Product inhibition of cellulases studied with 14C-labeled cellulose substrates

Abstract

Background: As a green alternative for the production of transportation fuels, the enzymatic hydrolysis of lignocellulose and subsequent fermentation to ethanol are being intensively researched. To be economically feasible, the hydrolysis of lignocellulose must be conducted at a high concentration of solids, which results in high concentrations of hydrolysis end-products, cellobiose and glucose, making the relief of product inhibition of cellulases a major challenge in the process. However, little quantitative information on the product inhibition of individual cellulases acting on cellulose substrates is available because it is experimentally difficult to assess the hydrolysis of the heterogeneous polymeric substrate in the high background of added products.

Results: The cellobiose and glucose inhibition of thermostable cellulases from Acremonium thermophilum, Thermoascus aurantiacus, and Chaetomium thermophilum acting on uniformly 14C-labeled bacterial cellulose and its derivatives, 14C-bacterial microcrystalline cellulose and 14C-amorphous cellulose, was studied. Cellulases from Trichoderma reesei were used for comparison. The enzymes most sensitive to cellobiose inhibition were glycoside hydrolase (GH) family 7 cellobiohydrolases (CBHs), followed by family 6 CBHs and endoglucanases (EGs). The strength of glucose inhibition followed the same order. The product inhibition of all enzymes was relieved at higher temperatures. The inhibition strength measured for GH7 CBHs with low molecular-weight model substrates did not correlate with that measured with 14C-cellulose substrates.

Conclusions: GH7 CBHs are the primary targets for product inhibition of the synergistic hydrolysis of cellulose. The inhibition must be studied on cellulose substrates instead of on low molecular-weight model substrates when selecting enzymes for lignocellulose hydrolysis. The advantages of using higher temperatures are an increase in the catalytic efficiency of enzymes and the relief of product inhibition.

Figure 1

Synergistic hydrolysis of ¹⁴C-BC by GH7 CBHs at different temperatures. ¹⁴C-BC (0.25 mg ml^-1) was incubated with 0.25 μM CBH, supplemented with 0.025 μM EG (TrCel5A) and 0.06 μM N188BG, at 25°C (□), 35°C (Δ), 40°C (◊), 50°C (×), and 60°C (○). CBH was (A)TrCel7A, (B)TaCel7A, (C)AtCel7A, and (D)CtCel7A.

Figure 2

Irreversible inactivation of TrCel7A is not responsible for the decreased hydrolysis rates at higher temperatures. ¹⁴C-BC (0.25 mg ml^-1) was incubated with 0.25 μM TrCel7A, supplemented with 0.025 μM EG (TrCel5A) and 0.06 μM N188BG. Temperature was 40°C (◊) and 55°C (□). In one trial, the hydrolysis was conducted at 55°C for 30 min, and then the temperature was decreased (indicated by arrowhead) to 40°C (■).

Figure 3

Inhibition of GH7 CBHs by cellobiose. ¹⁴C-BC (0.25 mg ml^-1) was incubated with a mixture of 0.25 μM CBH and 0.025 μM EG (TrCel5A) at 35°C. The concentration of cellobiose added was 0 mM + 0.06 μM N188BG (◊), 0 mM (♦), 0.5 mM (Δ), 1.0 mM (+), 2.0 mM (○), 5.0 mM (×) or 10 mM (□). CBH was (A)TrCel7A, (B)TaCel7A, (C)AtCel7A, and (D)CtCel7A.

Figure 4

Analysis of the inhibition of GH7 CBHs by cellobiose. (A) Data for the hydrolysis of ¹⁴C-BC by the mixture of TrCel7A and TrCel5A at 35°C (Figure 3A) were rearranged in the coordinates (D_CB/D_CB=0) versus [cellobiose], where D_CB and D_CB=0 represent the degree of conversion of ¹⁴C-BC in the presence and absence of cellobiose, respectively. The ratio of (D_CB/D_CB=0) was found after different times of hydrolysis, which were 2 min (◊), 5 min (□), 10 min (Δ), 20 min (○), and 30 min (×). (B) Data for the hydrolysis of ¹⁴C-BC by the mixture of CBH and TrCel5A at 35°C (Figure 3) in the coordinates (D_CB/D_CB=0) versus [cellobiose]. (D_CB/D_CB=0) values for all hydrolysis time points are shown. CBH was TrCel7A (□), TaCel7A, (◊), AtCel7A (Δ), and CtCel7A (×). Solid lines are from the non-linear regression according to Equation 5.

