Synergistic Hydrolysis of Cellulose by a Blend of Cellulase-Mimicking Polymeric Nanoparticle Catalysts

Abstract

Enzyme-like catalysts by design have been a long sought-after goal of chemists but difficult to realize due to the challenges in the construction of multifunctionalized active sites with accurately positioned catalytic groups for complex substrates. Hydrolysis of cellulose is a key step in biomass utilization and requires multiple enzymes to work in concert to overcome the difficulty associated with hydrolyzing the recalcitrant substrate. We here report methods to construct synthetic versions of these enzymes through covalent molecular imprinting and strategic postmodification of the imprinted sites. The synthetic catalysts cleave a cellulose chain endolytically at multiple positions or exolytically from the nonreducing end by one or three glucose units at a time, all using the dicarboxylic acid motif found in natural cellulases. By mimicking the endocellulase, exocellulase, and β-glucosidase, the synthetic catalysts hydrolyze cellulose in a synergistic manner, with an activity at 90 °C in pH 6.5 buffer more than doubled that of Aspergillus niger cellulase at pH 5 and 37 °C and 44% of that of a commercial cellulase blend (from Novozyme). As robust cross-linked polymeric nanoparticles, the synthetic catalysts showed little changes in activity after preheating at 90 °C for 3 days and could be reused, maintaining 76% of activity after 10 reaction cycles.

Figure 1.

Effects of solution pH on the hydrolysis of cellulose by synthetic endocellulases. [Sigmacell cellulose] = 5.0 mg/mL. [catalyst] = 2.0 mg/mL.

Figure 2.

Michaelis-Menten plot for cellobiose hydrolysis by (a) NP₁-CH₂CH₂CO₂H and (b) in NP₆-CO₂H 10 mM MES buffer (pH 6.5) at 90 °C. [NP] = 10.0 μM.

Figure 3.

Binding free energies of NP hosts for cello-oligomers in 10 mM MES buffer at pH 6.5 at 298 K determined by ITC.

Figure 4.

Michaelis-Menten plot for the hydrolysis of cellotetraose by NP₇-(CO₂H)₂ in pH 6.5 buffer at 60 °C. [NP] = 10.0 μM.

Figure 5.

Effects of solution pH on cellulose hydrolysis by the NP catalyst blend. [Sigmacell cellulose] = 5.0 mg/mL. [Total catalysts] = 2.0 mg/mL with a 1:1:2 ratio of NP₆-CO₂H, NP₇-(CO₂H)₂, and NP₁₁-(CO₂H)₂.

Figure 6.

Recyclability of NP catalyst blend for cellulose hydrolysis in 60 °C NaOAc buffer (pH 6.5).

Scheme 1.

Preparation of endocellulase-mimicking NP₁-CO₂H (a) and NP₁-CH₂CH₂CO₂H (b). The surface ligands are omitted for clarity.

Scheme 2.

Preparation of NP₆-CO₂H from template 6.

Scheme 3.

Preparation of exocellulase-mimicking NP₇-CO₂H. The surface ligands are omitted for clarity.

Scheme 4.

Preparation of β-glucosidase-mimicking NP₁₁-(CO₂H)₂. The surface ligands are omitted for clarity.