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Quantitative colorimetric measurement of cellulose degradation under microbial culture conditions

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Abstract

We have developed a simple, rapid, quantitative colorimetric assay to measure cellulose degradation based on the absorbance shift of Congo red dye bound to soluble cellulose. We term this assay “Congo Red Analysis of Cellulose Concentration,” or “CRACC.” CRACC can be performed directly in culture media, including rich and defined media containing monosaccharides or disaccharides (such as glucose and cellobiose). We show example experiments from our laboratory that demonstrate the utility of CRACC in probing enzyme kinetics, quantifying cellulase secretion, and assessing the physiology of cellulolytic organisms. CRACC complements existing methods to assay cellulose degradation, and we discuss its utility for a variety of applications.

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References

  • Amann E, Ochs B, Abel KJ (1988) Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene 69:301–315

    Article  CAS  Google Scholar 

  • Anthon GE, Barrett DM (2002) Determination of reducing sugars with 3-methyl-2-benzothiazolinonehydrazone. Anal Biochem 305(2):287–289

    Article  CAS  Google Scholar 

  • Béguin P, Aubert JP (1994) The biological degradation of cellulose. FEMS Microbiol Rev 13(1):25–58

    Article  Google Scholar 

  • Bertani G (1951) Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol 62(3):293–300

    CAS  Google Scholar 

  • Bertani G (2004) Lysogeny at mid-twentieth century: P1, P2, and other experimental systems. J Bacteriol 186(3):595–600

    Article  CAS  Google Scholar 

  • Carere C, Sparling R, Cicek N, Levin D (2008) Third generation biofuels via direct cellulose fermentation. Int J Mol Sci 9(7):1342–1360

    Article  CAS  Google Scholar 

  • Carroll A, Somerville C (2009) Cellulosic biofuels. Annu Rev Plant Biol 60(1):165–182

    Article  CAS  Google Scholar 

  • Chapon V, Simpson HD, Morelli X, Brun E, Barras F (2000) Alteration of a single tryptophan residue of the cellulose-binding domain blocks secretion of the Erwinia chrysanthemi Cel5 cellulase (ex-EGZ) via the type II system. J Mol Biol 303(2):117–123

    Article  CAS  Google Scholar 

  • DeBoy RT, Mongodin EF, Fouts DE, Tailford LE, Khouri H, Emerson JB, Mohamoud Y, Watkins K, Henrissat B, Gilbert HJ, Nelson KE (2008) Insights into plant cell wall degradation from the genome sequence of the soil bacterium Cellvibrio japonicus. J Bacteriol 190(15):5455–5463

    Article  CAS  Google Scholar 

  • Doner LW, Irwin PL (1992) Assay of reducing end-groups in oligosaccharide homologues with 2,2′-bicinchoninate. Anal Biochem 202(1):50–53

    Article  CAS  Google Scholar 

  • Du F, Wolger E, Wallace L, Liu A, Kaper T, Kelemen B (2010) Determination of product inhibition of CBH1, CBH2, and EG1 using a novel cellulase activity assay. Appl Biochem Biotech 161(1):313–317

    Article  CAS  Google Scholar 

  • Gardner JG, Keating DH (2010) Requirement of the type II secretion system for utilization of cellulosic substrates by Cellvibrio japonicus. Appl Environ Microbiol 76(15):5079–5087

    Article  CAS  Google Scholar 

  • Ghose TK (1987) Measurement of cellulase activities. Pure Appl Chem 59(2):257–268

    Article  CAS  Google Scholar 

  • He SY, Lindeberg M, Chatterjee AK, Collmer A (1991) Cloned Erwinia chrysanthemi out genes enable Escherichia coli to selectively secrete a diverse family of heterologous proteins to its milieu. Proc Natl Acad Sci USA 88(3):1079–1083

    Article  CAS  Google Scholar 

  • Heimann M, Reichstein M (2008) Terrestrial ecosystem carbon dynamics and climate feedbacks. Nature 451(7176):289–292

    Article  CAS  Google Scholar 

  • Helbert W, Chanzy H, Husum TL, Schülein M, Ernst S (2003) Fluorescent cellulose microfibrils as substrate for the detection of cellulase activity. Biomacromolecules 4(3):481–487

    Article  CAS  Google Scholar 

  • Howie AJ, Brewer DB, Howell D, Jones AP (2007) Physical basis of colors seen in Congo red-stained amyloid in polarized light. Lab Invest 88(3):232–242

    Article  Google Scholar 

  • Hu G, Heitmann JA, Rojas OJ (2009) Quantification of cellulase activity using the quartz crystal microbalance technique. Anal Chem 81(5):1872–1880

