Investigation of inhibitory effect of diospyros kaki leave and fruit extracts on gram (+) replicative DNA polymerase (POLC)

In this thesis, inhibition of DNA replicative polymerase activity, antimicrobial activity and chemical content of Diospyros kaki (D. kaki) were studied and its fruit and leaf extract were used. Bacillus subtilis (B. subtilis) replicative DNA polymerase IIIα (PolC) was used a model polymerase in assays. The gene encoding PolC was amplified by PCR and cloned into pET-100 TOPO vector to produce recombinant protein. Expression was achieved in E. coli BL21 and the recombinant protein was purified by using nickel affinity chromatography. In order to determine inhibition, methanol and DMSO extract of fruit and leaf of D. kaki were prepared. Methanol exctract was used for determination of inhibition effect on PolC. In the primer extension experiments, synthetic designed 45/20-mer DNA which contains fluorogenic primer was used. It is observed that, leaf extract of D. kaki plant has strong inhibition on the contrary fruit exctract on PolC. Prepared DMSO extract was used to determine antimicrobial activity. It is concluded that, leaf extract of D. kaki has antimicrobial activity both Gram negative and Gram positive bacteria. Phenolic compounds of leaves of D. kaki were obtained ether and methanol extracts by using HPLC-DAD. Finally, routine derivatives and kaempferol were found in the leaf extract of D. kaki.