Rational Design of a New Trypanosoma rangeli Trans-Sialidase for Efficient Sialylation of Glycans

Abstract

This paper reports rational engineering of Trypanosoma rangeli sialidase to develop an effective enzyme for a potentially important type of reactivity: production of sialylated prebiotic glycans. The Trypanosoma cruzi trans-sialidase and the homologous T. rangeli sialidase has previously been used to investigate the structural requirements for trans-sialidase activity. We observed that the T. cruzi trans-sialidase has a seven-amino-acid motif (197–203) at the border of the substrate binding cleft. The motif differs substantially in chemical properties and substitution probability from the homologous sialidase, and we hypothesised that this motif is important for trans-sialidase activity. The 197–203 motif is strongly positively charged with a marked change in hydrogen bond donor capacity as compared to the sialidase. To investigate the role of this motif, we expressed and characterised a T. rangeli sialidase mutant, Tr13. Conditions for efficient trans-sialylation were determined, and Tr13's acceptor specificity demonstrated promiscuity with respect to the acceptor molecule enabling sialylation of glycans containing terminal galactose and glucose and even monomers of glucose and fucose. Sialic acid is important in association with human milk oligosaccharides, and Tr13 was shown to sialylate a number of established and potential prebiotics. Initial evaluation of prebiotic potential using pure cultures demonstrated, albeit not selectively, growth of Bifidobacteria. Since the 197–203 motif stands out in the native trans-sialidase, is markedly different from the wild-type sialidase compared to previous mutants, and is shown here to confer efficient and broad trans-sialidase activity, we suggest that this motif can serve as a framework for future optimization of trans-sialylation towards prebiotic production.

Glycosynthesis in a waterworld: new insight into the molecular basis of transglycosylation in retaining glycoside hydrolases

“…Using an enzyme engineering approach it was shown that the loop in TctrS promotes transglycosylation, increasing product yield and reducing hydrolysis, effects that were attributed to a perturbation of the water-binding network [154]. Using an enzyme engineering approach it was shown that the loop in TctrS promotes transglycosylation, increasing product yield and reducing hydrolysis, effects that were attributed to a perturbation of the water-binding network [154].…”

Separation of 3′-sialyllactose and lactose by nanofiltration: A trade-off between charge repulsion and pore swelling induced by high pH

“…It contained 5.7% (w/w) of covalently linked sialic acid and was used as the donor molecule for enzymatic production of 3 0 -sialyllated-lactose (with lactose as acceptor) using an engineered sialidase, Tr13, from Trypanosoma rangeli reacting for 60 min at 30°C and pH 6.5. The Tr13 enzyme was produced in Pichia pastoris and purified as described previously [2]. The enzyme and cGMP residues were removed from the post reaction mixture by ultrafiltration using a 10 kDa commercially available Alfa Laval RC70PP membrane before the sample was frozen and stored until it was used for filtration experiments.…”

“…Commercially sustainable processes for production of HMOs are therefore of great interest for both society and the dairy industry. 3 0 -sialyllactose (SL) is an HMO molecule which is currently produced from lactose and casein glyco macro peptide (cGMP) via an enzymatic reaction that relies on the presence of a large excess of lactose [2][3][4]. Once produced, the separation and purification of SL from lactose is a crucial step, as the SL added to the commercial formula should be as pure as possible.…”

“…Traditionally, when the production of SL has been performed at small scale, separation of lactose and SL has been achieved by anion exchange chromatography [5,4,3,2]. However, considering the economy of a large scale production process and the fact that anion exchange chromatography relies on the addition of buffers and eluents -which would then need to be removed before the SL can be considered for consumption -an alternative process for the efficient purification of SL from lactose is necessary for the addition of SL to infant formula to become a reality.…”

Loop Protein Engineering for Improved Transglycosylation Activity of a β‐N‐Acetylhexosaminidase

“…Our key idea was to engineer loop structures near the active site with the aim to confer water exclusion during catalysis to enhance transglycosylation. This strategy was recently successfully applied to improve the trans‐sialylation of a sialidase . However, as opposed to the design of the trans‐sialylation loop, for which a native trans‐sialidase structure was available (i.e., the Trypanosoma cruzi trans‐sialidase), there were no immediate real transhexosaminidase template enzymes available for engineering.…”