New perspective on glycoside hydrolase binding to lignin from pretreated corn stover

Abstract

Background: Non-specific binding of cellulases to lignin has been implicated as a major factor in the loss of cellulase activity during biomass conversion to sugars. It is believed that this binding may strongly impact process economics through loss of enzyme activities during hydrolysis and enzyme recycling scenarios. The current model suggests glycoside hydrolase activities are lost though non-specific/non-productive binding of carbohydrate-binding domains to lignin, limiting catalytic site access to the carbohydrate components of the cell wall.

Results: In this study, we have compared component enzyme affinities of a commercial Trichoderma reesei cellulase formulation, Cellic CTec2, towards extracted corn stover lignin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and p-nitrophenyl substrate activities to monitor component binding, activity loss, and total protein binding. Protein binding was strongly affected by pH and ionic strength. β-d-glucosidases and xylanases, which do not have carbohydrate-binding modules (CBMs) and are basic proteins, demonstrated the strongest binding at low ionic strength, suggesting that CBMs are not the dominant factor in enzyme adsorption to lignin. Despite strong adsorption to insoluble lignin, β-d-glucosidase and xylanase activities remained high, with process yields decreasing only 4-15 % depending on lignin concentration.

Conclusion: We propose that specific enzyme adsorption to lignin from a mixture of biomass-hydrolyzing enzymes is a competitive affinity where β-d-glucosidases and xylanases can displace CBM interactions with lignin. Process parameters, such as temperature, pH, and salt concentration influence the individual enzymes' affinity for lignin, and both hydrophobic and electrostatic interactions are responsible for this binding phenomenon. Moreover, our results suggest that concern regarding loss of critical cell wall degrading enzymes to lignin adsorption may be unwarranted when complex enzyme mixtures are used to digest biomass.

Keywords: Biomass; Cellulase; Enzyme binding; Glycoside hydrolase; Lignin; Pretreatment.

Fig. 1

Current paradigm of carbohydrate binding modules (CBMs) having the highest affinity toward lignin. Here Cel7A with a CBM adsorbs to lignin and is sequestered away from the cellulose, rendering it ineffective. Other enzymes without CBMs do not adsorb to lignin

Fig. 2

Lignin–cellulase relationships between physiochemical properties of proteins and substrates; as well as the parameters of the biomass conversion process are shown

Fig. 3

a SDS-PAGE gel comparing the supernatant and lignin pellet from CTec2 with the different molecular regions highlighted (rose = high MW, green = mid MW, purple = low MW). The gel has the following lane assignment: 1 Molecular weight standard, 2 CTec2 control, 3 Unbound fraction, and 4 Bound fraction. b Bar graph showing the activity of CTec2 on the different pNP-substrates

Fig. 4

a SDS-PAGE gel comparing the proteins bound to lignin at different lignin concentrations with the different molecular regions highlighted (red = HMW, green = Mid-MW, and purple = LMW). b pNP activities in unbound fractions versus lignin concentration indicating increasing binding as the concentration of lignin is increased

Fig. 5

a Percent of theoretical glucose conversion by the unbound protein fraction of Cellic CTec2 exposed to different levels of insoluble lignin. b Cellobiose concentration in the digestion supernatant

Fig. 6

SDS-PAGE and line cuts at a pH 4.8 and b pH 6.8. c Unbound fraction pNP activities at pH 4.8 and 6.0

Fig. 7

a SDS-PAGE comparing the commercial cellulase proteins bound to lignin as a function of pH and NaCl concentration (lanes 2–11). Lane 1—molecular weight standard. Lane 12—enzyme control. b Unbound pNP activities as a function of binding pH

Fig. 8

a Percent of cellulose conversion. b Percent cellobiose concentration

Fig. 9

Proposed model of higher lignin-affinity enzymes (β-d-glucosidases and xylanases) displacing CBM-bound cellobiohydrolase from lignin, allowing higher rates of cellulose hydrolysis while retaining functional activity of the bound enzymes