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

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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