A proteomics strategy to discover beta-glucosidases from Aspergillus fumigatus with two-dimensional page in-gel activity assay and tandem mass spectrometry.
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. beta-Glucosidase catalyzes the final step in conversion of glucose polymers to glucose. Despite the importance, only a few beta-glucosidases are commercially available, and more efficient ones are clearly needed. We developed a proteomics strategy aiming to discover beta-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 beta-glucosidases was confirmed by tandem mass spectrometry. Two novel beta-glucosidases of A. fumigatus were identified by this in situ activity staining method, and the gene coding for a novel beta-glucosidase ( EAL88289 ) was cloned and heterologously expressed. The expressed beta-glucosidase showed far superior heat stability to the previously characterized beta-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 beta-glucosidases in a high throughput fashion.
Kim
Kee-Hong
KH
Novozymes, Inc., 1445 Drew Avenue, Davis California 95618, USA.
Brown
Kimberly M
KM
Harris
Paul V
PV
Langston
James A
JA
Cherry
Joel R
JR
D000595:Amino Acid Sequence; D001232:Aspergillus fumigatus / Q000201:enzymology* / Q000235:genetics; D044602:Cellulases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D040901:Proteomics; D053719:Tandem Mass Spectrometry
D044602:Cellulases