Investigation of the transglycosylation potential of ß-Galactosidase from Aspergillus oryzae in the presence of the ionic liquid [Bmim][PF6

Abstract

Finding efficient and simple methods for the synthesis of oligosaccharides is a relevant question for both pharmaceutical and food industry. An interesting route is transglycosylation by glycosidases due to their availability and cheap substrates. In this work transglycosylation by means of ß-galactosidase from Aspergillus oryzae was investigated at pH 4.5 using ONPG as glycosyl donor and several saccharides in different concentrations as acceptor. As the influence of ionic liquids was often described to be beneficial for synthesis or specificity of glycosidases, the ionic liquid [Bmim][Pf6] was investigated in an emulsion with aqueous buffer. Additionally, a fed-batch mode is presented here, including the ionic liquid as extraction phase. Reaction products were analyzed via HPLC and thin-layer chromatography.By use of transglycosylation, oligosaccharides were produced in high yields, often above 90%, both in aqueous buffer and the system using buffer and ionic liquid. The application of [Bmim][Pf6] lead to an altered substrate specificity of ß-galactosidase: Depending on the system applied, preferentially di- or trisaccharides were produced, partly in contrast to the synthesis in aqueous buffer. Using a new model for the transglycosylation with ONPG reaction the observed reaction courses were well described.The fed-batch mode, applying ONPG above solubility and using [Bmim][Pf6] as extraction phase for ONP, strongly increased and product concentration. In this context it was observed that the absorption spectrum of ONP in [Bmim][Pf6] is altered in the presence of an aqueous phase, even though [Bmim][Pf6] is not mixable with water.The findings in this work enable a better understanding of the influence of ionic liquids on enzyme activity and can help to make glycosidases an even more interesting tool in saccharide synthesis.