| Our country is a leading country in producing poultry blood all over the world. The poultry blood is hard to be preserved. The efficiency utilization of poultry is not high. Using microorganism to decompose poultry blood is recognized as a comprehensive method to utilize the resource well. Compared with other methods, using microorganism needs less invests and causes less energy consumption. Furthermore, it can produce high content of protein, peptide and amino acid. Through many years'selection and breeding by our research team, the strains of Bacillus pumilus CN8 were obtained and used for the porcine blood hemoglobin degradation.In this experiment, Bacillus pumilus CN8 produce highly efficient Enzyme-degrading of porcine hemoglobin, which can be utilized by porcine hemoglobin. Taken ammonium sulfate out of the precipitation, the culture liquid is centrifuged at 10000 r/min for 15min, then ammonium sulfate is added into the supernatant to a final concentration of 40% to precipitate the others. After centrifugation, the concentration of ammonium sulfate is increased to 60% to get crude protease. Bacillus pumilus CN8 protease is purified to homogeneity from liquor by ultrafiltration, ammonium sulfate precipitation, dialysis, DEAE-Cellulose-52 gel filtration. The Bacillus pumilus CN8 protease of electrophoresis pure is collected. The specific activity of the enzyme is 686.66 U/mg. Purification times and recovery rate of enzyme are 13.2 and 35.0% respectively. The molecular weight of the purified enzyme is estimated to be 86.92 KDa.After studying on the enzymatic properties of Bacillus pumilus CN8 protease, the characterization of crude enzyme is close to the pure enzyme. Results can be concluded as follow, the protease performed the maximal activity at pH 7.3 and 42℃. The Bacillus pumilus CN8 which belonged to the topical neutral protease showed a higher enzyme activity when the pH was between 6.5 and 9.5. The Bacillus pumilus CN8 protease was stable when pH was between 6.5 and 9.5 and temperature was between 30℃and 45℃. Effect of different metal ions on Bacillus pumilus CN8 protease stability was various. The protease could be slightly activated by K+. The enzyme activity could be completely inhibited by Cu2+, Mn2+, Sn2+, Cd2+, in spite of the lower or higher concentration. The inhibition of protease activity would enhanced with the increase of these metal ions concentration. Zn2+ has no effect on the enzyme activity in a low concentration, as the Zn2+ concentration increases, the inhibition of protease activity increase slightly. Other ions such as Fe2+, Na+, Ca2+, Mg2+ had no significant activation or inhibition of the protease. K+, Mg2+ had a protective effect on the enzyme activity. Cu2+, Mn2+, Sn2+, Cd2+ had a strong inhibitory effect on the protease activity. Fe2+, Na+, Ca2+, Zn2+had little effect on the protease activity. The enzyme activity could be completely inhibited by Glycerin. Tween 80 had little impact on enzyme activity. Span 20 and Tween 20 could promote the protease activity.Through the study it can be seen that, within a Bacillus pumilus CN8 strains, protease activity showed 1400U/mL. The soluble protein and free amino acid contents in hemoglobin degradation products 5.02%,34.6 mg/100 mL, respectively. The degradation yield of hemoglobin was 59.12%. The fermentation broth contained in the sterilized fermentation medium has some changes in structure and quality, after treated by the strains of Bacillus pumilus CN8. When tested by UV scanning, the degradation of hemoglobin decomposed into peptides, oligopeptides or amino acids and other substances among the 250nm-280nm wavelength, hemoglobin molecules structure has changed. When tested by Fourier transform infrared absorption spectrometer, the structure of hemoglobin also changed by acillus pumilus CN8 strain. The result mensurated by amino acid automatic analyzer also illustrates that the strains of Bacillus pumilus CN8 have some degradation affect on pig's blood hemoglobin. When tested by SEM micro-structural scanning, the result shows that hemoglobin particles become small, honeycomb-like structure disappeared, the surface of the porous structure no longer exists. The micro structure of hemoglobin has the fundamental nature of the change.This research has systematically studied the dynamics of hydrolysis on porcine hemoglobin by Bacillus pumilus CN8 protease, Neutral protease and Pepsin. It aimed at initial velocity of enzymolysis under different substrate concentration, temperature and pH. The optimal temperature and pH for 10 min reactions are concluded as follow:37℃and pH 7.0, for Bacillus pumilus CN8 protease; 45℃and pH 7.3, for neutral protease; 30℃and pH 1.5, for Pepsin. The order of Km value for the three proteases is Bacillus pumilus CN8 protease< Pepsin Pepsin>Neutral protease.
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