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Akira Isogai - One of the best experts on this subject based on the ideXlab platform.
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Preparation of oxidized Celluloses in a NaBr/NaClO system using 2-azaadamantane N-oxyl (AZADO) derivatives in water at pH 10
Cellulose, 2018Co-Authors: Hiromasa Hondo, Tsuguyuki Saito, Akira IsogaiAbstract:Catalytic oxidation using N-oxyl radicals in water at pH 10 was applied to wood Cellulose to determine the influence of different N-oxyl radical chemical structures on the reaction kinetics and structures of oxidized Celluloses. 2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO), 2-azaadamantane N-oxyl (AZADO), 1-methyl-AZADO (1-Me-AZADO), 9-azanoradamantane N-oxyl (Nor-AZADO), and 1,5-dimethyl-9-azanoradamantane N-oxyl (DMN-AZADO) were used as oxidation catalysts in a NaBr/NaClO system in water at room temperature and pH 10. The reaction time required for complete oxidation of wood Cellulose by 5 mmol/g of NaClO using AZADO, 1-Me-AZADO, or Nor-AZADO was less than 20 min, while DMN-AZADO and TEMPO required 70 min and 120 min, respectively. The sodium carboxylate contents of the oxidized Celluloses prepared using TEMPO and DMN-AZADO were 1.41 and 1.45 mmol/g, respectively, which were higher than those of oxidized Celluloses prepared using other AZADO derivatives. The original Cellulose I crystal structure, crystallinity, and crystal size of wood Cellulose were mostly maintained in all oxidized Celluloses, with oxidation selectively occurring at C6–OH groups on the crystalline Cellulose microfibril surfaces. Oxidized Celluloses prepared using DMN-AZADO and post-reduced with NaBH4 showed high carboxylate contents of ~ 1.41 mmol/g and the heist viscosity-average degrees of polymerization of ~ 800.
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influence of drying of chara Cellulose on length length distribution of microfibrils after acid hydrolysis
International Journal of Biological Macromolecules, 2018Co-Authors: Yoshiki Horikawa, Tsuguyuki Saito, Akira Isogai, Junji Sugiyama, Michiko Shimizu, Tomoya ImaiAbstract:Abstract Chara is a genus of freshwater alga that is evolutionarily observed at the aquatic-terrestrial boundary, whose Cellulose microfibrils are similar to those of terrestrial plants regarding the crystallinity and biosynthesis of Cellulose. Oven-dried and never-dried Celluloses samples were prepared from chara. Terrestrial plant Cellulose samples were used as references. The lengths and length distributions of oven-dried and never-dried chara Cellulose microfibrils after acid hydrolysis with or without pretreatment by 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-mediated oxidation, which was used for efficient fibrillation of acid-hydrolyzed products, were observed by transmission electron microscopy. All terrestrial plant Celluloses and oven-dried chara Cellulose had short nanocrystal-like morphologies of 100–300 nm in length after acid hydrolysis. In contrast, the never-dried chara Cellulose had much longer microfibrils of ∼970 nm in length after acid hydrolysis. These results indicated that disordered regions present periodically along the Cellulose microfibrils, which cause the formation of Cellulose nanocrystals after acid hydrolysis, are not present in inherent chara Cellulose microfibrils in water, but are formed artificially under drying or dehydration conditions.
