Skip to main content
SpringerLink
Log in

Partial Purification and Characterization of Fibrinolytic Enzymes from Yellow Mealworm

  • Published:
International Journal of Peptide Research and Therapeutics Aims and scope Submit manuscript

Abstract

Two proteins with fibrinolytic activity were partially purified from yellow mealworm (Tenebrio molitor) by ammonium sulfate precipitation between 30 and 70% saturation, gel filtration on Sephacryl-S200-HR, ion exchange chromatography on DEAE-Sepharose-FF and metal chelate on Cu–HiTrap–IMAC–FF, but the enzymes had not been completely separated from each other. The two partially purified fibrinolytic enzymes were designated as TMFE-I and TMFE-II (Tenebrio molitor fibrinolytic enzyme) with molecular weights of 27.5 and 24.9 kDa by SDS-PAGE individually. The partially purified solution of TMFE-I and TMFE-II was considerably stable in the range of pH 5–10 and characterized by pH optimum of the enzymatic activity at 8.0. Thermal stability of TMFE was excellent at 45°C and below. The K M value was 0.26 mM for amidolysis of Bz–Arg–pNA. According to inhibitor analysis by fibrin plate method, phenylmethylsulfonyl fluoride and tosyl-lysine chloromethyl ketone inactivated TMFE almost completely, but trans-(epoxysuccinyl)-l-leucylamino-4-guanidinobutane (E-64) and EDTA had little effect on their fibrinolytic activity. According to metal ion analysis by fibrin plate method, the effect of metal ions on activity of TMFE showed a great difference. Na+, K+ and Zn2+ had little effect on the activity of TMFE. Mg2+ and Cu2+ showed inhibition effect on the fibrinolytic activity of TMFE, but Ca2+ increased the fibrinolytic activity of TMFE at final concentration varying from 0 to 30 mM.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Astrup T, Mullertz S (1952) The fibrin plate method for estimating fibrinolytic activity. Arch Biochem Biophys 40:346–351

    Article  PubMed  CAS  Google Scholar 

  • Bradford MM (1976) Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  • Choi NS, Kim SH (2000) Two fibrin zymography methods for analysis of plasminogen activators on gels. Anal Biochem 281:236–238

    Article  PubMed  CAS  Google Scholar 

  • Erlanger BF, Cohen W, Kokowsky N (1961) Preparation and properties of 2 new chromogenic substrates of trypsin. Arch Biochem Biophys 95:271–278

    Article  PubMed  CAS  Google Scholar 

  • Ferreira AHP, Marana SR, Terra WR, Ferreira C (2001) Purification, molecular cloning, and properties of a beta-glycosidase isolated from midgut lumen of tenebrio molitor (coleoptera) larvae. Insect Biochem Mol Biol 31:1065–1076

    Article  PubMed  CAS  Google Scholar 

  • Fraenkel G, Blewett M, Coles M (1950) The nutrition of the mealworm, tenebrio molitor L (tenebrionidae, coleoptera). Physiol Zool 23:92–108

    PubMed  CAS  Google Scholar 

  • Genta FA, Blanes L, Cristofoletti PT (2006) do Lago CL, Terra WR, Ferreira C. Purification, characterization and molecular cloning of the major chitinase from tenebrio molitor larval midgut. Insect Biochem Mol Biol 36:789–800

    Article  PubMed  CAS  Google Scholar 

  • Genta FA, Bragatto I, Terra WR, Ferreira C (2009) Purification, characterization and sequencing of the major beta-1,3-glucanase from the midgut of tenebrio molitor larvae. Insect Biochem Mol Biol 39:861–874

    Article  PubMed  CAS  Google Scholar 

  • Hacke W, Kaste M, Fieschi C, Toni D, Lesaffre E, Vonkummer R, Boysen G, Bluhmki E, Hoxter G, Mahagne MH et al (1995) Intravenous thrombolysis with recombinant tissue-plasminogen activator for acute hemispheric stroke—the European Cooperative Acute Stroke Study (ecass). JAMA 274:1017–1025

    Article  PubMed  CAS  Google Scholar 

  • Heyneman RA (1965) Final purification of a latent phenolase with mono- and diphenoloxidase activity from tenebrio molitor. Biochem Biophys Res Commun 21:162–169

    Article  PubMed  CAS  Google Scholar 

  • Ilan J (1975) Similarities in properties and a functional difference in purified leucyl-transfer-rna synthetase isolated from 2 developmental stages of tenebrio molitor. Dev Biol 42:64–74

    Article  PubMed  CAS  Google Scholar 

  • Jang KS, Cho MY, Choi HW, Lee KM, Kim MH, Lee YU, Kurata S, Natori S, Lee BL (1998) Purification and characterization of a 25 kda cathepsin l-like protease from the hemocyte of coleopteran insect, tenebrio molitor larvae. J Biochem Mol Biol 31:364–369

