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

Abstract

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...

Specificity of O -glycosylation in enhancing the stability and cellulose binding affinity of Family 1 carbohydrate-binding modules

“…We report affinity enhancements of similar magnitudes as the enhancements reported by Takashima et al (50) and Linder et al (12), with affinity of CBMs 13 and 17 approaching or within range of K ads values obtained for both the whole TrCel7A or the multiglycosylated TrCel7A linker CBM domain (21,(52)(53)(54)(55), suggesting that glycoengineering of Family 1 CBMs is a viable strategy for activity enhancement of cellulases. Moreover, the O-linked glycosylation sites studied here are highly conserved across Family 1 CBMs (23), suggesting that the glycosylation-enhancing properties observed here likely occur throughout this ubiquitous CBM family.…”

Synergism of Glycoside Hydrolase Secretomes from Two Thermophilic Bacteria Cocultivated on Lignocellulose

“…Taking the protein concentration from the tube without Avicel as the total protein, the concentrations of bound protein were obtained by subtracting the protein concentration of the sample with Avicel from the total-protein concentration. For determination of the binding constant of the proteins at different temperatures, the Langmuir equation [q ad ϭ q max ϫ q/(K d ϩ q)] was used (54). In this equation, q ad represents the amount of bound protein (nmol of protein per g of Avicel), q is the free protein (M), K d is the dissociation constant (M), and q max is the maximal amount of protein bound to Avicel; ␣ is the partition coefficient, or q max /K d ratio.…”

Next-generation ammonia pretreatment enhances cellulosic biofuel production

“…1,2 Cellulose treatment by liquid ammonia has been used industrially to improve textile fiber properties at sub-zero temperatures and low pressures. 3,4 Effective CIII formation during ammonia pretreatment of lignocellulosic biomass requires the substrate to have low moisture content and the usage of high ammonia-to-biomass a Department of Chemical Engineering and Materials Science, and Department of…”

“…High water concentration during pretreatment impedes CIII formation and reverts the ammonia-cellulose complex back into the allomorph CI. 2,5,7 Other ammonia-based pretreatments such as ammonia fiber expansion (AFEXt, trade mark of MBI International, Lansing, MI) and ammonia-recycle percolation (ARP) do not lead to CIII formation because they employ high moisture contents and/or low ammonia-to-biomass ratios. In the case of AFEX, heat generated by the exothermal reaction between ammonia and water is used to reach temperatures up to 140 1C.…”