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changes in the degree of polymerization of wood Celluloses during dilute acid hydrolysis and tempo mediated oxidation formation mechanism of disordered regions along each Cellulose microfibril
International Journal of Biological Macromolecules, 2017Co-Authors: Ryunosuke Funahashi, Morihiko Yokoi, Kunihiko Daido, Toru Inamochi, Reina Tanaka, Yoshiki Horikawa, Tsuguyuki Saito, Akira IsogaiAbstract:Abstract Most commercially available plant Celluloses, such as kraft pulps and cotton Celluloses, have so-called leveling-off degrees of polymerization (LODPs) when subjected to dilute acid hydrolysis. The formation of LODPs is hypothesized to be caused by disordered regions that are present periodically along each Cellulose microfibril in plant Celluloses. Here, we prepared never-dried wood Cellulose, and wood Celluloses at different drying stages, and subjected them to dilute acid hydrolysis. The viscosity average degrees of polymerization (DPv) of the wood Celluloses decreased in DPv with increasing dilute acid-hydrolysis times. However, the DPv values after 4 h of acid hydrolysis differed from those of softwood bleached kraft pulp (SBKP), which showed typical patterns of LODPs. Thus, the disordered regions corresponding to LODPs that were observed for SBKP are probably not synthesized in the native wood. Instead, such disordered regions are formed secondarily or artificially during the isolation/purification and/or drying processes of plant Cellulose fibers. The results of the dilute acid hydrolysis and the 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-mediated oxidation of SBKP and softwood unbleached soda-anthraquinone pulp showed that the structures of disordered regions can be controlled via the preparation and drying conditions of wood Cellulose used as starting materials.
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Cellulose nanofibers prepared by tempo mediated oxidation of native Cellulose
Biomacromolecules, 2007Co-Authors: Tsuguyuki Saito, Satoshi Kimura, Yoshiharu Nishiyama, Akira IsogaiAbstract:Never-dried and once-dried hardwood Celluloses were oxidized by a 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-mediated system, and highly crystalline and individualized Cellulose nanofibers, dispersed in water, were prepared by mechanical treatment of the oxidized Cellulose/water slurries. When carboxylate contents formed from the primary hydroxyl groups of the Celluloses reached approximately 1.5 mmol/g, the oxidized Cellulose/water slurries were mostly converted to transparent and highly viscous dispersions by mechanical treatment. Transmission electron microscopic observation showed that the dispersions consisted of individualized Cellulose nanofibers 3−4 nm in width and a few microns in length. No intrinsic differences between never-dried and once-dried Celluloses were found for preparing the dispersion, as long as carboxylate contents in the TEMPO-oxidized Celluloses reached approximately 1.5 mmol/g. Changes in viscosity of the dispersions during the mechanical treatment corresponded with th...
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tempo mediated oxidation of native Cellulose microscopic analysis of fibrous fractions in the oxidized products
Carbohydrate Polymers, 2006Co-Authors: Tsuguyuki Saito, Junji Sugiyama, Yusuke Okita, Akira IsogaiAbstract:Abstract The 2,2,6,6-tetramethylpiperidine-1-oxy radial (TEMPO)-mediated oxidation was applied to aqueous suspensions of cotton linters, ramie and spruce holoCellulose at pH 10.5, and water-insoluble fractions of the TEMPO-oxidized Celluloses collected by filtration with water were analyzed by optical and transmission electron microscopy and others. The results showed that both fibrous forms and microfibrillar nature of the original native Celluloses were maintained after the TEMPO-mediated oxidation, even though carboxylate and aldehyde groups of 0.67–1.16 and 0.09–0.21 mmol/g, respectively, were introduced into the water-insoluble fractions. Neither crystallinity nor crystal size of Cellulose I of the original native Celluloses was changed under the conditions adopted in this study. Carboxylate groups in the TEMPO-oxidized ramie were mapped by labeling with lead ions as their counter ions. The transmission electron micrographs indicated that some heterogeneous distribution of carboxylate groups along each Cellulose microfibril or each bundle of Cellulose microfibrils seemed to be present in the TEMPO-oxidized Celluloses.
Tsuguyuki Saito - One of the best experts on this subject based on the ideXlab platform.
