Towards practical time-of-flight secondary ion mass spectrometry lignocellulolytic enzyme assays

Biotechnology for Biofuels

Robyn E Goacher²  Alex Yi-Lin Tsai¹  Emma R Master² 

[1] Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario, Canada

[2] Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada

关键词: Cellulase;    Extractives;    Enzymes;    Lignocellulose;    ToF-SIMS;   

Others  :  797904
DOI  :  10.1186/1754-6834-6-132

 received in 2013-07-22, accepted in 2013-09-12,  发布年份 2013

【 摘 要 】

Background

Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) is a surface sensitive mass spectrometry technique with potential strengths as a method for detecting enzymatic activity on solid materials. In particular, ToF-SIMS has been applied to detect the enzymatic degradation of woody lignocellulose. Proof-of-principle experiments previously demonstrated the detection of both lignin-degrading and cellulose-degrading enzymes on solvent-extracted hardwood and softwood. However, these preliminary experiments suffered from low sample throughput and were restricted to samples which had been solvent-extracted in order to minimize the potential for mass interferences between low molecular weight extractive compounds and polymeric lignocellulose components.

Results

The present work introduces a new, higher-throughput method for processing powdered wood samples for ToF-SIMS, meanwhile exploring likely sources of sample contamination. Multivariate analysis (MVA) including Principal Component Analysis (PCA) and Multivariate Curve Resolution (MCR) was regularly used to check for sample contamination as well as to detect extractives and enzyme activity. New data also demonstrates successful ToF-SIMS analysis of unextracted samples, placing an emphasis on identifying the low-mass secondary ion peaks related to extractives, revealing how extractives change previously established peak ratios used to describe enzyme activity, and elucidating peak intensity patterns for better detection of cellulase activity in the presence of extractives. The sensitivity of ToF-SIMS to a range of cellulase doses is also shown, along with preliminary experiments augmenting the cellulase cocktail with other proteins.

Conclusions

These new procedures increase the throughput of sample preparation for ToF-SIMS analysis of lignocellulose and expand the applications of the method to include unextracted lignocellulose. These are important steps towards the practical use of ToF-SIMS as a tool to screen for changes in plant composition, whether the transformation of the lignocellulose is achieved through enzyme application, plant mutagenesis, or other treatments.

