Molecular characterization and optimization of enzymes involved in glycosaminoglycan biosynthesis

Glycosaminoglycans are biological active polysaccharides composed of repeating disaccharides composed of a hexuronic acid and a hexosamine. They have various pharmaceutical applications and traditionally this type of molecule is isolated from animal tissue. Since extraction from animal derivatives has serious limitations for the production of a large variety of defined glycosaminoglycans, there is a general interest in developing alternative systems enabling a more tightly controlled synthesis. During this research project we explored alternative ways of controlled chemo-enzymatic synthesis ofmonodisperse and uniform glycosaminoglycans for pharmaceutical applications, with a main focus on heparin. Heparin is a highly sulfated and complex glycosaminoglycan which is worldwide used as an anticoagulant to prevent blood clotting during surgery.Upon closer investigation of the heparin biosynthesis the D-glucuronyl C5-epimerase was recognized as a key enzyme, as this is the only reaction that cannot be done chemically. Two alternative approaches were taken to get closer to an industrial applicable enzyme; improvement of the animal heparin sulfate D-glucuronyl C5-epimerase and identification and isolation of novel candidate C5-epimerases from prokaryotes. Both approaches gave functional production of C5-epimerases.&nbsp