Environmental Engineering Reference
In-Depth Information
away from the cell wall matrix, or choosing a thermostable enzyme that is inactive under plant
growth conditions. The E1 endoglucanase, a thermostable enzyme from
A. cellulolyticus
, is one
of the most well studied of all of the cellulase enzymes and has been expressed in many different
plant species with various subcellular localizations. The fact that high expression levels of E1 are
achieved even when localized to the apoplast is likely because of the high temperature optimum
(80°C) as well as the fact that generally single enzymes are not sufficient to cause hydrolysis of cel-
lulose within the cell wall matrix. Cleavage of full-length enzymes can sometimes be a problem; for
example, cleavage of full-length E1 occurred in several of the expression systems (Dai et al. 2000a;
Hood et al. 2007; Sun et al. 2007) and was also cleaved in barley (Nuutila et al. 1999).
Although most of the expression studies performed to date have focused on stably transformed
nuclear or chloroplast transformation, our group has been focusing on transient expression in har-
vested plant tissues using vacuum agroinfiltration (McDonald et al. 2008). This method, which
allows for the use of green agricultural wastes, is rapid compared with the time it takes to gener-
ate (and to be approved through the regulatory approval process) a GM bioenergy feedstock, and
expression of the enzyme is performed in a contained environment. We have produced full-length
active E1 in harvested
N. benthamiana
leaves at levels of approximately 4 mg/kg FW 6 days after
agroinfiltration.
In many of the studies reported, the authors have demonstrated that the plant-made enzymes,
when supplemented with the additional enzymes necessary to complete the hydrolysis, are capa-
ble of hydrolyzing substrates to generate glucose. In most cases the enzymes did not need to be
purified—a crude extract could be used directly. For example, Oraby et al. (2007) showed that
TSP extracts from rice leaves expressing the E1 catalytic domain supplemented with commercial
β-glucosidase were able to hydrolyze realistic substrates, AFEX-treated corn stover, and AFEX-
treated rice straw into glucose with 30% and 22% conversion, respectively, demonstrating the
feasibility of using plant-expressed enzymes as additives for biological deconstruction of biomass
feedstocks. Ransom et al. (2007) did a similar study using TSP extracts from corn leaves express-
ing the E1 catalytic domain supplemented with commercial β-glucosidase on AFEX-pretreated
corn stover as a feedstock and demonstrated that sugar production increased with increasing TSP
concentration. Jung et al. (2010) also showed that crude extracts from transgenic tobacco producing
β-glucosidase (BglB), when mixed with extracts from transgenic tobacco producing Cel5A, could
hydrolyze cellulose in pretreated rice straw. Verma et al. (2010) demonstrated that cocktails com-
posed of mixing various plant-produced enzymes and cell-wall-modifying proteins could be used
to produce fermentable sugars from pine wood and citrus waste.
Reuse of enzymes is important to the overall economic feasibility of biological methods for
lignocellulosic conversion to biofuels. Liu et al. (1997) showed that a xylanase fused to oleosin
(proteins that localize to the periphery of oil bodies) successfully targeted the enzyme to oil bodies
where it retained its activity. This allowed the use of the enzyme in an “immobilized” oil body for-
mat, and they also showed that the enzymes could also be recycled through floatation centrifugation
and reused several times.
4.4 conclusIons
There has been significant progress in the last 10-15 years in the development of plant-based sys-
tems for production of cell-wall-degrading enzymes and cell-wall-modifying proteins. Various
expression technologies, plant promoters, plant hosts, and subcellular targeting methods have been
investigated. It is clear that functional enzymes can be produced in plants in such a way that they do
not harm the plant and that crude protein extracts can be used for lignocellulose hydrolysis. In the
short term, these approaches are likely to lead to more efficient, cost-effective, and environmentally
friendly sources of enzymes for biofuel production and in the longer term they could lead to trig-
gered self-deconstructing bioenergy crops.
