Identification of proteins involved in starch and polygalacturonic acid degradation using LC/MS

Raimonda Petkauskaite 1 , Dangiras Lukosius 1 , Janusz Dębski 2 , Andrius Jasilionis 1 , Michał Dadlez 2 , Ieva Kieraite 1 , Ana Timonina 1  and Nomeda Kuisiene 1
  • 1 Department of Microbiology and Biotechnology, Faculty of Natural Sciences, Vilnius University, LT-03101, Vilnius, Lithuania
  • 2 Mass Spectrometry Laboratory, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106, Warsaw, Poland


Plant biomass in the form of cheap wastes, such as straw, corn stalks, wood chips, sawdust, bagasse, pomace, etc., is abundant throughout the world. To convert these wastes into the useful value-added compounds microbial enzymes are the preferred choice. In this paper, we identify enzymes involved in the degradation of starch and polygalacturonic acid using liquid chromatography/mass spectrometry based analysis. We analysed total protein from soil and compost samples. Extracellular proteins from enrichment cultures were analysed in parallel and used as controls in the sample preparation and identification of proteins. In general, both protein sequence coverage and the number of identified peptides were higher in the samples obtained from the enrichment cultures than from the total protein from soil and compost. The influence of the nature of gel (zymography vs. SDS/polyacrylamide) was negligible. Thus, starch and polygalacturonic acid degradation associated proteins can be directly excised from the zymograms without the need to align zymograms with the SDS/polyacrylamide gels. A range of starch and polygalacturonic acid degradation associated enzymes were identified in both total protein samples and extracellular proteins from the enrichment cultures. Our results show that proteins involved in starch and polygalacturonic acid degradation can be identified by liquid chromatography/mass spectrometry from the complex protein mixtures both with and without cultivation of microorganism

If the inline PDF is not rendering correctly, you can download the PDF file here.

  • [1] Akpinar O., Gunay K., Yilmaz Y., Levent O., Bostanci S., Enzymatic processing and antioxidant activity of agricultural waste autohydrolysis liquors, BioRes., 2010,

  • [2] Mussatto S.I., Ballesteros L.F., Martins S., Teixeira J.A., Use of agro-industrial wastes in solid-state fermentation processes, In: Show K.-Y. (Ed.), Industrial Waste, InTech, 2012

  • [3] Sweeney M.T., Xu F., Biomass converting enzymes as industrial biocatalysts for fuels and chemicals: recent developments, Catalysts, 2012,

  • [4] Turner P., Mamo G., Nordberg Karlsson E., Potential and utilization of thermophiles and thermostable enzymes in biorefining, Microb. Cell. Fact., 2007,

  • [5] Ruijssenaars H.J., Hartman S., Plate screening methods for the detection of polysaccharaseproducing microorganisms, Appl. Microbiol. Biotechnol., 2001, 55, 143–149

  • [6] Pang H., Zhang P., Duan C.J., Mo X.C., Tang J.L., Feng J.X., Identification of cellulase genes from the metagenomes of compost soils and functional characterization of one novel endoglucanase, Curr. Microbiol., 2009, 58, 404–408

  • [7] Badel S., Laroche C., Gardarin C., Petit E., Bernardi T., Michaud P., A new method to screen polysaccharide cleavage enzymes, Enzyme Microb. Technol., 2011, 48, 248–252

  • [8] Fer M., Préchoux A., Leroy A., Sassi J.-F., Lahaye M., Boisset C., et al., Medium-throughput profiling method for screening polysaccharide-degrading enzymes in complex bacterial extracts, J. Microbiol. Methods, 2012, 55, 222–229

  • [9] Peterson R., Grinyer J., Joss J., Khan A., Nevalainen H., Fungal proteins with mannanase activity identified directly from a Congo Red stained zymogram by mass spectrometry, J. Microbiol. Methods, 2009, 79, 374–377

  • [10] Geib S.M., Tien M., Hoover K., Identification of proteins involved in lignocellulose degradation using in gel zymogram analysis combined with mass spectroscopy-based peptide analysis of gut proteins from larval Asian longhorned beetles, Anoplophora grabripennis, Insect Sci, 2010, 17, 253–264

  • [11] Cheng H.-L., Tsai C.-Y., Chen H.-J., Yang S.-S., Chen Y.-C., The identification, purification, and characterization of STXF10 expressed in Streptomyces thermonitrificans NTU-88, Appl. Microbiol. Biotechnol., 2009, 82, 681–689

  • [12] Murase A., Yoneda M., Ueno R., Yonebayashi K., Isolation of extracellular protein from greenhouse soil, Soil Biol. Biochem., 2003, 35, 733–736

  • [13] Takao M., Nakaniwa T., Yoshikawa K., Terashita T., Sakai T., Purification and characterization of thermostable pectate lyase with protopectinase activity from thermophilic Bacillus sp. TS 47, Biosci. Biotechnol. Biochem., 2001, 64, 2360–2367

  • [14] Al-Qodah Z., Production and characterization of thermostable α-amylase by thermophilic Geobacillus stearothermophilus, Biotechnol. J., 2006, 1, 850–857

  • [15] Laemmli U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 1970, 227, 680–685

  • [16] Tamura K., Peterson D., Peterson N., Stecher G., Nei M., Kumar S., MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods, Mol. Biol. Evol., 2011, 28, 2731–2739

  • [17] Higdon R., Kolker E., A predictive model for identifying proteins by a single peptide match, Bioinformatics, 2007, 23, 277–280


Journal + Issues

Open Life Sciences (previously Central European Journal of Biology) is a fast growing OA journal, devoted to scholarly research in all areas of life sciences. The journal assures top quality of published data through critical peer review, editorial involvement throughout the whole publication process. Thanks to OA it also offers unrestricted access to published articles for all users.