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Takao Ojima - One of the best experts on this subject based on the ideXlab platform.
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characterization of a ghf45 Cellulase akeg21 from the common sea hare aplysia kurodai
Frontiers in Chemistry, 2014Co-Authors: Akira Inoue, Mohammad Matiur Rahman, Takao OjimaAbstract:The common sea hare Aplysia kurodai is known to be a good source for the enzymes degrading seaweed polysaccharides. Recently four Cellulases, i.e., 95 kDa, 66 kDa, 45 kDa and 21 kDa enzymes, were isolated from A. kurodai (Tsuji et al., PLoS ONE, 8, e65418, 2013). The former three Cellulases were regarded as glycosyl-hydrolase-family 9 (GHF9) enzymes, while the 21 kDa Cellulase was suggested to be a GHF45 enzyme. The 21 kDa Cellulase was significantly heat stable, and appeared to be advantageous in performing heterogeneous expression and protein-engineering study. In the present study, we determined some enzymatic properties of the 21 kDa Cellulase and cloned its cDNA to provide the basis for the protein engineering study of this Cellulase. The purified 21 kDa enzyme, termed AkEG21 in the present study, hydrolyzed carboxymethyl cellulose with an optimal pH and temperature at 4.5 and 40oC, respectively. AkEG21 was considerably heat-stable, i.e., it was not inactivated by the incubation at 55oC for 30 min. AkEG21 degraded phosphoric-acid-swollen cellulose producing cellotriose and cellobiose as major end products but hardly degraded oligosaccharides smaller than tetrasaccharide. This indicated that AkEG21 is an endolytic -1,4-glucanase (EC 3.2.1.4). A cDNA of 1,013 bp encoding AkEG21 was amplified by PCR and the amino-acid sequence of 197 residues was deduced. The sequence comprised the initiation Met, the putative signal peptide of 16 residues for secretion and the catalytic domain of 180 residues, which lined from the N-terminus in this order. The sequence of the catalytic domain showed 47-62% amino-acid identities to those of GHF45 Cellulases reported in other mollusks. Both the catalytic residues and the N-glycosylation residues known in other GHF45 Cellulases were conserved in AkEG21. Phylogenetic analysis for the amino-acid sequences suggested the close relation between AkEG21 and fungal GHF45 Cellulases.
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isolation and primary structure of a Cellulase from the japanese sea urchin strongylocentrotus nudus
Biochimie, 2007Co-Authors: Yukiko Nishida, Kenichi Suzuki, Yuya Kumagai, Hiroyuki Tanaka, Akira Inoue, Takao OjimaAbstract:Abstract Glycoside-hydrolase-family 9 (GHF9) Cellulases are known to be widely distributed in metazoa. These enzymes have been appreciably well investigated in protostome invertebrates such as arthropods, nematodes, and mollusks but have not been characterized in deuterostome invertebrates such as sea squirts and sea urchins. In the present study, we isolated the Cellulase from the Japanese purple sea urchin Strongylocentrotus nudus and determined its enzymatic properties and primary structure. The sea urchin enzyme was extracted from the acetone-dried powder of digestive tract of S. nudus and purified by conventional chromatographies. The purified enzyme, which we named SnEG54, showed a molecular mass of 54 kDa on SDS–PAGE and exhibited high hydrolytic activity toward carboxymethyl cellulose with an optimum temperature and pH at 35 °C and 6.5, respectively. SnEG54 degraded cellulose polymer and cellooligosaccharides larger than cellotriose producing cellotriose and cellobiose but not these small cellooligosaccharides. From a cDNA library of the digestive tract we cloned 1822-bp cDNA encoding the amino-acid sequence of 444 residues of SnEG54. This sequence showed 50–57% identity with the sequences of GHF9 Cellulases from abalone, sea squirt, and termite. The amino-acid residues crucial for the catalytic action of GHF9 Cellulases are completely conserved in the SnEG54 sequence. An 8-kbp structural gene fragment encoding SnEG54 was amplified by PCR from chromosomal DNA of S. nudus. The positions of five introns are consistent with those in other animal GHF9 Cellulase genes. Thus, we confirmed that the sea urchin produces an active GHF9 Cellulase closely related to other animal Cellulases.
