Enhancing the enzymatic hydrolysis of lignocellulosic biomass by increasing the carboxylic acid content of the associated lignin

Nakagame, Seiji; Chandra, Richard P.; Kadla, John F.; Saddler, Jack N.

doi:10.1002/bit.22981

Cited by 231 publications

(147 citation statements)

References 42 publications

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“…It is known that cellulases can nonproductively bind to lignin through hydrophobic interactions through either the CBM or the exposed CD active site aromatic residues (28)(29)(30). We find that, although equivalent glucan hydrolysis yields for pretreated lignocellulose and model cellulosic substrates were achieved within 48 h, less than 25-60% of cellulases were desorbed back into solution for the former.…”

Section: Discussionmentioning

confidence: 78%

Increased enzyme binding to substrate is not necessary for more efficient cellulose hydrolysis

Gao

Chundawat

Sethi

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

142

147

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Substrate binding is typically one of the rate-limiting steps preceding enzyme catalytic action during homogeneous reactions. However, interfacial-based enzyme catalysis on insoluble crystalline substrates, like cellulose, has additional bottlenecks of individual biopolymer chain decrystallization from the substrate interface followed by its processive depolymerization to soluble sugars. This additional decrystallization step has ramifications on the role of enzyme-substrate binding and its relationship to overall catalytic efficiency. We found that altering the crystalline structure of cellulose from its native allomorph I β to III I results in 40-50% lower binding partition coefficient for fungal cellulases, but surprisingly, it enhanced hydrolytic activity on the latter allomorph. We developed a comprehensive kinetic model for processive cellulases acting on insoluble substrates to explain this anomalous finding. Our model predicts that a reduction in the effective binding affinity to the substrate coupled with an increase in the decrystallization procession rate of individual cellulose chains from the substrate surface into the enzyme active site can reproduce our anomalous experimental findings.iological conversion of lignocellulosic biomass to fuels and chemicals has attracted tremendous interest because of its potential to address problems associated with climate change, energy security, and rural economic development. However, the transition from a petroleum-to a biomass-based economy is not easily accomplished. Biomass recalcitrance to biological conversion is one of the major hindrances to the production of cheap biofuels (1). Cellulose (a β-1,4-glucose polymer) is the most abundant organic molecule in plant cell walls that is recalcitrant to enzymatic hydrolysis because of its highly self-associated (through hydrogen bonding and stacking forces) and microfibrillar nature. Cellulose fibrils are hydrolyzed by a suite of enzymes called cellulases that can be endo-(cleave midchain glycosidic bonds) or exoactive (processively cleave glycosidic bonds starting at chain ends). Endo-(like endoglucanase I or EG-I; also known as Cel7B) and exocellulases (like cellobiohydrolases I and II or CBH-I and CBH-II, respectively; also known as Cel7A and Cel6A, respectively) are the two major components of aerobic fungal secretomes (e.g., like Trichoderma reesei) active on lignocellulose. However, the inherent disadvantages of processivity to polysaccharide hydrolysis (2) and the high abundance of processive enzymes necessary for efficient lignocellulose hydrolysis (3) suggest the need to better understand and eventually overcome the factors contributing to biomass recalcitrance.Most Trichoderma cellulases are two domain proteins consisting of a carbohydrate binding module (CBM) and a catalytic domain (CD). CBMs are known to facilitate cellulase binding to cellulose primarily through interactions between the glucopyranose rings and conserved aromatic residues (4). The mechanism of cellulose deconstruction into sugars by p...

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Section: Discussionmentioning

confidence: 78%

Increased enzyme binding to substrate is not necessary for more efficient cellulose hydrolysis

Gao

Chundawat

Sethi

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

142

147

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“…The cellulose surface can be blocked by lignin, which decreases cellulose accessibility. Enzymes can also be irreversibly adsorbed to the lignin surface and are thus no longer available for cellulose degradation (Nakagame et al 2011). It is expected that waste paper pulp becomes more hydrolyzable with decreasing lignin content, as previously observed by several researchers (Yoshida et al 2008).…”

Section: Enzymatic Hydrolysis Of Various Fraction Pulps Of Waste Papermentioning

confidence: 60%

Comparative Study of Enzymatic Hydrolysis Properties of Pulp Fractions from Waste Paper

