Problems and Solutions of Zymography Techniques

자이모그라피 기술의 문제점과 해결

  • Kang, Dae-Ook (Department of Bio Health Science, Changwon National University) ;
  • Choi, Nack-Shick (Department of Bio Health Science, Changwon National University)
  • 강대욱 (창원대학교 생명보건학부) ;
  • 최낙식 (창원대학교 생명보건학부)
  • Received : 2019.12.09
  • Accepted : 2019.12.26
  • Published : 2019.12.30


Enzymes are widely used in industrial applications such as detergents, food, feed production, pharmaceuticals and medical applications and major contributors to clean industrial products and processes. To screen, identify, and characterize the enzymes the zymography techniques are routinely used. The zymography technique is a simple, sensitive, and quantifiable technique that is widely used to detect functional enzymes following electrophoretic separation in sodium dodecyl sulfate (SDS)-polyacrylamide gels. The method is a versatile two-stage technique involving protein separation by electrophoresis followed by the detection of enzyme activity in polyacrylamide gels under non-reducing conditions. It is based on SDS-polyacrylamide gel (PAG) copolymerization with substrates, which are degraded by the hydrolytic enzymes restored in enzyme reaction buffer after the electrophoretic separation. Any kind of biological sample can be applied and analyzed on zymography, including culture supernatants of microbes, plants extracts, blood, tissue culture fluids, enzymes in foods extracts and metaproteome. The advantage of zymography is that it is possible to directly detect the protein with activity on the electrophoretic gel as well as confirm the activity at the nanogram level. Thus, this zymography technology can be applied in various fields. However, these advantages are rather disadvantageous and can often lead to experimental errors. In this review, the advantages, disadvantages, and problem solving of zymography technique are described.


  1. Brown, T. L., Yet, M. G. and Wold, F. 1982. Substrate-containing gel electrophoresis: sensitive detection of amylolytic, nucleolytic, and proteolytic enzymes. Anal. Biochem. 122, 164-172
  2. Choi, N. S., Choi, J. H., Kim, B. H., Han, Y. J., Kim, J. S., Lee, S. G. and Song, J. J. 2009. Mixed-substrate (glycerol tributyrate and fibrin) zymography for simultaneous detection of lipolytic and proteolytic enzymes on a single gel. Electrophoresis 30, 2232-2237.
  3. Choi, N. S., Choi, J. H., Yoon, J. H., Lee, S. G. and Song, J. J. 2009. Identification of a serine protease from a Bacillus sp. using multiple loading of O'Farrell-type isoelectric focusing slab two-dimensional gel. Biotechnol. Lett. 31, 975-978.
  4. Choi, N. S., Kim, B. H., Park, C. S., Han, Y. J., Lee, H. W., Choi, J. H., Lee, S. G. and Song, J. J. 2009. Multiple-layer substrate zymography for detection of several enzymes in a single sodium dodecyl sulfate gel. Anal. Biochem. 386, 121-122.
  5. Choi, N. S. and Kim, S. H. 1999. Application of fibrin zymography for determining the optimum culture time for protease activity. Biotechnol. Techniq. 13, 899-901.
  6. Choi, N. S. and Kim, S. H. 2000. Two fibrin zymography methods for analysis of plasminogen activators on gels. Anal. Biochem. 281, 236-238.
  7. Choi, N. S., Song, J. J., Chung, D. M., Kim, Y. J., Maeng, P. J. and Kim, S. H. 2009. Purification and characterization of a thermo acid-stable fibrinolytic enzyme from Staphylococcus sp. strain AJ isolated from Korean salted-fermented Anchovy-joet. J. Ind. Microbiol. Biotechnol. 36, 417-426.
  8. Choi, N. S., Jeong, S. Y., Yang, H. J., Ahn, K. H., Park, C. S., Kim, C. Y., Kim, J. S., Yoon, B. D. and Kim, M. S. 2010. Activity assay for nisin-like acidic bacteriocins using an optimal pH-conditioned gel matrix. Anal. Biochem. 397, 259-261.
  9. Chung, D. M., Kim, K. E., Ahn, K. H., Park, C. S., Kim, D. H., Koh, H. B., Chun, H. K., Yoon, B. D., Kim, H. J., Kim, M. S. and Choi, N. S. 2011. Silver-stained fibrin zymography: separation of proteases and activity detection using a single substrate-containing gel. Biotechnol. Lett. 33, 1663-1666.