Figure 5

Relative strength of cellobiose inhibition of GH7 CBHs depends on the substrate.K_i values measured for MUL hydrolysis and IC₅₀ values measured for the hydrolysis of ¹⁴C-BC, both at 35°C and 50°C, were taken from Table 2 and Table 1, respectively. CBH was TrCel7A (□), TaCel7A, (◊), AtCel7A (Δ), and CtCel7A (×).

Figure 6

Inhibition of GH7 CBHs by glucose. ¹⁴C-BC (0.25 mg ml^-1) was incubated with a mixture of 0.25 μM CBH and 0.025 μM EG (TrCel5A) at 35°C. The concentration of added glucose was as follows: 0 M + 0.06 μM N188BG (◊), 0 M (♦), 0.05 M (Δ), 0.125 M (+), 0.25 M (○), 0.5 M (×) or 1.0 M (□). CBH was as follows: (A)TrCel7A, (B)AtCel7A, and (C)CtCel7A. (D) Hydrolysis data in the coordinates (D_Glc/D_Glc=0) versus [glucose], where D_Glc and D_Glc=0 represent the degree of conversion of ¹⁴C-BC in the presence and absence (+N188BG series) of added glucose, respectively. (D_Glc/D_Glc=0) values for all hydrolysis time points are shown. CBH was TrCel7A (□), AtCel7A (Δ), and CtCel7A (×).

Figure 7

Inhibition of GH6 CBHs by cellobiose. (A) ¹⁴C-BC (0.25 mg ml^-1) was incubated with a mixture of 0.25 μM TrCel6A and 0.025 μM EG (TrCel5A) at 25°C. (B and C) ¹⁴C-BMCC (0.25 mg ml^-1) was incubated with 0.25 μM TrCel6A (panel B) or with 0.25 μM CtCel6A (panel C) at 50°C. The concentration of added cellobiose was 0 mM + 0.06 μM N188BG (◊), 1.0 mM (*), 5.0 mM (+), 10 mM (□), 20 mM (Δ), 50 mM (×), or 100 mM (○). (D) Hydrolysis data (from panels A-C) in the coordinates (D_CB/D_CB=0) versus [cellobiose], where D_CB and D_CB=0 represent the degree of conversion of ¹⁴C-cellulose in the presence and absence of cellobiose, respectively. The average (D_CB/D_CB=0) values over hydrolysis time points are plotted. Solid lines are from the non-linear regression according to Equation 5. TrCel6A + TrCel5A on ¹⁴C-BC (□), TrCel6A on ¹⁴C-BMCC (◊), and CtCel6A on ¹⁴C-BMCC (×).

Figure 8

Inhibition of GH6 CBHs by glucose. (A) ¹⁴C-BMCC (0.25 mg ml^-1) was incubated with 0.25 μM TrCel6A (opened symbols) or with 0.25 μM CtCel6A (filled symbols) 50°C. The concentration of added glucose was as follows: 0 M + 0.06 μM N188BG (◊,♦), 0.25 M (□, ■), 0.5 M (Δ, ▲), or 1.0 M (○, ●). (B) Hydrolysis data in coordinates (D_Glc/D_Glc=0) versus [glucose] where D_Glc and D_Glc=0 represent the degree of conversion of ¹⁴C-BMCC in the presence and absence of added glucose, respectively. (D_Glc/D_Glc=0) values for all hydrolysis time points are shown. Solid lines are from the non-linear regression according to Equation 5 (the terms referring to cellobiose were replaced with corresponding terms for glucose). CBH was TrCel6A (◊) or CtCel6A (×).

Figure 9

Inhibition of EGs by cellobiose. (A) ¹⁴C-amorphous cellulose (0.5 mg ml^-1) was incubated with 2.5 nM TrCel7B at 35°C. The concentration of added cellobiose was 0 mM (◊), 75 mM (□), 150 mM (×), or 225 mM (○). (B) Hydrolysis data from panel A and Additional file 1: Figure S5 in the coordinates (D_CB/D_CB=0) versus [cellobiose] where D_CB and D_CB=0 represent the degree of conversion of ¹⁴C-cellulose in the presence and absence of cellobiose, respectively. Average (D_CB/D_CB=0) values over hydrolysis time points are plotted. The solid line is from the non-linear regression according to Equation 5. TrCel7B (◊), TrCel5A (□), and TrCel12A (○).