Globin Protein Extraction, Preparation Of ACE Inhibitor From Globin By Enzymatic Hydrolysis And Enzyme Kinetics Studies

The pork is a main meat source in our country, however, the utilization of pig blood is very low in the majority of meat slaughterhouses, and basically abandoned . Hemoglobin is an essential element in pig blood, and rich of iron .The globin extracted from hemoglobin is a very good protein. The peptides derived from the globin through hydrolyzing has a higher ACE inhibition activity.In this paper, firstly, by globin extraction test, identify that 0.3% sodium citrate, four times acetone and 2% NaAc were the optimal parameters; At this point globin yield was 29.03g/l(the grams of the globin can be extracted from one liter of fresh pig blood).The Alcalase, pepsin, trypsinase and papain were individual and double applied to hydrolyze the globin.The reaction time,quantity of enzyme addition, substrate concentration, temperature and pH effected the hydrolysis degree of globin.The globin hydrolyzed by four enzymes individual and double had different degree of ACE inhibitory activity and had different hydrolysis degrees. Through TCA method and the RP-HLPC method, the hydrolysis degree and the degree of ACE inhibitory activity were determined.In individual enzyme hydrolysis, Hydrolyzate by pepsine hydrolysis had the highest degree of ACE inhibitory activity, up to 94.63 %, alcalase hydrolysis had higher degree of ACE inhibitory activity, up to 94.49% , and 14.42% of hydrolysis degree. In double enzyme hydrolysis,the hydrolysis of combination of Pepsin and Alkaline had the highest ACE inhibitory activity, up to 94.82%.And the double enzyme hydrolysis test needed shorter time than the individual, with greater efficiency.In kinetics of enzyme-catalyzed reactions test,though Lineweaver-Burk method, Km and Vmax were obtained,though animal protein-the globin as hydrolysis substrate, with a view to identifying the optimal reaction conditions enzyme.At pH2.5,Vmax of pepsin hydrolysis achieve the maxmum value for 0.190mg/ml·min, Km lower, so this enzyme molecules had stronger affinity on the substrate;Km of Alcalase in the 50℃to achieve the minimum value for 13.00 mg / ml, 0.142 mg/ml·min of the Vmax.The results of Papain enzyme kinetics test than Alcalase and pepsin the were poorer.The combination of Pepsin and Alkaline hydrolysating globin was more suitable, Vmax of 0.237mg/ml·min, Km of 6.916 mg/ml.