Li¹,

Yu²,

Sun³

et al. 2015

BioResources

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As a lignocellulosic material, wastepaper is a potential material for ethanol production. However, little research on the enzymatic hydrolysis of wastepaper pulp has been conducted. In this study, the enzymatic hydrolysis of different waste pulp fractions (R80 represents greater than 80-mesh wastepaper pulp, R80-180 represents the range of 80-to 180-mesh wastepaper pulp, and R180 represents smaller than 180-mesh waste paper pulp) were carried out at 50 °C, pH 4.8, for 96 h, with a substrate concentration of 5% (w/v) and cellulase loading of 18 FPU/g cellulose. In terms of the specific surface area, fiber structure, and surface morphology, R80-180 had the highest affinity to cellulase and therefore the highest glucose yield of 80.33%. R180 had the lowest glucose yield (55.36%) because of its high ash content (21.36%), which reduced the adsorption of cellulase to cellulose. The enzymatic hydrolysis of R80 mixed with R80 or R80-180 was also studied. Results indicated that adding R80-180 increased the glucose yield of R80. The highest glucose yield (82.57%) was obtained when 15% R80-180 was mixed with R80. However, the glucose content decreased when R180 was mixed with R80 because of its high ash content.

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“…At initiation, the phase angle of this mixture was high but soon fell to near solid-like state, similar to that of the Avicel with xylan mixture, but then started to increase at a rate similar to that of Avicel, i.e., the hydrolysis process was more rapid in the presence of lignin. At low ICs, the addition of extracted lignin to an enzymatic hydrolysis slurry had shown to increase sugar yield from enzymatic hydrolysis (Nakagame et al, 2011). At high IC, improved rheology can be another motive for such additions.…”

Section: Rheological Testing Was Applicable With Other Enzymes and Sumentioning

confidence: 99%

In Situ Rheological Method to Evaluate Feedstock Physical Properties Throughout Enzymatic Deconstruction

et al. 2018

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Feedstock physical properties determine not only downstream flow behavior, but also downstream process yields. Enzymatic treatment of pretreated feedstocks is greatly dependent on upstream feedstock physical properties and choice of pre-processing Technologies. Currently available enzyme assays have been developed to study biomass slurries at low concentrations of ≤1% w/w. At commercially relevant biomass concentrations of ≥15% w/w, pretreated feedstocks have sludge-like properties, where low free water restricts movement of unattached enzymes. This work is an account of the various steps taken to develop a method that helps identify the time needed for solid-like biomass slurries transition into liquid-like states during enzymatic hydrolysis. A pre-processing technology that enables feedstocks in achieving this transition sooner will greatly benefit enzyme kinetics and thereby overall process economics. Through this in situ rheological properties determining method, we compared a model feedstock, Avicel ® PH101 cellulose, with acid pretreated corn stover. Novozymes Cellic ® CTec2 (80 mg protein/g glucan) can reduce 25% (w/w) Avicel from solid-like to liquid-like state in 5.5 h, as the phase angles rise beyond 45 • at this time. The same slurry needed 5.3 h to achieve liquid-like state with Megazyme endoglucanase (40 mg protein/g glucan). After 10.8 h, CTec2 slurry reached a phase angle of 89 • or complete liquid-like state but Megazyme slurry peaked only to 64.7 • , possibly due to inhibition by cello-oligomers. Acid pretreated corn stover at 30% (w/w) with a CTec2 protein loading of 80 mg/g glucan exhibited a solid-like to liquid-like transition at 37.8 h, which reflects the combined inhibition of low water activity and presence of lignin. The acid pretreated slurry also never achieved complete liquid-like state due to the presence of biomass residue. This method is applicable in several scenarios comparing varying combinations of pre-processing technologies, feedstock types, pretreatment chemistries, and enzymes. Using this method, we can generate a process chain with optimal flow behavior at commercially-relevant conditions.

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Enhancing the enzymatic hydrolysis of lignocellulosic biomass by increasing the carboxylic acid content of the associated lignin

Cited by 231 publications

References 42 publications

Increased enzyme binding to substrate is not necessary for more efficient cellulose hydrolysis

Increased enzyme binding to substrate is not necessary for more efficient cellulose hydrolysis

Comparative Study of Enzymatic Hydrolysis Properties of Pulp Fractions from Waste Paper

In Situ Rheological Method to Evaluate Feedstock Physical Properties Throughout Enzymatic Deconstruction

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