Rangées de graines.. © INRA, Elena Schweitzer © Fotolia

Our results

Contents
  1. Introduction
  2. Human milk digestion in the preterm infant: impact of technological treatments
  3. Research & Innovation 2017 - For Food and Biobased Products
  4. The way in which proteins aggregate when heated may change their sensitising potency
  5. Enhancing the viability of spray-dried probiotic bacteria by stimulating their stress tolerance
  6. To stick or not to stick? Pulling pili sheds new light on biofilm formation
  7. When biopolymers selfassemble: a balance between energy and entropy.
  8. Mimicking the gastrointestinal digestion in a lab-on-a-chip:the microdigester
  9. How a milk droplet becomes a powder grain
  10. Research & Innovation 2016 - For Food and Bioproducts
  11. A new process for the biorefining of plants
  12. Under the UV light : the bacterial membrane
  13. Reverse engineering or how to rebuild ... bread!
  14. Green Chemistry: a step towards lipid production in yeast
  15. Individually designed neo-enzymes for antibacterial vaccines
  16. Multi-scale mechanical modelling: from the nanometric scale to the macroscopic properties of bread crumb
  17. Minimill: 500 g to assess the milling value of soft wheats
  18. Microbial production of lipids for energy or chemical purposes
  19. The discrete role of ferulic acid in the assembly of lignified cell wall
  20. Eco-design of composites made from wood co-products
  21. Analysis of volatile compounds enables the authentication of a poultry production system
  22. Nanoparticles as capping agents for biopolymers microscopy
  23. Pasteurisation, UHT, microfiltration...All the processes don't affect the nutritional quality of milk in the same way
  24. Integration of expert knowledge applied to cheese ripening
  25. Controlling cheese mass loss during ripening
  26. The shape memory of starch
  27. Research & Innovation 2015 - For Food & Biobased Products
  28. Behaviour of casein micelles during milk filtering operations
  29. Overaccumulation of lipids by the yeast S. cerevisiae for the production of biokerosine
  30. Sequential ventilation in cheese ripening rooms: 50% electrical energy savings
  31. An innovative process to extract bioactive compounds from wheat
  32. Diffusion weighted MRI: a generic tool for the microimaging of lipids in food matrices
  33. Characterization of a major gene of anthocyanin biosynthesis in grape berry
  34. New enzyme activity detectors made from semi-reflective biopolymer nanolayers
  35. Improving our knowledge about the structure of the casein micelle
  36. Heating milk seems to favour the development of allergy in infants
  37. Fun with Shape
  38. Using volatile metabolites in meat products to detect livestock contamination by environmental micropollutants
  39. SensinMouth, when taste makes sense
  40. A decision support system for the fresh fruit and vegetable chain based on a knowledge engineering approach
  41. SOLEIL casts light on the 3D structure of proteins responsible for the stabilisation of storage lipids in oilseed plants
  42. A close-up view of the multi-scale protein assembly process
  43. Controlling the drying of infant dairy products by taking water-constituent interactions into account
  44. Polysccharide nanocrystals to stabilise pickering emulsions
  45. Discovery of new degradative enzymes of plant polysaccharides in the human intestinal microbiome
  46. A durum wheat flour adapted for the production of traditional baguettes
  47. Virtual modelling to guide the construction of « tailored-made » enzymes
  48. How far can we reduce the salt content of cooked meat products?
  49. Diffusion of organic substances in polymer materials: beyond existing scaling laws
  50. Smart Foams : various ways to destroy foams on demand !
  51. Dates, rich in tannins and yet neither bitter nor astringent
  52. Sodium content reduction in food
  53. Research & Innovation 2014

Individually designed neo-enzymes for antibacterial vaccines

Shigellosis is one of the most severe enteric bacterial infections to strike in third world countries. Its prevention focuses on, among other things, the design of antibacterial glycoconjugate vaccines based on the use of synthetic oligosides that mimic the molecular motives found at the surface of pathogenic bacteria. These complex carbohydrates are capable of stimulating an immune response, but their synthesis through chemical means is sometimes difficult. In view of these difficulties, researchers have devised new chemo-enzymatic synthesis pathways. To do this, they succeeded in building new enzymes that are perfectly adapted for the catalysis of targeted synthesis reactions. This is the very first example of the creation of a new specificity for this family of biocatalysts. This approach underlines the remarkable potential of new protein engineering techniques used in combination with molecular modelling approaches to create specifically tailored enzymes, particularly for the development of new chemo-enzymatic synthesis pathways.

