Abstract
A sialidase (EC 3.2.1.18; GH 33) from non-pathogenic Trypanosoma rangeli has been engineered with the aim of improving its transsialylation activity. Recently, two engineered variants containing 15 and 16 amino acid substitutions, respectively, were found to exhibit significantly improved transsialylation activity: both had a 14 times higher ratio between transsialylation and hydrolysis products compared to the first reported mutant TrSA5mut. In the current work, these two variants, Tr15 and Tr16, were characterized in terms of pH optimum, thermal stability, effect of acceptor-to-donor ratio, and acceptor specificity for transsialylation using casein glycomacropeptide (CGMP) as sialyl donor and lactose or other human milk oligosaccharide core structures as acceptors. Both sialidase variants exhibited pH optima around pH 4.8. Thermal stability of each enzyme was comparable to that of previously developed T. rangeli sialidase variants and higher than that of the native transsialidase from T. cruzi (TcTS). As for other engineered T. rangeli sialidase variants and TcTS, the acceptor specificity was broad: lactose, galactooligosaccharides (GOS), xylooligosaccharides (XOS), and human milk oligosaccharide structures lacto-N-tetraose (LNT), lacto-N-fucopentaose (LNFP V), and lacto-N-neofucopentaose V (LNnFP V) were all sialylated by Tr15 and Tr16. An increase in acceptor-to-donor ratio from 2 to 10 had a positive effect on transsialylation. Both enzymes showed high preference for formation α(2,3)-linkages at the non-reducing end of lactose in the transsialylation. Tr15 was the most efficient enzyme in terms of transsialylation reaction rates and yield of 3’-sialyllactose. Finally, Tr15 was immobilized covalently on glyoxyl-functionalized silica, leading to a 1.5-fold increase in biocatalytic productivity (mg 3’-sialyllactose per mg enzyme) compared to free enzyme after 6 cycles of reuse. The use of glyoxyl-functionalized silica proved to be markedly better for immobilization than silica functionalized with (3-aminopropyl)triethoxysilane (APTES) and glutaraldehyde, which resulted in a biocatalytic productivity which was less than half of that obtained with free enzyme.
Highlights
Human milk oligosaccharides (HMOs) designate a unique family of bioactive lactose-based molecules present in human breast milk (5–15 g/L), which are known to be of major importance for infant health and development [1]
The use of glyoxyl-functionalized silica proved to be markedly better for immobilization than silica functionalized with (3-aminopropyl)triethoxysilane (APTES) and glutaraldehyde, which resulted in a biocatalytic productivity which was less than half of that obtained with free enzyme
The difference in transsialylation yield was not significant between the two recycling methods (Figure 7), but it is evident that some activity was lost in the centrifugation method, where the biocatalyst was separated from the reaction mixture after each cycle: for the glyoxyl method, the 3’SL concentration obtained in AIMS Molecular Science the sixth cycle was around 70% of that obtained in the first cycle, whereas it was only around 50% for the (3-aminopropyl)triethoxysilane GA (APTES) method (Figure 6)
Summary
Human milk oligosaccharides (HMOs) designate a unique family of bioactive lactose-based molecules present in human breast milk (5–15 g/L), which are known to be of major importance for infant health and development [1]. As they are virtually absent from bovine milk, which is used for production of infant formula, production of HMOs receives increasing attention for commercial use as well as for functional studies. Introducing five mutations affecting the binding site and the sialic acid binding
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