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Project Summary Abstract Zymtronix is developing a series of highly tunable materials and processes for universal enzyme immobilization based on hierarchical magnetic metamaterialsThis enzyme immobilization platform is being designed to quickly find the optimal conditions to immobilize single and full systems of enzymesIt affords enzyme stabilitymaximal use of substratesincluding co factorsand recent developments imparts modularity for flow processesFor this phase I projectZymtronix proposes to develop its technology to improve the efficiency of defined glycan productionfocusing on human milk oligosaccharidesHMOswith optimized full enzyme systems immobilized on macroporous scaffolds specifically designed for continuous flow processingLibraries of large and complex glycans have been made availablethanks to NIH s Common Fund programAccelerating Translation of GlycoscienceIntegration and Accessibilitylaunched inCurrentlythe primary hurdle towards the broader application of complex synthetic glycans in researchpreclinical and commercial applications is their availability and affordabilityZymtronix s technology allows maintaining high activities and co immobilizing multiple enzymes to mimic live cell reactions and combined in situ production of active sugarstransfer of the sugar for de novo synthesiswhile ensuring the reuse of the enzymes and substoichiometric use of cofactors for significant cost reductionFor this Phase I we will demonstrate key steps for the production of Human milk oligosaccharidesHMOsthe third largest component of breast milk that are particularly commercially relevant glycan modelsWhile some simple probiotic HMOs can be effectively produced via fermentation for infant formulaFLDPcomplex and branched prophylactic HMOs are elusive and have been marginally produced at high cost via chemocatalysisZymtronix s technology solution will significantly improve HMO production efficiency while imparting significant cost savingsThe Phase I proofof concept will primarily focus onkey steps of glycan functionalization with fucose and sialic acid activation and transfer onto HMO backbones with immobilized enzymesSpecific Aims for this Phase I project includeProduce two key activated sugars CMP Sialic acid and GDP L fucose with immobilized enzymesPerform the fucosylation and siallyation of lactose and lactose N tetraose with immobilized enzymesDemonstrate fucosylation of Lactose directly with L fucose with an immobilized three enzyme system including GTP recyclingFor Phase IIwe proposed to expand our continuous flow approach for gram scale synthesis of a glycan libraryandgtDPconsisting oflinearbiantennaryandtriantennary HMOs and go beyond HMOs with glycosylation of active small molecules Project Narrative Glycans are complex carbohydrate structures that are the predominant molecules on the cell surface or in milk and serve as the first point of contact between cellsthe extracellular matrix and pathogensWhile their role as mediators of cellular interaction is still poorly understoodit is crucial to improve the accessibility and affordability of these moleculesZymtronixnovel modular method and processes for producing complex HMOsand other defined glycanswill enhance production efficiency of complex carbohydrates while lowering costspotentially allowing for development of improved infant formulas and other bioactive glycosylated molecules