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In this study, Rana oil as the substrate, and papain, alkaline protease, bromelain, pepsin and compound protease enzymatic experiments, and by measuring the enzyme activity of each enzyme, after hydrolysis the degree of hydrolysis of the substrate (DH) and the the enzymatic solution polypeptide content (TCA-SN) and free amino nitrogen content to determine the effect of the enzymatic hydrolysis of various enzymes. The results show that higher than other protease papain and alkaline protease enzyme, respectively for 510U/mg and 350U/mg; higher degree of hydrolysis of 12.3% and 11.8%, respectively; relatively high yield of acid soluble polypeptides, respectively. In this study was 43.7% and 50.3%, respectively; and free amino nitrogen content in the hydrolyzate, respectively for 0.42mg/mL and 0.56mg/mL; comprehensive consideration of papain and the alkaline protease enzymatic Rana oil preparation activity peptide. Using the double enzyme combination controllable enzyme technology Rana oil hydrolysis. First papain hydrolysis of the substrate, and the use of single-factor test effects of substrate concentration, the amount of enzyme hydrolysis temperature, pH and enzymatic hydrolysis time on the degree of hydrolysis of the substrate and product of the acid-soluble peptide content and Screening the Box-Behnken central composite design and response surface analysis of its impact significant factors, the main factors affecting the enzymatic reaction polynomial regression modeling and optimization, to determine the best papain enzymatic substrate The conditions were: temperature 60 ° C, 1% of the pH value of 6.5, substrate concentration, enzyme amount (E / S) of 1.5%, enzymatic hydrolysis time 3.0h. On the basis of the enzymatic hydrolysis, further alkaline protease hydrolysis, and the same on the basis of single factor experiments, using the Box-Behnken central composite design and response surface analysis to determine the optimum conditions for alkaline protease digestion : the amount of the enzyme (E / S) 0.875%, temperature 50 ° C, pH value of 8.0, the hydrolysis time 3.0h. The degree of hydrolysis of the substrate and the acid-soluble peptide content 18.4% and 78.6%, respectively, 5.6% and 30.2%, respectively, than the single enzymatic hydrolysis. Enzymatic purified by Sephadex G-15 gel column separation, papain hydrolysates from peptide molecular weight between 300-1355Da, 41.3% of the total elution peak area; 38% greater than 1355Da; less than 300Da 20.7%. Alkaline protease hydrolysates from peptide molecular weight between 300-1355Da, 38.2% of the total elution peak area; 42.6% greater than 1355Da; less than 300Da accounted for 19.2%. Dual-enzyme combination of Hydrolysates polypeptide molecular weight between 300-1355Da, 52.6% of the total elution peak; greater than 1355Da accounted for 31.5%; less than 300Da accounted for 15.9%. Substance distributed between 300-1355Da, by HPLC analysis, three different levels of the chromatographic peaks in the peak pattern appears, retention time sequentially 21.837min, 23.300min and 24.499min, wherein the retention time in the 23.300min when the corresponding material content Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis, the hydrolyzate contains four different molecular weight peptides, molecular weight: 712.4Da, 826.6Da, 1101.2Da and 1326.1Da.
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