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Preparation of oxidized Celluloses in a NaBr/NaClO system using 2-azaadamantane N-oxyl (AZADO) derivatives in water at pH 10
Cellulose, 2018Co-Authors: Hiromasa Hondo, Tsuguyuki Saito, Akira IsogaiAbstract:Catalytic oxidation using N-oxyl radicals in water at pH 10 was applied to wood Cellulose to determine the influence of different N-oxyl radical chemical structures on the reaction kinetics and structures of oxidized Celluloses. 2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO), 2-azaadamantane N-oxyl (AZADO), 1-methyl-AZADO (1-Me-AZADO), 9-azanoradamantane N-oxyl (Nor-AZADO), and 1,5-dimethyl-9-azanoradamantane N-oxyl (DMN-AZADO) were used as oxidation catalysts in a NaBr/NaClO system in water at room temperature and pH 10. The reaction time required for complete oxidation of wood Cellulose by 5 mmol/g of NaClO using AZADO, 1-Me-AZADO, or Nor-AZADO was less than 20 min, while DMN-AZADO and TEMPO required 70 min and 120 min, respectively. The sodium carboxylate contents of the oxidized Celluloses prepared using TEMPO and DMN-AZADO were 1.41 and 1.45 mmol/g, respectively, which were higher than those of oxidized Celluloses prepared using other AZADO derivatives. The original Cellulose I crystal structure, crystallinity, and crystal size of wood Cellulose were mostly maintained in all oxidized Celluloses, with oxidation selectively occurring at C6–OH groups on the crystalline Cellulose microfibril surfaces. Oxidized Celluloses prepared using DMN-AZADO and post-reduced with NaBH4 showed high carboxylate contents of ~ 1.41 mmol/g and the heist viscosity-average degrees of polymerization of ~ 800.
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influence of drying of chara Cellulose on length length distribution of microfibrils after acid hydrolysis
International Journal of Biological Macromolecules, 2018Co-Authors: Yoshiki Horikawa, Tsuguyuki Saito, Akira Isogai, Junji Sugiyama, Michiko Shimizu, Tomoya ImaiAbstract:Abstract Chara is a genus of freshwater alga that is evolutionarily observed at the aquatic-terrestrial boundary, whose Cellulose microfibrils are similar to those of terrestrial plants regarding the crystallinity and biosynthesis of Cellulose. Oven-dried and never-dried Celluloses samples were prepared from chara. Terrestrial plant Cellulose samples were used as references. The lengths and length distributions of oven-dried and never-dried chara Cellulose microfibrils after acid hydrolysis with or without pretreatment by 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-mediated oxidation, which was used for efficient fibrillation of acid-hydrolyzed products, were observed by transmission electron microscopy. All terrestrial plant Celluloses and oven-dried chara Cellulose had short nanocrystal-like morphologies of 100–300 nm in length after acid hydrolysis. In contrast, the never-dried chara Cellulose had much longer microfibrils of ∼970 nm in length after acid hydrolysis. These results indicated that disordered regions present periodically along the Cellulose microfibrils, which cause the formation of Cellulose nanocrystals after acid hydrolysis, are not present in inherent chara Cellulose microfibrils in water, but are formed artificially under drying or dehydration conditions.
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changes in the degree of polymerization of wood Celluloses during dilute acid hydrolysis and tempo mediated oxidation formation mechanism of disordered regions along each Cellulose microfibril
International Journal of Biological Macromolecules, 2017Co-Authors: Ryunosuke Funahashi, Morihiko Yokoi, Kunihiko Daido, Toru Inamochi, Reina Tanaka, Yoshiki Horikawa, Tsuguyuki Saito, Akira IsogaiAbstract:Abstract Most commercially available plant Celluloses, such as kraft pulps and cotton Celluloses, have so-called leveling-off degrees of polymerization (LODPs) when subjected to dilute acid hydrolysis. The formation of LODPs is hypothesized to be caused by disordered regions that are present periodically along each Cellulose microfibril in plant Celluloses. Here, we prepared never-dried wood Cellulose, and wood Celluloses at different drying stages, and subjected them to dilute acid hydrolysis. The viscosity average degrees of polymerization (DPv) of the wood Celluloses decreased in DPv with increasing dilute acid-hydrolysis times. However, the DPv values after 4 h of acid hydrolysis differed from those of softwood bleached kraft pulp (SBKP), which showed typical patterns of LODPs. Thus, the disordered regions corresponding to LODPs that were observed for SBKP are probably not synthesized in the native wood. Instead, such disordered regions are formed secondarily or artificially during the isolation/purification and/or drying processes of plant Cellulose fibers. The results of the dilute acid hydrolysis and the 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-mediated oxidation of SBKP and softwood unbleached soda-anthraquinone pulp showed that the structures of disordered regions can be controlled via the preparation and drying conditions of wood Cellulose used as starting materials.