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Isolation and primary structure of a Cellulase from the Japanese sea urchin Strongylocentrotus nudus.
Biochimie, 2007Co-Authors: Yukiko Nishida, Kenichi Suzuki, Yuya Kumagai, Hiroyuki Tanaka, Akira Inoue, Takao OjimaAbstract:Glycoside-hydrolase-family 9 (GHF9) Cellulases are known to be widely distributed in metazoa. These enzymes have been appreciably well investigated in protostome invertebrates such as arthropods, nematodes, and mollusks but have not been characterized in deuterostome invertebrates such as sea squirts and sea urchins. In the present study, we isolated the Cellulase from the Japanese purple sea urchin Strongylocentrotus nudus and determined its enzymatic properties and primary structure. The sea urchin enzyme was extracted from the acetone-dried powder of digestive tract of S. nudus and purified by conventional chromatographies. The purified enzyme, which we named SnEG54, showed a molecular mass of 54kDa on SDS-PAGE and exhibited high hydrolytic activity toward carboxymethyl cellulose with an optimum temperature and pH at 35 degrees C and 6.5, respectively. SnEG54 degraded cellulose polymer and cellooligosaccharides larger than cellotriose producing cellotriose and cellobiose but not these small cellooligosaccharides. From a cDNA library of the digestive tract we cloned 1822-bp cDNA encoding the amino-acid sequence of 444 residues of SnEG54. This sequence showed 50-57% identity with the sequences of GHF9 Cellulases from abalone, sea squirt, and termite. The amino-acid residues crucial for the catalytic action of GHF9 Cellulases are completely conserved in the SnEG54 sequence. An 8-kbp structural gene fragment encoding SnEG54 was amplified by PCR from chromosomal DNA of S. nudus. The positions of five introns are consistent with those in other animal GHF9 Cellulase genes. Thus, we confirmed that the sea urchin produces an active GHF9 Cellulase closely related to other animal Cellulases.
David Wilson - One of the best experts on this subject based on the ideXlab platform.
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evidence for a novel mechanism of microbial cellulose degradation
Cellulose, 2009Co-Authors: David WilsonAbstract:There are two well studied mechanisms that are used by cellulolytic microorganisms to degrade the cellulose present in plant cell walls and a third less well studied oxidative mechanism used by brown rot fungi. The well studied mechanisms use Cellulases to hydrolyze the β-1,4 linkages present in cellulose, however the way in which Cellulases are presented to the environment are quite different for each mechanism. Most aerobic microorganisms secrete a set of Cellulases outside the cell (free Cellulase mechanism) while most anaerobic microorganisms produce large multi enzyme complexes on their outer surface (cellulosomal mechanism). Their genomic sequences suggest that the aerobic bacterium, Cytophaga hutchinsonii and the anaerobic bacterium, Fibrobacter succinogenes, do not use either of these mechanisms for degrading cellulose, as these organisms only code for normal endoCellulases not for processive Cellulases like exoCellulases and processive endoCellulases which are used in both of the well studied mechanisms.
