Preparation Of Rana Oviduct Micromolecule Peptide And Research For Its Molecular Weight Distribution

In this study, Rana oviduct was the substrate, caroid, alkaline proteinase, extranase, pepsase and comp-protease were employed to hydrolyze substrate, the effect of enzymolysis was determined by determining enzyme activity, substrate's degree of hydrolysis and content of acid-soluble peptide,free amino acids in the digest. The result was: the enzyme activity of caroid and alkaline proteinase were higher than the other three enzymes, 510U/mg, 350U/mg respectively; degree of hydrolysis was higher, 12.3% and 11.8% respectively; the content of acid-soluble peptide content was higher relatively, 43.7% and 50.3% respectively; The content of free amino acids was lower relatively, 0.42mg/mL and 0.56% respectively. In summary, caroid and alkaline proteinase were emplyed to prepare active peptide.Rana oviduct was hydrolyzed by combination of two enzymes. Firstly, substrate was hydrolyzed by employing caroid, the effect to degree of hydrolysis and free amino acids in digest by substrate concentration,enzyme amount, hydrolytic temperature, pH and hydrolytic time were determined by single factor experiment, important factors were screened by single factor experiment. The best technology condition by Caroid was: temperature 60℃, pH 6.5, concentration of substract 1%, enzyme amount 1.5%, hydrolytic time 3.0h. On the base of the above experiment, alkaline proteinase was empolyed to hydrolysis again. Firstly, single factor was employed, than the best technology condition by alkaline proteinase was: temperature 50℃, pH 8.0, enzyme amount 0.875%, hydrolytic time 3.0h. Substrate's degree of hydrolysis and content of acid-soluble peptide in digest reached 18.4% and 78.6% respectively, which was 5.6% and 30.2% higher than single enzyme hydrolization.The digest was segregated by Sephadex G-15 gel column. Polypeptide molecular weight between 300-1355Da occupied 41.3% of all eluting peak area in digest by caroid hydrolization, higher than 1355Da occupied 38%, lower than 300Da occupied 20.7%. Polypeptide molecular weight between 300-1355Da occupied 38.2% of all eluting peak area in digest by alkaline proteinase hydrolization, higher than 1355Da occupied 42.6%, lower than 300Da occupied 19.2%. Polypeptide molecular weight between 300-1355Da occupied 52.6% of all eluting peak area in digest by combination of the above two enzymes hydrolization, higher than 1355Da occupied 31.5%, lower than 300Da occupied 15.9%.High-performance liquid chromatography and laser desorption time-of-flight mass spectrometry were employed to analyze and detecte the digest. There have been three chromatographic peak, retention time was 21.837 min, 23.300 min and 24.499 min by turns. The content of material which retention time was 23.300 was maximal. There were four molecular weigh peptide, molecular weighs were 712.4 Da, 826.6 Da, 1101.2 Da and 1326.1 Da respectively.