Design of Trypanosoma rangeli sialidase mutants with improved trans-sialidase activity

Nyffenegger, Christian; Nordvang, Rune Thorbjørn; Jers, Carsten; Meyer, Anne S.; Mikkelsen, Jørn Dalgaard

doi:10.1371/journal.pone.0171585

Cited by 17 publications

(28 citation statements)

References 21 publications

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“…For HEX2, all the loop‐engineered enzymes had profoundly decreased activity (Table ). Nevertheless, these enzymes were also characterized towards their transglycosylation ability, as it is well known that decreasing the hydrolytic activity can lead to an increased transglycosylation yield …”

Section: Resultsmentioning

confidence: 99%

“…Considering that the global market for foods and beverages containing pre‐ and probiotics is estimated to be approximately US$ 15.4 billion and with dairy‐based foods and beverages constituting over half of this market, the potential for commercial production of human milk oligosaccharides and other prebiotic oligosaccharides is very appealing. Biosynthetic synthesis (analytical scale) of 3′‐sialyllactose and 6′‐sialyllactose, both considered as fundamental HMO structures with particular functions, has been achieved by using different types of one‐step enzymatic routes . In contrast, reports on the biocatalytic production of HMO backbone blueprint structures of LNT2 by using lactose and naturally occurring food‐grade oligosaccharides such as chitin derivatives as N ‐acetylglucosamine donors, and not p ‐nitrophenol‐modified β‐ N ‐acetylglucosamine or other unnatural saccharide derivatives as donor substrates, are scarce.…”

Section: Resultsmentioning

confidence: 99%

“…A minimum of three of the microbial sources of the enzyme sequences (AAT89596.1, AGW40804.1, ABY25113.1) appear to be either plant or fish pathogens (Table ). In general, pathogenic organisms are often a potential source of transglycosidases, because transglycosylation may be used by these microbes to produce camouflage saccharide structures by utilization of host glycans to facilitate invasion of the host; secondly, charged loops were previously shown to alter glycosidase activity towards transglycosylation activity . Lastly, preliminary studies with HotSpot Wizard 2.0 identified R355 as a potential hotspot for mutations.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2018

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Certain enzymes of the glycoside hydrolase family 20 (GH20) exert transglycosylation activity and catalyze the transfer of β-N-acetylglucosamine (GlcNAc) from a chitobiose donor to lactose to produce lacto-N-triose II (LNT2), a key human milk oligosaccharide backbone moiety. The present work is aimed at increasing the transglycosylation activity of two selected hexosaminidases, HEX1 and HEX2, to synthesize LNT2 from lactose and chitobiose. Peptide pattern recognition analysis was used to categorize all GH20 proteins in subgroups. On this basis, we identified a series of proteins related to HEX1 and HEX2. By sequence alignment, four additional loop sequences were identified that were not present in HEX1 and HEX2. Insertion of these loop sequences into the wild-type sequences induced increased transglycosylation activity for three out of eight mutants. The best mutant, HEX1 , had a transglycosylation yield of LNT2 on the donor that was nine times higher than that of the wild-type enzyme. Homology modeling of the enzymes revealed that the loop insertion produced a more shielded substrate-binding pocket. This shielding is suggested to explain the reduced hydrolytic activity, which in turn resulted in the increased transglycosylation activity of HEX1 .

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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

et al. 2018

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“…Over the last decade, protein engineering has been established as a strong tool for improving the transglycosylation efficiency of HMO-relevant glycosyl hydrolases [54,55,59,67,73,74,75,76,77]. We also review a few approaches to improve transglycosylation efficiency that have not yet been reported for HMO synthesis but could be relevant for the future of the field.…”