【 授权许可】

   
2013 Goacher et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

【 参考文献 】

• [1]Goacher RE, Edwards EA, Mims CA, Master ER: Application of ToF-SIMS for the detection of enzyme activity on solid wood substrates. Anal Chem 2012, 84:4443-4452.
• [2]Kleen M: Surface lignin and extractives on hardwood RDH kraft pulp chemically characterized by ToF-SIMS. Holzforschung 2005, 59:481-487.
• [3]Kokkonen P, Fardim P, Holmbom B: Surface distribution of extractives on TMP handsheets analyzed by ESCA, ATR-IR, ToF-SIMS and ESEM. Nordic Pulp & Paper Res J 2004, 19:318-324.
• [4]Koljonen K, Oesterberg M, Kleen M, Fuhrmann A, Stenius P: Precipitation of lignin and extractives on kraft pulp: effect on surface chemistry, surface morphology and paper strength. Cellulose 2004, 11:209-224.
• [5]Fardim P, Holmbom B: ToF-SIMS imaging: a valuable chemical microscopy technique for paper and paper coatings. Appl Surf Sci 2005, 249:393-407.
• [6]Matsushita Y, Suzuki A, Sekiguchi T, Saito K, Imai T, Fukushima K: Mapping of the cationic starch adsorbed on pulp fibers by ToF-SIMS. Appl Surf Sci 2008, 255:1022-1024.
• [7]Fardim P, Holbom B: Chemical microscopy of extractives on fiber and paper surfaces. In Characterization of Lignocellulosic Materials. Edited by Hu TQ. Oxford: Blackwell Publishing Ltd; 2008:101-118.
• [8]Tokareva EN, Pranovich AV, Holmbom BR: Characteristic fragment ions from lignin and polysaccharides in ToF-SIMS. Wood Sci Technol 2011, 45:767-785.
• [9]Tokareva EN, Fardim P, Pranovich AV, Fagerholm HP, Daniel G, Holmbom B: Imaging of wood tissue by ToF-SIMS: critical evaluation and development of sample preparation techniques. Appl Surf Sci 2007, 253:7569-7577.
• [10]Jung S, Foston M, Kalluri UC, Tuskan GA, Ragauskas AJ: 3D Chemical image using TOF-SIMS revealing the biopolymer component spatial and lateral distributions in biomass. Angew Chem Int Edit 2012, 51:12005-12008.
• [11]Saito K, Mitsutani T, Imai T, Matsushita Y, Fukushima K: Discriminating the indistinguishable sapwood from heartwood in discolored ancient wood by direct molecular mapping of specific extractives using time-of-flight secondary ion mass spectrometry. Anal Chem 2008, 80:1552-1557.
• [12]Matsushita Y, Jang I-C, Imai T, Takama R, Saito K, Masumi T, Lee S-C, Fukushima K: Distribution of extracts including 4,8-dihydroxy-5-methoxy-2-naphthaldehyde in diospyros kaki analyzed by gas chromatography–mass spectrometry and time-of-flight secondary ion mass spectrometry. Holzforschung 2012, 66:705-709.
• [13]Brabander DJ, Keon N, Stanley RHR, Hemond HF: Intra-ring variability of Cr, as, Cd, and Pb in red oak revealed by secondary ion mass spectrometry: implications for environmental biomonitoring. Proc Natl Acad Sci USA 1999, 96:14635-14640.
• [14]Saito K, Watanabe Y, Shirakawa M, Matsushita Y, Imai T, Koike T, Sano Y, Funada R, Fukazawa K, Fukushima K: Direct mapping of morphological distribution of syringyl and guaiacyl lignin in the xylem of maple by time-of-flight secondary ion mass spectrometry. Plant J 2012, 69:542-552.
• [15]Jung S, Foston M, Sullards MC, Ragauskas AJ: Surface characterization of dilute acid pretreated populus deltoides by ToF-SIMS. Energy Fuels 2010, 24:1347-1357.
• [16]Mou H-Y, Orblin E, Kruus K, Fardim P: Topochemical pretreatment of wood biomass to enhance enzymatic hydrolysis of polysaccharides to sugars. Bioresource Technol 2013, 142C:540-545.
• [17]Mahajan S, Jeremic D, Goacher RE, Master ER: Mode of coniferous wood decay by the white-rot fungus phanerochaete carnosa as elucidated by FT-IR and ToF-SIMS. Appl Microbiol Biot 2012, 94:1303-1311.
• [18]Goacher RE, Jeremic D, Master ER: Expanding the library of secondary ions that distinguish lignin and polysaccharides in time-of-flight secondary Ion mass spectrometry analysis of wood. Anal Chem 2011, 83:804-812.
• [19]Lee JLS, Gilmore IS: The application of multivariate data analysis techniques in surface analysis. In Surface Analysis. The Principal Techniques. 2nd edition. Edited by Vickerman JC, Gilmore IS. West Sussex, UK: John Wiley & Sons, Ltd; 2009.
• [20]Fardim P, Gustafsson J, von Schoultz S, Peltonen J, Holmbom B: Extractives on fiber surfaces investigated by XPS, ToF-SIMS and AFM. Colloids Surf A 2005, 255:91-103.
• [21]Strabala TJ, MacMillan CP: The arabidopsis wood model—the case for the inflorescence stem. Plant Sci 2013, 210:193-205.
• [22]Ninomiya S, Ichiki K, Yamada H, Nakata Y, Seki T, Aoki T, Matsuo J: Molecular depth profiling of multilayer structures of organic semiconductor materials by secondary ion mass spectrometry with large argon cluster ion beams. Rapid Commun Mass Spec 2009, 23:3264-3268.
• [23]Pachuta SJ, Vlasak PR: Post-acquisition mass resolution improvement in time-of-flight secondary Ion mass spectrometry. Anal Chem 2012, 84:1744-1753.
• [24]Yang B, Wyman CE: BSA treatment to enhance enzymatic hydrolysis of cellulose in lignin containing substrates. Biotechnol Bioeng 2006, 94:611-617.
• [25]Hagerman AE, Butler LG: Protein precipitation method for the quantitative determination of tannins. J Agr Food Chem 1978, 26:809-812.
• [26]Keenan MR, Kotula PG: Accounting for poisson noise in the multivariate analysis of ToF-SIMS spectrum images. Surf Interface Anal 2004, 36:203-212.