Rational design of an improved transglucosylase for production of the rare sugar nigerose

Franceus, Jorick; Dhaene, Shari; Decadt, Hannes; Vandepitte, J; Caroen, Jurgen; Eycken, Johan Van der; Beerens, Koen; Desmet, Tom

doi:10.1039/c9cc01587f

Cited by 29 publications

(23 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…All chemicals were of analytical grade and were obtained from Sigma-Aldrich/Merck (Darmstadt, Germany) or Carbosynth (Berkshire, United Kingdom), except for psicose (Izumori Lab, Kagawa University, Kagawa, Japan), glycerol (Chem-Lab, Zedelgem, Belgium), tagatose (Nutrilab, Giessen, The Netherlands) and trehalose (Cargill, Vilvoorde, Belgium). Kojibiose, nigerose and βGlc1P were produced in-house according to the procedures of Beerens et al [ 51 ], Franceus et al [ 52 ] and Van der Borght et al [ 53 ], respectively.…”

Section: Methodsmentioning

confidence: 99%

Discovery of a Kojibiose Hydrolase by Analysis of Specificity-Determining Correlated Positions in Glycoside Hydrolase Family 65

et al. 2021

Self Cite

View full text Add to dashboard Cite

The Glycoside Hydrolase Family 65 (GH65) is an enzyme family of inverting a-glucoside phosphorylases and hydrolases that currently contains 10 characterized enzyme specificities. However, its sequence diversity has never been studied in detail. Here, an in-silico analysis of correlated mutations was performed, revealing specificity-determining positions that facilitate annotation of the family’s phylogenetic tree. By searching these positions for amino acid motifs that do not match those found in previously characterized enzymes from GH65, several clades that may harbor new functions could be identified. Three enzymes from across these regions were expressed in E. coli and their substrate profile was mapped. One of those enzymes, originating from the bacterium Mucilaginibacter mallensis, was found to hydrolyze kojibiose and a-1,2-oligoglucans with high specificity. We propose kojibiose glucohydrolase as the systematic name and kojibiose hydrolase or kojibiase as the short name for this new enzyme. This work illustrates a convenient strategy for mapping the natural diversity of enzyme families and smartly mining the ever-growing number of available sequences in the quest for novel specificities.

show abstract

Section: Methodsmentioning

confidence: 99%

Discovery of a Kojibiose Hydrolase by Analysis of Specificity-Determining Correlated Positions in Glycoside Hydrolase Family 65

et al. 2021

Self Cite

View full text Add to dashboard Cite

show abstract

“…In a different study, the Q345F variant was further engineered in order to improve the activity and selectivity of SP towards nigerose formation without the need for a cosolvent. A quadruple mutant (R135Y-D342G-Y344Q-Q345F) was designed that forms nigerose with greater selectivity and a 68-fold improved catalytic efficiency in aqueous solution [77]. Molecular dynamics simulations suggested that the acceptor site of the quadruple mutant is more flexible than the one of the Q345F mutant, offering a possible explanation for its enhanced properties.…”

Section: Engineering Of Specificity or Selectivitymentioning

confidence: 99%

Sucrose Phosphorylase and Related Enzymes in Glycoside Hydrolase Family 13: Discovery, Application and Engineering

Franceus

Desmet

2020

IJMS

Self Cite

View full text Add to dashboard Cite

Sucrose phosphorylases are carbohydrate-active enzymes with outstanding potential for the biocatalytic conversion of common table sugar into products with attractive properties. They belong to the glycoside hydrolase family GH13, where they are found in subfamily 18. In bacteria, these enzymes catalyse the phosphorolysis of sucrose to yield α-glucose 1-phosphate and fructose. However, sucrose phosphorylases can also be applied as versatile transglucosylases for the synthesis of valuable glycosides and sugars because their broad promiscuity allows them to transfer the glucosyl group of sucrose to a diverse collection of compounds other than phosphate. Numerous process and enzyme engineering studies have expanded the range of possible applications of sucrose phosphorylases ever further. Moreover, it has recently been discovered that family GH13 also contains a few novel phosphorylases that are specialised in the phosphorolysis of sucrose 6F-phosphate, glucosylglycerol or glucosylglycerate. In this review, we provide an overview of the progress that has been made in our understanding and exploitation of sucrose phosphorylases and related enzymes over the past ten years.

show abstract

“…‘Rare sugars’, which are defined by the International Society of Rare Sugars as ‘monosaccharides and their derivatives that are present in limited quantities in nature’, may be promising sugar replacers, based on studies reporting low caloric content and reduced glycemic responses for rare sugars, such as allulose and tagatose, compared to conventional sugars [ 8 , 9 ]. Recently, rare sugars also attracted attention due to innovative biocatalysis strategies that allow efficient production of these otherwise insufficiently available sugars, including biosynthesis of the rare glucobioses kojibiose [ 10 ] and nigerose [ 11 ] (visualized in Figure 1 ) from widely available sugars using (engineered) sucrase phosphorylase [ 12 , 13 ]. These innovations allow further research on the metabolic health impact of rare sugars, which is still in an early stage, especially for rare disaccharides [ 14 ].…”

Section: Introductionmentioning

confidence: 99%

Metabolism and Health Effects of Rare Sugars in a CACO-2/HepG2 Coculture Model

et al. 2022

Self Cite

View full text Add to dashboard Cite

Non-alcoholic fatty liver disease (NAFLD) has become the most prevalent liver disease worldwide and is impacted by an unhealthy diet with excessive calories, although the role of sugars in NAFLD etiology remains largely unexplored. Rare sugars are natural sugars with alternative monomers and glycosidic bonds, which have attracted attention as sugar replacers due to developments in enzyme engineering and hence an increased availability. We studied the impact of (rare) sugars on energy production, liver cell physiology and gene expression in human intestinal colorectal adenocarcinoma (Caco-2) cells, hepatoma G2 (HepG2) liver cells and a coculture model with these cells. Fat accumulation was investigated in the presence of an oleic/palmitic acid mixture. Glucose, fructose and galactose, but not mannose, l-arabinose, xylose and ribose enhanced hepatic fat accumulation in a HepG2 monoculture. In the coculture model, there was a non-significant trend (p = 0.08) towards higher (20–55% increased) median fat accumulation with maltose, kojibiose and nigerose. In this coculture model, cellular energy production was increased by glucose, maltose, kojibiose and nigerose, but not by trehalose. Furthermore, glucose, fructose and l-arabinose affected gene expression in a sugar-specific way in coculture HepG2 cells. These findings indicate that sugars provide structure-specific effects on cellular energy production, hepatic fat accumulation and gene expression, suggesting a health potential for trehalose and l-arabinose, as well as a differential impact of sugars beyond the distinction of conventional and rare sugars.

show abstract

Rational design of an improved transglucosylase for production of the rare sugar nigerose

Abstract: Sucrose phosphorylase was redesigned to vastly improve its ability to produce a promising rare sugar from abundant bulk sugars.

Cited by 29 publications

References 23 publications

Discovery of a Kojibiose Hydrolase by Analysis of Specificity-Determining Correlated Positions in Glycoside Hydrolase Family 65

Discovery of a Kojibiose Hydrolase by Analysis of Specificity-Determining Correlated Positions in Glycoside Hydrolase Family 65

Sucrose Phosphorylase and Related Enzymes in Glycoside Hydrolase Family 13: Discovery, Application and Engineering

Metabolism and Health Effects of Rare Sugars in a CACO-2/HepG2 Coculture Model

Contact Info

Product

Resources

About