Poster Presentation 1B-36
The Effect of Glycosylation
on the Thermostability and Activity of Trichoderma reesei
Cel7A (CBH I)
Tina Jeoh,*
William S. Adney, Yat-Chen
Chou, David Templeton,
John O. Baker,
and Michael E. Himmel
National Renewable Energy Laboratory
Phone: (303)384-7777
Fax: (303)384-7752
E-mail: tina_jeoh@nrel.gov
Trichoderma reesei Cel7A (CBH I) is generally considered to be one of the most important industrial cellulase component enzymes due to its efficiency at hydrolyzing insoluble cellulose. In its native form, Cel7A is a glycoprotein, consisting of four putative N-glycosylation sites at positions 45, 64, 270 and 384 on the catalytic domain
. Only three of the
four sites (45, 270 and 384), however, have been found to be glycosylated. It has
been shown that the extent of glycosylation of Cel7A
can vary when expressed in heterologous host cells,
such as insect cells or other fungi. For
example, recombinant T. reesei Cel7A expressed
in Aspergillus awamori
was determined to have five times more glycan content
than native T. reesei Cel7A (5.47 ± 0.16 % and
0.84 ± 0.17 % glycan for A. awamori
expressed rCel7A and native T. reesei Cel7A,
respectively). Binding and hydrolysis
studies using fluorescence-labeled Cel7A and rCel7A, however, showed no
activity differences between the two cellulase forms
when acting on bacterial cellulose. Glycosylation is known to affect the secretion, thermal
stability, and activity of enzymes, but little is known about the effect of glycosylation on Cel7A.
We present a preliminary study of the effect of glycosylation
on the thermal stability and cellulose hydrolysis kinetics displayed by native
and various engineered forms of T. reesei
Cel7A, including three mutants: N45A, N270A, and N384A. Tools used to compare these enzyme forms
include differential scanning microcalorimetry for
overall thermal tolerance, titration microcalorimetry
for binding enthalpies, and hydrolysis kinetics on bacterial cellulose.