牛羊胰酶提取、胰蛋白酶纯化及其酶学特性研究
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摘要
牛羊在人类食物的地位及产生的经济价值很高，但每年肉制品加工产生大量的不可食用的胰脏约有4.472-8.944×10~4t。它不但含有大量的酶，而且易自溶，造成环境污染。所以探讨牛羊胰脏的开发利用研究具有重要的意义。采取新颖的实验设计和数理统计方法，研究其胰酶提取工艺，并用凝胶过滤的方法研究牛羊胰蛋白酶分离纯化，且较深入地研究了牛羊胰蛋白酶的生物学特性。为减少环境污染和牛羊胰脏生物资源开发利用提供理论依据。文中主要述及了以下几方面的研究结果：
1.牛胰酶壳聚糖絮凝法提取工艺流程确定和参数优化。采用Plackett-Burman设计对影响牛胰酶活力和提取率的13个因子进行筛选，用方差分析筛选出主效因素（p＜0.05）：壳聚糖、CaCl_2浓度、胰腺与十二指肠配比和激活pH。在此基础上，再运用均匀设计对以上四个显著因子的最佳水平范围进行研究，通过偏最小二乘法（PLS）建立回归方程求解得到牛胰酶提取最适条件为：壳聚糖0.17%、CaCl_20.06%、胰腺与十二指肠配比8.26:1、激活pH5.37。此条件下验证实测值胰蛋白酶、胰脂肪酶、胰淀粉酶活力和胰酶提取率分别为3408.71u/g、33.11ku/g、23.93ku/g和13.22%，与回归方程理论预测值相比，相对误差分别为7.15%、8.56%、10.63%、1.93%。用两种激活剂和壳聚糖絮凝法提取牛胰酶工艺合理，具有可行性。且采用Plackett-Burman和均匀设计相结合并运用PLS回归分析效果显著。
2.羊胰酶提取工艺流程确定和参数优化。在单因素实验基础上，再运用均匀设计法研究激活时间、异丙醇浓度、提取pH和CaCl_2对羊胰酶提取工艺的影响。以胰蛋白酶、胰淀粉酶、胰脂肪酶活力和胰酶提取率为响应指标，通过PLS回归分析法对羊胰酶提取工艺的影响因素进行优化组合，并建立了具有提取条件的数学模型。结果表明：提取最适条件为：激活时间12h、CaCl_20.08%、提取pH6.34、沉淀和提取异丙醇浓度分别为41%和12.5%，此条件下验证实测值胰蛋白酶、胰淀粉酶、胰脂肪酶的活力和胰酶提取率分别为3062.78u/g、30.06ku/g、32.37Ku/g和6.16%，与数学模型方程的理论预测值相比，相对误差分别为8.11%、1.28%、3.55%和5.29%。该工艺合理，具有可行性。进一步验证了均匀设计在多因素多水平试验设计的优越性和PLS对多因素多指标的综合分析数据结果可靠。
3.分别用2种胰酶提取方法及工艺参数对牛羊胰脏进行交换试验验证，结果发现羊胰酶壳聚糖絮凝法和牛胰酶异丙醇提取法所得胰酶活力均超过中华人民共和国药典规定的指标，以最低的胰蛋白酶活力作比较，牛羊胰蛋白酶分别3668u/g、2151u/g；超过药典规定6.1倍、3.5倍。干燥失重、残留脂肪在药典规定的指标以下，具有特殊的肉臭味；异丙醇提取胰酶呈灰白色粉末，而壳聚糖絮凝法提取产品则呈淡黄色，胰蛋白酶、胰淀粉酶的活力比同一种类异丙醇法提取胰酶制品的活力低，但羊胰淀粉酶和胰脂肪酶活力相反；在胰酶提取操作方法相同的条件下，用胰蛋白酶活力做参照比较，猪牛羊三种酶活力平均分别为4754.5u/g、3538u/g、2607u/g，猪胰酶的活力最高，羊胰酶活力最小，牛胰酶制品的活力介于二者之间。
4．胰蛋白酶分离纯化。牛羊胰酶粉分别经过胰蛋白酶粗提、过滤、离心、饱和硫酸铵分级沉淀、丙酮沉淀、凝胶过滤等一系列纯化后，牛羊胰蛋白酶的纯化倍数和蛋白质回收率分别达到19.8、23.2；38.2%、13.6%。2种纯化的蛋白酶在SDS-PAGE电泳胶片上显示单条带，其相对分子量分别约为24kDa和27kDa。纯化后牛羊胰蛋白酶均能水解TAME，且被PMSF、SBTI和TLCK强烈抑制，抑制率分别在81-98%、74-96%的范围，不被EDTA抑制，所以结合其相对分子质量大小、水解特性、抑制特性，可以确定这是2种胰蛋白酶。
5．胰蛋白酶的生物特性。在波长247nm下，牛羊胰蛋白酶TAME底物时，2种胰蛋白酶的最适温度及稳定温度范围相同，分别是60℃、20-50℃；最适作用pH分别是8.0、9.0，酸碱稳定性范围各为pH8-12和pH7-12；说明2种酶在中性和碱性条件稳定；且CaCl_2对其胰蛋白酶均具有稳定性，不同浓度的NaCl对胰蛋白酶的活力有降低作用。
6．胰蛋白酶动力学特性。研究表明牛羊胰蛋白酶的米氏常数Km分别是0.28μmmol/L、0.53μmmol/L，2种胰蛋白酶对TAME有很好的亲和力，牛胰蛋白酶对底物TAME的亲和力比羊胰蛋白酶高。同时还发现二者对TAME的亲和力比BAPNA高。其催化效率Kcat、生理转化效率Kcat/Km分别为7.905s~(-1)、7.702s~(-1)；28.23s~(-1)μmol~(-1)、14.53s~(-1)μmol~(-1)。明显高于已报道的其它哺乳动物胰蛋白酶。
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.
