Improvement of trans-sialylation versus hydrolysis activity of an engineered sialidase from Trypanosoma rangeli by use of co-solvents
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Biocatalytic trans-sialylation is relevant for the design of biomimetic oligosaccharides such as human milk oligosaccharides. t-Butanol and ionic liquids, EAN (ethylammonium nitrate), [MMIm][MeSO4] (1,3-dimethylimidazolium methyl sulfate), and [C2OHMIm][PF6] (1-(2-hydroxyethyl)-3-methylimidazolium hexafluorophosphate), were examined as co-solvents for the improvement of the synthesis versus hydrolysis ratio in the trans-sialylation of lactose, catalysed by an engineered sialidase from Trypanosoma rangeli. The use of 25 % (v/v) t-butanol as co-solvent significantly increased 3′-sialyllactose production by 40 % from 1.04 ± 0.09 to 1.47 ± 0.01 mM. The synthesis versus hydrolysis ratio increased correspondingly by 1.2-times. 1–2.5 % (v/v) EAN or [C2OHMIm][PF6] improved the synthesis versus hydrolysis ratio up to 2.5-times but simultaneously decreased the 3′-sialyllactose yield, probably due to enzyme inactivation caused by the ionic liquid. [MMIm][MeSO4] had a detrimental effect on the trans-sialylation yield and on the ratio between synthesis and hydrolysis.
Keywordst-Butanol Casein glycomacropeptide (CGMP) Co-solvent Ionic liquids 3′-Sialyllactose trans-Sialidase Trypanosoma rangeli
We acknowledge the Danish “Strategic Research Council (DSF)” for financial support to the strategic project “Enzymatic production of human milk oligosaccharides”. Arla Foods amba is acknowledged for the the CGMP gift. We thank Tim Ståhlberg and Saravanamurugan Shunmugavel (Centre for Catalysis and Sustainable Chemistry, DTU Chemistry) for preparation of the ionic liquid [C2OHMIm][PF6]. Jesper Holck and Stine Jørgensen (Center for BioProcess Engineering, DTU Chemical Engineering) are thanked for analysing the sialic acid content in the CGMP used in this study.
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