A Mechanism Study Of Pseudomonas Cepacia Lipase’s Stereorecognition Towards Chiral Primary Alcohol Via Chemical Modification

The aim of this dissertation is to analyze the stereorecognition mechanism of Pseudomonas cepacia lipase(PcL) towards primary alcohols via the method of chemical modification combined with protein mass spectrometry analysis and molecular modeling of PcL.The main contents of this work were as follows:The systems of kinetic resolution of different chiral alcohols were established. The results showed that the enantioselectivity changed with the structure of substrates, For secondary alcohols, the E value was at a high level; for primary alcohols, when chiral primary alcohols with non-acyloxy oxygen atom were able to be resoluted by PcL with a reasonably high enantioselectivity, but those without the extra oxygen not, the stereorecognition did not obey the empirical rule that the secondary-alcohol did.The amino acid residues that can be modified of PcL were analyzed.The results showed that the enantioselectivity of primary alcohols catalysed by tyrosine modified PcL had a significant change. The stronger the ability to form hydrogen bond between phenolic hydroxyl of Tyr and primary alcohol, the higher E value was.Inactivation curve of Tyr modification of PcL was studied.The structure of modified PcL was characterized by circular dichroism and MALDI-TOF mass measurements. The results showed that the secondary structure of tyrosine modified lipase changed, and Tyr29residue which was located at bingding pocket of PcL was modified.Molecular modeling of tyrosine modified PcL and unmodified PcL were investigated. The results showed that the formation of the hydrogen bond between phenolic hydroxyl group of Tyr and the non-acyloxy oxygen of substrate did affect the enantioselectivity. So hydrogen bond affect was one of the chiral recognition mechanisms of PcL toward primary alcohols.