Biotechnological production; protein engineering

使用聚唾液酸（polySia）作为支架的源材料需要以连续和受控的方式保证大规模可用性的生产工艺。结合WP 1中的工作，将利用酶反应器技术实现这一任务。考虑到实际和财务方面，生产链中至少需要包括两个酶促步骤。这些是通过CMP-唾液酸合成酶将唾液酸活化为CMP-唾液酸和通过聚唾液酸转移酶催化的聚合物的产生。编码这些酶的基因已经从不同的细菌和脊椎动物物种中克隆，并且可以在细菌或昆虫细胞中表达为活性重组蛋白。技术化学研究所（TCI）提供的生物反应器技术将使各种规模的酶生产和纯化成为可能。有机化学研究所（OCI）开发的PassflowTM技术为优化酶反应器提供了平台。为了使用WP 2中生产的唾液酸衍生物建立修饰的polySia的酶促合成，将在Abteilung Zelluläre Chemie（ZCh）确定相应酶对这些和现有衍生物的底物特异性。为了扩大酶的底物谱，将进行基于结构的以及随机诱变方法。

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.