Study Of Salvianolic Acid-esterase Gene Cloning

In this paper,purification,characterization and gene sequence of salvianolic acid-esterase,which Salvianolic acid B can hydrolyze into danshensu and Przewalskinic acid A,were studied.Through the experiments,the optimal liquid fermentation conditions of the enzyme were determined.The best proportion of inducer was:Salvia miltiorrhiza lixivium:bran-lixiviu=l:1.The best training time for strains was 144 hours.The enzyme cultured from Absidia sp.D30s strain was purified by sephadex chromatography and DEAE-cellulose ion exchange chromatography.The molecular weight of the purified enzyme was about 37 kDa based on SDS-PAGE.The optimal temperature of salvianolic acid-esterase and pH were 40? and 5.0 respectively,and the range of temperature stabilization was from 20?to 50?,pH 3.0-6.0.Mg2+,Ca2+,K+ and Na2+ barely affected the activity of the enzyme,but it would be suppressed by high concentration of Zn2+.While salvianolic acid-esterase was inhibited by Cu2+ and Fe3+.After western blotting,the N-terminal sequence of salvianolic acid-esterase was ALTXQVSAPI(X was unknown).The total RNA of Absidia sp.D30s was isolated.The method was that the degenerate primers were designed according to the N-terminal sequence of salvianolic acid-esterase,then extracting total RNA from Absidia sp.D30s strain and immediately doing the RT-PCR reaction.And to use bioinformatics tools analyzing the sequences of genes,constructing the phylogenetic trees.The result was that salvianolic acid-esterase had high affinity with peptidase which was cultured by Aspergillus oryzae.