Abstract
Accurate protein quantification is necessary in many of the steps during the enzymatic hydrolysis of pretreated lignocellulosic biomass, from the fundamental determination of enzyme kinetics to techno-economic assessments, such as the use of enzyme recycling strategies, evaluation of enzyme costs, and the optimization of various process steps. In the work described here, a modified, more rapid ninhydrin-based protein quantification assay was developed to better quantify enzyme levels in the presence of lignocellulosic biomass derived compounds. The addition of sodium borohydride followed by acid hydrolysis at 130 °C greatly reduced interference from monosaccharides and oligosaccharides and decreased the assay time 6-fold. The modified ninhydrin assay was shown to be more accurate as compared to various traditional colorimetric protein assays when commercial cellulase enzyme mixtures were quantified under typical pretreated lignocellulosic biomass enzymatic hydrolysis conditions. The relatively short assay time and microplate-reading capability of the modified assay indicated that the method could likely be used for high-throughput protein determination.
Similar content being viewed by others
References
Klein-Marcuschamer, D., Oleskowicz-Popiel, P., Simmons, B. A., & Blanch, H. W. (2012). The challenge of enzyme cost in the production of lignocellulosic biofuels. Biotechnology and Bioengineering, 109, 1083–1087.
Davis, R., Tao, L., Tan, E.C.D., Biddy, M.J., Beckham, G.T., Scarlata, C., et al. (2013). Process design and economics for the conversion of lignocellulosic biomass to hydrocarbons: dilute-acid and enzymatic deconstruction of biomass to sugars and biological conversion of sugars to hydrocarbons. Technical report, National Renewable Energy Laboratory. Golden, CO.
Humbird, D., Davis, R., Tao, L., Kinchin, C., Hsu, D., Aden, A., et al. (2011). Process design and economics for biochemical conversion of lignocellulosic biomass to ethanol: dilute-acid pretreatment and enzymatic hydrolysis of corn stover. Technical report, National Renewable Energy Laboratory. Golden, CO.
Ghose, T. K. (1987). Measurement of cellulase activities. Pure and Applied Chemistry, 59, 257–268.
Adney, W. S., Dowe, N., Jennings, E. W., Mohagheghi, A., Yarbrough, J., & McMillan, J. D. (2012). Assessing the protein concentration in commercial enzyme preparations. Methods in Molecular Biology, 908, 169–180.
Arantes, V., & Saddler, J. N. (2011). Cellulose accessibility limits the effectiveness of minimum cellulase loading on the efficient hydrolysis of pretreated lignocellulosic substrates. Biotechnology for Biofuels, 4, 3.
McMillan, J. D., Jennings, E. W., Mohagheghi, A., & Zuccarello, M. (2011). Comparative performance of precommercial cellulases hydrolyzing pretreated corn stover. Biotechnology for Biofuels, 4, 29.
Gupta, R., & Lee, Y. Y. (2013). In C. E. Wyman (Ed.), Aqueous pretreatment of plant biomass for biological and chemical conversion to fuels and chemicals (pp. 261–279). Chichester: Wiley.
Adney, W. S., Mohagheghi, A., Thomas, S. R., & Himmel, M. E. (1996). In J. N. Saddler & M. H. Penner (Eds.), Enzymatic degradation of insoluble carbohydrates. Washington: American Chemical Society.
Schilling, E. D., Burchill, P. I., & Clayton, R. A. (1963). Anomalous reactions of ninhydrin. Analytical Biochemistry, 5, 1–6.
Walker, J. M. (2002). The protein protocols handbook. Totowa: Humana Press.
Zhang, Y. H., & Lynd, L. R. (2004). Toward an aggregated understanding of enzymatic hydrolysis of cellulose: noncomplexed cellulase systems. Biotechnology and Bioengineering, 88, 797–824.
Aguiar, A., & Ferraz, A. (2007). Fe3+- and Cu2+-reduction by phenol derivatives associated with Azure B degradation in Fenton-like reactions. Chemosphere, 66, 947–954.
Chauvet, J., & Lamy, F. (1990). Determination of soluble lignin and proteins in the presence of each other. Analytica Chimica Acta, 235, 299–306.
Chylenski, P., Felby, C., Ostergaard, H. M., Gama, M., & Selig, M. J. (2012). Precipitation of Trichoderma reesei commercial cellulase preparations under standard enzymatic hydrolysis conditions for lignocelluloses. Biotechnology Letters, 34, 1475–1482.