Glycochemistry: laying the groundwork for the design of antibacterial vaccines   

Shigellosis is one of the most severe enteric bacterial infections to strike in third world countries.  Its prevention focuses on, among other things, the design of first-generation antibacterial glycoconjugate vaccines that use the antigenic properties of synthetic oligosides that mimic the lipopolysaccharide of the bacteria responsible for the infection.  
The chemical synthesis of these oligosides is difficult. Enzyme catalysts have been proposed as a possible alternative to get around these difficulties. Nevertheless, the specificity of wild type enzymes is not always compatible with the transformation of non-natural substrates that carry the functional groups enabling their eventual chemical modification.   
The development of new chemo-enzymatic synthesis pathways therefore requires the generation of new enzymes that are perfectly adapted to the desired function.  Within this perspective, the strategy implemented in partnership with L. Mulard (Pasteur Institute, Paris) within the framework of the OPTIGluc project funded by the French National Research Agency (ANR), consisted in designing chemo-enzymatic synthesis pathways for osidic antigens that include a 1,2-cis enzymatic glucosylation step.
Since none of the available wild-type enzymes was able to effectively catalyse the reaction, the challenge consisted in creating new enzymes using semi-rational engineering.  

Obtaining the glucidic component of vaccines though chemo-enzymatic synthesis

The chemo-enzymatic synthesis of two selected antigen patterns involves the production of glucosylated derivatives of α-D-GlcNAc-OAll (allyl 2-N-acetyl-2-deoxy-α-D-glucopyranoside) and the α-L-Rha-OMe (methyl α-L-rhamnopyranoside). To carry out these syntheses, we chose the amylosucrase from Neisseria polysaccharea, a transglucosidase that uses sucrose, an abundant and very inexpensive substrate, as a glucose donor.  The natural enzyme catalyses the synthesis of the allyl-α-D-Glcp-(1→4)-α-D-GlcNAc, with a yield of less than 6% and does not recognise the α-methyl-L-rhamnose to produce the target methyl-α-D-Glcp-(1→3)-α-L-Rhap.
The re-engineering of the catalyst presented a dual challenge: conserving its specificity towards the donor substrate and creating efficient catalysts adapted to the regiospecific glucosylation of the acceptors. An integrated re-engineering strategy of the acceptor binding site that combined molecular modelling with semi-random mutagenesis was adopted. Using in silico analysis of the enzyme:non-natural acceptor complexes, seven target amino acids were identified and then systematically replaced by 19 possible amino acid substitutes. The library obtained, with a total of 133 mutants, was screened for the two reactions of interest. Several mutants capable of recognising the methyl α-L-rhamnopyranoside and of glucosylating it could therefore be isolated.
Targeted enzyme reaction in the chemo-enzymatic synthesis pathway of antigenic patterns mimicking the lipopolysaccharide of S. flexneri bacteria, serotypes 3a and 1b.

Figure 1: Targeted enzyme reaction in the chemo-enzymatic synthesis pathway of antigenic patterns mimicking the lipopolysaccharide of S. flexneri bacteria, serotypes 3a and 1b.. © INRA
Figure 1: Targeted enzyme reaction in the chemo-enzymatic synthesis pathway of antigenic patterns mimicking the lipopolysaccharide of S. flexneri bacteria, serotypes 3a and 1b. © INRA

This is the very first time that a new specificity has been created for this family of enzymes and for α-transglucosidases in general. Concerning the glucosylation of the allyl 2-N-acetyl-2-deoxy-α-D-glucopyranoside, mutants 130 times more effective than the wild type enzyme were obtained that glucosylate this acceptor with yields over 80%. These neo-enzymes were used in programmed chemo-enzymatic pathways, allowing the successful validation of the strategy initially proposed. Two patents were filed at the end of this study.

New prospects for human health

Focusing on Shigella flexneri, a target of major interest to human health, our approach shows the relevance of innovative synthesis strategies in the area of glycochemistry, using specifically designed enzymes. Our efforts will now be concentrated on the structural analysis of improved variants and the development of innovative virtual screening techniques that will make it possible to optimise the creation of neo-enzymes.

Partners

Institut Pasteur, Biomolecule chemistry Unit (URA CNRS 2128)

See also

  • Champion E, André I, Moulis C, Boutet J, Descroix K, Morel S, Monsan P, Mulard LA, Remaud-Simeon M. 2009a. Design of alpha-transglucosidases of controlled specificity for programmed chemoenzymatic synthesis of antigenic oligosaccharides. J Am Chem Soc 131(21):7379-89. (JCR = 8.091)
  • Champion E, André I, Mulard LA, Monsan P, Remaud-Siméon M, Morel S. 2009b. Synthesis of L-rhamnose and N-acetyl-D-glucosamine derivatives entering in the composition of bacterial polysaccharides by use of glucansucrases . J Carbohydr Chem 28:142-159. (JCR = 1.114)
  • CHAMPION E., ANDRÉ I., MOULIS C., MOREL S., MONSAN P., BOUTET J., MULARD L., REMAUD-SIMÉON M. (2008) Mutants of Glycoside hydrolases and uses thereof for synthesizing complex oligosaccharides and disaccharide intermediates. EP08 290 238.8// PCT/IB2009/005343
  • CHAMPION E., ANDRÉ I., MOULIS C., MOREL S., MONSAN P., DESCROIX K., MULARD L., REMAUD-SIMÉON M. (2008) Mutants of Amylosucrase and uses thereof for synthesizing complex oligosaccharides and disaccharide intermediates. EP08 290 237.0// PCT/IB2009/005325