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cooperative and competitive binding in synergistic mixtures of thermobifida fusca Cellulases cel5a cel6b and cel9a
Biotechnology Progress, 2002Co-Authors: Tina Jeoh, David Wilson, Larry P. WalkerAbstract:Synergism between Cellulases facilitates efficient hydrolysis of microcrystalline cellulose. We hypothesize that the effects of synergism, observed as enhanced extents of hydrolysis, are related to Cellulase binding to the substrate in mixtures. In this study, direct measurements of bound concentrations of fluorescence-labeled T. fusca Cel5A, Cel6B, and Cel9A on bacterial microcrystalline cellulose were used to study binding behaviors of Cellulases in binary component reactions. The accuracy of the determination of fluorescence-labeled Cellulase concentrations in binary component mixtures was in the range of 7-9%. Data at 5 °C show that binding levels of Cellulases in mixture reactions are only 22-70% of the binding levels in single component reactions. At 50 °C, however, most of the Cellulase components in the same mixtures bound to extents of 40-126% higher than in the corresponding single component reactions. The degrees of synergistic effect (DSE) observed for the reactions at 50 °C were greater than 1, indicating that the components in the mixture acted synergistically, whereas DSE 1. We conclude that the lower extents of binding at 5 °C are due to competition for binding sites by the Cellulase components in the mixtures and the enhanced binding extents at 50 °C are due to increased availability of binding sites on the substrates brought about by the higher extents of hydrolysis.
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structure and mechanism of endo exoCellulase e4 from thermomonospora fusca
Nature Structural & Molecular Biology, 1997Co-Authors: Joshua Sakon, David Wilson, Diana C Irwin, P A KarplusAbstract:Cellulase E4 from Thermomonospora fusca is unusual in that it has characteristics of both exo- and endo-Cellulases. Here we report the crystal structure of a 68K M(r) fragment of E4 (E4-68) at 1.9 A resolution. E4-68 contains both a family 9 catalytic domain, exhibiting an (alpha/alpha)6 barrel fold, and a family III cellulose binding domain, having an antiparallel beta-sandwich fold. While neither of these folds is novel, E4-68 provides the first Cellulase structure having interacting catalytic and cellulose binding domains. The complexes of E4-68 with cellopentaose, cellotriose and cellobiose reveal conformational changes associated with ligand binding and allow us to propose a catalytic mechanism for family 9 enzymes. We also provide evidence that E4 has two novel characteristics: first it combines exo- and endo-activities and second, when it functions as an exo-Cellulase, it cleaves off cellotetraose units.
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binding capacities for thermomonospora fusca e3 e4 and e5 the e3 binding domain and trichoderma reesei cbhi on avicel and bacterial microcrystalline cellulose
Bioresource Technology, 1997Co-Authors: M K Bothwell, S D Daughhetee, G Y Chaua, David Wilson, Larry P. WalkerAbstract:Abstract Equilibrium binding of Thermomonospora fusca E 3 , E 4 and E 5 , the E 3 binding domain (CBDE 3 ), and Trichoderma reesei CBHI on Avicel PH102 and bacterial microcrystalline cellulose (BMCC) was studied. The maximum adsorption levels, E b,m , for all four Cellulases and the binding domain were 9–30 times higher on BMCC than on Avicel. The association constants for the individual Cellulases were dependent upon the substrate; however, no obvious patterns were noted. A comparison of the T. fusca E bm s showed a decreasing power function relationship between molecular weight and maximum adsorption levels. This was particularly true for the Cellulases binding on Avicel. The T. fusca binding results strongly suggest that binding capacity is a function of the Cellulase size and the pore structure of the cellulose.
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binding reversibility and surface exchange of thermomonospora fusca e3 and e5 and trichoderma reesei cbhi
Enzyme and Microbial Technology, 1997Co-Authors: M K Bothwell, David Wilson, Diana C Irwin, Larry P. WalkerAbstract:Abstract The sorption of Thermomonospora fusca E 3 and E 5 and Trichoderma reesei CBHI Cellulases on bacterial microcrystalline cellulose (BMCC) was investigated to measure binding reversibility and surface exchange. The adsorption and desorption curves for CBHI were comparable; this suggests a totally reversible binding process. By contrast, the desorption isotherms of E 3 and E 5 did not retrace their respective adsorption curves; instead, hysteresis loops were formed. The calculated percent reversibilities for E 3 , E 5 , and CBHI were 73 ± 7%, 80 ± 7%, and 95 ± 7%, respectively. Surface exchange studies indicated that the adsorbed and free Cellulases were exchanging at the cellulose surface. Despite this confirmation of molecular exchange, the extent of exchange did not reach the level that would be predicted if the preadsorbed Cellulase were to equally redistribute between the free and bound states. The percent of exchange for E 3 and E 5 decreased with increasing initial total Cellulase concentration (E t ) while that for CBHI showed no dependence on E t .
