Extraction,Purification And Identification Of Alpha-amylase Inhibitor And Lectin From Phaseolus Vulgaris

As one of the special agricultural crops in Xinjiang,phaseolus vulgaris is a rare food with high-potassium,high-magnesium and low-sodium.It is especially suitable for heart disease,arteriosclerosis,hyperlipidemia,hypokalemia,and salt-free patients.At present,researches on phaseolus vulgaris focus on simple processing and physicochemical properties,lack of deep processing technology,and low resource utilization rate.Therefore,how to make full use of phaseolus vulgaris and improve its added value has become a problem to be solved in the development of bean industry.Alpha-amylase inhibitor(?-AI)is a glycoside hydrolase inhibitor.It mainly inhibits the activity of saliva and pancreatic alpha amylase to impede the digestion and absorption of starch and other carbohydrates consumed by the human body.It does not affect the absorption and metabolism of other nutrients,but also reduces the intake of sugar,reduces the synthesis of fat,plays a role in hypoglycemic and weight loss.At present,?-AI is widely used in medicine,pharmacy,agriculture and other fields.It can be used to control diabetes,high blood lipids,high blood pressure,and it can also be used as a biopharmaceutical as an insect-resistant gene,has a broad application prospects.Lectin is a non-immunogenic,non-enzymatic protein or glycoprotein that contains one or more proteins that can specifically identifiable and reversibly bound monosaccharides or polysaccharides.It is widely distributed in natural organisms.At present,it has been isolated from animals,plants,microorganisms,viruses,and fungi,and can be used in cell agglutination,induction of apoptosis,anti-fungal,immune regulation,anti-tumor,mitosis promotion,and biological nitrogen fixation.In this experiment,?-AI and lectin were extracted from phaseolus vulgaris.After preliminary separation and identification,the main conclusions showed follows:(1)The response surface software was used to optimize four factors,such as extraction volume(A),pH(B),extraction time(C),and extraction temperature(D).It was determined that?-AI had the highest inhibitory activity under the conditions of extract volume 200 mL,pH 3.5,extraction time 2.3 h,extraction temperature 43°C.The crude extract was precipitated by 20%-60%ammonium sulfate for Sephadex G-50gel chromatography and DEAE A-50 anion exchange chromatography.The purified?-AI inhibition rate of the purified bean reached(94.73±0.52)%and the purification multiple was 1.15.Using SDS-PAGE electrophoresis to determine the target molecular weight of about 17 kDa,and determined by LC-MS/MS to determine the isolated protein is phaseolus vulgaris?-AI.(2)The response surface software was used to optimize the three factors,such as feed-liquid ratio(A),pH(B),and extraction time(C).It was determined that lectin had the highest specific activity under the conditions of feed-liquid ratio(g/mL)1:13,pH 7.4 The extraction time was 18.5 h.The crude extract was precipitated by 30%-70%ammonium sulfate for Sephadex G-50 gel chromatography and DEAE A-50anion exchange chromatography.The purified lectin-specific activity of cowpea reached(59.93±0.31)×10~3HU/mg and the purification multiple was 4.52.Using SDS-PAGE electrophoresis to determine the target molecular weight of about 31 kDa,and determined by LC-MS/MS to determine the isolated protein is phaseolus vulgaris lectin.