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http://dx.doi.org/10.5352/JLS.2019.29.12.1408

Problems and Solutions of Zymography Techniques  

Kang, Dae-Ook (Department of Bio Health Science, Changwon National University)
Choi, Nack-Shick (Department of Bio Health Science, Changwon National University)
Publication Information
Journal of Life Science / v.29, no.12, 2019 , pp. 1408-1414 More about this Journal
Abstract
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.
Keywords
Binding mode; diagonal zymography; electrotransfer; enzyme; zymography;
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1 Lantz, M. S. and Ciborowski, P. 1994. Zymographic techniques for detection and characterization of microbial proteases. Methods Enzymol. 235, 563-594.   DOI
2 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.   DOI
3 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.   DOI
4 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.   DOI
5 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.
6 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.   DOI
7 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.   DOI
8 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.   DOI
9 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.   DOI
10 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.   DOI
11 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.   DOI
12 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.   DOI
13 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.   DOI
14 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.   DOI
15 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.   DOI
16 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.
17 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.   DOI
18 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.   DOI
19 Choi, N. S. and Kim, S. H. 2000. Two fibrin zymography methods for analysis of plasminogen activators on gels. Anal. Biochem. 281, 236-238.   DOI
20 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.   DOI
21 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.   DOI
22 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   DOI
23 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.
24 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.   DOI
25 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.   DOI
26 Granelli-Piperno, A. and Reich, E. 1978. A study of proteases and protease-inhibitor complexs in biological fluids. J. Exp. Med. 148, 223-234.   DOI
27 Gross, J. and Lapiere, C. M. 1962. Collagenolytic activity in amphibian tissues: a tissue culture assay. Proc. Natl. Acad. Sci. USA. 48, 1014-1022.   DOI
28 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.   DOI
29 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.   DOI
30 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.   DOI
31 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.   DOI
32 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.   DOI
33 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.   DOI
34 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.   DOI
35 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.   DOI
36 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.   DOI
37 Kim, S. H. and Choi, N. S. 1999. Electrophoretic analysis of protease inhibitors in fibrin zymography. Anal. Biochem. 270, 179-181.   DOI
38 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.   DOI
39 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.   DOI
40 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.
41 Laemmli, U. K. 1970. Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 227, 680-685.   DOI