Cellobiohydrolase I as a chiral sector

Sammanfattning: The protein cellobiohydrolase I (CBH I) immobilized on silica particles has been used as a chiral selector in liquid chromatography (LC) and the free enzyme has served as a chiral complexing agent in the background electrolyte in capillary electrophoresis (CE) using the partial tilling technique.The CE systems were much more efficient and more solutes could be enantioseparated than in the LC systems. Due to the high enantioselectivity, a few substances could be completely enantioresolved in an injected CBH I zone that was shorter than the sample zone. The affinity for the amino alcohol propranolol was very high at neutral pH and in order to displace this solute for detection, a plug of the disaccharide cellobiose was injected after the sample zone. The high affinity of propranolol combined with the displacing ability of cellobiose made it possible to preconcentrate the solute prior to enantioseparation by compression of long dilute sample zones.The mechanisms of retention and enantioselectivity in LC have been studied using charged and uncharged additives in the mobile phase. Site-specific mutation and covalent modification of the protein were also used to elucidate retention mechanisms. The enantioselective sites of amino alcohols and the acid warfarin overlap in the vicinity of the catalytically active site of CBH I. The main binding mechanism for both amines and acids was electrostatic attraction. Interestingly, both the retention and the enantioselectivity increased upon changing from potassium to sodium as mobile phase cation. The thermodynamics of the system was studied by microcalorimetry and LC. Both enantiomers of the amino alcohol alprenolol bind to CBH I with an endothermic enthalpy change. Hence, the complexation is entropy driven.Molecular mechanics calculations were carried out on the complex between propranolol and CBH I. The bound conformation of (S)-piopranolol differs both in geometry and energy compared to the preferred conformation in water solution. The ability of different parameter setups to reproduce the experimental structure of the (S)-propranolol - CBH I complex was investigated. One setup was chosen to suggest possible conformations of the (R)-enantiomer. The water structure in the bindning site seems to be an important factor in the complexation mechanism.