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Cellulose nanofibers prepared by tempo mediated oxidation of native Cellulose
Biomacromolecules, 2007Co-Authors: Tsuguyuki Saito, Satoshi Kimura, Yoshiharu Nishiyama, Akira IsogaiAbstract:Never-dried and once-dried hardwood Celluloses were oxidized by a 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-mediated system, and highly crystalline and individualized Cellulose nanofibers, dispersed in water, were prepared by mechanical treatment of the oxidized Cellulose/water slurries. When carboxylate contents formed from the primary hydroxyl groups of the Celluloses reached approximately 1.5 mmol/g, the oxidized Cellulose/water slurries were mostly converted to transparent and highly viscous dispersions by mechanical treatment. Transmission electron microscopic observation showed that the dispersions consisted of individualized Cellulose nanofibers 3−4 nm in width and a few microns in length. No intrinsic differences between never-dried and once-dried Celluloses were found for preparing the dispersion, as long as carboxylate contents in the TEMPO-oxidized Celluloses reached approximately 1.5 mmol/g. Changes in viscosity of the dispersions during the mechanical treatment corresponded with th...
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tempo mediated oxidation of native Cellulose microscopic analysis of fibrous fractions in the oxidized products
Carbohydrate Polymers, 2006Co-Authors: Tsuguyuki Saito, Junji Sugiyama, Yusuke Okita, Akira IsogaiAbstract:Abstract The 2,2,6,6-tetramethylpiperidine-1-oxy radial (TEMPO)-mediated oxidation was applied to aqueous suspensions of cotton linters, ramie and spruce holoCellulose at pH 10.5, and water-insoluble fractions of the TEMPO-oxidized Celluloses collected by filtration with water were analyzed by optical and transmission electron microscopy and others. The results showed that both fibrous forms and microfibrillar nature of the original native Celluloses were maintained after the TEMPO-mediated oxidation, even though carboxylate and aldehyde groups of 0.67–1.16 and 0.09–0.21 mmol/g, respectively, were introduced into the water-insoluble fractions. Neither crystallinity nor crystal size of Cellulose I of the original native Celluloses was changed under the conditions adopted in this study. Carboxylate groups in the TEMPO-oxidized ramie were mapped by labeling with lead ions as their counter ions. The transmission electron micrographs indicated that some heterogeneous distribution of carboxylate groups along each Cellulose microfibril or each bundle of Cellulose microfibrils seemed to be present in the TEMPO-oxidized Celluloses.
Lee R Lynd - One of the best experts on this subject based on the ideXlab platform.
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OPTIMIZATION OF AFFINITY DIGESTION FOR THE ISOLATION OF CELLULOSOMES FROM Clostridium thermocellum
Preparative Biochemistry & Biotechnology, 2013Co-Authors: Lois A. St Brice, Xiongjun Shao, Javier A. Izquierdo, Lee R LyndAbstract:The affinity digestion process for cellulase purification consisting of binding to amorphous Cellulose, and amorphous Cellulose hydrolysis in the presence of dialysis (Morag et al., 1991), was optimized to obtain high activity recoveries and consistent protein recoveries in the isolation of Clostridium thermocellum cellulase. Experiments were conducted using crude supernatant prepared from C. thermocellum grown on either Avicel or cellobiose. While no difference was observed between Avicel-grown or cellobiose-grown cellulase in the adsorption step, differences were observed during the hydrolysis step. The optimal amorphous Cellulose loading was found to be 3 mg amorphous Cellulose per milligram supernatant protein. At this loading, 90–100% of activity in the crude supernatant was adsorbed. Twenty-four-hour incubation with the amorphous Cellulose during the adsorption stage was found to result in maximal and stable adsorption of activity to the substrate. By fitting the adsorption data to the Langmuir mode...
