Isolation And Purification And Gene Cloning Of Salvianolic Acid-esterase

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.After 3-steps,a salvianolic acid-esterase was purified from the Absidia sp.D30s culture by the methods of molecular sieve chromatography and ion exchange chromatography.The new purified enzyme is consisted with two subnits on SDS-PAGE,the molecular weight of the subunit I was about 38 kDa based on SDS-PAGE,the molecular weight of the subunit II was about 18 kDa based on SDS-PAGE.The optimal temperature of salvianolic acid-esterase and pH were 40? and 5.0 respectively,Mg2+,Ca2+,K+and Na+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 the subunit I was as GLTTQKSAPWGLGSI,the N-terminal sequence of the subunit II was as VKAVAVLRGDSKISG.According to the N-terminal amino-acid sequence of enzyme subnits,the degenerate primers were designed,then immediately doing the RT-PCR reaction.Designing the primers on the basis of above sequence,the complete gene sequence was amplified by RACE.The sequence length of subunit I is 1829 bp,the sequence length of the cDNA is 1212 bp;the sequence length of subunit II is 764 bp,the sequence length of the cDNA is 465 bp.