Selective oligosaccharide production
For many biotechnological processes, a large part of the manufacturing costs and a large part of the use of resources is needed for product recovery and purification. Integration of synthesis and in-situ product removal can improve the product yield and thereby reduce manufacturing costs. By using a selective biocomplexant, with a non-specific biocatalyst, a highly selective process can be developed. Because of the wide diversity of biocomplexants found in nature, the process can be made very flexible resulting in the development of a generic process.
Figure 1: Adapted from: M.E. Bruins et al. (2003) Oligosaccharide synthesis by the hyperthermostable ß-glucosidase from Pyrococcus furiosus: kinetics and modelling. Enzyme and Microbial Technology 33(1):3-11Oligosaccharides, for example, are often manufactured through enzymatic synthesis. The enzymatic synthesis using mono and/or disaccharides as substrates results in the production of a wide range of oligosaccharides, of diverse chain length, monomer sequence and chain morphology. Since specific oligosaccharide synthesis reactions are not yet cost effective due to the lack of diversity of the available catalysts the oligosaccharides has to be purified to obtain pure oligosaccharides. Fig. 1 shows a typically enzymatic reaction where oligosaccharides are synthesized, but the just formed product is hydrolyzed resulting in poor yields. When the just formed product is removed out of the reaction, much higher yields can be obtained as seen in figure 2.
Figure 2: Adapted from: M.A. Boon et al.(2000) Enzymatic synthesis of oligosaccharides: Product removal during a kinetically controlled reaction. Biotechnol. Bioeng. 70:411-420 In this project we are using an enzymatic synthesis of oligosaccharides as a model system. This model system has to be integrated with a highly specific separation of oligosaccharides, to show the increased conversion of substrate into product. In nature, lectins are common biocomplexants. In a sustainable process the biocomplexants have to be removed from the reaction mixture in order to regenerate the biocomplexant. Immobilization of the biocomplexant on magnetic beads is considered as an option. In this way the complex of biocomplexants and bound product can easily be picked out from the reaction mixture using only magnetic fields. The molecules can easily be transported for regeneration when the magnetic field is switched off (see figure 3). Extremes of pH, chaotropic salts, ionic strength and temperature shocks can dissociate the product from the biocomplexant, mostly by denaturating the biocomplexant. For a sustainable process the product has to be dissociated without denaturating the biocomplexant. Therefore, milder dissociation conditions has to be found.
Figure 3:Integration of a non-specific enzymatic reaction and a highly specific separation using biocomplexants immobilized on magnetic particles. After loading, the biocomplexants are removed from the reaction mixture using magnetic fields.