Lack of fundamental understanding of cellulase interactions with different plant cell wall components during cellulose saccharification hinders progress toward achieving an economic production of biofuels from renewable plant biomass. Here, chemical force microscopy (CFM) was utilized to quantify the interactions between two surfaces that model either hydrophilic or hydrophobic functional groups of cellulases and a set of lignocellulosic substrates prepared through Kraft, sulfite, or organosolv pulping with defined chemical composition. The measured forces were then decoupled into specific and nonspecific components using the Poisson statistical approach. Heterogeneities in the distributions of forces as a function of the pretreatment method were mapped. Our results showed that hydrophobic domains and chemical moieties involved in hydrogen bonding and polar interactions were homogeneously distributed on all substrates but with distribution densities that varied with the type of the pretreatment method used to prepare substrates. In addition, we showed that increasing surface lignin coverage increased the heterogeneity of the substrates. When forces were decoupled, our results indicated that xylan reduced the strength of hydrogen bonding between the hydrophilic model surface and substrates. Permanent dipole-dipole interactions dominated the adhesion of the hydrophilic model surface to lignosulfonates, whereas hydrophobic interactions facilitated the adhesion of the hydrophobic model surface to Kraft lignin. We further showed that the structure of lignin determines the type of forces that dominate lignocellulosic interactions with other surfaces. Our findings suggest that nonproductive binding of cellulases to lignocellulosic biomass can be reduced by altering the hydrophobicity and/or chemical moieties involved in the polar interactions and by utilizing organosolv as a pretreatment method.

Tropomodulin family of proteins includes several isoforms of tropomodulins (Tmod) and leiomodins (Lmod). These proteins can sequester actin monomers or nucleate actin polymerization. Although it is known that their actin-binding properties are isoform-dependent, knowledge on how they vary in strengths of interactions with G-actin is missing. While it is confirmed in many studies that Tmods have two actin-binding sites, information on number and location of actin-binding sites in Lmod2 is controversial. We used atomic force microscopy to study interactions between G-actin and proteins of the tropomodulin family. Unbinding forces between G-actin and Tmod1, Tmod2, Tmod3, or Lmod2 were quantified. Our results indicated that Tmod1 and Tmod3 had unimodal force distributions, Tmod2 had a bimodal distribution and Lmod2 had a trimodal distribution. The number of force distributions correlates with the proteins' abilities to sequester actin or to nucleate actin polymerization. We assigned specific unbinding forces to the individual actin-binding sites of Tmod2 and Lmod2 using mutations that destroy actin-binding sites of Tmod2 and truncated Lmod2. Our results confirm the existence of the N-terminal actin-binding site in Lmod2. Altogether, our data demonstrate how the differences between the number and the strength of actin-binding sites of Tmod or Lmod translate to their functional abilities.

The lack of fundamental understanding of the types of forces that govern how cellulose-degrading enzymes interact with cellulosic and noncellulosic components of lignocellulosic surfaces limits the design of new strategies for efficient conversion of biomass to bioethanol. In a step to improve our fundamental understanding of such interactions, nanoscale forces acting between a model cellulase-a carbohydrate-binding module (CBM) of cellobiohydrolase I (CBH I)-and a set of lignocellulosic substrates with controlled composition were measured using atomic force microscopy (AFM). The three model substrates investigated were kraft (KP), sulfite (SP), and organosolv (OPP) pulped substrates. These substrates varied in their surface lignin coverage, lignin type, and xylan and acetone extractives' content. Our results indicated that the overall adhesion forces of biomass to CBM increased linearly with surface lignin coverage with kraft lignin showing the highest forces among lignin types investigated. When the overall adhesion forces were decoupled into specific and nonspecific component forces via the Poisson statistical model, hydrophobic and Lifshitz-van der Waals (LW) forces dominated the binding forces of CBM to kraft lignin, whereas permanent dipole-dipole interactions and electrostatic forces facilitated the interactions of lignosulfonates to CBM. Xylan and acetone extractives' content increased the attractive forces between CBM and lignin-free substrates, most likely through hydrogen bonding forces. When the substrates treated differently were compared, it was found that both the differences in specific and nonspecific forces between lignin-containing and lignin-free substrates were the least for OPP. Therefore, cellulase enzymes represented by CBM would weakly bind to organosolv lignin. This will facilitate an easy enzyme recovery compared to other substrates treated with kraft or sulfite pulping. Our results also suggest that altering the surface hydrophobicity and the surface energy of lignin that facilitates the LW forces should be a priori to avoid nonproductive binding of cellulase to kraft lignin.