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Consolidated bioprocessing of cellulosic biomass: an update
Current Opinion in Biotechnology, 2005Co-Authors: Lee R Lynd, John Mcbride, Mark LaserAbstract:Biologically mediated processes seem promising for energy conversion, in particular for the conversion of lignocellulosic biomass into fuels. Although processes featuring a step dedicated to the production of cellulase enzymes have been the focus of most research efforts to date, consolidated bioprocessing (CBP) – featuring cellulase production, Cellulose hydrolysis and fermentation in one step – is an alternative approach with outstanding potential. Progress in developing CBP-enabling microorganisms is being made through two strategies: engineering naturally occurring cellulolytic microorganisms to improve product-related properties, such as yield and titer, and engineering non-cellulolytic organisms that exhibit high product yields and titers to express a heterologous cellulase system enabling Cellulose utilization. Recent studies of the fundamental principles of microbial Cellulose utilization support the feasibility of CBP.
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toward an aggregated understanding of enzymatic hydrolysis of Cellulose noncomplexed cellulase systems
Biotechnology and Bioengineering, 2004Co-Authors: Yiheng Percival Zhang, Lee R LyndAbstract:Information pertaining to enzymatic hydrolysis of Cellulose by noncomplexed cellulase enzyme systems is reviewed with a particular emphasis on development of aggregated understanding incorporating substrate features in addition to concentration and multiple cellulase compo- nents. Topics considered include properties of Cellulose, adsorption, Cellulose hydrolysis, and quantitative models. A classification scheme is proposed for quantitative models for enzymatic hydrolysis of Cellulose based on the number of solubilizing activities and substrate state variables in- cluded. We suggest that it is timely to revisit and reinvig- orate functional modeling of Cellulose hydrolysis, and that this would be highly beneficial if not necessary in order to bring to bear the large volume of information available on cellulase components on the primary applications that
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toward an aggregated understanding of enzymatic hydrolysis of Cellulose noncomplexed cellulase systems
Biotechnology and Bioengineering, 2004Co-Authors: Yiheng Percival Zhang, Lee R LyndAbstract:Information pertaining to enzymatic hydrolysis of Cellulose by noncomplexed cellulase enzyme systems is reviewed with a particular emphasis on development of aggregated understanding incorporating substrate features in addition to concentration and multiple cellulase components. Topics considered include properties of Cellulose, adsorption, Cellulose hydrolysis, and quantitative models. A classification scheme is proposed for quantitative models for enzymatic hydrolysis of Cellulose based on the number of solubilizing activities and substrate state variables included. We suggest that it is timely to revisit and reinvigorate functional modeling of Cellulose hydrolysis, and that this would be highly beneficial if not necessary in order to bring to bear the large volume of information available on cellulase components on the primary applications that motivate interest in the subject.
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Quantitative determination of cellulase concentration as distinct from cell concentration in studies of microbial Cellulose utilization: Analytical framework and methodological approach
Biotechnology and Bioengineering, 2002Co-Authors: Lee R Lynd, Yiheng ZhangAbstract:In analyzing microbial Cellulose utilization, it would be useful to independently measure the mass concentration of cells and cellulase enzymes. Such measurements would allow investigation of the allocation of cellular resources between synthesis of cells and cellulase, in vivo cell- and cellulase-specific Cellulose hydrolysis rates, and bioenergetics. Methodological protocols are not established for independent determination of cell and cellulase concentrations for the common case in which a substantial fraction of cellulase is attached to the cell surface. Alternative analytical approaches by which to develop such protocols are examined from the perspective of error minimization. For cell concentration measurement, acceptable accuracy is expected when the concentrations of a cell-specific component (e.g., DNA) is determined or when total protein is determined in conjunction with a measurement specific to cellulase. For cellulase concentration measurement, acceptable accuracy is expected when a measurement specific to cellulase such as ELISA is used. Several analytical approaches are rejected based on large expected errors.
