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Qualitative and quantitative NMR in optimization of methyl β-D-fructofuranoside synthesis catalyzed by invertase glycoforms

Glycoforms of invertase that exhibit high stability in hydrophilic organic solvent was purified from a crude mixture of a multitude of invertase N-glycoforms synthesized by Saccharomyces cerevisiae. Purification was achieved by specially designed sub- fractionation procedure on anion-exchange chromatographic monolithic supports. Among separated N-glycoforms (EINV1-EINV4), only N- glycoform EINV1 exhibited 2-5 times higher stability in 30% methanol. Difference of glycoforms stability in the presence of high methanol concentration was used to improve the efficiency and yield of methyl β-D- fructofuranoside (MF) synthesis. Formation of MF in the reaction mixtures was confirmed by COSY, HMBC and HMQC. Kinetic of MF formation was assessed by quantitative NMR. NMR analysis provided data that enabled identification of optimal reaction time and calculation of reaction yield. The efficiency and yield of MF synthesis was improved more than 50% when the most stabile N- glycoform was utilized. These data underline the importance of analysis of glycan structures attached to glycoproteins, demonstrates different impact of N-glycans on invertase stability in hydrophilic organic solvent reaction environment, and provide a platform for improvement of yield of biocatalytic processes by selection of N- glycoforms.

N- glycosylation ; enzyme stability ; glycoform selection ; organic solvent ; methyl β-D-fructofuranoside ; enzymatic alkylation

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