Separation and quantification of cellulases and hemicellulases by capillary electrophoresis.
Cellulases and hemicellulases are two classes of enzymes produced by filamentous fungi and secreted into the cultivation medium. Both classes of enzymes consist of a subset of classes of which the fungi produce several enzymes with varying molecular mass and pI but similar enzymatic activities. Current methods are limited in their ability to quantify all of these enzymes when all are present simultaneously in a mixture. Five different cellulases (two cellobiohydrolases and three endoglucanases) and one hemicellulase (endoxylanase) were separated using capillary electrophoresis (CE) in a fused silica capillary at pH values close to neutral. The improvement of the separation of these six proteins by the addition of alpha,omega-diaminoalkanes with chain lengths from three to seven carbon units was investigated. Dynamically coating the capillary with 1,3-diaminopropane resulted in separation of the six enzymes and the reproducibility of the migration times was between 0.6 and 1.9%. Two cases-quantitative determination of the enzyme concentrations in cultivation samples and investigation of adsorption of the enzymes onto cellulose-demonstrated the advantages and perspectives of CE analysis of these broad groups of enzymes.
Center for Process Biotechnology, BioCentrum-DTU, Technical University of Denmark, Building 223, DK-2800 Kgs, Lyngby, Denmark.
Henning
H
Jørgensen
Jörg P
JP
Kutter
Lisbeth
L
Olsson
D000327:Adsorption; D000473:Alkanes / Q000737:chemistry; D044602:Cellulases / Q000032:analysis* / Q000302:isolation & purification; D002482:Cellulose / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods; D006026:Glycoside Hydrolases / Q000032:analysis* / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008970:Molecular Weight; D010407:Penicillium / Q000201:enzymology / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000473:Alkanes; D002482:Cellulose; D044602:Cellulases; D006026:Glycoside Hydrolases; C023305:hemicellulase