Environmental Engineering Reference
In-Depth Information
contributing to the cellulase improvement effort with support from DOE. In 1998, J. Sakon
and colleagues at the University of Arkansas showed that performance of a ternary system
was improved 13 percent by site-directed modification of one active site amino acid in
Acidothermus E1; currently they are pursuing E1 mutations that modify the biomass
interactive surface.
2.3.2. Chimeric cellulase systems
. Cellulase components from diverse organisms,
primarily bacteria and fungi, are being combined in ways that yield overall improved activity.
Baker and colleagues have successfully combined bacterial and fungal cellulases in vitro [40],
showing that these mixtures can be competitive with a native ternary system from T. reesei
[41]. Work with expansins, proteins that enable extension of plant cell walls during plant cell
growth, has also shown enhancement of hydrolysis of microcrystalline cellulose in a mixed
Trichoderma cellulase preparation [42]. The initial approaches to developing artificial
cellulase systems, still instructive after nine years, are reviewed in [43].
2.3.3. Heterologous expression
. The next logical step in chimeric cellulase systems is the
cloning of cellulases from one organism into another; this avenue is being explored as well, as
shown by the expression of the T. reesei cellobiohydrolase I in Pichia pastoris [44].
Especially important for commercial production, cellulases are being expressed in plants such
as tobacco and potatoes, potentially providing more abundant sources of the enzymes [45].
Another innovative approach to the heterologous expression of cellulases is the
expression of heat-activated cellulases within biomass crops themselves, with the idea that the
plants grow normally until harvested and exposed to elevated temperatures, at which point
heat- activated cellulases hydrolyze the cellulose without need for externally added enzymes
[46].
2.3.4. Cellulase performance assays
. Convenient, accurate, efficient assays are central to
the development of any new technology. The diafiltration saccharification assay (DSA)
developed at the NREL produces precise, detailed progress curves for enzymatic
saccharification of cellulosic materials under conditions that mimic those of SSF. From this
method, it is possible to describe the performance of a given cellulase preparation over a wide
range of loading and reaction times with comparatively little data [47, 48, 3].
2.3.5. Proteomic analysis, microarray analysis, and modeling
. Proteomics is an emerging
set of techniques that has proven extremely useful in understanding the interactions of
multienzyme systems. Hydrolysis of complex organic substrates is an ideal candidate for
proteomic analysis, as it involves a number of enzymes: β3-1,4-endoglucanases, β3-1,4-
cellobiohydrolases, xylanases, β3-glucosidases, α -L-arabinofuranosidase, acetyl xylan
esterase, β3- mannanase, and α-glucuronidase in T. reesei, for example. At the NREL, the
expression of these enzymes is being investigated under various conditions by proteomic
methods and compared to corresponding enzyme activities using the DSA assay [3].
To reveal gene expression responses to environmental conditions in both wild-type and
genetically engineered microbes, microarray analysis is underway and could become a
valuable industrial tool for evaluation of new recombinant organisms [49]. Mathematical
molecular analysis is also being employed to gain greater understanding of structure-function
relationships to complement the physiological understanding provided by proteomic and
microarray analysis. Current work includes molecular mechanics efforts by Brady and
colleagues at Cornell University as well as Palma and colleagues; cellulase crystallization
work is also in progress by a number of groups [50-53].
