Clone, Expression, Purification And Crystallographic Analysis Of Fatty Acid Synthase And Glycosyl Pyrophosphorylase

Many microbiology enzymes have great potential in industrial application and have played very important roles in human life. Studies on the Physiochemical Characteristics,biological activity and catalytic mechanism of enzymes are important for their possible wider applications. Nowadays, enzymes have been commonly applied in food fermentation and biomedical regent development.In this thesis, two enzymes were selected to probe the crystal structures in order to provide clue to understand their function and catalytic mechanism, including a putative fatty acid synthetase PFS (PF2046) from Pyrococcus furio sus and a pyrophosphorylase FKP from Bacteroides.fragilis 9343. Both of these genes were cloned into the expression vector pMCSG7 and used E.coli BL21 (DE3) as a host for large-scale protein expression. The bacteria were broken and the soluble portion was purified by nickel affinity column, followed by ion-exchange and size-exclusion chromatography. The purity of the two enzymes was above 95%. Protein crystallization was performed by using commercial screening kits. PFS's structure was solved through the B-factor sharpening method when protein crystal was big enough for data collection. FKP's small crystal was diffracted to 3.5 ? and was very close to structure solving. Structure and biochemistry characterization of these two enzymes will provide people more information to develop new food additives or their extensive application in food and related industry.