A Study Of The Important Amino Acid Lys29at The Active Site Of The Cyanobacteria FBP/SBPase And The Screening Of Its Inhibitors

Recently, the increasing eutrophication of the water resource in China caused the frequent occurrence of blue-green algae bloom and brought great harm to the human life and the ecological environment. Thus, prevention and control of blue-green algae is extremely urgent. FBP/SBPase is the very important regulation of enzyme in the Cyanobacteria photosynthesis system, and the potential role of target for the green germicide which is research and development of environmental friendly, efficiently, high selective.In previous studies, we reasonably constructed the three-dimensional structure of Cyanobacteria FBP/SBPase. we discovered an important amino acid Lys29, which could possess a specific activity three times of wild type after being changed into Ala (A) through mutation.Based on the previous research, the thesis is mainly conducte the following two aspects of the work:(1) To study the important amino acid Lys29at the active site on the Cyanobacteria FBP/SBPase.To change the K29into nine mutants (K29D, K29Y, K29P, K29S, K29R, K29G, K29Q, K29T, K29F) through mutation and express&purify the protein of the nine mutants. To pick out the three mutants (K29A, K29D, K29S) whose activities are higher than wild-type protein (WT) and which has the different hydrophobic property, acid-base property and polarity when they are changed into amino acids through mutation after determination of their activity. Then study the K29from the five aspects affecting the enzyme activity, namely activity, enzyme kinetics, thermal stability, stability and pH. The study has shown the specific activities of the K29A, K29D and k29S are two to three times than that of WT, According to the parameters of enzyme kinetics, we can see that mutants with the substrate binding affinity do not have big difference with the wild-type. It means the reason for the mutant activity increased is when catalytic reaction, mutate the29th amino acid K to A、D、S is better for the release of the catalytic product;although the mutant activity increased, the stability, Thermal stability,and the pH influence of enzyme activity is worse than WT. it means organism preferred K in29th amino acid.(2) Take the Cyanobacteria FBP/SBPase as a target to screen the inhibitors for the analogues of D13and D7series.Screen inhibitors by acting the compounds on blue-green algae FBP/SBPase, and conducting algae level screening for the compounds with inbibitional effects. Five compounds with good inhibit effects are screened out:D13-2, D13M1, D7S5, D7-TQD004, D13-TQD170. The EC50of these compounds against FACHB912and PCC6803are below lOppm. The EC50of D13-2, D13M1, D7S5, D7-TQD004, D13-TQD170against FACHB912are6.06ppm,0.48ppm,4.6ppm,7.1ppm,1.57ppm respectively; the EC50of D13-2, D13M1, D7S5, D7-TQD004, D13-TQD170against PCC6803are3.1ppm,3.7ppm,2.9ppm,5.9ppm,7.01ppm respectively. Among them, the EC50of D13M1against FACHB912is at the same level as CuSO4.