Section: Improved Transglycosylation Through Protein Engineeringmentioning

confidence: 99%

Synthesis of Human Milk Oligosaccharides: Protein Engineering Strategies for Improved Enzymatic Transglycosylation

et al. 2019

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Human milk oligosaccharides (HMOs) signify a unique group of oligosaccharides in breast milk, which is of major importance for infant health and development. The functional benefits of HMOs create an enormous impetus for biosynthetic production of HMOs for use as additives in infant formula and other products. HMO molecules can be synthesized chemically, via fermentation, and by enzymatic synthesis. This treatise discusses these different techniques, with particular focus on harnessing enzymes for controlled enzymatic synthesis of HMO molecules. In order to foster precise and high-yield enzymatic synthesis, several novel protein engineering approaches have been reported, mainly concerning changing glycoside hydrolases to catalyze relevant transglycosylations. The protein engineering strategies for these enzymes range from rationally modifying specific catalytic residues, over targeted subsite −1 mutations, to unique and novel transplantations of designed peptide sequences near the active site, so-called loop engineering. These strategies have proven useful to foster enhanced transglycosylation to promote different types of HMO synthesis reactions. The rationale of subsite −1 modification, acceptor binding site matching, and loop engineering, including changes that may alter the spatial arrangement of water in the enzyme active site region, may prove useful for novel enzyme-catalyzed carbohydrate design in general.

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“…However, the enzyme constitutes an important virulence factor in T. cruzi (19). Although mutants of the nonpathogenic Trypanosoma rangeli sialidase (TrSA) have been developed, they show relatively low trans-sialidase activity (20,21). Additionally, the native TcTS and mutant TrSA form only ␣2-3 linkages.…”

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confidence: 99%

Enzymatic Synthesis of 6′-Sialyllactose, a Dominant Sialylated Human Milk Oligosaccharide, by a Novel exo -α-Sialidase from Bacteroides fragilis NCTC9343

Guo

Chen

et al. 2018

Appl Environ Microbiol

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Gut bacteria provide a rich source of glycosidases that can recognize and/or hydrolyze glycans for nutrition. Interestingly, some glycosidases have also been found to catalyze transglycosylation reactions and thus can be used for oligosaccharide synthesis. In this work, six putative and one known-α-sialidase genes-three from NCTC9343, three from ATCC 13124, and one known from JCM1254-were subjected to gene cloning and heterogeneous expression in The recombinant enzymes were purified, characterized for substrate specificity, and screened for transglycosylation activity. A sialidase, named BfGH33C, from NCTC9343 was found to possess excellent transglycosylation activity for the synthesis of sialylated human milk oligosaccharide. The native BfGH33C was a homodimer with a molecular weight of 113.6 kDa. The and values for 4-methylumbelliferyl-acetyl-α-d-neuraminic acid and sialic acid dimer were determined to be 0.06 mM and 283.2 s, and 0.75 mM and 329.6 s, respectively. The enzyme was able to transfer sialyl from sialic acid dimer or oligomer to lactose with high efficiency and strict α2-6 regioselectivity. The influences of the initial substrate concentration, pH, temperature, and reaction time on transglycosylation were investigated in detail. Using 40 mM sialic acid dimer (or 40 mg/ml oligomer) and 1 M lactose (pH 6.5) at 50°C for 10 min, BfGH33C could specifically produce 6'-sialyllactose, a dominant sialylated human milk oligosaccharide, at a maximal conversion ratio above 20%. It provides a promising alternative to the current chemical and enzymatic methods for obtaining sialylated oligosaccharides. Sialylated human milk oligosaccharides are significantly beneficial to the neonate, as they play important roles in supporting resistance to pathogens, gut maturation, immune function, and brain and cognitive development. Therefore, access to the sialylated oligosaccharides has attracted increasing attention both for the study of saccharide functions and for the development of infant formulas that could mimic the nutritional value of human milk. Nevertheless, nine-carbon sialic acids are rather complicated for the traditional chemical modifications, which require multiple protection and deprotection steps to achieve a specific glycosidic bond. Here, the -α-sialidase BfGH33C synthesized 6'-sialyllactose in a simple step with high transglycosylation activity and strict regioselectivity. Additionally, it could utilize oligosialic acid, which was newly prepared in an easy, economical way to reduce the substrate cost, as a glycosyl donor. All the studies laid a foundation for the practical use of BfGH33C in large-scale synthesis of sialylated oligosaccharides in the future.

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Design of Trypanosoma rangeli sialidase mutants with improved trans-sialidase activity

Cited by 17 publications

References 21 publications

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

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

Synthesis of Human Milk Oligosaccharides: Protein Engineering Strategies for Improved Enzymatic Transglycosylation

Enzymatic Synthesis of 6′-Sialyllactose, a Dominant Sialylated Human Milk Oligosaccharide, by a Novel exo -α-Sialidase from Bacteroides fragilis NCTC9343

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