  10. Duarte, J. G., Leone-lgnacio, K., da Silva, J. A. C., Fernandez-Lafuente, R. and Freire, D. M. G. 2016. Rapid determination of the synthetic activity of lipases/esterases via transesterification and esterification zymography. Fuel 177, 123-129.
  11. Granelli-Piperno, A. and Reich, E. 1978. A study of proteases and protease-inhibitor complexs in biological fluids. J. Exp. Med. 148, 223-234.
  12. Gross, J. and Lapiere, C. M. 1962. Collagenolytic activity in amphibian tissues: a tissue culture assay. Proc. Natl. Acad. Sci. USA. 48, 1014-1022.
  13. Haddar, A., Agrebi, R., Bougatef, A., Hmidet, N., Sellami-Kamoun, A. and Nasri, M. 2009. Two detergent stable alkaline serine-proteases from Bacillus mojavensis A21: Purification, characterization and potential application as a laundry detergent additive. Bioresour. Technol. 100, 3366-3373.
  14. Hammami, A., Hamdi, M., Abdelhedi, O., Jridi, M., Nasri, M. and Bayoudh, A. 2017. Surfactant- and oxidant-stable alkaline proteases from Bacillus invictae: Characterization and potential applications in chitin extraction and as a detergent additive. Int. J. Biol. Macromol. 96, 272-281.
  15. Heussen, C. and Dowdle, E. B. 1980. Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates. Anal. Biochem. 102, 196-202.
  16. Hibbs, M. S., Hasty, K. A., Seyer, J. M., Kang, A. H. and Mainardi, C. L. 1985. Biochemical and immunological characterization of the secreted forms of human neutrophil gelatinase. J. Biol. Chem. 260, 2493-2500.
  17. Hmidet, N., Jemil, N. and Masri, M. 2019. Simultaneous production of alkaline amylase and biosurfactant by Bacillus methylotrophicus DCS1: application as detergent additive. Biodegradation 30, 247-258.
  18. Houde, M., De Bruyne, G., Bracke, M., Ingelman-sundberg, M., Skoglund, G., Masure, S., Van Damme, J. and Opdenakker, G. 1993. Differential regulation of gelatinase B and tissue-type plasminogen activator expression in human Bowes melanoma cells. Int. J. Cancer 53, 395-400.
  19. Kang, S. J., Choi, N. S., Choi, J. H., Lee, J. S., Yoon, J. H. and Song, J. J. 2009. Brevundimonas naejangsanensis sp. nov., a proteolytic bacterium isolated from soil, and reclassification of Mycoplana bullata into the genus Brevundimonas as Brevundimonas bullata comb. nov. Int. J. Syst. Evol. Microbiol. 59, 3155-3160.
  20. Katrina, M. H., Penheiter, A. R., Gathman, A. C. and Lilly, W. W. 1996. Anomalous Estimation of Protease Molecular Weights Using Gelatin-Containing SDS-PAGE. Anal. Biochem. 233, 140-142.
  21. Kim, S. H. and Choi, N. S. 1999. Electrophoretic analysis of protease inhibitors in fibrin zymography. Anal. Biochem. 270, 179-181.
  22. Kim, S. H., Choi, N. S. and Lee, W. Y. 1998. Fibrin zymography: a direct analysis of fibrinolytic enzymes on gels. Anal. Biochem. 263, 115-116.
  23. Kobayashi, T., Kakizaki, I. and Nakamura, T. 2019. Proteoglycan-substrate gel zymography for the detection of chondroitin sulfate-degrading enzymes. Anal. Biochem. 568, 51-52.
  24. Kocabay, S., Cetinkaya, S., Akkaya, B. and Yenidunya, A. F. 2016. Characterization of thermostable ${\beta}$-amylase isozymes from Lactobacillus fermentum. Int. J. Biol. Macromol. 93, 195-202.
  25. Kurz, L., Hernandez, Z., Contreras, L. M. and Wilkesman, J. 2017. Sequential detection of thermophilic lipase and protease zymography. In Zymography: Methods in Mol. Biol. (pp. 271-277): Springer.