    CAS  Google Scholar 

  • Kim SH, Choi NS, Lee WY (1998) Fibrin zymography: A direct analysis of fibrinolytic enzymes on gels. Anal Biochem 263:115–116

    Article  PubMed  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during assembly of head of bacteriophage-t4. Nature 227:680–685

    Article  PubMed  CAS  Google Scholar 

  • Levinsky H, Birk Y, Applebaum SW (1977) Isolation and characterization of a new trypsin-like enzyme from tenebrio molitor L larvae. Int J Pept Protein Res 10:252–264

    Article  PubMed  CAS  Google Scholar 

  • Loncar N, Vujcic Z, Bozic N, Ivanovic J, Nenadovic V (2010) Purification and properties of trypsin-like enzyme from the midgut of morimus funereus (coleoptera, cerambycidae) larvae. Arch Insect Biochem Physiol 74:232–246

    Article  PubMed  CAS  Google Scholar 

  • Mihara H, Sumi H, Yoneta T, Mizumoto H, Ikeda R, Seiki M, Maruyama M (1991) A novel fibrinolytic enzyme extracted from the earthworm, lumbricus rubellus. Jpn J Physiol 41:461–472

    Article  PubMed  CAS  Google Scholar 

  • Moon HJ, Lee SY, Kurata S, Natori S, Lee BL (1994) Purification and molecular cloning of cDNA for an inducible antibacterial protein from larvae of the coleopteran, tenebrio molitor. J Biochem 116:53–58

    PubMed  CAS  Google Scholar 

  • Oliveira MGA, De Simone SG, Xavier LP, Guedes RNC (2005) Partial purification and characterization of digestive trypsin-like proteases from the velvet bean caterpillar, anticarsia gemmatalis. Comp Biochem Physiol B 140:369–380

    Article  PubMed  CAS  Google Scholar 

  • Park YD, Kim JW, Min BG, Seo JW, Jeong JM (1998) Rapid purification and biochemical characteristics of lumbrokinase III from earthworm for use as a fibrinolytic agent. Biotechnol Lett 20:169–172

    Article  CAS  Google Scholar 

  • Silva LB, Reis AP, Pereira EJG, Oliveira MGA, Guedes RNC (2010) Partial purification and characterization of trypsin-like proteinases from insecticide-resistant and -susceptible strains of the maize weevil, sitophilus zeamais. Comp Biochem Physiol B 155:12–19

    Article  PubMed  CAS  Google Scholar 

  • Tomchaney AP, Morris JP, Kang SH, Duman JG (1982) Purification, composition, and physical properties of a thermal hysteresis antifreeze protein from larvae of the beetle, tenebrio molitor. Biochemistry 21:716–721

    Article  PubMed  CAS  Google Scholar 

  • Tsybina TA, Dunaevsky YE, Belozersky MA, Zhuzhikov DP, Oppert B, Elpidina EN (2005) Digestive proteinases of yellow mealworm (tenebrio molitor) larvae: Purification and characterization of a trypsin-like proteinase. Biochemistry (Moscow) 70:300–305

    Article  CAS  Google Scholar 

  • Vinokurov KS, Elpidina EN, Oppert B, Prabhakar S, Zhuzhikov DP, Dunaevsky YE, Belozersky MA (2006) Diversity of digestive proteinases in tenebrio molitor (coleoptera : Tenebrionidae) larvae. Comp Biochem Physiol B 145:126–137

    Article  PubMed  CAS  Google Scholar 

  • Walton PL (1966) An improved fibrin plate method for assay of plasminogen activators. Clin Chim Acta 13:680–684

    Article  PubMed  CAS  Google Scholar 

  • Yaginuma T, Mizuno T, Mizuno C, Ikeda M, Wada T, Hattori K, Yamashita O, Happ GM (1996) Trehalase in the spermatophore from the bean shaped accessory gland of the male mealworm beetle, tenebrio molitor: Purification, kinetic properties and localization of the enzyme. J Comp Physiol B 166:1–10

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the Nature Science Foundation of China (No. 30772739) and the “211” Project of Faculty of Chemical Engineering and Light Industry, Guangdong University of Technology (No. 20091015).

Author information

Authors and Affiliations

  1. Department of Biological Engineering, Faculty of Chemical Engineering and Light Industry, Guangdong University of Technology, Guangzhou, 510006, China

    Ming Xing Huang, Yun Ye, Ya Xiong Chen & Ya Li Han

Authors
  1. Ming Xing Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yun Ye
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ya Xiong Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ya Li Han
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ming Xing Huang.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Huang, M.X., Ye, Y., Chen, Y.X. et al. Partial Purification and Characterization of Fibrinolytic Enzymes from Yellow Mealworm. Int J Pept Res Ther 18, 153–161 (2012). https://doi.org/10.1007/s10989-012-9288-x

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10989-012-9288-x

Keywords

Search

Navigation