Larry P. Walker - One of the best experts on this subject based on the ideXlab platform.
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cooperative and competitive binding in synergistic mixtures of thermobifida fusca Cellulases cel5a cel6b and cel9a
Biotechnology Progress, 2002Co-Authors: Tina Jeoh, David Wilson, Larry P. WalkerAbstract:Synergism between Cellulases facilitates efficient hydrolysis of microcrystalline cellulose. We hypothesize that the effects of synergism, observed as enhanced extents of hydrolysis, are related to Cellulase binding to the substrate in mixtures. In this study, direct measurements of bound concentrations of fluorescence-labeled T. fusca Cel5A, Cel6B, and Cel9A on bacterial microcrystalline cellulose were used to study binding behaviors of Cellulases in binary component reactions. The accuracy of the determination of fluorescence-labeled Cellulase concentrations in binary component mixtures was in the range of 7-9%. Data at 5 °C show that binding levels of Cellulases in mixture reactions are only 22-70% of the binding levels in single component reactions. At 50 °C, however, most of the Cellulase components in the same mixtures bound to extents of 40-126% higher than in the corresponding single component reactions. The degrees of synergistic effect (DSE) observed for the reactions at 50 °C were greater than 1, indicating that the components in the mixture acted synergistically, whereas DSE 1. We conclude that the lower extents of binding at 5 °C are due to competition for binding sites by the Cellulase components in the mixtures and the enhanced binding extents at 50 °C are due to increased availability of binding sites on the substrates brought about by the higher extents of hydrolysis.
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binding capacities for thermomonospora fusca e3 e4 and e5 the e3 binding domain and trichoderma reesei cbhi on avicel and bacterial microcrystalline cellulose
Bioresource Technology, 1997Co-Authors: M K Bothwell, S D Daughhetee, G Y Chaua, David Wilson, Larry P. WalkerAbstract:Abstract Equilibrium binding of Thermomonospora fusca E 3 , E 4 and E 5 , the E 3 binding domain (CBDE 3 ), and Trichoderma reesei CBHI on Avicel PH102 and bacterial microcrystalline cellulose (BMCC) was studied. The maximum adsorption levels, E b,m , for all four Cellulases and the binding domain were 9–30 times higher on BMCC than on Avicel. The association constants for the individual Cellulases were dependent upon the substrate; however, no obvious patterns were noted. A comparison of the T. fusca E bm s showed a decreasing power function relationship between molecular weight and maximum adsorption levels. This was particularly true for the Cellulases binding on Avicel. The T. fusca binding results strongly suggest that binding capacity is a function of the Cellulase size and the pore structure of the cellulose.
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binding reversibility and surface exchange of thermomonospora fusca e3 and e5 and trichoderma reesei cbhi
Enzyme and Microbial Technology, 1997Co-Authors: M K Bothwell, David Wilson, Diana C Irwin, Larry P. WalkerAbstract:Abstract The sorption of Thermomonospora fusca E 3 and E 5 and Trichoderma reesei CBHI Cellulases on bacterial microcrystalline cellulose (BMCC) was investigated to measure binding reversibility and surface exchange. The adsorption and desorption curves for CBHI were comparable; this suggests a totally reversible binding process. By contrast, the desorption isotherms of E 3 and E 5 did not retrace their respective adsorption curves; instead, hysteresis loops were formed. The calculated percent reversibilities for E 3 , E 5 , and CBHI were 73 ± 7%, 80 ± 7%, and 95 ± 7%, respectively. Surface exchange studies indicated that the adsorbed and free Cellulases were exchanging at the cellulose surface. Despite this confirmation of molecular exchange, the extent of exchange did not reach the level that would be predicted if the preadsorbed Cellulase were to equally redistribute between the free and bound states. The percent of exchange for E 3 and E 5 decreased with increasing initial total Cellulase concentration (E t ) while that for CBHI showed no dependence on E t .
