Studies On The Purification, Characterization And Application Of Baicalin Glucosidase

Glycosidase all so called glycoside hydrolases,which is an enzyme that catalyzes by internally or extrinsically tangent the hydrolysis of a bond joining a sugar of a glycoside to monosaccharide,oligosaccharide or complex carbohydrates.It is distributed extensively the natural world,and play a significant role in keeping the basic training of biosystem.As a kind of instrumental enzyme which catalyze new pattern glycoside compound,it is efficient,stabile,tolerated,adaptive,suitable.This reduce the cost,sublime plurinatality,improve quality.Baicalin glucosidase hydrolyzes the glucuronide bond of baicalin to form baicalein,aglycone with ideal bioavailability and biological activity.To obtain bioactive substance by enzymolysis,and the reaction is high performance,gentle,convenient,protection environmental,which becomes hot spot in investigation.This subject establish purification technology route of baicalin glucosidase,and then study on its characterization,and furtherly application reseach.Using means of collection,dislysis,DEAE-sephacel ion exchange chromatography,G-200 gel column chromatography to purify target component from raw material,the purified enzyme shows a single component on HPLC and cellulose-acetafolic cellulose membtane;a 57.12%yield of enzyme with a specific activity of 1295.33 U/mg was obtained,establish a enzyme activity detection method which product baicalein,average recovery is 100.7%;calculate Km to baicalin is 57.4mmol/L by Linewear-Burk graphical method;and its optimum pH is 5.8; optimum temperature is 45℃;optimum ionic concentration is 1mol/L;optimum concentration of substrate is 40mmol/L,and enzyme concentration to reaction velocity offer a favourable linear correlation;the native enzyme has a molecular weight of 230kD,and the disoxidative molecular weight is about 55kD by SDS-polyacrylamide gel electrophoresis.We suggested that the enzyme exists as a homotetramer composed of four identical 55-kD subunits,detect the isoelctric point of this enzyme is pH 5.4;analysis the enzyme's secondary structure by circular dichroism spectra,and theβ-turn and andom coil have a higher ratio;and product the immobilized enzyme by sodium polymannuronate and gelatin respectively,sodium polymannuronate immobilized enzyme is a optimum temperature of 55℃,and optimum pH of 6.4;gelatin immobilized enzyme is a optimum temperature of 47℃,and optimum pH of 6.2;meanwhile,hydrolyze aurantiamarin with this enzyme, and the resulte is negative.