Thermostable β-galactosidases for the synthesis of human milk oligosaccharides

Zeuner, Birgitte; Nyffenegger, Christian; Mikkelsen, Jørn Dalgaard; Meyer, Anne S.

doi:10.1016/j.nbt.2016.01.003

Cited by 41 publications

(45 citation statements)

References 19 publications

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“…The reaction yield at 95 °C was three times higher than that obtained at 60 °C (32.01 and 10.58% respectively). Similar effect was observed by Zeuner et al [30] who reported a sixfold increase of the reaction yield (from 0.9 to 5.4%) in the N-acetyllactosamine synthesis catalyzed by βgalactosidase from Pyrococcus furiosus when the temperature was raised from 40 to 90 °C. In another work, the conversion yield of GOS in the presence of β-galactosidase from Bacillus circulans was three times greater at 60 °C than at 25 °C (5 and 15 g/L, respectively) [31].…”

Section: Synthesis Of Fucosylated Oligosaccharidessupporting

confidence: 86%

Improvement of Fucosylated Oligosaccharides Synthesis by α-L-Fucosidase from Thermotoga maritima in Water-Organic Cosolvent Reaction System

Robles-Arias

Garcı́a-Garibay

Alatorre‐Santamaría

et al. 2021

Appl Biochem Biotechnol

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The effects of water activity (a w ), pH, and temperature on transglycosylation activity of α-L-fucosidase from Thermotoga maritima in the synthesis of fucosylated oligosaccharides were evaluated using different water -organic cosolvent reaction systems. The optimum conditions of transglycosylation reaction were the pH range between 7 and 10, and temperature 90-95°C. The addition of organic cosolvent decreased α-L-fucosidase transglycosylation activity in the following order: acetone > dimethyl sulfoxide (DMSO) > acetonitrile (0.51 > 0.42 > 0.18 mM/h). However, the presence of DMSO and acetone enhanced enzyme-catalyzed transglycosylation over hydrolysis as demonstrated by the obtained transglycosylation/hydrolysis rate (r T/H ) values of 1.21 and 1.43, respectively. The lowest r T/H was calculated for acetonitrile (0.59), though all cosolvents tested improved the transglycosylation rate in comparison to a control assay (0.39). Overall, the study allowed the production of fucosylated oligosaccharides in water-organic cosolvent reaction media using α-L-fucosidase from Thermotoga maritima as biocatalyst.

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Section: Synthesis Of Fucosylated Oligosaccharidessupporting

confidence: 86%

Improvement of Fucosylated Oligosaccharides Synthesis by α-L-Fucosidase from Thermotoga maritima in Water-Organic Cosolvent Reaction System

Robles-Arias

Garcı́a-Garibay

Alatorre‐Santamaría

et al. 2021

Appl Biochem Biotechnol

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“…Hence, a large incentive for biosynthetic production of authentic HMO structures for use in, for example, infant formulae or as functional food ingredients, exist. Lacto‐ N ‐triose II (LNT2, GlcNAcβ1‐3Galβ1‐4Glc, Scheme ) is a central HMO backbone moiety that is particularly relevant to synthesize not only because of its reported putative prebiotic effects in itself but notably because LNT2 can serve as a backbone precursor for further chain‐elongation reactions in biomimetic HMO production . Biocatalytic synthesis of LNT2 on the basis of the addition of N ‐acetylglucosamine to lactose can be accomplished either by β‐ N ‐acetylglucosaminyltransferases or by transglycosylation catalyzed by the β‐ N ‐acetylhexosaminidases of glycosyl hydrolase family 20 (GH20) .…”

Section: Introductionmentioning

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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“…However, the strongest tool to efficiently improve transglycosylation activity and/or diminish hydrolytic activity in GHs is protein engineering. Enzymatic transglycosylation has shown great potential in HMO synthesis, especially for sialylation or fucosylation of lactose or LNT [42,48,51,52,53,54,55,56,57], but also for synthesis of HMO core structures [58,59,60,61]. This technology holds great potential to expand the current limited industrial-scale HMO portfolio, but much of this potential relies on either the discovery of novel transglycosidases or protein engineering of the enzymes for improved transglycosylation efficiency.…”

Section: Routes To Hmo Production Outside the Mammary Glandmentioning

confidence: 99%

“…Using one of the most efficient commercial β-galactosidase preparations—Biolacta, which contains several Bacillus circulans β-galactosidases [151]—a 19% yield of LNnT was obtained from a reaction with Lac and LNT2 [61] (Table 1). A one-pot cascade reaction from Lac to LNnT was not accomplished due to low LNT2 yields [60]. β-Galactosidase-catalyzed production of LNT from LNT2 has only been reported with a o -nitrophenyl galactoside donor using a GH35 β1,3-galactosidase from B. circulans [61].…”

Section: Abundant Natural Substrates From Dairy and Agro-industriamentioning

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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Thermostable β-galactosidases for the synthesis of human milk oligosaccharides

Cited by 41 publications

References 19 publications

Improvement of Fucosylated Oligosaccharides Synthesis by α-L-Fucosidase from Thermotoga maritima in Water-Organic Cosolvent Reaction System

Improvement of Fucosylated Oligosaccharides Synthesis by α-L-Fucosidase from Thermotoga maritima in Water-Organic Cosolvent Reaction System

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

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

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