Purification And Characterization Of Salvianolic Acid-esterase

Salvianolic acid B is one of the water-soluble components in Salvia miltiorrhiza,but after oral intake,salvianolic acid B is hydrolyzed into danshensu and low-polymeric-salvianolic acid,which can be easily absorbed and utilized.In this paper,salvianolic acid-esterase from microorganism Absidia sp.D38s strain,which could hydrolyze salvianolic acid B to danshensu and low-polymeric-salvianolic acid,was studied.In addition,the hydrolyzation of salvianolic acid-esterase was researched.The crude salvianolic acid B,whose content is 10%,was purified by SP207 macroporous resin.After that the purity of Salvianolic acid B reached to 72.32%.The enzyme cultured from Absidia sp.D38 strain was purified to one band in PAGE after buffer extraction,(NH4)2SO4 precipitation and ion exchange chromatography.The molecular weight of the purified enzyme was about 36 kDa based on SDS-PAGE.The optimal temperature of salvianolic acid-esterase and pH were 35? and 5.0,respectively;and the range of stabilization was under 50?,pH 3.0-6.0.Salvianolic acid B was precipitated by Cu2+ or Fe3+;the activity was promoted by low concentration of Mg2+,Ca2+,K+,Na+ and Zn2+,but with the concentration increasing,promotion changeed into inhibition.The Km was 8.62 mmol/L,and the Vm was 27.03 mmol/(L-h).The enzyme reaction characteristics of the enzyme showed that salvianolic acid B enzyme could not hydrolyze the pNPP;it could cleave the ester linkage of salvianolic acid B.6.4g salvianolic acid B was hydrolyzed by salvinolic acid-esrerase under the conditions of that substrate concentration 16 mg/mL,35?,pH 5.0 and 14h,16.11 g crude product was obtained.After separation by Sepabeads SP207 macroporous resin,5.68 g danshensu monomer eluted by water was obtained and its yield was 35.26%.6.80 g low-polymeric-salvianolic acid eluted by ethanol was obtained and its yield was 42.21%.The molecular weight of danshensu and low-polymeric-salvianolic acid,which were the hyderlysis products,were respectively 198 and 358 by UPLC-MS.And the molecular formula of low-polymeric-salvianolic acid was C18H14O8,and its system name was 8-hydroxyl-3-carboxy-2-(3,4-dihydroxy)phenyl-1-benzopyran-5-propenoic acid.