    Article  CAS  Google Scholar 

  • Keen NT, Dahlbeck D, Staskawicz B, Belser W (1984) Molecular cloning of pectate lyase genes from Erwinia chrysanthemi and their expression in Escherichia coli. J Bacteriol 159(3):825–831

    CAS  Google Scholar 

  • Klunk WE, Pettegrew JW, Abraham DJ (1989) Quantitative evaluation of Congo red binding to amyloid-like proteins with a beta-pleated sheet conformation. J Histochem Cytochem 37(8):1273–1281

    Article  CAS  Google Scholar 

  • Kongruang S, Han MJ, Breton CI, Penner MH (2004) Quantitative analysis of cellulose-reducing ends. Appl Biochem Biotechnol 113–116:213–231

    Article  Google Scholar 

  • Leschine SB (1995) Cellulose degradation in anaerobic environments. Annu Rev Microbiol 49(1):399–426

    Article  CAS  Google Scholar 

  • Lever M (1972) A new reaction for colorimetric determination of carbohydrates. Anal Biochem 47(1):273–279

    Article  CAS  Google Scholar 

  • Lindner WA, Dennison C, Quicke GV (1983) Pitfalls in the assay of carboxymethylcellulase activity. Biotech Bioeng 25(2):377–385

    Article  CAS  Google Scholar 

  • Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS (2002) Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol Rev 66(3):506–577

    Article  CAS  Google Scholar 

  • Maglione G, Russell J, Wilson D (1997) Kinetics of cellulose digestion by Fibrobacter succinogenes S85. Appl Environ Microbiol 63(2):665–669

    CAS  Google Scholar 

  • Marais JP, De Wit JL, Quicke GV (1966) A critical examination of the Nelson-Somogyi method for the determination of reducing sugars. Anal Biochem 15(3):373–381

    Article  CAS  Google Scholar 

  • Park SR, Cho SJ, Kim MK, Ryu SK, Lim WJ, An CL, Hong SY, Kim JH, Kim H, Yun HD (2002) Activity enhancement of Cel5Z from Pectobacterium chrysanthemi PY35 by removing C-terminal region. Biochem Biophys Res Comm 291(2):425–430

    Article  CAS  Google Scholar 

  • Py B, Isabelle B-G, Haiech J, Chippaux M, Barras F (1991) Cellulase EGZ of Erwinia chrysanthemi: structural organization and importance of His98 and Glu133 residues for catalysis. Protein Eng 4(3):325–333

    Article  CAS  Google Scholar 

  • Teather RM, Wood PJ (1982) Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl Environ Microbiol 43(4):777–780

    CAS  Google Scholar 

  • Vlasenko EY, Ryan AI, Shoemaker CF, Shoemaker SP (1998) The use of capillary viscometry, reducing end-group analysis, and size exclusion chromatography combined with multi-angle laser light scattering to characterize endo-1,4-β-glucanases on carboxymethylcellulose: a comparative evaluation of the three methods. Enzym Microb Technol 23(6):350–359

    Article  CAS  Google Scholar 

  • Waffenschmidt S, Jaenicke L (1987) Assay of reducing sugars in the nanomole range with 2,2′-bicinchoninate. Anal Biochem 165(2):337–340

    Article  CAS  Google Scholar 

  • Wilson DB (2008) Three microbial strategies for plant cell wall degradation. Ann NY Acad Sci 1125:289–297

    Article  CAS  Google Scholar 

  • Wilson DB (2011) Microbial diversity of cellulose hydrolysis. Curr Opin Microbiol 14(3):259–263

    Article  CAS  Google Scholar 

  • Wood PJ (1980) Specificity in the interaction of direct dyes with polysaccharides. Carbohydr Res 85(2):271–287

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We thank Matthew DeLisa and Nicole Perna for providing plasmids and genomic DNA derived from D. dadantii and Paul Weimer for guidance in framing the manuscript. This work was funded by the DOE Great Lakes Bioenergy Research Center (DOE BER Office of Science DE-FC02-07ER64494).

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, 5445 Microbial Sciences Building, 1550 Linden Dr., Madison, WI, 53706, USA

    Rembrandt J. F. Haft

  2. Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, 3554 Microbial Sciences Building, 1550 Linden Dr., Madison, WI, 53706, USA

    Jeffrey G. Gardner & David H. Keating

Authors
  1. Rembrandt J. F. Haft
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  2. Jeffrey G. Gardner
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  3. David H. Keating
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Correspondence to David H. Keating.

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Haft, R.J.F., Gardner, J.G. & Keating, D.H. Quantitative colorimetric measurement of cellulose degradation under microbial culture conditions. Appl Microbiol Biotechnol 94, 223–229 (2012). https://doi.org/10.1007/s00253-012-3968-5

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  • DOI: https://doi.org/10.1007/s00253-012-3968-5

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