The combination of plant-expressed cellobiohydrolase and low dosages of cellulases for the hydrolysis of sugar cane bagasse

Abstract

Background: The expression of biomass-degrading enzymes (such as cellobiohydrolases) in transgenic plants has the potential to reduce the costs of biomass saccharification by providing a source of enzymes to supplement commercial cellulase mixtures. Cellobiohydrolases are the main enzymes in commercial cellulase mixtures. In the present study, a cellobiohydrolase was expressed in transgenic corn stover leaf and assessed as an additive for two commercial cellulase mixtures for the saccharification of pretreated sugar cane bagasse obtained by different processes.

Results: Recombinant cellobiohydrolase in the senescent leaves of transgenic corn was extracted using a simple buffer with no concentration step. The extract significantly enhanced the performance of Celluclast 1.5 L (a commercial cellulase mixture) by up to fourfold on sugar cane bagasse pretreated at the pilot scale using a dilute sulfuric acid steam explosion process compared to the commercial cellulase mixture on its own. Also, the extracts were able to enhance the performance of Cellic CTec2 (a commercial cellulase mixture) up to fourfold on a range of residues from sugar cane bagasse pretreated at the laboratory (using acidified ethylene carbonate/ethylene glycol, 1-butyl-3-methylimidazolium chloride, and ball-milling) and pilot (dilute sodium hydroxide and glycerol/hydrochloric acid steam explosion) scales. We have demonstrated using tap water as a solvent (under conditions that mimic an industrial process) extraction of about 90% recombinant cellobiohydrolase from senescent, transgenic corn stover leaf that had minimal tissue disruption.

Conclusions: The accumulation of recombinant cellobiohydrolase in senescent, transgenic corn stover leaf is a viable strategy to reduce the saccharification cost associated with the production of fermentable sugars from pretreated biomass. We envisage an industrial-scale process in which transgenic plants provide both fibre and biomass-degrading enzymes for pretreatment and enzymatic hydrolysis, respectively.

Keywords: Biomass; Cellobiohydrolase; Cellulase; Enzymatic hydrolysis; Pretreatment; Saccharification; Sugar cane; Transgenic.

Figure 1

Preparation and analysis of corn stover leaf extracts containing recombinant CBH. Extracts were prepared at 16:1, 12:1, and 8:1 buffer-to-dry mass ratios. Extracts were prepared from non-transgenic corn stover leaf under the same conditions as negative controls. Cellulase activity was measured by monitoring the ability of extracts to release 4-methylumbelliferone (4-Mu) from 4-methylumbelliferyl-β-D-lactopyranoside (MUL) at pH 4.75 and 40°C, relative to a 4-Mu standard curve. Activity is presented as micromoles 4-Mu released per minute per milligram of protein and compared to the total MULase specific activity in Celluclast 1.5 L (C). All samples were analysed in triplicate, and error bars represent standard deviation.

Figure 2

SDS-PAGE analysis of corn stover leaf extracts. Transgenic corn stover leaf extracts prepared at 16:1 (lane 2), 12:1 (lane 3), and 8:1 (lane 4) buffer-to-dry mass ratios were resolved using NuPAGE® 4-16% Bis-Tris gel (Invitrogen) with MES SDS buffer. Extracts from non-transgenic corn stover leaf prepared under corresponding conditions (lanes 5-7) were resolved as negative controls. SeeBlue® Plus2 (Invitrogen) was used as the size standard (lane 1). Equal volumes (10 μL) of extracts were analysed.

Figure 3

Saccharification of H ₂ SO ₄ steam exploded bagasse by mixtures of Celluclast 1.5 L and corn stover leaf extracts. Celluclast 1.5 L at dosages of 4 (circles), 6 (triangles), 10 (squares), and 20 (diamonds) FPU/g glucan were supplemented with MULase activity from transgenic corn stover leaf extracts containing recombinant CBH (black symbols) and used to saccharify H₂SO₄ steam exploded bagasse for 24 h. The numeral 1on the x-axis represents the total MULase activity present in the indicated dosage of Celluclast 1.5 L. Values above 1 indicate the addition of corn stover leaf extract containing recombinant CBH to Celluclast 1.5 L in units of MULase activity equal to the total MULase activity in Celluclast 1.5 L at each of the indicated dosages. Each reaction was supplemented with 50 μg β-glucosidase/g glucan. Equal volumes of extracts from non-transgenic corn stover leaves prepared under the same conditions were assessed for comparison (open symbols). Glucose release from cellulose was monitored using a colorimetric (GOPOD Format) assay and the results reported as the percentage of glucan converted to glucose. All samples were analysed in triplicate, and error bars represent standard deviation.