Yiheng Percival Zhang - One of the best experts on this subject based on the ideXlab platform.
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evaluations of Cellulose accessibilities of lignoCelluloses by solute exclusion and protein adsorption techniques
Biotechnology and Bioengineering, 2012Co-Authors: Qianqian Wang, Yiheng Percival Zhang, Z He, Yonghao NiAbstract:Cellulose accessibilities of a set of hornified lignocellulosic substrates derived by drying the never dried pretreated sample and a set of differently pretreated lodge- pople pine substrates, were evaluated using solute exclusion and protein adsorption methods. Direct measurements of cellulase adsorption onto Cellulose surface of the set of pretreated substrates were also carried out using an in situ UV-Vis spectrophotometric technique. The Cellulose accessibilities measured by the solute exclusion and a cellu- lose-binding module (CBM)-containing green fluorescent protein (TGC) adsorption methods correlate well for both sets of samples. The substrate enzymatic digestibilities (SEDs) of the hornified substrates are proportional to the measured Cellulose accessibilities. Approximately over 90% of the SED was contributed by the accessible pore surfaces of the hornified substrates, suggesting that the substrate exter- nal surface plays a minor role contributing to Cellulose accessibility and SED. The Cellulose accessibilities of the pretreated substrates correlated well with the amounts of cellulase adsorbed. The SEDs of these substrates directly correlated with the amounts of adsorbed cellulase. Biotechnol. Bioeng. 2012;109: 381-389. 2011 Wiley Periodicals, Inc.
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toward an aggregated understanding of enzymatic hydrolysis of Cellulose noncomplexed cellulase systems
Biotechnology and Bioengineering, 2004Co-Authors: Yiheng Percival Zhang, Lee R LyndAbstract:Information pertaining to enzymatic hydrolysis of Cellulose by noncomplexed cellulase enzyme systems is reviewed with a particular emphasis on development of aggregated understanding incorporating substrate features in addition to concentration and multiple cellulase compo- nents. Topics considered include properties of Cellulose, adsorption, Cellulose hydrolysis, and quantitative models. A classification scheme is proposed for quantitative models for enzymatic hydrolysis of Cellulose based on the number of solubilizing activities and substrate state variables in- cluded. We suggest that it is timely to revisit and reinvig- orate functional modeling of Cellulose hydrolysis, and that this would be highly beneficial if not necessary in order to bring to bear the large volume of information available on cellulase components on the primary applications that
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toward an aggregated understanding of enzymatic hydrolysis of Cellulose noncomplexed cellulase systems
Biotechnology and Bioengineering, 2004Co-Authors: Yiheng Percival Zhang, Lee R LyndAbstract:Information pertaining to enzymatic hydrolysis of Cellulose by noncomplexed cellulase enzyme systems is reviewed with a particular emphasis on development of aggregated understanding incorporating substrate features in addition to concentration and multiple cellulase components. Topics considered include properties of Cellulose, adsorption, Cellulose hydrolysis, and quantitative models. A classification scheme is proposed for quantitative models for enzymatic hydrolysis of Cellulose based on the number of solubilizing activities and substrate state variables included. We suggest that it is timely to revisit and reinvigorate functional modeling of Cellulose hydrolysis, and that this would be highly beneficial if not necessary in order to bring to bear the large volume of information available on cellulase components on the primary applications that motivate interest in the subject.
Ingeol Choi - One of the best experts on this subject based on the ideXlab platform.
