Bioconversion of lignocellulose forms the basis for renewable, advanced biofuels, and bioproducts. Mechanisms of hydrolysis of cellulose by cellulases have been actively studied for nearly 70 years with significant gains in understanding of the cellulolytic enzymes. Yet, a full mechanistic understanding of the hydrolysis reaction has been elusive. We present a review to highlight new insights gained since the most recent comprehensive review of cellulose hydrolysis kinetic models by Bansal et al. (2009) Biotechnol Adv 27:833-848. Recent models have taken a two-pronged approach to tackle the challenge of modeling the complex heterogeneous reaction-an enzyme-centric modeling approach centered on the molecularity of the cellulase-cellulose interactions to examine rate limiting elementary steps and a substrate-centric modeling approach aimed at capturing the limiting property of the insoluble cellulose substrate. Collectively, modeling results suggest that at the molecular-scale, how rapidly cellulases can bind productively (complexation) and release from cellulose (decomplexation) is limiting, while the overall hydrolysis rate is largely insensitive to the catalytic rate constant. The surface area of the insoluble substrate and the degrees of polymerization of the cellulose molecules in the reaction both limit initial hydrolysis rates only. Neither enzyme-centric models nor substrate-centric models can consistently capture hydrolysis time course at extended reaction times. Thus, questions of the true reaction limiting factors at extended reaction times and the role of complexation and decomplexation in rate limitation remain unresolved. Biotechnol. Bioeng. 2017;114: 1369-1385. © 2017 Wiley Periodicals, Inc.
Alkaline pretreatment using sodium hydroxide offers a means to extract lignin and acetate from lignocellulosic biomass, in turn enabling higher enzymatic digestibility of the remaining polysaccharides and production of a lignin-enriched stream for potential valorization. Key criteria for alkaline pretreatment processes, which are important for commercial feasibility, include the minimization of carbohydrate loss during pretreatment and the ability to capture carbon lost to the liquor stream, much of which will be feedstock dependent. Here, we present a comprehensive study of alkaline pretreatment of switchgrass over NaOH loadings from 35 to 140 mg NaOH/g dry switchgrass and with a constant charge of 0.2% anthraquinone for pretreatment temperatures between 100 and 160°C for 30 min. Full compositional analysis of the pretreated solids are reported as a function of pretreatment severity, along with the yields of each biomass component present in the process streams generated during pretreatment (pretreated solid, liquor, and wash fraction). The pretreated solids are further characterized through crystallinity measurements and electron microscopy. Additionally, enzymatic digestions of the residual solids are performed over a range of enzyme loadings for varying pretreatment severities. These results are compared to our recent work with alkaline pretreatment of corn stover using the ratio of lignin fractionation to carbohydrate retention (in the solids after pretreatment), which highlights the greater recalcitrance of switchgrass relative to corn stover. Specifically, compared to corn stover, switchgrass requires approximately twice the NaOH loading to achieve identical delignification and high enzymatic digestibility. From this work, the optimal pretreatment conditions for switchgrass are suggested to be 154 mg NaOH/ g dry switchgrass at 130°C for 30 min at temperature. The results from these bench-scale experiments will serve as a guide to scale up processes for the optimization of lignin removal while minimizing carbohydrate loss during alkaline pretreatment.
Background: Molecular-scale mechanisms of the enzymatic breakdown of cellulosic biomass into fermentable sugars are still poorly understood, with a need for independent measurements of enzyme kinetic parameters. We measured binding times of cellobiohydrolase Trichoderma reesei Cel7A (Cel7A) on celluloses using wild-type Cel7A (WT intact), the catalytically deficient mutant Cel7A E212Q (E212Q intact) and their proteolytically isolated catalytic domains (CD) (WT core and E212Q core , respectively). The binding time distributions were obtained from time-resolved, super-resolution images of fluorescently labeled enzymes on cellulose obtained with total internal reflection fluorescence microscopy. Results: Binding of WT intact and E212Q intact on the recalcitrant algal cellulose (AC) showed two bound populations: ~ 85% bound with shorter residence times of < 15 s while ~ 15% were effectively immobilized. The similarity between binding times of the WT and E212Q suggests that the single point mutation in the enzyme active site does not affect the thermodynamics of binding of this enzyme. The isolated catalytic domains, WT core and E212Q core , exhibited three binding populations on AC: ~ 75% bound with short residence times of ~ 15 s (similar to the intact enzymes), ~ 20% bound for < 100 s and ~ 5% that were effectively immobilized. Conclusions: Cel7A binding to cellulose is driven by the interactions between the catalytic domain and cellulose. The cellulose-binding module (CBM) and linker increase the affinity of Cel7A to cellulose likely by facilitating recognition and complexation at the substrate interface. The increased affinity of Cel7A to cellulose by the CBM and linker comes at the cost of increasing the population of immobilized enzyme on cellulose. The residence time (or inversely the dissociation rates) of Cel7A on cellulose is not catalysis limited.
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