  26. Laemmli, U. K. 1970. Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 227, 680-685.
  27. Lantz, M. S. and Ciborowski, P. 1994. Zymographic techniques for detection and characterization of microbial proteases. Methods Enzymol. 235, 563-594.
  28. Masure, S., Billiau, A., Van Damme, J. and Opdenakker, G. 1990. Human hepatoma cells produce an 85 kDa gelatinase regulated by phorbol 12-myristate 13-acetate. Biochim. Biophys. Acta 1054, 317-325.
  29. Masure, S., Proost, P., Van Damme, J. and Opdenakker, G. 1991. Purification and identification of 91-kDa neutrophil gelatinase. Release by the activating peptide interleukin-8. Eur. J. Biochem. 198, 391-398.
  30. Paemen, L., Martens, E., Narga, K., Masure, S., Roets, E., Hoogmartens, J. and Opdenakker, G. 1996. The gelatinase inhibitory activity of tetracyclines and chemically modified tetracycline analogues as measured by a novel microtiter assay for inhibitors. Biochem. Pharmacol. 52, 105-111.
  31. Park, C. S., Kang, D. O., Lee, W. Y., Chun, S. S., Lim, S. Y., Moon, J. Y., Kim, D. H. and Choi, N. S. 2015. Identification of two types binding modes using reverse or diagonal electrophoretic zymography. Aca. J. Biotechnol. 3, 52-55.
  32. Park, C. S., Yang, H. J., Kim, D. H., Kang, D. O., Kim, M. S. and Choi, N. S. 2012. A screening method for ${\beta}$-glucan hydrolase employing trypan blue-coupled ${\beta}$-glucan agar plate and ${\beta}$-glucan zymography. Biotechnol. Lett. 34, 1073-1077.
  33. Park, J. W., Cho, S. Y. and Choi, S. J. 2008. Purification and characterization of hepatic lipase from Todarodes pacificus. BMB Reports 41, 254-258.
  34. Park, S. G., Kho, C. W., Cho, S., Lee, D. H., Kim, S. H. and Park, B. C. 2002. A functional proteomic analysis of secreted fibrinolytic enzymes from Bacillus subtilis 168 using a combined method of two-dimensional gel electrophoresis and zymography. Protomics 2, 206-211.<206::AID-PROT206>3.0.CO;2-5
  35. Phitsuwan, P., Tachaapaikoon, C., Kosugi, A., Mori, Y., Kyu, K. L. and Ratanakhanokchai, K. 2010. A Cellulolytic and Xylanolytic Enzyme Complex from an Alkalothermoanaerobacterium, Tepidimicrobium xylanilyticum BT14. J. Microbiol. Biotechnol. 20, 893-903.
  36. Picart, P., Diaz, P. and Pastor, F. I. J. 2007. Cellulases from two Penicillium sp. strains isolated from subtropical forest soil: production and characterization. Lett. Appl. Microbiol. 45, 108-113.
  37. Pillai, P., Mandge, S. and Archana, G. 2011. Statistical optimization of production and tannery applications of a keratinolytic serine protease from Bacillus subtilis P13. Proc. Biochem. 46, 1110-1117.
  38. Schwarz, W. H., Bronnenmeier, K., Grabnitz, F. and Staudenbauer, W. L. 1987. Activity staining of cellulases in polyacrylamide gels containing mixed linkage ${\beta}$-glucans. Anal. Biochem. 164, 72-77.
  39. Sookkheo, B., Sinchaikul, S., Phutrakul, S. and Chen, S. T. 2000. Purification and Characterization of the Highly Thermostable Proteases from Bacillus stearothermophilus TLS33. Prot. Exp. Purif. 20, 142-151.
  40. Su, L. J., Liu, H., Li, Y., Zhang, H. F., Chen, M., Gao, X. H., Wang, F. Q. and Song, A. D. 2014. Cellulolytic activity and structure of symbiotic bacteria in locust guts. Gen. Mol. Res. 13, 7926-7936.
  41. Tapizquent, M., Fernandez, M., Barreto, G., Hernandez, Z., Contreras, L. M., Kurz, L. and Wilkesman, J. 2017. Zymography detection of a bacterial extracellular thermoalkaline esterase/lipase activity. In Zymography: Methods in Mol. Biol. (pp. 295-300): Springer.