胰蛋白酶激活条件优化及其在细胞培养中的应用

doi:10.3969/j.issn.1673-1689.2017.12.012

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胰蛋白酶激活条件优化及其在细胞培养中的应用
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                        10.3969/j.issn.1673-1689.2017.12.012
                    
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Activate Condition Optimization of Trypsin and Its Application in Animal Cell Culture

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基于猪胰脏提取胰蛋白酶，以酶活力为衡量指标，在单因素试验的基础上通过正交试验探讨了胰蛋白酶原的激活条件，并通过离子交换层析纯化胰蛋白酶，最后探讨胰蛋白酶对BHK-21和Vero两种贴壁依赖性细胞的消化效果。结果表明，胰蛋白酶原最佳的激活条件为：激活剂浓度0.2 mol/L，pH 8.5，16 ℃下激活1.5 h，酶活力达到2 953.68 U/mg。通过DEAE sepharose FF纯化后其酶活力达到3 980.42 U/mg，纯化倍数达到2.26；细胞消化结果表明，用其消化BHK-21细胞和Vero细胞并连续10代，平均消化时间和细胞增殖浓度与Hyclone和Gibco产品没有明显的差异（p＞0.05），每代细胞形态良好，生长正常。该工艺简单，激活耗时短，易于产业化，为胰蛋白酶的国产化和进一步研究奠定理论依据。

Abstract:

Trypsin was extracted from porcine pancreas，enzyme activity was used as the measurement index. On the basis of single factor experiment the orthogonal test was used to determine the optimization of parameters of trypsinogen activation. At the same time，the trypsin was purified using ion exchange chromatography. The effects of digestive BHK-21 and Vero cells were investigated. The results showed that the optimum conditions for activation of trypsinogen were as follows：activator concentration 0.2 mol/L，activation pH 8.5，16 ℃ for 1.5 h. Under these conditions，enzyme activity of trypsin was 2 953.68 U/mg. Trypsin was purified to activity get 3 980.42 U/mg with purification fold of 2.26 times onto a DEAE-sepharose-FF column. The cell culture results showed that BHK-21 and Vero cells were digestived 10 generation. The growth and proliferate well，every generation cell morphology was normal. The digestion time and cell proliferation concentration had no obvious difference with the products of Hyclone and Gibco. In conclusion，the process is simple and easy to be industrialized with short activate time. The study can be served as a scientific basis for the further exploitation，utilization and localization of trypsin.

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李明生,靳冬武,张健,冯玉萍,郑荣,张磊,李倬,马忠仁.胰蛋白酶激活条件优化及其在细胞培养中的应用[J].食品与生物技术学报,2017,36(12):1304-1310.

LI Mingsheng, JIN Dongwu, ZHANG Jian, FENG Yuping, ZHENG Rong, ZHANG Lei, LI Zhuo, MA Zhongren. Activate Condition Optimization of Trypsin and Its Application in Animal Cell Culture[J]. Journal of Food Science and Biotechnology,2017,36(12):1304-1310.

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在线发布日期: 2018-01-08
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