Adsorption on a functionalized surface can be an effective way of purifying polyphenols from complex plant extracts. Polymeric resins that rely on hydrophobic interactions suffer from low selectivity, weak affinity towards polyphenols, and lack tunability therefore making the purification of polyphenols less efficient. In this study, a purification process for the recovery of polyphenols from grape pomace extract was successfully developed using hydrogen bonding affinity ligands grafted on silica particles and PEG-assisted elution solvents. Bare silica (SiO2) and polyethylene glycol (mPEG)-grafted silica microparticles with molecular weights of 2000 and 5000 were tested to determine their polyphenol binding and release characteristics. Functionalizing the surface of bare silica with mPEG ligands increased the adsorption capacity by 7.1- and 11.4-fold for mPEG-2000 and mPEG-5000 compared to bare silica particles, respectively. This was likely due to the introduction of more polyphenol binding sites with mPEG functionalization. Altering the molecular weight (MW) of mPEG grafted on silica surfaces provided tunability in the adsorption capacity. A complete recovery of polyphenols (~99.9%) from mPEG-grafted silica particles was achieved by utilizing PEG–ethanol or PEG–water cosolvent systems. Recovered polyphenols showed up to ~12-fold antioxidant activity compared to grape pomace extract. This study demonstrates that mPEG-grafted silica particles and elution of polyphenols with PEG cosolvents can potentially be used for large-scale purification of polyphenols from complex plant extracts and simplify the use of polyphenols, as PEG facilitates remarkable solvation and is an ideal medium for the final formulation of polyphenols.

The effects of the morphology and conformations of the surface biopolymers present on lignocellulosic biomass as well as their steric hindrance on enzymatic adsorption to biomass surfaces remain elusive. In a step to better understand these effects, nanoscale steric forces between a model surface that represents the hydrophobic residues of a cellulase enzyme and a set of reference lignocellulosic substrates were measured using atomic force microscopy (AFM) in liquid media. The reference substrates investigated were prepared by kraft, sulfite, and organosolv pulping pretreatment methods and varied in their surface lignin, xylan, and acetone extractives' contents. Measured steric forces were quantified through fitting to a model developed to describe polyelectrolytes brushes in terms of a brush thickness and a brush grafting density. Our data indicated that cellulose microfibrils extend from the microfibril matrix leading to a long-range steric repulsion and low attractive forces to the hydrophobic model of the enzyme, suggesting that steric hindering can be a possible mechanism for nonproductive binding of enzymes to cellulose. When the amount of xylan increased in the absence of lignin, steric repulsions between the hydrophobic model of the enzyme, and biomass biopolymers decreased as a result of collapsed cellulose microfibrils and adhesion forces increased. This suggests that leaving a small amount of xylan after biomass pretreatment can help improve enzymatic binding to cellulose. Irrespective of the type of lignin present on biomass, grafting densities increased and brush thicknesses decreased compared to those of lignin-free substrates. When compared to lignin-free substrates, lignin-containing substrates had higher attractive forces and lower steric repulsive forces. In addition, AFM images of the reference substrates in the wet and dry states showed that lignin precipitates on the biomass surface where kraft lignin had the highest particle size leading to a limited accessibility of the enzyme to the cellulose in biomass. When the effects of lignin precipitate size, the adhesion force, and steric forces on nonproductive enzymatic binding were all considered, our results indicate that organosolv pretreatment should be the treatment of choice to minimize enzymatic nonproductive binding to lignin.