Detection of Multiple Enzymes in Fermentation Broth Using Single PAGE Analysis

  • K. Divakar
  • J. Deepa Arul Priya
  • G. Panneer Selvam
  • M. Suryia Prabha
  • Ashwin Kannan
  • G. Nandhini Devi
  • Pennathur GautamEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1853)


Activity staining or zymography is a technique to detect enzymes based on their function/activity toward a specific substrate. Multiple enzyme-producing microbes secrete enzymes along with other proteins at varying time points during fermentation. The technique of zymography can be used to detect functionality of enzymes in complex protein/other enzyme mixtures. The protein bands corresponding to specific enzyme among other enzymes/proteins can be located by polyacrylamide gel electrophoresis (PAGE) followed by zymogram analysis. This can be employed to locate the secretion pattern of protein/enzyme from intracellular region to extracellular medium. Here we describe simple method for detection and cellular localization of esterases and protease secreted by single microbial strain in one PAGE gel.

Key words

Zymogram Multiple enzymes Fermentation broth PAGE 



We thank Department of Biotechnology and Department of Science and Technology (DST), Government of India, for their continuous support to carry out research in our lab. K. Divakar thanks DST, Govt. of India for funding through DST-INSPIRE Faculty award and research grant (IFA14-ENG-87).


  1. 1.
    Vandooren J, Geurts N, Martens E, Van den Steen PE, Opdenakker G (2013) Zymography methods for visualizing hydrolytic enzymes. Nat Methods 10:211–220CrossRefPubMedGoogle Scholar
  2. 2.
    Choi NS et al (2009) Multiple-layer substrate zymography for detection of several enzymes in a single sodium dodecyl sulfate gel. Anal Biochem 386:121–122CrossRefPubMedGoogle Scholar
  3. 3.
    Choi NS et al (2009) Mixed-substrate (glycerol tributyrate and fibrin) zymography for simultaneous detection of lipolytic and proteolytic enzymes on a single gel. Electrophoresis 30:2234–2237CrossRefPubMedGoogle Scholar
  4. 4.
    Carvalho NB, de Souza RL (2008) Sequential production of amylolytic and lipolytic enzymes by bacterium strain isolated from petroleum contaminated soil. Appl Biochem Biotechnol 150:25–32CrossRefPubMedGoogle Scholar
  5. 5.
    Divakar K, Suryia Prabha M, Devi GN, Gautam P (2016) Kinetic characterisation and fed-batch fermentation for maximal simultaneous production of esterase and protease from Lysinibacillus fusiformis AU01. Prep Biochem Biotechnol 47:323–332CrossRefPubMedGoogle Scholar
  6. 6.
    Divakar K, Sujatha V, Barath S, Srinath K, Gautam P (2011) In-gel staining of proteins in native poly acryl amide gel electrophoresis using Tetrakis(4-sulfonato phenyl)porphyrin. Anal Sci 27:101–103CrossRefPubMedGoogle Scholar
  7. 7.
    Divakar K, Deepa Arul Priya J, Gautam P (2010) Purification and characterization of thermostable organic solvent-stable protease from Aeromonas veronii PG01. J Mol Catal B Enzym 66:311–318CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • K. Divakar
    • 1
  • J. Deepa Arul Priya
    • 2
  • G. Panneer Selvam
    • 2
  • M. Suryia Prabha
    • 2
  • Ashwin Kannan
    • 3
  • G. Nandhini Devi
    • 2
  • Pennathur Gautam
    • 2
    • 4
    Email author
  1. 1.Department of BiotechnologyNational Institute of TechnologyWarangalIndia
  2. 2.Centre for BiotechnologyAnna UniversityChennaiIndia
  3. 3.Department of Chemical EngineeringVisvesvaraya National Institute of TechnologyNagpurIndia
  4. 4.AU-KBC Research CentreAnna UniversityChennaiIndia

Personalised recommendations