Folin, O., & Denis, W. (1912). On phosphotungstic-phosphomolybdic compounds as color reagents. Journal of Biological Chemistry, 12, 239–243.
Martinez, A., Rodriguez, M. E., York, S. W., Preston, J. F., & Ingram, L. O. (2000). Use of UV absorbance to monitor furans in dilute acid hydrolysates of biomass. Biotechnology Progress, 16, 637–641.
Turunen, J., & Turunen, K. (1967). Some factors causing colour in acetate pulp and cellulose acetate. Pure and Applied Chemistry, 67, 555–562.
**menes, E., Kim, Y., Mosier, N., Dien, B., & Ladisch, M. (2011). Deactivation of cellulases by phenols. Enzyme and Microbial Technology, 48, 54–60.
D’Aniello, A., D’Onofrio, G., Pischetola, M., & Strazzullo, L. (1985). Effect of various substances on the colorimetric amino acid-ninhydrin reaction. Analytical Biochemistry, 144, 610–611.
Friedman, M. (2004). Applications of the ninhydrin reaction for analysis of amino acids, peptides, and proteins to agricultural and biomedical sciences. Journal of Agricultural and Food Chemistry, 52, 385–406.
Kang, S. U., & Lubec, G. (2011). Determination of in-gel protein concentration by a ninhydrin-based method. Proteomics, 11, 481–484.
Marks, D. L., Buchsbaum, R., & Swain, T. (1985). Measurement of total protein in plant samples in the presence of tannins. Analytical Biochemistry, 147, 136–143.
Starcher, B. (2001). A ninhydrin-based assay to quantitate the total protein content of tissue samples. Analytical Biochemistry, 292, 125–129.
Haven, M. O., & Jorgensen, H. (2013). The challenging measurement of protein in complex biomass-derived samples. Applied Biochemistry and Biotechnology, 172, 1–15.
Abdek-Akher, M., Hamilton, J. K., & Smith, F. (1951). The reduction of sugars with sodium borohydride. Journal of the American Chemical Society, 73, 4691–4692.
Fountoulakis, M., & Lahm, H. (1998). Hydrolysis and amino acid composition analysis of proteins. Journal of Chromatography A, 826, 109–134.
Chiou, S., & Wang, K. (1989). Peptide and protein hydrolysis by microwave irradiation. Journal of Chromatography B: Biomedical Sciences and Applications, 491, 424–431.
Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J. and Templeton, D. (2006). Determination of sugars, byproducts, and degradation products in liquid fraction process samples. Technical report, National Renewable Energy Laboratory. Golden, CO.
Zhang, Q., Zhang, J., Shen, J., Silva, A., Dennis, D., & Barrow, C. (2006). A simple 96-well microplate method for estimation of total polyphenol content in seaweeds. Journal of Applied Phycology, 18, 445–450.
Ng, T. K., & Zeikus, J. G. (1986). Synthesis of [14C]cellobiose with clostridium thermocellum cellobiose phosphorylase. Applied and Environmental Microbiology, 52, 902–904.
Zhu, Z., Sathitsuksanoh, N., & Percival Zhang, Y. H. (2009). Direct quantitative determination of adsorbed cellulase on lignocellulosic biomass with its application to study cellulase desorption for potential recycling. Analyst, 134, 2267–2272.
Banka, R. R., & Mishra, S. (2002). Adsorption properties of the fibril forming protein from Trichoderma reesei. Enzyme and Microbial Technology, 31, 784–793.
Nieves, R. A., Ehrman, C. I., Adney, W. S., Elander, R. T., & Himmel, M. E. (1997). Survey and analysis of commercial cellulase preparations suitable for biomass conversion to ethanol. World Journal of Microbiology and Biotechnology, 14, 301–304.
Noble, J. E., & Bailey, M. J. (2009). Quantitation of protein. Methods in Enzymology, 463, 73–95.
Acknowledgments
We want to thank our colleague, Dr. **guang Hu, for all of his invaluable help. The Natural Sciences and Engineering Research Council of Canada (NSERC) and the Bioconversion network financially supported this work. We also thank our colleagues at Novozymes for their generous donation of enzyme preparations.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Mok, Y.K., Arantes, V. & Saddler, J.N. A NaBH4 Coupled Ninhydrin-Based Assay for the Quantification of Protein/Enzymes During the Enzymatic Hydrolysis of Pretreated Lignocellulosic Biomass. Appl Biochem Biotechnol 176, 1564–1580 (2015). https://doi.org/10.1007/s12010-015-1662-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12010-015-1662-7