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