A High Throughput Assay of Lichenase Activity with Congo Red Dye in Plants

Abstract

Background : To elucidate the functional role of regulatory sequences and contexts identified and predicted during omics studies in the complex mechanisms of gene expression regulation, experimental verification methods in a plant cell are required. For this, the method of transient expression of reporter genes fused with the tested sequences is widely used. Several reporter systems are available that have shown good performance in the studies including the thermostable lichenase Clostridium thermocellum . However, each reporter system has its own limitations with respect to the quantification of the protein product of a reporter gene and, in particular, for the use of high-throughput approaches. Results: In this study, we report the design a high throughput fluorometric method for quantification of thermostable lichenase C. thermocellum using Congo red and further experimental verification of its relevance and efficiency in assessment of the functional role of regulatory sequences in the plant cell. Conclusion: The specific interaction between the dye Congo red and β -D-glucans formed the background for designing a high-throughput fluorometric assay for quantification of C. thermocellum thermostable lichenase as a reporter protein for plants. This assay (i) makes it possible to precisely measure the amount of reporter protein in a plant sample; (ii) has shown a high sensitivity for quantification of thermostable lichenase; (iii) is more time- and cost-efficient as compared with the Somogyi–Nelson assay; and (iv) is to the least degree dependent on the presence of the tested buffer components as compared with the Somogyi–Nelson assay.

The synergism of lytic polysaccharide monooxygenases with lichenase and their co-immobilization on silica nanospheres for green conversion of lichen biomass

“…Lichenan (β-1,3-1,4-glucan) is a linear polysaccharide derived from lichen, accounting for approximately 62% of the total carbohydrates in lichen, but the high viscosity of lichens in water results in low utilization ( 1 ). Lichenase (EC 3.2.1.73) could cleave linear lichenan consisting of β-1,3 and β-1,4 bonds with strict specificity for the β-1,4 glycosidic bond adjacent to the 3-O-glucopyranose residue ( 2 – 4 ), which can reduce viscosity for processing into biofuels (i.e., ethanol) or high value-added products ( 5 , 6 ). Lichenase plays a promising role in the green conversion of lichen biomass ( 7 , 8 ).…”