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

Background
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 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 solution
d. Enzyme activity - space-time yields of products and economic use of starting material as defined by end user.



Project Aim and Description
This 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.