Cloning And Expression Of The E. Coli AHAS â…  And The Studies On Its Catalytic Function And Mechanism

Acetolactate synthase (AHAS) is the first enzyme unique to the biosynthesis of the branched chain amino acids. It can be used as a target for screening the herbicide. Because of its low soluble expression and easy deactivation, it has a great significance to study its preparation and the preservation methods. AHAS can take pyruvate,2-ketobutyric acid or benzaldehyde as its substrates, which laids a good foundation for the development of its functionality. Currently, there still exist so many issues of the ThDP-dependent enzymes. As one of a ThDP-dependent enzymes, it’s very helpful to studied the catalytic mechanism of AHAS. Focusing on AHAS Ⅰ, we not only studied the cloning, expression and purification of it, and also explored the function and the catalytic mechanism of it in this study.In this study, we select pGEX-4T-1as an expression vector to construct the pGEX-4T-1-livB recombinant plasmid which contains the catalytic subunit of acetolactate synthase. The plasmid was transformed into E. coli BL21(DE3) strain and the expression of the target enzyme was induced with IPTG. The recombinant AHAS Ⅰ was purified with glutathione Sepharose column. The results of SDS-PAGE and Western blot showed we obtained the recombinant AHAS Ⅰ with a purity over95%, and the molecular weight of it was about80KDa. In addition, the soluble expression of the enzyme was greatly improved, and the concentration of the fusion protein was0.52mg/mL. It could be because of the advantages of pGEX-4T-1expression vector and the PBs buffer used, the stability of the AHAS Ⅰ acquired was improved greatly, and its activity could be kept for at least eight months.The function of AHAS Ⅰ was also analyzed. Due to its broad spectrum of substrates, we selected pyruvate,2-ketobutyrate and benzaldehyde as its substrates, respectively. First, we used AHAS Ⅰ to synthesis the food flavors2,3-butanedione and2,3-pentanedione, the yield of2,3-butanedione could reach76%. Then, we utilized AHAS Ⅰ to synthesize the important pharmaceutical intermediate L-phenylacetylcarbinol (L-PAC). After optimizing the reaction conditions, such as the reaction temperature, the pH of the reaction medium, reaction time, and the extracting solvents, L-PAC could be obtained in a yield of72%and high purity (>95%). Since the enzymatic procedure established in this thesis can produce L-PAC in high purity and good yield, it is thus a promising choice to prepare this compound.Additionally, we also investigated the catalytic mechanism of AHAS Ⅰ in the research. Based on the data we got, we believed that there should be a third resonant structure of the reaction intermediate hydroxyethyl thiamine pyrophosphate (HEThDP) besides the reported enamine resonant structure and C2-a-alkenyl carbanion resonant structure, which might be named enol resonant structure. This result was similar to the other ThDP dependent enzymes which we studied before.