1.牛胰酶壳聚糖絮凝法提取工艺流程确定和参数优化。采用Plackett-Burman设计对影响牛胰酶活力和提取率的13个因子进行筛选，用方差分析筛选出主效因素（p＜0.05）：壳聚糖、CaCl_2浓度、胰腺与十二指肠配比和激活pH。在此基础上，再运用均匀设计对以上四个显著因子的最佳水平范围进行研究，通过偏最小二乘法（PLS）建立回归方程求解得到牛胰酶提取最适条件为：壳聚糖0.17%、CaCl_20.06%、胰腺与十二指肠配比8.26:1、激活pH5.37。此条件下验证实测值胰蛋白酶、胰脂肪酶、胰淀粉酶活力和胰酶提取率分别为3408.71u/g、33.11ku/g、23.93ku/g和13.22%，与回归方程理论预测值相比，相对误差分别为7.15%、8.56%、10.63%、1.93%。用两种激活剂和壳聚糖絮凝法提取牛胰酶工艺合理，具有可行性。且采用Plackett-Burman和均匀设计相结合并运用PLS回归分析效果显著。
2.羊胰酶提取工艺流程确定和参数优化。在单因素实验基础上，再运用均匀设计法研究激活时间、异丙醇浓度、提取pH和CaCl_2对羊胰酶提取工艺的影响。以胰蛋白酶、胰淀粉酶、胰脂肪酶活力和胰酶提取率为响应指标，通过PLS回归分析法对羊胰酶提取工艺的影响因素进行优化组合，并建立了具有提取条件的数学模型。结果表明：提取最适条件为：激活时间12h、CaCl_20.08%、提取pH6.34、沉淀和提取异丙醇浓度分别为41%和12.5%，此条件下验证实测值胰蛋白酶、胰淀粉酶、胰脂肪酶的活力和胰酶提取率分别为3062.78u/g、30.06ku/g、32.37Ku/g和6.16%，与数学模型方程的理论预测值相比，相对误差分别为8.11%、1.28%、3.55%和5.29%。该工艺合理，具有可行性。进一步验证了均匀设计在多因素多水平试验设计的优越性和PLS对多因素多指标的综合分析数据结果可靠。
3.分别用2种胰酶提取方法及工艺参数对牛羊胰脏进行交换试验验证，结果发现羊胰酶壳聚糖絮凝法和牛胰酶异丙醇提取法所得胰酶活力均超过中华人民共和国药典规定的指标，以最低的胰蛋白酶活力作比较，牛羊胰蛋白酶分别3668u/g、2151u/g；超过药典规定6.1倍、3.5倍。干燥失重、残留脂肪在药典规定的指标以下，具有特殊的肉臭味；异丙醇提取胰酶呈灰白色粉末，而壳聚糖絮凝法提取产品则呈淡黄色，胰蛋白酶、胰淀粉酶的活力比同一种类异丙醇法提取胰酶制品的活力低，但羊胰淀粉酶和胰脂肪酶活力相反；在胰酶提取操作方法相同的条件下，用胰蛋白酶活力做参照比较，猪牛羊三种酶活力平均分别为4754.5u/g、3538u/g、2607u/g，猪胰酶的活力最高，羊胰酶活力最小，牛胰酶制品的活力介于二者之间。
4．胰蛋白酶分离纯化。牛羊胰酶粉分别经过胰蛋白酶粗提、过滤、离心、饱和硫酸铵分级沉淀、丙酮沉淀、凝胶过滤等一系列纯化后，牛羊胰蛋白酶的纯化倍数和蛋白质回收率分别达到19.8、23.2；38.2%、13.6%。2种纯化的蛋白酶在SDS-PAGE电泳胶片上显示单条带，其相对分子量分别约为24kDa和27kDa。纯化后牛羊胰蛋白酶均能水解TAME，且被PMSF、SBTI和TLCK强烈抑制，抑制率分别在81-98%、74-96%的范围，不被EDTA抑制，所以结合其相对分子质量大小、水解特性、抑制特性，可以确定这是2种胰蛋白酶。
5．胰蛋白酶的生物特性。在波长247nm下，牛羊胰蛋白酶TAME底物时，2种胰蛋白酶的最适温度及稳定温度范围相同，分别是60℃、20-50℃；最适作用pH分别是8.0、9.0，酸碱稳定性范围各为pH8-12和pH7-12；说明2种酶在中性和碱性条件稳定；且CaCl_2对其胰蛋白酶均具有稳定性，不同浓度的NaCl对胰蛋白酶的活力有降低作用。
6．胰蛋白酶动力学特性。研究表明牛羊胰蛋白酶的米氏常数Km分别是0.28μmmol/L、0.53μmmol/L，2种胰蛋白酶对TAME有很好的亲和力，牛胰蛋白酶对底物TAME的亲和力比羊胰蛋白酶高。同时还发现二者对TAME的亲和力比BAPNA高。其催化效率Kcat、生理转化效率Kcat/Km分别为7.905s~(-1)、7.702s~(-1)；28.23s~(-1)μmol~(-1)、14.53s~(-1)μmol~(-1)。明显高于已报道的其它哺乳动物胰蛋白酶。
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.
引文
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