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engineering Cellulase mixtures by varying the mole fraction of thermomonospora fusca e5 and e3 trichoderma reesei cbhi and caldocellum saccharolyticum β glucosidase
Biotechnology and Bioengineering, 1993Co-Authors: Larry P. Walker, David Wilson, C D Belair, Diana C IrwinAbstract:In this study, different mole fractions of pure Thermomonospora fusca E5 and E3, plus Trichoderma reesei CBHI were studied for reducing sugar production at 2 h, degree of synergism, and cellulose binding. In addition, the effects of introducing the Caldocellum saccharolyticum β-glucosidase into this Cellulase system were investigated. The Cellulases used were purified to homogeneity. Avicel PH 102 (4% w/w solution in 0.05 sodium acetate pH 5.5 buffer) was the substrate. Reactions were run at 50°C for 2 h using total Cellulase concentrations of 8.3 or 12.2 μM. A bimixture of T. fusca E3 and T. reesei CBHI was very effective in hydrolyzing microcrystalline cellulose (9.1% conversion). The addition of endoglucanase E5 to the mixture only increased conversion to 9.8%. However, when both E5 and β-glucosidase were added, conversion increased to 14%. It was also observed that increasing total Cellulase concentration beyond 8.3 μM did little to increase percent conversion of cellulose into glucose. The results of the binding studies indicate no competition for binding sites between the endo- and exoCellulases. © 1993 John Wiley & Sons, Inc.
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enzymatic hydrolysis of cellulose an overview
Bioresource Technology, 1991Co-Authors: Larry P. Walker, David WilsonAbstract:Abstract Despite the fact that the world community is no longer preoccupied with fossil fuel shortages, there is still considerable research and development directed toward understanding and commercializing enzymatic hydrolysis of cellulose. These efforts have ranged from applied work on bioreactors to basic research focusing on the detailed molecular mechanisms of hydrolysis. Studies on cellulose pretreatment have provided considerable insights into the influence of crystallinity and specific surface area on the rate and extent of hydrolysis. In addition these studies have demonstrated that the economics of enzymatic hydrolysis processes can be significantly improved by pretreatment. However, the major drawback to commercialization of enzymatic hydrolysis processes continues to be the relatively low hydrolysis rates achieved and the high cost of the enzymes. Research efforts directed toward understanding and manipulating Cellulase systems suggest that greater Cellulase activity can be achieved through optimizing the mix of Cellulases used in hydrolysis and through protein engineering of Cellulases. Investigators are probing into the synergistic mechanisms observed in several Cellulase systems in an effort to demonstrate that optimal combinations of Cellulases can be obtained that will yield increased rates and extents of hydrolysis. These studies and other studies of Cellulases have yielded considerable insights into the role that Cellulase binding plays in synergism. Better understanding of the molecular mechanisms at work in hydrolysis will make it possible to use the revolutionary tools of cloning and site directed mutagenesis to modify Cellulase systems so as to improve the conversion efficiency and economics of enzymatic hydrolysis processes.
Shishir P S Chundawat - One of the best experts on this subject based on the ideXlab platform.
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restructuring the crystalline cellulose hydrogen bond network enhances its depolymerization rate
Journal of the American Chemical Society, 2011Co-Authors: Shishir P S Chundawat, Giovanni Bellesia, Nirmal Uppugundla, Leonardo Da Costa Sousa, Albert M Cheh, Umesh P Agarwal, Christopher M Bianchetti, George N Phillips, Paul Langan, Venkatesh BalanAbstract:Conversion of lignocellulose to biofuels is partly inefficient due to the deleterious impact of cellulose crystallinity on enzymatic saccharification. We demonstrate how the synergistic activity of Cellulases was enhanced by altering the hydrogen bond network within crystalline cellulose fibrils. We provide a molecular-scale explanation of these phenomena through molecular dynamics (MD) simulations and enzymatic assays. Ammonia transformed the naturally occurring crystalline allomorph Iβ to IIII, which led to a decrease in the number of cellulose intrasheet hydrogen bonds and an increase in the number of intersheet hydrogen bonds. This rearrangement of the hydrogen bond network within cellulose IIII, which increased the number of solvent-exposed glucan chain hydrogen bonds with water by ∼50%, was accompanied by enhanced saccharification rates by up to 5-fold (closest to amorphous cellulose) and 60–70% lower maximum surface-bound Cellulase capacity. The enhancement in apparent Cellulase activity was attrib...
