Biocatalytic production of 3′-sialyllactose by use of a modified sialidase with superior trans-sialidase activity

Michalak, Malwina; Larsen, Dorte Møller; Jers, Carsten; Almeida, J. R. M. de; Willer, Martin; Li, Haiying; Kirpekar, Finn; Kjaerulff, Louise; Gotfredsen, Charlotte Held; Nordvang, Rune Thorbjørn; Meyer, Anne S.; Mikkelsen, Jørn Dalgaard

doi:10.1016/j.procbio.2013.10.023

Cited by 35 publications

(38 citation statements)

References 31 publications

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“…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%

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%

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

et al. 2018

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“…In trans-glycosylation reactions using glycosyl hydrolases, careful control of reaction time is often important due the inherent hydrolase activity that affects yield by degradation of both donor and product molecules [ 8 ]. It has also been demonstrated for other hydrolases that a high concentration of acceptor can improve transglycosylation yields [ 12 , 34 ]. We therefore set up reactions with a higher acceptor:donor ratio (100 mM lactose as acceptor and 25 mM pNP-Fuc as donor) and followed the reactions in time course-experiments.…”

Section: Resultsmentioning

confidence: 99%

Novel α-L-Fucosidases from a Soil Metagenome for Production of Fucosylated Human Milk Oligosaccharides

et al. 2016

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This paper describes the discovery of novel α-L-fucosidases and evaluation of their potential to catalyse the transglycosylation reaction leading to production of fucosylated human milk oligosaccharides. Seven novel α-L-fucosidase-encoding genes were identified by functional screening of a soil-derived metagenome library and expressed in E. coli as recombinant 6xHis-tagged proteins. All seven fucosidases belong to glycosyl hydrolase family 29 (GH 29). Six of the seven α-L-fucosidases were substrate-inhibited, moderately thermostable and most hydrolytically active in the pH range 6–7, when tested with para-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as the substrate. In contrast, one fucosidase (Mfuc6) exhibited a high pH optimum and an unusual sigmoidal kinetics towards pNP-Fuc substrate. When tested for trans-fucosylation activity using pNP-Fuc as donor, most of the enzymes were able to transfer fucose to pNP-Fuc (self-condensation) or to lactose. With the α-L-fucosidase from Thermotoga maritima and the metagenome-derived Mfuc5, different fucosyllactose variants including the principal fucosylated HMO 2’-fucosyllactose were synthesised in yields of up to ~6.4%. Mfuc5 was able to release fucose from xyloglucan and could also use it as a fucosyl-donor for synthesis of fucosyllactose. This is the first study describing the use of glycosyl hydrolases for the synthesis of genuine fucosylated human milk oligosaccharides.

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“…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.…”

Section: Introductionmentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

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

Nordvang

Luo

Zeuner

et al. 2014

Separation and Purification Technology

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Biocatalytic production of 3′-sialyllactose by use of a modified sialidase with superior trans-sialidase activity

Cited by 35 publications

References 31 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

Novel α-L-Fucosidases from a Soil Metagenome for Production of Fucosylated Human Milk Oligosaccharides

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

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