦芽寡糖转移到线性受体，以及(Iv)水解。从机理上讲，这些反应都是相似的，因为它们涉及到立体选择性的α-葡萄糖基转移到酒精受体上，要么是葡萄糖苷(I-III)，要么是水(IV)。这些酶在生物技术中的一个特别吸引人的特点是糖基转移反应(I-III)，因为它们允许廉价和丰富的起始原料形成新的糖苷键，即天然存在的糖苷，例如淀粉，而不需要昂贵的激活。研究表明，烷基葡萄糖苷等“非天然”受体可以在反应中用作受体(Ii)以可持续的方式生成新的表面活性剂。抗坏血酸已被用作生成2-O-α-D-葡萄糖基-L-抗坏血酸(AA-2G)的受体，由于其抗还原和氧化以及其容易被α-葡萄糖苷酶降解以释放L-抗坏血酸和葡萄糖而在食品、医药和化妆品中具有商业用途。为了利用这些机会，将解决以下挑战：a.选择性-生成仅在(I-IV)之间调节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.酶的生产--找到可以规模化生产的酶(类似于目前的CGTase)c.酶的稳定性--由最终用户定义的催化剂的寿命，包括固定化和溶解。酶活性-最终用户定义的产品的时空产率和原料的经济使用

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