Controlling the size and linkage type of biopolymers derived from sucrose

Please click Download on the upper right corner to see the full description

Bacterial α-Glucan and Branching Sucrases from GH70 Family: Discovery, Structure–Function Relationship Studies and Engineering

International audienceGlucansucrases and branching sucrases are classified in the family 70 of glycoside hydrolases. They are produced by lactic acid bacteria occupying very diverse ecological niches (soil, buccal cavity, sourdough, intestine, dairy products, etc.). Usually secreted by their producer organisms, they are involved in the synthesis of α-glucans from sucrose substrate. They contribute to cell protection while promoting adhesion and colonization of different biotopes. Dextran, an α-1,6 linked linear α-glucan, was the first microbial polysaccharide commercialized for medical applications. Advances in the discovery and characterization of these enzymes have remarkably enriched the available diversity with new catalysts. Research into their molecular mechanisms has highlighted important features governing their peculiarities thus opening up many opportunities for engineering these catalysts to provide new routes for the transformation of sucrose into value-added molecules. This article reviews these different aspects with the ambition to show how they constitute the basis for promising future developments

Natural and engineered transglycosylases: Green tools for the enzyme-based synthesis of glycoproducts

International audienceAn increasing number of transglycosylase-based processes provide access to oligosaccharides or glycoconjugates, some of them reaching performance levels compatible with industrial developments. Nevertheless, the full potential of transglycosylases has not been explored because of the challenges in transforming a glycoside hydrolase into an efficient transglycosylase. Advances in studying enzyme structure/function relationships, screening enzyme activity, and generating synthetic libraries guided by computational protein design or machine learning methods should considerably accelerate the development of these catalysts. The time has now come for researchers to uncover their possibilities and learn how to design and precisely refine their activity to respond more rapidly to the growing demand for well-defined glycosidic structures

Synthetic Derivatives of (+)- epi -α-Bisabolol Are Formed by Mammalian Cytochromes P450 Expressed in a Yeast Reconstituted Pathway

International audienceIdentification of the enzyme(s) involved in complex biosynthetic pathways can be challenging. An alternative approach might be to deliberately diverge from the original natural enzyme source and use promiscuous enzymes from other organisms. In this paper, we have tested the ability of a series of human and animal cytochromes P450 involved in xenobiotic detoxification to generate derivatives of (+)-epi-alpha-bisabolol and attempt to produce the direct precursor of hernandulcin, a sweetener from Lippia dulcis for which the last enzymatic steps are unknown. Screening steps were implemented in vivo in S. cerevisiae optimized for the biosynthesis of oxidized derivatives of (+)-epi-alpha-bisabolol by coexpressing two key enzymes: the (+)-epi-alpha-bisabolol synthase and the NADPH cytochrome P450 reductase. Five out of 25 cytochromes P450 were capable of producing new hydroxylated regioisomers of (+)-epi-alpha-bisabolol. Of the new oxidized bisabolol products, the structure of one compound, 14-hydroxy-(+)-epi-alpha-bisabolol, was fully elucidated by NMR while the probable structure of the second product was determined. In parallel, the production of (+)-epi-alpha-bisabolol derivatives was enhanced through the addition of a supplementary genomic copy of (+)-epi-alpha-bisabolol synthase that augmented the final titer of hydroxylated product to 64 mg/L. We thus demonstrate that promiscuous drug metabolism cytochromes P450 can be used to produce novel compounds from a terpene scaffold

Homopolysaccharides from lactic acid bacteria

International audienceIn addition to heteropolysaccharides of complex structure, lactic bacteria produce a variety of homopolysaccharides containing only either d-fructose or d-glucose. These fructans and glucans have a common feature in being synthesized by extracellular transglycosylases (glycansucrases) using sucrose as glycosyl donor. The energy of the osidic bond of sucrose enables the efficient transfer of a d-fructosyl or d-glucosyl residue via the formation of a covalent glycosyl-enzyme intermediate. In addition to the synthesis of high molecular weight homopolysaccharides, glycansucrases generally catalyse the synthesis of low molecular weight oligosaccharides or glycoconjugates when efficient acceptors, like maltose, are added to the reaction medium. While the enzymatic synthesis of fructans (levan and inulin) is poorly documented at the molecular level, the field of Streptococcus and Leuconostoc glucansucrases (glucosyltransferases and dextransucrases) has been well studied, both at the mechanistic and gene structure levels

A specific oligosaccharide-binding site in the alternansucrase catalytic domain mediates alternan elongation

Microbial ?-glucans produced by GH70 (glycoside hydrolase family 70) glucansucrases are gaining importance because of the mild conditions for their synthesis from sucrose, their biodegradability, and their current and anticipated applications that largely depend on their molar mass. Focusing on the alternansucrase (ASR) fromLeuconostoc citreumNRRL B-1355, a well-known glucansucrase catalyzing the synthesis of both high- and low-molar-mass alternans, we searched for structural traits in ASR that could be involved in the control of alternan elongation. The resolution of five crystal structures of a truncated ASR version (ASR?2) in complex with different gluco-oligosaccharides pinpointed key residues in binding sites located in the A and V domains of ASR. Biochemical characterization of three single mutants and three double mutants targeting the sugar-binding pockets identified in domain V revealed an involvement of this domain in alternan binding and elongation. More strikingly, we found an oligosaccharide-binding site at the surface of domain A, distant from the catalytic site and not previously identified in other glucansucrases. We named this site surface-binding site (SBS) A1. Among the residues lining the SBS-A1 site, two (Gln(700)and Tyr(717)) promoted alternan elongation. Their substitution to alanine decreased high-molar-mass alternan yield by a third, without significantly impacting enzyme stability or specificity. We propose that the SBS-A1 site is unique to alternansucrase and appears to be designed to bind alternating structures, acting as a mediator between the catalytic site and the sugar-binding pockets of domain V and contributing to a processive elongation of alternan chains

New Efficient Recombinant Expression System To Engineer Candida antarctica Lipase B▿ †

Here, we report the use of Yarrowia lipolytica as a versatile expression host for developing protein engineering approaches to modify the properties of Candida antarctica lipase B. A reliable screening protocol was defined and validated using a saturation mutagenesis library, yielding mutants displaying higher catalytic efficiencies than the wild-type enzyme

A Robust and Efficient Production and Purification Procedure of Recombinant Alzheimers Disease Methionine-Modified Amyloid-β Peptides.

An improved production and purification method for Alzheimer's disease related methionine-modified amyloid-β 1-40 and 1-42 peptides is proposed, taking advantage of the formation of inclusion body in Escherichia coli. A Thioflavin-S assay was set-up to evaluate inclusion body formation during growth and optimize culture conditions for amyloid-β peptides production. A simple and fast purification protocol including first the isolation of the inclusion bodies and second, two cycles of high pH denaturation/ neutralization combined with an ultrafiltration step on 30-kDa cut-off membrane was established. Special attention was paid to purity monitoring based on a rational combination of UV spectrophotometry and SDS-PAGE analyses at the various stages of the process. It revealed that this chromatography-free protocol affords good yield of high quality peptides in term of purity. The resulting peptides were fully characterized and are appropriate models for highly reproducible in vitro aggregation studies

Correction: A Robust and Efficient Production and Purification Procedure of Recombinant Alzheimers Disease Methionine-Modified Amyloid-β Peptides.

[This corrects the article DOI: 10.1371/journal.pone.0161209.]