Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Vandooren J, Geurts N, Martens E, Van den Steen PE, Opdenakker G (2013) Zymography methods for visualizing hydrolytic enzymes. Nat Methods 10:211–220
Choi NS et al (2009) Multiple-layer substrate zymography for detection of several enzymes in a single sodium dodecyl sulfate gel. Anal Biochem 386:121–122
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 30:2234–2237
Carvalho NB, de Souza RL (2008) Sequential production of amylolytic and lipolytic enzymes by bacterium strain isolated from petroleum contaminated soil. Appl Biochem Biotechnol 150:25–32
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 47:323–332
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 27:101–103
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 66:311–318
Acknowledgments
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).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Divakar, K. et al. (2018). Detection of Multiple Enzymes in Fermentation Broth Using Single PAGE Analysis. In: Kurien, B., Scofield, R. (eds) Protein Gel Detection and Imaging. Methods in Molecular Biology, vol 1853. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8745-0_16
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8745-0_16
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8744-3
Online ISBN: 978-1-4939-8745-0
eBook Packages: Springer Protocols