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restructuring the crystalline cellulose hydrogen bond network enhances its depolymerization rate
Journal of the American Chemical Society, 2011Co-Authors: Shishir P S Chundawat, Giovanni Bellesia, Nirmal Uppugundla, Leonardo Da Costa Sousa, Albert M Cheh, Umesh P Agarwal, Christopher M Bianchetti, George N Phillips, Dahai Gao, Paul LanganAbstract:Conversion of lignocellulose to biofuels is partly inefficient due to the deleterious impact of cellulose crystallinity on enzymatic saccharification. We demonstrate how the synergistic activity of Cellulases was enhanced by altering the hydrogen bond network within crystalline cellulose fibrils. We provide a molecular-scale explanation of these phenomena through molecular dynamics (MD) simulations and enzymatic assays. Ammonia transformed the naturally occurring crystalline allomorph I(β) to III(I), which led to a decrease in the number of cellulose intrasheet hydrogen bonds and an increase in the number of intersheet hydrogen bonds. This rearrangement of the hydrogen bond network within cellulose III(I), which increased the number of solvent-exposed glucan chain hydrogen bonds with water by ~50%, was accompanied by enhanced saccharification rates by up to 5-fold (closest to amorphous cellulose) and 60-70% lower maximum surface-bound Cellulase capacity. The enhancement in apparent Cellulase activity was attributed to the "amorphous-like" nature of the cellulose III(I) fibril surface that facilitated easier glucan chain extraction. Unrestricted substrate accessibility to active-site clefts of certain endoCellulase families further accelerated deconstruction of cellulose III(I). Structural and dynamical features of cellulose III(I), revealed by MD simulations, gave additional insights into the role of cellulose crystal structure on fibril surface hydration that influences interfacial enzyme binding. Subtle alterations within the cellulose hydrogen bond network provide an attractive way to enhance its deconstruction and offer unique insight into the nature of cellulose recalcitrance. This approach can lead to unconventional pathways for development of novel pretreatments and engineered Cellulases for cost-effective biofuels production.
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the quest for alternatives to microbial Cellulase mix production corn stover produced heterologous multi Cellulases readily deconstruct lignocellulosic biomass into fermentable sugars
Journal of Chemical Technology & Biotechnology, 2011Co-Authors: Sanghyuck Park, Robab Sabzikar, Shishir P S Chundawat, Callista Ransom, Chuansheng Mei, Bruce E Dale, Mariam B SticklenAbstract:BACKGROUND: Production of cellulosic ethanol is still expensive compared with corn (maize) grain ethanol due to the high costs of bulk production of microbial Cellulases. At least three Cellulases including endo-Cellulase, exo-Cellulase and cellobiase are needed to convert cellulosic biomass into fermentable sugars. All these Cellulases could be self-produced within cells of transgenic bio-energy crops. The production of heterologous Acidothermus cellulolyticus (E1) endo-Cellulase in endoplasmic reticulum and mitochondria of green tissues of transgenic corn plants was recently reported, and it was confirmed that the heterologous E1 converts cellulose into fermentable sugars. RESULTS: Biologically active A. cellulolyticus E1, Trichoderma reesei 1,4-β-cellobiohydrolases I (CBH I) exo-Cellulase and bovine rumen Butyrivibrio fibrisolvens cellobiase were expressed in corn plant endoplasmic reticulum (ER), apoplast (cell wall areas) and vacuole respectively. Results show that the ratio 1:4:1 (E1:CBH I:cellobiase) of crude heterologous Cellulases is ideal for converting ammoniafiber explosion (AFEX) pretreated corn stover into fermentable sugars. CONCLUSIONS: Corn plants that express all three biologically active heterologous Cellulases within their cellulosic biomass to facilitate conversion of pretreated corn stover into fermentable sugars is a step forward in the quest for alternatives to the present microbial Cellulase mix production for cellulosic biofuels. c � 2011 Society of Chemical Industry
Paul Langan - One of the best experts on this subject based on the ideXlab platform.
