A proteomics strategy to discover β-glucosidases from Aspergillus fumigatus with two-dimensional page in-gel activity assay and tandem mass spectrometry
Author:
Kim KH, Brown KM, Harris PV, Langston JA, Cherry JR
Date: 11 December 2007
Abstract:
Economically competitive production of ethanol from lignocellulosic biomass by enzymatic hydrolysis and fermentation is currently limited, in part, by the relatively high cost and low efficiency of the enzymes required to hydrolyze cellulose to fermentable sugars. Discovery of novel cellulases with greater activity could be a critical step in overcoming this cost barrier. β-Glucosidase catalyzes the final step in conversion of glucose polymers to glucose. Despite the importance, only a few β-glucosidases are commercially available, and more efficient ones are clearly needed. We developed a proteomics strategy aiming to discover β-glucosidases present in the secreted proteome of the cellulose-degrading fungus Aspergillus fumigatus. With the use of partial or complete protein denaturing conditions, the secretory proteome was fractionated in a 2DGE format and beta-glucosidase activity was detected in the gel after infusion with a substrate analogue that fluoresces upon hydrolysis. Fluorescing spots were subjected to tryptic-digestion, and identification as β-glucosidases was confirmed by tandem mass spectrometry. Two novel β-glucosidases of A. fumigatus were identified by this in situ activity staining method, and the gene coding for a novel β-glucosidase ( EAL88289 ) was cloned and heterologously expressed. The expressed β-glucosidase showed far superior heat stability to the previously characterized β-glucosidases of Aspergillus niger and Aspergillus oryzae. Improved heat stability is important for development of the next generation of saccharifying enzymes capable of performing fast cellulose hydrolysis reactions at elevated temperatures, thereby lowering the cost of bioethanol production. The in situ activity staining approach described here would be a useful tool for cataloguing and assessing the efficiency of β-glucosidases in a high throughput fashion.
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