中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (25): 4627-4630.doi: 10.3969/j.issn.1673-8225.2010.25.017

• 骨髓干细胞 bone marrow stem cells • 上一篇    下一篇

重组降血压肽VLPVPR酶解制备工艺优化及降解原因分析

肖  平1，孙海燕2，刘  冬2，周丽珍2，李  艳2   

  1. 1广西大学轻工与食品工程学院，广西壮族自治区南宁市  530004；2深圳职业技术学院，广东省深圳市  518055
  • 出版日期:2010-06-18 发布日期:2010-06-18
  • 通讯作者: 孙海燕，博士，副教授，深圳职业技术学院，广东省深圳市 518055
  • 作者简介:肖 平★，男，1985年生，江西省安福县人，汉族，广西大学在读硕士，主要从事食品生物技术研究。 xiaoping0305@126.com
  • 基金资助:

    广东省科技厅科技攻关项目(粤科计字[2006]119号)。

Enzymolysis technology optimization and degradation reasons of recombinant antihypertensive peptides VLPVPR

Xiao Ping1, Sun Hai-yan2, Liu Dong2, Zhou Li-zhen2, Li Yan2   

  1. 1 College of Light Industry and Food Engineering, Guangxi University, Nanning  530004, Guangxi Zhuang Autonomous Region, China; 2 Shenzhen Polytechnic, Shenzhen  518055, Guangdong Province, China
  • Online:2010-06-18 Published:2010-06-18
  • Contact: Sun Hai-yan, Doctor, Associate professor, Shenzhen Polytechnic, Shenzhen 518055, Guangdong Province, China
  • About author:Xiao Ping★, Studying for master’s degree, College of Light Industry and Food Engineering, Guangxi University, Nanning 530004, Guangxi Zhuang Autonomous Region, China xiaoping0305@126.com
  • Supported by:

    the Science and Technology Development Program of Guangdong Provincial Science and Technology Department, No. [2006]119*

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377

被引次数

2

摘要：

背景：采用基因工程技术与酶解法制备重组降血压肽VLPVPR，酶解产物为多肽混合物，易受微生物侵染而发生降解。
目的：分析胰蛋白酶酶解制备重组降血压肽VLPVPR过程中影响VLPVPR稳定性的因素，并对酶解工艺进行了优化。
方法：采用反相高效液相色谱法检测酶解产物中降血压肽VLPVPR 的含量。工程菌表达产物经0.22 μm膜过滤除菌，观察其酶解过程中降血压肽VLPVPR含量随时间的变化情况，与不过滤除菌酶解过程进行比较，探讨杂菌污染和工程菌自身蛋白水解酶对VLPVPR降解的影响。采用正交试验对温度、pH、酶浓度和时间等酶解工艺参数进行优化。
结果与结论：降血压肽VLPVPR标准品与胰蛋白酶作用4 h，含量基本不发生变化，差异无显著性意义(P > 0.05)，说明胰蛋白酶不能再进一步降解VLPVPR。经过过滤除菌后的细胞破碎上清液在酶解过程中的VLPVPR含量在达到最大后迅速降低，未经过滤除菌的样品中的降血压肽比过滤除菌的样品降解得更快，差异有显著性意义（P < 0.05），表明工程菌释放的胞内酶和杂菌污染是导致VLPVPR降解的主要原因。通过正交试验优化了酶解制备重组降血压肽VLPVPR的最佳条件：温度30 ℃，pH9.5，胰蛋白酶终浓度180 U/mL，酶解时间1 h。

关键词: 降血压肽, 制备, 酶解, 降解, 生物材料与生物技术

Abstract:

BACKGROUND: The recombinant antihypertensive peptide, Val-Lys-Pro-Val-Pro-Arg (VLPVPR), was prepared using genetic engineering technology and enzymolysis method. The enzymolysis products are polypeptide compound, which is prone to be infested and deteriorated by microorganism.
OBJECTIVE: To analyze enzymolysis technology and degradation causes of the recombinant antihypertensive peptide, VLPVPR, during trypsin-catalyzed hydrolysis process.
METHODS: The content of antihypertensive peptide, VLPVPR, was detected by reversed phase high-performance liquid chromatography. The engineering germ products were sterilized by 0.22 μm membrane filter, and the content information of VLPVPR in the enzymolysis process was observed. Compared with non-filtered sterilization enzymatic process, the degradation of endoenzymes released from the engineered strain cells and bacterial contamination on VLPVPR was approached. The enzymolysis technology conditions, such as temperature, pH, final concentration of trypsin, and duration, were optimized by orthogonal experiment.
RESULTS AND CONCLUSION: The content of VLPVPR standard remained unchanged after 4 hours’ action with trypsin, which showed no significant difference (P > 0.05), demonstrating that VLPVPR could not be degraded by trypsin. The VLPVPR of the cell disruption which had been sterilized was degraded rapidly after reaching the maximum value, and the VLPVPR of the sample without sterilized was degraded faster, which showed significant difference (P < 0.05). Results showed the endoenzymes released from the engineered strain cells and bacterial contamination is the main reasons for VLPVPR degradation during preparation process. The best hydrolyzing condition of trypsin was optimized by orthogonal experiment: 30 ℃, pH 9.5, final concentration of trypsin at 180 U/mL, and reaction time for 1 hour.

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