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农学学报

农学学报 ›› 2025, Vol. 15 ›› Issue (4): 61-68.doi: 10.11923/j.issn.2095-4050.cjas2024-0044

• 农业工程 农业机械 生物技术 食品科学 • 上一篇    下一篇

赤子爱胜蚯蚓激酶的提取工艺及酶学性质研究

张红艳1(), 殷红2, 韩姗姗2, 张强1, 辛东林1, 冉淦侨1()   

  1. 1 陕西省生物农业研究所/陕西省秦岭生态安全重点实验室，西安 710043
    2 陕西省酶工程技术研究中心，西安 710043
  • 收稿日期:2024-03-11 修回日期:2024-08-15 出版日期:2025-04-20 发布日期:2025-04-17
  • 通讯作者:
    冉淦侨，女，1984年出生，重庆人，副研究员，博士，研究方向：生物农业。通信地址：710043 陕西省西安市新城区咸宁中路125号，E-mail：。
  • 作者简介:

    张红艳，女，1978年出生，河南沁阳人，助理研究员，在读硕士，研究方向：生物农业。通信地址：710043 陕西省西安市新城区咸宁中路125号，E-mail：。

  • 基金资助:
    陕西省自然科学基金项目“甘露聚糖对木质纤维材料中纤维素水解的影响及作用机制”(2023-JC-QN-0254)

Study on Extraction Technology and Enzyme Properties of Lumbrokinase from Eisenia foetida

ZHANG Hongyan1(), YIN Hong2, HAN Shanshan2, ZHANG Qiang1, XIN Donglin1, RAN Ganqiao1()   

  1. 1 Bio-Agriculture Institute of Shaanxi /Shaanxi Key Laboratory of Qinling Ecological Security, Xi'an 710043
    2 Shaanxi Enzyme Engineering Technology Research Center, Xi’an 710043
  • Received:2024-03-11 Revised:2024-08-15 Online:2025-04-20 Published:2025-04-17

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摘要：

对比赤子爱胜蚯蚓中提取蚓激酶的不同工艺及参数，分析其酶学性质，促进蚯蚓加工副产物的开发和利用。结合纤维蛋白平板酶活测定、聚丙烯酰铵凝胶电泳(SDS-PAGE)以及液质联用技术(HPLC-MS)对蚓激酶的提取工艺进行研究。得到较佳提取工艺为：以干蚯蚓粉做原料，采用5倍质量比的10 mmol/L Tris-HCl缓冲液浸提离心2次后合并上清液；粗提液采用80%硫酸铵饱和度盐析，10~200 kD孔径的膜分离方法浓缩，膜循环过滤时间≤50 min；不添加保护剂上机冷冻干燥。此提取干燥工艺条件下得到酶活性为201.8万U/g的冻干粉。酶学性质研究表明；该酶包含6种蚓激酶特异性蛋白，相对分子量在24.7~26.3 kD之间；酶液最适pH 7.2~8.0；较稳定区间为pH 7.4~8.8；酶液最适温度区间为50℃以下，稳定温度区间为60℃以下；4℃条件下储藏80 d，酶活保持率可达90%。硫酸铵盐析-冷冻干燥法可以从赤子爱胜蚯干粉中提取得到酶活稳定的蚓激酶产品，实验结果可为蚯蚓的高值化利用提供一定的参考依据。

关键词: 赤子爱胜蚯蚓, 蚓激酶, 提取, 硫酸铵盐析, 冷冻干燥, 酶学性质, 高值化利用

Abstract:

This study compared different methods and parameters for extracting earthworm fibrinolytic enzyme (EFE) from Eisenia fetida, analyzed the enzyme properties, and promoted the development and utilization of earthworm processing byproducts. The extraction process of EFE was studied by combining fibrin plate enzyme activity assay, dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and high performance liquid chromatography-mass spectrometry (HPLC-MS). The optimal extraction process was as follows: using dry earthworm powder as raw material, extract with 5 times the mass ratio of 10 mmol/L Tris-HCl buffer, centrifuge twice and combine the supernatants; precipitate the crude extract with 80% ammonium sulfate, concentrate with a membrane separation method with a pore size of 10k-200 kD, and limit the membrane circulation filtration to ≤50 min; freeze-dry without adding a protective agent. Under this extraction and drying process, the enzyme activity of the freeze-dried powder was 201.8 million U/g. Enzyme characterization studies revealed that the enzyme comprised six earthworm-specific proteins with molecular weights ranging from 24.7 kD to 26.3 kD. The optimal pH range for the enzyme solution is 7.2 to 8.0, with a relatively stable pH range of 7.4 to 8.8. The optimal temperature for the enzyme solution is below 50℃, and it remains stable below 60℃. After 80 days of storage at 4℃, the enzyme activity retention rate can reach 90%. The ammonium sulfate-freeze drying method can extract enzymatically stable lumbrokinase products from dried E. fetida powder. The experimental results provide a reference for the high utilization of earthworms.

Key words: Eisenia fetida, lumbrokinase, extraction, ammonium sulfate, freeze drying, enzymatic properties, high value utilization