Biotechnological production; protein engineering

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The use of polysialic acids (polySia) as source materials for scaffolds requires production processes that guarantee availability at large scale in a continuous and controlled manner. Work combined in WP1 will realize this task by enzyme-reactor technology. Considering practical and financial aspects at least two enzymatic steps need to be included in the production chain. These are the activation of sialic acids to CMP-sialic acids by CMP-sialic acid synthetase and the production of the polymer catalyzed by a polysialyltransferase. The genes encoding these enzymes have been cloned from different bacteria and vertebrate species and can be expressed as active recombinant proteins in either bacterial or insect cells. The bioreactor technology available at the Institut für Technische Chemie (TCI) will enable enzyme production and purification at various scales. The PassflowTM technology developed at the Institut für Organische Chemie (OCI) provides the platform for optimized enzyme reactors. To set up the enzymatic synthesis of modified polySia using sialic acid derivatives produced in WP2, the substrate specificity of the respective enzymes towards these and already existing derivatives will be determined at the Abteilung Zelluläre Chemie (ZCh). With the goal to expand the enzymes' substrate spectra, structure-based as well as random mutagenesis approaches will be carried out.