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restructuring the crystalline cellulose hydrogen bond network enhances its depolymerization rate
Journal of the American Chemical Society, 2011Co-Authors: Shishir P S Chundawat, Giovanni Bellesia, Nirmal Uppugundla, Leonardo Da Costa Sousa, Albert M Cheh, Umesh P Agarwal, Christopher M Bianchetti, George N Phillips, Paul Langan, Venkatesh BalanAbstract:Conversion of lignocellulose to biofuels is partly inefficient due to the deleterious impact of cellulose crystallinity on enzymatic saccharification. We demonstrate how the synergistic activity of Cellulases was enhanced by altering the hydrogen bond network within crystalline cellulose fibrils. We provide a molecular-scale explanation of these phenomena through molecular dynamics (MD) simulations and enzymatic assays. Ammonia transformed the naturally occurring crystalline allomorph Iβ to IIII, which led to a decrease in the number of cellulose intrasheet hydrogen bonds and an increase in the number of intersheet hydrogen bonds. This rearrangement of the hydrogen bond network within cellulose IIII, which increased the number of solvent-exposed glucan chain hydrogen bonds with water by ∼50%, was accompanied by enhanced saccharification rates by up to 5-fold (closest to amorphous cellulose) and 60–70% lower maximum surface-bound Cellulase capacity. The enhancement in apparent Cellulase activity was attrib...
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restructuring the crystalline cellulose hydrogen bond network enhances its depolymerization rate
Journal of the American Chemical Society, 2011Co-Authors: Shishir P S Chundawat, Giovanni Bellesia, Nirmal Uppugundla, Leonardo Da Costa Sousa, Albert M Cheh, Umesh P Agarwal, Christopher M Bianchetti, George N Phillips, Dahai Gao, Paul LanganAbstract:Conversion of lignocellulose to biofuels is partly inefficient due to the deleterious impact of cellulose crystallinity on enzymatic saccharification. We demonstrate how the synergistic activity of Cellulases was enhanced by altering the hydrogen bond network within crystalline cellulose fibrils. We provide a molecular-scale explanation of these phenomena through molecular dynamics (MD) simulations and enzymatic assays. Ammonia transformed the naturally occurring crystalline allomorph I(β) to III(I), which led to a decrease in the number of cellulose intrasheet hydrogen bonds and an increase in the number of intersheet hydrogen bonds. This rearrangement of the hydrogen bond network within cellulose III(I), which increased the number of solvent-exposed glucan chain hydrogen bonds with water by ~50%, was accompanied by enhanced saccharification rates by up to 5-fold (closest to amorphous cellulose) and 60-70% lower maximum surface-bound Cellulase capacity. The enhancement in apparent Cellulase activity was attributed to the "amorphous-like" nature of the cellulose III(I) fibril surface that facilitated easier glucan chain extraction. Unrestricted substrate accessibility to active-site clefts of certain endoCellulase families further accelerated deconstruction of cellulose III(I). Structural and dynamical features of cellulose III(I), revealed by MD simulations, gave additional insights into the role of cellulose crystal structure on fibril surface hydration that influences interfacial enzyme binding. Subtle alterations within the cellulose hydrogen bond network provide an attractive way to enhance its deconstruction and offer unique insight into the nature of cellulose recalcitrance. This approach can lead to unconventional pathways for development of novel pretreatments and engineered Cellulases for cost-effective biofuels production.