Increased yield of enzymatic synthesis by chromatographic selection of different N‐glycoforms of yeast invertase

Abstract

Invertases are glycosidases applied for synthesis of alkyl glycosides that are important and effective surfactants. Stability of invertases in the environment with increased content of organic solvent is crucial for increase of productivity of glycosidases. Their stability is significantly influenced by N-glycosylation. However, yeast N-glycosylation pathways may synthesize plethora of N-glycan structures. A total natural crude mixture of invertase glycoforms (EINV) extracted from Saccharomyces cerevisiae was subfractionated by anionexchange chromatography on industrial monolithic supports to obtain different glycoforms (EINV1-EINV3). Separated glycoforms exhibited different stabilities in water-alcohol solutions that are in direct correlation with the amount of phosphate bound to N-glycans.Observed differences in stability of different invertase glycoforms were used to improve productivity of methyl β-D-fructofuranoside (MF) synthesis. The efficiency and yield of MF synthesis was improved more than 50% when the most stabile glycoform bearing the lowest amount of phosphorylated N-glycans is selected and utilized. These data underline the importance of analysis of glycan structures attached to glycoproteins, demonstrates different impact of N-glycans on the surface charge and enzyme stability in regard to particular reaction environment, and provide a platform for improvement of yield of industrial enzymatic synthesis by chromatographic selection of glycoforms on monolithic supports.

A monolithic stationary phase with dendritic nanostructures for the separation of PEGylated proteins

“…Similar results were reported by Andjelković et al. using a CIM‐QA monolithic support for the separation of invertase glycoforms [33, 34]. If so, this phenomenon might be caused by the increase in hydrophobic surface area, conferred by the natural architecture of the dendrons, as well as by the differences in the hydrophobic surface between isomers conferred by the PEG molecules.…”

“…Thus, it seems that the dendronized monolith not only separates PEGylated species of RNase A but is able to separate positional isomers of mono-PEGylated RNase A, which is quite relevant to the field. Similar results were reported by Andjelković et al using a CIM-QA monolithic support for the separation of invertase glycoforms [33,34]. If so, this phenomenon might be caused by the increase in hydrophobic surface area, conferred by the natural architecture of the dendrons, as well as by the differences in the hydrophobic surface between isomers conferred by the PEG molecules.…”