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Study on Extraction of Bovine and Ovine Pancreatins and Enzymology Characteristics of Purification Trypsins

Author: LiJunLan
Tutor: YuQunLi
School: Gansu Agricultural University
Course: Animal Food and Nutrition
Keywords: Bovine and Ovine Pancreatins Extraction Trypsins Purification Enzymology characteristics
CLC: TQ925
Type: PhD thesis
Year: 2011
Downloads: 235
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Abstract


Flocks and herds have a high status and economic value in human food. But a large amount of inedible pancreas about4.472-8.944×104t in manufactured meat every year. It not only contains a lot of enzymes but also easy to autolysis very quickly post-mortem due to enzymes leaking from the digestive organs, and resulting in environmental pollution. Therefore, the development and utilization of bovine and ovine pancreas has significant. It was reported the process of extraction pancreatin from bovine and ovine pancreases by novel experimental design and mathematical statistical methods, and separation and purification of the trypsin with the gel filtration And it was thoroughly study of the biological characteristics of bovine and ovine trypsin. Provide a theoretical basis for reduce environmental pollution and exploitation of biological resources pancreas. This paper mainly addressed the following aspects of the research results:1. Extraction bovine pancreatin process flow and parameter optimization with chitosan flocculation method. Plackett-Burman Design was undertaken to evaluate the effect of the thirteen factors on the activity of pancreatin and extraction rate. Main factors were selected by analysis of variance(p<0.05):chitosan, CaCl2concentration, the pancreas and ratio of the pancreas and duodenum and activation pH. On this basis, a uniform design method was used to study the scope of the optimal level above the four significant factors, and obtained the optimal conditions for extraction bovine pancreatin by partial least squares regression (PLS) equation:chitosan0.17%, CaCl20.06%, ratio8.26:1of pancreas and duodenum and the activation pH5.37. Under these conditions, validation actual value of trypsin, lipase, amylase and extraction rates were3408.71u/g,33.11ku/g,23.93ku/g and13.22%, respectively, and the relative errors of the regression equation theoretical prediction were7.15%,8.56%,10.63%,1.93%, respectively. It was feasible that process of extraction bovine pancreatin with the method of two activators and chitosan flocculation. The results were obvious by the method of combining Plackett-Burman and uniform design and PLS regression analysis.2. Extraction ovine pancreatin process flow and parameter optimization with isopropanol. After the process of extraction ovine pancreatin was slight modified with isopropyl alcohol. Based on single factor experiments, Studing activation time, isopropyl alcohol concentration, extraction pH and CaCl2affect on the ovine pancreatin extraction process with a uniform design. With the response result of trypsin, amylase, lipase and extraction rate, the impact factors of ovine pancreatin extracton process were optimized by PLS, and established the mathematical model with the extraction conditions. The results showed that:The optimal conditions of extracton ovine pancreatin were activation time12h, CaCl20.08%, precipitation isopropanol concentration41%, extraction pH6.34, and Extract isopropanol concentration12.5%. Under these conditions, verified the actual value of trypsin, amylase, lipase and extraction rate were3062.78u/g,30.06ku/g,32.37Ku/g and6.16%, respectively, the relative errors of the mathematical model equations theoretical prediction were7.15%,8.56%,10.63%,1.93%, respectively. The process is feasible and further verified superiority of uniform design in multi-factors and multi-level experimental design, and the data result was reliable by multi-factors and Multi index composite analysis with PLS.3. On the exchange of experimental verification of extraction pancreatin from bovine and ovine pancreas with chitosan flocculation and isopropyl alcohol method, respectively, and processes parameter. The results showed that pancreatin activity exceeds the targets set in the Chinese Pharmacopoeia. Bovine and ovine pancreatins were3668u/g and2151u/g respectively, which exceed6.1times and3.5times targets set in the Pharmacopoeia compare to the lowest activity of trypsin, respectively. Loss on drying and residual fat of pancreases were low the standards of Pharmacopoeia, which had a special no smell of corruption. Pancreatin with isopropyl alcohol extraction was gray powder, and with chitosan flocculation was pale yellow. Activity of trypsin and amylase with chitosan flocculation extraction was lower than activity with isopropyl alcohol in the same pancreatin, but the ovine amylase and lipase was contrary. Under the same extract conditions, The activity of porcine pancreatin was highest and ovine was smallest among porcine, bovine and ovine pancreatin activity with trypsin activity as the reference, which average activity of porcine, bovine and ovine pancreatin were4754.5u/g,3538u/g and2607u/g, respectively.4. A series purification of trypsins from bovine and ovine pancreatin after crude extraction, filtration, centrifugation, saturated sulfate ammonium fractionation precipitation, acetone precipitation and gel filtration, respetively, the purification factor of trypsins and recovery of protein were19.8,23.2;38.2%,13.6%, respectively. Purified two trypsins showed a single band on SDS-PAGE electrophoresis film, and approximately molecular weights were24kDa and27kDa, respectively. Trypsins could both hydrolyze TAME, and was strongly inhibited by PMSF, SBTI and TLCK. Which inhibit ratio were81-98%and74-96%, respetively, and was not inhibited by EDTA. Therefore, two enzymes were serine trypsins by molecular weight, hydrolysis properties, and inhibited characteristics.5. The optimume and stability temperatur range of bovine and ovine trypsins were60℃and20-50℃, respectively, when trypsins hydrolyze TAME substrate under the absorbance at274nm wavelength; Optimum pH were8.0and9.0, respectively, and PH stability range were8-12and7-12, respectively. The result showed that bovine and ovine trypsins were stability in neutral and alkaline conditions and two typsins has stability in CaCl2solution, and the trypsin activity could be decreased in different concentration NaCl.6. Kinetic studies had shown that Michaelis constant Km of trypsins were0.28μmmol/L and0.53μmmol/L, respectively. They had a good affinity for substrate TAME, and bovine trypsin had a higher affinity for TAME than did ovine trypsin. Meanwhile, trypsins had a higher affinity for TAME than did BAPNA, of which the Kcat and Kcat/Km was7.905s-1,7.702s-1,28.23s-μmol-1and14.53s-μmol-1respectively. It was significantly higher than those reported in other mammal’s trypsin.

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