Viper Enzymatic Preparation Process Of The Active Peptide And Its Chemical Composition

Agkistrodon halys (Palls) or Agkistrodon biomboffi Boie is belong to the viperidae.Agkistrodon halys was first recorded by"BIELU"."Benchaojingjizhu"recorded that the Agkistrodon halys have yellow and black skin, yellow beak jaw, strong toxicity."Compendium of Materia Medica"recorded that Agkistrodon halys is with qufeng and detoxication effect, which is used for Rheumatism,Leprosy,M. scrofulaceum,Sore furuncle,Scabies,Hemorrhoids,cancer. etc. .Immemorial, The viper was used for strong tonic and enhancing physical. Now the viper is maily used as tonic drugs. In china the Agkistrodon halys was maily produced in Northeast and rich in natural resource.This paper mainly takes advantage of controlled hydrolysis techniques,ultrafiltration and gel column chromatography with viper protein to study the method of producting the bioactive peptites. In addition using clearing hydroxyl radical (? OH) in vitro and experiment of anti-fatigue to screening the best method.In the controlled enzymatic hydrolysis process, with papain and neutral protease to consist complex enzymes to catalyze the hydrolysis reaction. The mainly purpose is to study the impact of the hydrolysis system pH,hydrolysis time,the proportion of complex composition and amount of protease,hydrolysis temperature and hydrolysis time on enzymatic effect. According the the degree of hydrolysis the best way to hydrolysis is hydrolysis temperature being 50℃,enzyme and substrate concentration ratio [E] / [S] being 6%,pH value being 7.5,reaction time being 5h and compound enzyme (papain: neutral protease) ratio of 1:6 at the fixed 3% concentration of substrate. In such condition, the degree of hydrolysis is 21.6 %, Yield is 51.58%. But the result of clearing hydroxyl radical in vitro is show that the ratio of scavenging hydroxyl radical does not increase as the increase of the degree of hydrolysis. By variance analysis and responsed curve. The best method of hydrolysis is to be show that hydrolysis temperature is 45℃,pH value being 7.0,reaction time being 5h,enzyme and substrate concentration ratio [E] / [S] being 6% and compound enzyme (papain: neutral protease) ratio of 1:5 at the fixed 3% concentration of substrate. In such condition, the degree of hydrolysis is 19.28% and the the value of IC50 is 3.70mg/ml, which was used to measured the capacity of scavenging hydroxyl radical. The anti-fatigue experiment shows that using the the best method to produce the bioactive peptite which can prolong the time of mice swimming.Active peptide dose being related to the mice swimming time. In male rat's organ inspection test the index of kidney,prostate,testis and epididymis are all Significant. the result of experiment show that Agkistrodon peptide hydrolysis have better biological activity of anti-fatigue impotence.Ultrafiltration separation method showed that increasing the hydrolysis degree of hydrolyzate can increase the rate of filtration, membrane performance and transmission components.Meanwhile, reducting the rejection rate of components and conforming the hydrolysis of membrane components with role of separation of different molecular weight. In this study, the main application of the membrane MWCO 3000Da to collect the following molecular weight 3000Da peptide hydrolysis.Gel exclusion method studies have shown that large molecular weight range of short peptides at an earlier time period to be eluted by mobile phase, low molecular weight range of components after elution, thus the use of gel-exclusion chromatography can be enrichment and purification of different molecular weight range of short peptides.In the study SephadexG-25,SephadexLH-20,SephadexG-15 were usded to get active FractionⅠ.ODS-C8 and ODS-C18 are the RP silica gel,which are usded to purifity the FrⅠ.At end the monomer ,AP.Ⅰis purificied.Using the ESI-MS/MS the Structure of Molecular is distinguished.MW=245,the Chemical equation is H-LEU-ASN-OH.The use of the ESI-MS is to analysis the active pate Fr-Ⅰ, H-LEU-ASN-OH,H-LYS-GLU-OH,H-ASN-GLU-OH,H-ARG-ASP-OH,H-ASN-VAL-OH,H-PHE-ASN-OH,H-ARG-GLU-OH and H–ASN—VAL—ALA-OH were identifided.