Figure 4

Saccharification of pretreated bagasse by mixtures of Cellic CTec2 and transgenic corn stover leaf extracts. (a) H₂SO₄ steam explosion. (b) NaOH steam explosion. (c) Glycerol/HCl steam explosion. (d) EC/EG. (e) BMIMCl. (f) Ball-milling. Cellic CTec2 at a dosage of 2 FPU/g glucan was supplemented with MULase activity from transgenic corn stover leaf extract (black symbols) and used to saccharify pretreated bagasse for 24 h. Each unit on the x-axis represents the total MULase activity present in Cellic CTec2. Values above 1 indicate the addition of MULase activity from transgenic corn stover leaf extract (black symbols) to Cellic CTec2. Each additional unit of MULase activity supplied from transgenic corn stover leaf extract containing CBH is equal to the total MULase activity in Cellic CTec2 at a dosage of 2 FPU/g glucan. Each reaction was supplemented with 50 μg β-glucosidase/g glucan. Equal volumes of extracts from non-transgenic corn stover leaves prepared under the same conditions were assessed for comparison (open symbols). Glucose release from cellulose was monitored using a colorimetric (GOPOD) assay and the results reported as the percentage of glucan converted to glucose. All samples were analysed in triplicate, and error bars represent standard deviation.

Figure 5

Saccharification of pretreated bagasse by transgenic corn stover leaf extracts containing recombinant CBH. (a) Pretreated bagasse samples were saccharified for 24 h with extracts from transgenic corn stover leaf containing recombinant CBH. The numerals on the x-axis represent the total loading of MULase activity from transgenic corn stover leaf extract, in multiples of the total MULase activity present in Cellic CTec2 at a dosage of 2 FPU/g glucan. The transgenic-extract MULase loadings designated as “1 + 4” in this figure are each thus equal to the loadings of “additional” transgenic-extract MULase activity added to Cellic CTec2 in the loading designated as “1 + 5” in Figure 4. Each reaction was supplemented with 50 μg β-glucosidase/g glucan. Glucose release from glucan was monitored using a colorimetric (GOPOD) assay and the results reported as the percentage of glucan converted to glucose. All samples were analysed in triplicate, and error bars represent standard deviation. (b) Comparison between the ability of corn stover leaf extract containing recombinant CBH to hydrolyse pretreated bagasse and its ability to enhance the performance of a commercial cellulase mixture. The black line indicates equal performance alone and in combination with a commercial cellulase mixture.

Figure 6

Saccharification of pretreated bagasse with mixtures of Cellic CTec2 and transgenic corn stover leaf extract. (a) H₂SO₄ steam explosion. (b) NaOH steam explosion. (c) Glycerol/HCl steam explosion. (d) EC/EG. Pretreated bagasse samples were saccharified with a dosage of Cellic CTec2 that resulted in about 30% glucan conversion after 24 h. Cellic CTec2 at these dosages was supplemented with MULase activity from transgenic corn stover leaf extract containing recombinant CBH (black symbols). Values above 1 indicate the addition of corn stover leaf extract containing recombinant CBH to Cellic CTec2 in units of MULase activity equal to the total MULase activity in Cellic CTec2 at each of the indicated dosages. Equal volumes of extracts from non-transgenic corn stover leaves prepared under the same conditions were assessed for comparison (open symbols).

Figure 7

Model for “mixed delivery” of plant-expressed cellulase into a commercial sugar cane enzymatic hydrolysis system.

Figure 8

Extraction of CBH from transgenic corn stover leaf into tap water after mild disruption. Transgenic corn stover leaf containing recombinant CBH was disrupted with three (white squares), six (grey squares), or nine (black squares) passes through a cutting mill. Three water extracts were prepared for each level of tissue disruption. Water extracts were also prepared from corn stover leaf milled to a particle size of <2 mm and disrupted using a bead mill (dotted line). CBH activity was measured in the extracts by monitoring the ability of extracts to release 4-methylumbelliferone (4-Mu) from 4-methylumbelliferyl-β-D-lactopyranoside (MUL) at pH 4.75 and 40°C, relative to a 4-Mu standard curve. Activity is presented as micromoles 4-Mu released per min per mg of protein. All samples were analysed in triplicate, and error bars represent standard deviation.