Enzymatic Cascade Synthesis Provides Novel Linear Human Milk Oligosaccharides as Reference Standards for xCGE-LIF Based High-Throughput Analysis

Fischöder, Thomas; Cajic, Samanta; Reichl, Udo; Rapp, Erdmann; Elling, Lothar

doi:10.1002/biot.201800305

Cited by 17 publications

(16 citation statements)

References 79 publications

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“…With respect to the known specificity, the production of Lacto- N -triose II (GlcNAc-β1,3-Gal-β1,4Glc) has to be considered critically. The β3GlcNAcT is known for good conversion of longer poly -LacNAc type II motives [30,32] but also for a bad acceptance of structures without N -acetyl group [23,50]. Nevertheless, we were able to reach 92.3% conversion of lactose within 48 h and nearly full conversion (99.7%) after 68 h reaction time as exemplarily depicted in Figure S6 (Supplementary Materials).…”

Section: Resultsmentioning

confidence: 74%

“…The direct salvage enzyme cascades utilized in this study are characterized in previous works by great space-time yields and high stability under optimized reaction conditions [23,38,39]. Additionally, their application into a repetitive batch approach maximizes the enzyme productivities and reduces, therefore, enzyme preparation time and effort to the minimum (Supplementary Materials, Table S1).…”

Section: Resultsmentioning

confidence: 99%

“…Identification and annotation of OS were carried out primarily based on our internal HMOS database (comparison of migration times). Identification and annotation of synthesized HMOS by comparison of migration times with in-house database entries (consisting of commercially available standards and previously annotated structures) was feasible for all synthesized structures since a wide range of linear HMOS, even the ones with minor structural differences, were extensively characterized and annotated previously [23]. Compositions of products were verified by LC-ESI-MS and MALDI-TOF-MS (Supplementary Materials, Figures MS5–MS17 and MTS1–MTS7).…”

Section: Resultsmentioning

confidence: 99%

“…Beside chemical routes for the HMOS synthesis [20,21], the enzyme-mediated HMOS tailoring is a very straightforward strategy [7,21,22,23,24,25]. Leloir -glycosyltransferases are especially known for their quantitative substrate conversion, a very high specificity, and high regioselectivity by utilizing activated nucleotide sugars [26].…”

Section: Introductionmentioning

confidence: 99%

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Enzymatic Cascades for Tailored 13C6 and 15N Enriched Human Milk Oligosaccharides

et al. 2019

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Several health benefits, associated with human milk oligosaccharides (HMOS), have been revealed in the last decades. Further progress, however, requires not only the establishment of a simple “routine” method for absolute quantification of complex HMOS mixtures but also the development of novel synthesis strategies to improve access to tailored HMOS. Here, we introduce a combination of salvage-like nucleotide sugar-producing enzyme cascades with Leloir-glycosyltransferases in a sequential pattern for the convenient tailoring of stable isotope-labeled HMOS. We demonstrate the assembly of [13C6]galactose into lacto-N- and lacto-N-neo-type HMOS structures up to octaoses. Further, we present the enzymatic production of UDP-[15N]GlcNAc and its application for the enzymatic synthesis of [13C6/15N]lacto-N-neo-tetraose for the first time. An exemplary application was selected—analysis of tetraose in complex biological mixtures—to show the potential of tailored stable isotope reference standards for the mass spectrometry-based quantification, using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) as a fast and straightforward method for absolute quantification of HMOS. Together with the newly available well-defined tailored isotopic HMOS, this can make a crucial contribution to prospective research aiming for a more profound understanding of HMOS structure-function relations.

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Section: Resultsmentioning

confidence: 74%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Enzymatic Cascades for Tailored 13C6 and 15N Enriched Human Milk Oligosaccharides

et al. 2019

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“…Combined with chemical synthesis to equip the core lactose with a multifunctional anomeric linker, a microarray was created and utilized for assessing protein binding [25]. Similarly, a library of defined linear HMO structures was synthesized recently using a set of microbial and human GTs [46]. These are excellent examples of how GTs are particularly strong for synthesis of clearly defined HMO structures of varying length with different branching, fucosylation, and sialylation patterns for analytical purposes and especially for bioactivity studies.…”

Section: Routes To Hmo Production Outside the Mammary Glandmentioning

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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One‐Step Preparation of Cell‐Free ATP Regeneration Module Based on Non‐Oxidative Glycolysis Using Thermophilic Enzymes

2022

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Adenosine triphosphate (ATP) is an essential cofactor for energy‐dependent enzymatic reactions that occur during in vitro biochemical conversion. Recently, an enzyme cascade based on non‐oxidative glycolysis, which uses starch and orthophosphate as energy and phosphate sources, respectively, for the regeneration of ATP from adenosine diphosphate, has been developed (Wei et al., ChemCatChem 2018, 10, 5597–5601). However, the 12 enzymes required for this system hampered its practical usability and further testing potential. Here, we addressed this issue by constructing co‐expression vectors for the simultaneous gene expression of the 12 enzymes in a single expression strain. All enzymes were sourced from (hyper)thermophiles, which enabled a one‐step purification via a heat‐treatment process. We showed that the combination of the two enabled the ATP regeneration system to function in a single recombinant Escherichia coli strain. Additionally, this work provides a strategy to rationally design and control proteins expression levels in the co‐expression vectors.

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Enzymatic Cascade Synthesis Provides Novel Linear Human Milk Oligosaccharides as Reference Standards for xCGE-LIF Based High-Throughput Analysis

Cited by 17 publications

References 79 publications

Enzymatic Cascades for Tailored 13C6 and 15N Enriched Human Milk Oligosaccharides

Enzymatic Cascades for Tailored 13C6 and 15N Enriched Human Milk Oligosaccharides

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

One‐Step Preparation of Cell‐Free ATP Regeneration Module Based on Non‐Oxidative Glycolysis Using Thermophilic Enzymes

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