Immobilization of CMP‐Sialic Acid Synthetase and α2,3‐Sialyltransferase for Cascade Synthesis of 3′‐Sialyl β‐D‐Galactoside with Enzyme Reuse

Schelch, Sabine; Köszagová, Romana; Kuballa, Jürgen; Nidetzky, Bernd

doi:10.1002/cctc.202101860

Cited by 8 publications

(4 citation statements)

References 67 publications

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“…The oligosaccharide content change caused by adding sugar during processing has not yet been explained. From this study, it was speculated that monocrystal rock sugar (used in this study) could be hydrolyzed into glucose and fructose [ 28 , 29 , 30 , 31 ], in which the glucose could be dehydrated and condensed with galactose sialate by heat treatment to form 3′-sialyllactose. In sum, it showed that adding sugar (monocrystal rock sugar) could increase the nutritional value of edible bird’s nest products within the tested range.…”

Section: Resultsmentioning

confidence: 99%

Determination of 3′-Sialyllactose in Edible Bird’s Nests and the Effect of Stewing Conditions on the 3′-Sialyllactose Content of Edible Bird’s Nest Products

Zhang¹,

Liang²,

Bai³

et al. 2023

Molecules

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Sialyllactose is an acidic oligosaccharide that has an immune-protective effect against pathogens and contributes to developing the immune system and intestinal microbes. In this study, a method for the determination of 3′-sialyllactose by high-performance liquid chromatography tandem mass spectrometry was established. The sample was treated with 0.1% formic acid methanol solution, and the gradient elution was performed with 0.05% formic acid water and 0.1% formic acid acetonitrile. The hydrophilic liquid chromatographic column was used for separation. The results showed that the linearity was good in the concentration range of 1~160 μg/L. The limit of detection (LOD) and the limit of quantification (LOQ) of the method were 0.3 μg/kg and 1.0 μg/kg, the recovery range was 91.6%~98.4%, and the relative standard deviation (RSD) was 1.5%~2.2%. This method is fast and sensitive. In addition, the 3′-sialyllactose content in edible bird’s nest products produced by different processes was studied. It was found that within the tested range, 3′-sialyllactose in edible bird’s nest products increased with the intensity of stewing and increased with the addition of sugar. In short, the results provided a new method for detecting the nutritional value of edible bird’s nests, as well as a new direction for improving the nutritional value of edible bird’s nest products.

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Section: Resultsmentioning

confidence: 99%

Determination of 3′-Sialyllactose in Edible Bird’s Nests and the Effect of Stewing Conditions on the 3′-Sialyllactose Content of Edible Bird’s Nest Products

Zhang¹,

Liang²,

Bai³

et al. 2023

Molecules

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“…Utilizing a confinement and compartmentalization strategy enabled higher substrate concentrations to be used. Immobilization has also been used to improve the overall efficiency of sialyl galactoside synthesis using a two-step route from Neu5Ac . Initially, immobilization via N-terminus His-tags of CMP-sialic acid synthetase from Neisseria meningitis ( Nm CSS) and sialyltransferase from Pasteurella dagmatis ( Pd SiaT) yielded poor immobilizates in all instances (via affinity or epoxy on a range of commercial carriers).…”

Section: Using Biocatalysis To Provide the Building Blocks For Bioche...mentioning

confidence: 99%

“…Immobilization has also been used to improve the overall efficiency of sialyl galactoside synthesis using a two-step route from Neu5Ac. 124 Initially, immobilization via N-terminus His-tags of CMP-sialic acid synthetase from Neisseria meningitis ( Nm CSS) and sialyltransferase from Pasteurella dagmatis ( Pd SiaT) yielded poor immobilizates in all instances (via affinity or epoxy on a range of commercial carriers). Therefore, constructs containing the Z basic2 protein module (a 7 kDa, 58 amino acid module with a high charge ratio) were made and tested, affording high immobilization yields and full activity recovery on all of the carriers versus the native enzymes ( Figure 6 ).…”

Section: Using Biocatalysis To Provide the Building Blocks For Bioche...mentioning

confidence: 99%

Biocatalytic Approaches to Building Blocks for Enzymatic and Chemical Glycan Synthesis

Dolan

Cosgrove

Miller

2022

JACS Au

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While the field of biocatalysis has bloomed over the past 20−30 years, advances in the understanding and improvement of carbohydrate-active enzymes, in particular, the sugar nucleotides involved in glycan building block biosynthesis, have progressed relatively more slowly. This perspective highlights the need for further insight into substrate promiscuity and the use of biocatalysis fundamentals (rational design, directed evolution, immobilization) to expand substrate scopes toward such carbohydrate building block syntheses and/or to improve enzyme stability, kinetics, or turnover. Further, it explores the growing premise of using biocatalysis to provide simple, cost-effective access to stereochemically defined carbohydrate materials, which can undergo late-stage chemical functionalization or automated glycan synthesis/polymerization.

