Lignin triggers irreversible cellulase loss during pretreated lignocellulosic biomass saccharification

Abstract

Background: Non-productive binding of enzymes to lignin is thought to impede the saccharification efficiency of pretreated lignocellulosic biomass to fermentable sugars. Due to a lack of suitable analytical techniques that track binding of individual enzymes within complex protein mixtures and the difficulty in distinguishing the contribution of productive (binding to specific glycans) versus non-productive (binding to lignin) binding of cellulases to lignocellulose, there is currently a poor understanding of individual enzyme adsorption to lignin during the time course of pretreated biomass saccharification.

Results: In this study, we have utilized an FPLC (fast protein liquid chromatography)-based methodology to quantify free Trichoderma reesei cellulases (namely CBH I, CBH II, and EG I) concentration within a complex hydrolyzate mixture during the varying time course of biomass saccharification. Three pretreated corn stover (CS) samples were included in this study: Ammonia Fiber Expansion(a) (AFEX™-CS), dilute acid (DA-CS), and ionic liquid (IL-CS) pretreatments. The relative fraction of bound individual cellulases varied depending not only on the pretreated biomass type (and lignin abundance) but also on the type of cellulase. Acid pretreated biomass had the highest levels of non-recoverable cellulases, while ionic liquid pretreated biomass had the highest overall cellulase recovery. CBH II has the lowest thermal stability among the three T. reesei cellulases tested. By preparing recombinant family 1 carbohydrate binding module (CBM) fusion proteins, we have shown that family 1 CBMs are highly implicated in the non-productive binding of full-length T. reesei cellulases to lignin.

Conclusions: Our findings aid in further understanding the complex mechanisms of non-productive binding of cellulases to pretreated lignocellulosic biomass. Developing optimized pretreatment processes with reduced or modified lignin content to minimize non-productive enzyme binding or engineering pretreatment-specific, low-lignin binding cellulases will improve enzyme specific activity, facilitate enzyme recycling, and thereby permit production of cheaper biofuels.

Keywords: Cellulase adsorption; Cellulosic biofuels; Enzymatic saccharification; Lignin; Non-specific enzyme binding.

Figure 1

Hydrolysis yield (A, C) of glucan to glucose and percentage of free cellulases in the hydrolyzate supernatant (B, D) for crystalline (A-B) and amorphous (C-D) cellulose. Assays were carried out using a ternary cellulase cocktail of CBH I (blue squares), CBH II (green triangles), and EG I (red circles) for 48 h at 50°C. All assays were carried out in triplicate with mean values reported here. Standard deviations in all cases were less than 5%. Data for this figure have been reproduced from our previous study [46].

Figure 2

Hydrolysis yield (A) to glucose (G or G*) or xylose (X or X*) and percentage of free cellulases in the hydrolyzate supernatant (B) for IL-CS. Assays were carried out using a ternary cellulase cocktail of CBH I (blue squares), CBH II (green triangles), and EG I (red circles) either with (filled symbols with asterisk) or without (empty symbols with no asterisk) hemicellulase (endoxylanase and β-xylosidase) supplementation during the 48-h hydrolysis duration at 50°C. All assays were carried out in triplicate with mean values reported here. Standard deviations in all cases were less than 5%.

Figure 3

Hydrolysis yield (A) to glucose (G or G*) or xylose (X or X*) and percentage of free cellulases in the hydrolyzate supernatant (B) for AFEX-CS. Assays were carried out using a ternary cellulase cocktail of CBH I (blue squares), CBH II (green triangles), and EG I (red circles) either with (filled symbols with asterisk) or without (empty symbols with no asterisk) hemicellulase (endoxylanase and β-xylosidase) supplementation during the 48-h hydrolysis duration at 50°C. All assays were carried out in triplicate with mean values reported here. Standard deviations in all cases were less than 5%. Data for this figure have been reproduced from our previous study [46].

Figure 4

Hydrolysis yield (A) to glucose (G or G*) or xylose (X or X*) and percentage of free cellulases in the hydrolyzate supernatant (B) for DA-CS. Assays were carried out using a ternary cellulase cocktail of CBH I (blue squares), CBH II (green triangles), and EG I (red circles) either with (filled symbols with asterisk) or without (empty symbols with no asterisk) hemicellulase (endoxylanase and β-xylosidase) supplementation during the 48-h hydrolysis duration at 50°C. All assays were carried out in triplicate with mean values reported here. Standard deviations in all cases were less than 5%.

Figure 5

Free cellulase concentration in the hydrolyzate supernatant as a function of total lignin concentration. Data for free CBH I (blue squares), CBH II (green triangles), and EG I (red circles) after 48-h saccharification of Avicel (0 g/L lignin), IL-CS (0.58 g/L lignin), AFEX-CS (3.18 g/L lignin), and DA-CS (5.43 g/L lignin) at 50°C are reported here. All assays were carried out in triplicate with mean values shown here. Standard deviations in all cases were less than 5%.

Figure 6

Relative activity of CBH I (in blue), CBH II (in green), and EG I (in red) after incubation at 50°C for varying time periods. Activity assays were conducted on CMC (for EG I) or Avicel (CBH I and CBH II). Error bars indicate standard deviations (±σ) for reported mean values. All assays were carried out in triplicate. Data for this figure have been reproduced from our previous study [46].

Figure 7

Carbohydrate binding module (CBM) binds strongly to lignin isolated from AFEX-CS. Here, percentage of protein fluorescence lost (% RFU Lost) due to GFP-CBM1 (filled blue diamonds) and GFP (empty blue diamonds) binding to lignin isolated from AFEX-CS is shown. For subtractive mass balance binding experiments, protein concentration was held constant at 0.2 μM and measured after 1 h of protein-lignin binding at 22°C. Error bars indicate standard deviations (±σ) for reported mean values and in some cases are smaller than the symbols. All assays were carried out in triplicate on two separate days.