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optimization of synergism of a recombinant auxiliary activity 9 from chaetomium globosum with cellulase in Cellulose hydrolysis
Applied Microbiology and Biotechnology, 2015Co-Authors: In Jung Kim, Ingeol Choi, Ki Hyun Nam, Eun Ju Yun, Sooah Kim, Hak Jin Youn, Hee Jin Lee, Kyoung Heon KimAbstract:Auxiliary activity family 9 (AA9, formerly known as glycoside hydrolase family 61 or polysaccharide monooxygenase) is a group of fungal proteins that were recently found to have a significant synergism with cellulase in Cellulose hydrolysis via the oxidative cleavage of glycosidic bonds of Cellulose chains. In this study, we report the active expression of a recombinant fungal AA9 from Chaetomium globosum (CgAA9) in a bacterial host, Escherichia coli, and the optimization of its synergistic activity in Cellulose hydrolysis by using cellulase. The recombinant CgAA9 (0.9 mg/g Cellulose) exhibited 1.7-fold synergism in the hydrolysis of Avicel when incubated with 0.9 filter paper units of Celluclast 1.5 L/g Cellulose. The first study of the active expression of AA9 using a bacterial host and its synergistic optimization could be useful for the industrial application of AA9 for the saccharification of lignoCellulose.
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functional characterization of a bacterial expansin from bacillus subtilis for enhanced enzymatic hydrolysis of Cellulose
Biotechnology and Bioengineering, 2009Co-Authors: Won Gi Bang, Ingeol ChoiAbstract:: Expansin is a plant protein family that induces plant cell wall-loosening and Cellulose disruption without exerting Cellulose-hydrolytic activity. Expansin-like proteins have also been found in other eukaryotes such as nematodes and fungi. While searching for an expansin produced by bacteria, we found that the BsEXLX1 protein from Bacillus subtilis had a structure that was similar to that of a beta-expansin produced by maize. Therefore, we cloned the BsEXLX1 gene and expressed it in Escherichia coli to evaluate its function. When incubated with filter paper as a Cellulose substrate, the recombinant protein exhibited both Cellulose-binding and Cellulose-weakening activities, which are known functions of plant expansins. In addition, evaluation of the enzymatic hydrolysis of filter paper revealed that the recombinant protein also displayed a significant synergism when mixed with cellulase. By comparing the activity of a mixture of cellulase and the bacterial expansin to the additive activity of the individual proteins, the synergistic activity was found to be as high as 240% when filter paper was incubated with cellulase and BsEXLX1, which was 5.7-fold greater than the activity of cellulase alone. However, this synergistic effect was observed when only a low dosage of cellulase was used. This is the first study to characterize the function of an expansin produced by a non-eukaryotic source.
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functional characterization of a bacterial expansin from bacillus subtilis for enhanced enzymatic hydrolysis of Cellulose
Biotechnology and Bioengineering, 2009Co-Authors: Won Gi Bang, Ingeol ChoiAbstract:Expansin is a plant protein family that induces plant cell wall-loosening and Cellulose disruption without exerting Cellulose-hydrolytic activity. Expansin-like proteins have also been found in other eukaryotes such as nematodes and fungi. While searching for an expansin produced by bacteria, we found that the BsEXLX1 protein from Bacillus subtilis had a structure that was similar to that of a β-expansin produced by maize. Therefore, we cloned the BsEXLXl gene and expressed it in Escherichia coli to evaluate its function. When incubated with filter paper as a Cellulose substrate, the recombinant protein exhibited both Cellulose-binding and Cellulose-weakening activities, which are known functions of plant expansins. In addition, evaluation of the enzymatic hydrolysis of filter paper revealed that the recombinant protein also displayed a significant synergism when mixed with cellulase. By comparing the activity of a mixture of cellulase and the bacterial expansin to the additive activity of the individual proteins, the synergistic activity was found to be as high as 240% when filter paper was incubated with cellulase and BsEXLX1, which was 5.7-fold greater than the activity of cellulase alone. However, this synergistic effect was observed when only a low dosage of cellulase was used. This is the first study to characterize the function of an expansin produced by a non-eukaryotic source.