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“…Due to multiple arginine residues clustered on one of its sides, the Z basic2 exhibits highly localized, positive charge density. , These properties enable strong protein binding on anionic supports for enzyme immobilization. A number of studies have shown that enzyme fusions with Z basic2 can be immobilized with excellent efficiency, partly due to the fact that the immobilization is orientationally controlled; that is, surface tethering occurs mainly via the Z basic2 module. ,, The current study was performed with the idea that mechanically and dimensionally stronger NFC/CMC-based scaffolds differing in pore sizes, interconnected morphology or structure, and negative surface charge might present a new class of solid supports for the immobilization of Z basic2 enzymes. The enzymes used in this proof-of-principle analysis were from the class of sugar-nucleotide dependent (Leloir) glycosyltransferases.…”

Section: Introductionmentioning

confidence: 99%

3D Printed Porous Nanocellulose-Based Scaffolds As Carriers for Immobilization of Glycosyltransferases

Lackner

Liu

Štiglic

et al. 2022

ACS Appl. Bio Mater.

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Biocatalysis is increasingly becoming an alternative method for the synthesis of industrially relevant complex molecules. This can be realized by using enzyme immobilized polysaccharide-based 3D scaffolds as compatible carriers, with defined properties. Especially, immobilization of either single or multiple enzymes on a 3D printed polysaccharide scaffold, exhibiting well-organized interconnected porous structure and morphology, is a versatile approach to access the performance of industrially important enzymes. Here, we demonstrated the use of nanocellulose-based 3D porous scaffolds for the immobilization of glycosyltransferases, responsible for glycosylation in natural biosynthesis. The scaffolds were produced using an ink containing nanofibrillated cellulose (NFC), carboxymethyl cellulose (CMC), and citric acid. Direct-ink-writing 3D printing followed by freeze-drying and dehydrothermal treatment at elevated temperature resulted in chemically cross-linked scaffolds, featuring tunable negative charges (2.2−5.0 mmol/g), pore sizes (10−800 μm), fluid uptake capacity, and exceptional dimensional and mechanical stability in the wet state. The negatively charged scaffolds were applied to immobilize two sugar nucleotide-dependent glycosyltransferases (C-glycosyltransferase, Z basic2 -CGT; sucrose synthase, Z basic2 -SuSy), each harboring a cationic binding module (Z basic2 ) to promote charge-based enzyme adsorption. Both enzymes were immobilized at ∼30 mg of protein/g of dry carrier (∼20% yield), independent of the scaffold used. Their specific activities were 0.50 U/mg (Z basic2 -CGT) and 0.19 U/mg (Z basic2 -SuSy), corresponding to an efficacy of 37 and 18%, respectively, compared to the soluble enzymes. The glycosyltransferases were coimmobilized and shown to be active in a cascade reaction to give the natural C-glycoside nothofagin from phloretin (1.0 mM; ∼95% conversion). All enzyme bound scaffolds showed reusability of a maximum of 5 consecutive reactions. These results suggest that the 3D printed and cross-linked NFC/CMC-based scaffolds could present a class of solid carriers for enzyme (co)-immobilization, with promising applications in glycosyltransferase-catalyzed synthesis and other fields of biocatalysis.

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Immobilization of CMP‐Sialic Acid Synthetase and α2,3‐Sialyltransferase for Cascade Synthesis of 3′‐Sialyl β‐D‐Galactoside with Enzyme Reuse

Cited by 8 publications

References 67 publications

Determination of 3′-Sialyllactose in Edible Bird’s Nests and the Effect of Stewing Conditions on the 3′-Sialyllactose Content of Edible Bird’s Nest Products

Determination of 3′-Sialyllactose in Edible Bird’s Nests and the Effect of Stewing Conditions on the 3′-Sialyllactose Content of Edible Bird’s Nest Products

Biocatalytic Approaches to Building Blocks for Enzymatic and Chemical Glycan Synthesis

3D Printed Porous Nanocellulose-Based Scaffolds As Carriers for Immobilization of Glycosyltransferases

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