Surface analysis technique as a methodology for screening of lignocellulose-active enzymes

Sustainable production of fuels and chemicals from biomass (e.g. lignocellulose) is essential considering growing demand for alternatives to petrochemical commodities. While considerable effort has been devoted to genome and metagenome sequencing and discovery of new lignocellulolytic activities, a remaining limitation to enzyme development is the availability of robust enzyme screens on relevant biomass substrates.

Several methods have been described for screening enzymes by measuring monomers and oligomers released during lignocellulose hydrolysis. Dinitrosalicylic acid (DNS) assay is one such method that has been previously automated (1) and is particularly relevant for detecting enzymes that are active on solubilized polysaccharides. However, assays like DNS that detect soluble products, will miss activities that modify fibre composition without releasing products to the reaction mixture.  Moreover, assessment of both polysaccharide active and lignin active enzymes is difficult. Alternatively, time of flight-secondary ion mass spectrometry (ToF-SIMS) is a surface technique analysis that can be used to characterize solid biomass residuals after enzyme treatment. This technique has been recently adapted for 96-well enzymatic assays (2), which has increased the potential of this approach for enzyme screening.

Herein, we combined the analysis of (i) soluble sugars with an automated DNS assay and (ii) the surface of solid residues using ToF-SIMS, to screen the activity of more than 40 enzymes on powdered wood samples. Among other samples, enzymes from metagenome sequences that originated from a water waste-treatment plant were targeted.  

1 Navarro D et al, 2010.  Microb Cell Fact, 9:58.

2 Goacher RE et al, 2013. Biotech Biofuels, 6:132.