136 results on '"Saddler JN"'
Search Results
2. Cellulolytic enzyme system of Acetivibrio cellulolyticus
- Author
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Saddler Jn and Khan Aw
- Subjects
Cellobiose ,Glycoside Hydrolases ,Immunology ,Polyacrylamide ,Cellulase ,Biology ,Applied Microbiology and Biotechnology ,Microbiology ,Substrate Specificity ,chemistry.chemical_compound ,Enzyme system ,Genetics ,medicine ,Cellulose 1,4-beta-Cellobiosidase ,Cellulose ,Molecular Biology ,Polyacrylamide gel electrophoresis ,chemistry.chemical_classification ,Gram-Negative Anaerobic Bacteria ,Molecular mass ,beta-Glucosidase ,Acetivibrio cellulolyticus ,General Medicine ,Carboxymethyl cellulose ,Molecular Weight ,Enzyme ,chemistry ,Biochemistry ,biology.protein ,Electrophoresis, Polyacrylamide Gel ,Glucosidases ,medicine.drug - Abstract
Polyacrylamide gel electrophoresis of the cellulolytic system from culture supernates of Acetivibrio cellulolyticus showed the presence of four major enzymes: a β-glucosidase, an exoglucanase, and two endoglucanases. The relative proportions of these enzymes in the culture supernate were affected by the nature of the cellulosic substrate and by the length of the incubation period. The molecular weights of the cellulolytic enzymes were β-glucosidase, 81 000; exoglucanase, 38 000; endoglucanase C2, 33 000; and endoglucanase C3, 10 400, as estimated by their electrophoretic mobilities relative to proteins of known molecular weight. Treatment of the high molecular weight endoglucanase with SDS–mercaptoethanol led to reversible dissociation of the enzyme into polypeptide subunits similar to the low molecular weight endoglucanase. Endoglucanase activity could be assayed for directly using a novel method of incorporating carboxymethyl cellulose in the polyacrylamide gels. The molecular weights and functions of these enzymes are compared with those detected in culture filtrates of various fungi.
- Published
- 1981
3. Size-controlled synthesis of xylan micro / nanoparticles by self-assembly of alkali-extracted xylan.
- Author
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Zhang H, Johnson AM, Hua Q, Wu J, Liang Y, Karaaslan MA, Saddler JN, and Renneckar S
- Abstract
Valorization of underutilized biobased feedstocks like hetero-polysaccharides is critical for the development of the biorefinery concept. Towards this goal, highly uniform xylan micro/nanoparticles with a particle size ranging from 400 nm to 2.5 μm in diameter were synthesized by a facile self-assembly method in aqueous solutions. Initial concentration of the insoluble xylan suspension was utilized to control the particle size. The method utilized supersaturated aqueous suspensions formed at standard autoclaving conditions without any other chemical treatments to create the resulting particles as solutions cooled to room temperature. Processing parameters of the xylan micro/nanoparticles were systematically studied and correlated with both the morphology and size of xylan particles. By adjusting the crowding of the supersaturated solutions, highly uniform dispersions of xylan particles were synthesized of defined size. The xylan micro/nanoparticles prepared by self-assembly have a quasi-hexagonal shape, like a tile, and depending upon solution concentrations xylan nanoparticles with a thickness of <100 nm were achieved at high concentrations. Based on the usefulness of polysaccharide nanoparticles, like cellulose nanocrystals, these particles have potential for unique structures for hydrogels, aerogels, drug delivery, and photonic materials. This study highlights the formation of a diffraction grating film for visible light with these size-controlled particles., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Scott Renneckar reports financial support was provided by Canada Research Chairs. Huaiyu Zhang reports financial support was provided by China Scholarship Council. Scott Renneckar reports financial support was provided by Natural Sciences and Engineering Research Council of Canada. Scott Renneckar reports financial support was provided by Edwina and Paul Heller Memorial Fund., (Copyright © 2023 Elsevier Ltd. All rights reserved.)
- Published
- 2023
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4. The production of lactic acid from chemi-thermomechanical pulps using a chemo-catalytic approach.
- Author
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Wu J, Kim KH, Jeong K, Kim D, Kim CS, Ha JM, Chandra RP, and Saddler JN
- Subjects
- Biomass, Carbohydrates, Catalysis, Cellulose, Lactic Acid
- Abstract
Previous work has shown that sulfonation and oxidation of chemi-thermomechanical pulps (CTMPs) significantly enhanced enzyme accessibility to cellulose while recovering the majority of carbohydrates in the water-insoluble component. In the work reported here, modified (sulfonated and oxidized) CTMPs derived from hard-and-softwoods were used to produce a DL-mix of lactic acid via a chemo-catalytic approach using lanthanide triflate (Ln (OTf)
3 ) catalysts (Ln = La, Nd, Er, and Yb). It was apparent that sulfonation and oxidation of chemi-thermomechanical pulps (CTMPs) also enhanced Ln(OTf)3 catalyst accessibility to the carbohydrate components of the pulps, with the Er(OTf)3 catalysts resulting in significant lactic acid production. Under optimum conditions (250 °C, 60 min, 0.5 mmol catalyst g-1 biomass), 72% and 67% of the respective total carbohydrate present in the hard-and-softwood CTMPs could be converted to lactic acid compared to the respective 59% and 51% yields obtained after energy-intensive ball milling., (Copyright © 2021 Elsevier Ltd. All rights reserved.)- Published
- 2021
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5. Non-productive celluase binding onto deep eutectic solvent (DES) extracted lignin from willow and corn stover with inhibitory effects on enzymatic hydrolysis of cellulose.
- Author
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Song Y, Chandra RP, Zhang X, and Saddler JN
- Subjects
- Enzyme Inhibitors, Hydrolysis, Hydrophobic and Hydrophilic Interactions, Lignin chemistry, Lignin isolation & purification, Cellulases antagonists & inhibitors, Cellulose metabolism, Lignin pharmacology, Salix chemistry, Solvents chemistry, Zea mays chemistry
- Abstract
In this work, deep eutectic solvent (DES) was prepared by mixing choline chloride (ChCl) with lactic acid (LA), and effects of cellulase non-productive binding onto DES-extracted lignin from willow and corn stover on enzymatic hydrolysis of cellulose was investigated. The correlation between hydrolysis yield of cellulose and chemical features of lignin was evaluated, and a potential inhibitory mechanism was proposed. Condensation of lignin was observed during DES treatment, and these condensed aromatic structures had an increased tendency to adsorb enzymes through hydrophobic interactions. As well as hydrophobic interactions mediated by lignin condensation, an increase in phenolic hydroxyl groups resulted in a greater amount of hydrogen bonds between cellulases and lignin that appeared to inhibit enzymatic hydrolysis yields of cellulose (39.96-42.86 % to 31.96-32.68 %). Although large amounts of COOHs were generated, the elevated electrostatic repulsion as a result of ionic groups was insufficient to decrease non-productive adsorption., (Copyright © 2020 Elsevier Ltd. All rights reserved.)
- Published
- 2020
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6. Enhancing Enzyme-Mediated Cellulose Hydrolysis by Incorporating Acid Groups Onto the Lignin During Biomass Pretreatment.
- Author
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Wu J, Chandra RP, Takada M, Liu LY, Renneckar S, Kim KH, Kim CS, and Saddler JN
- Abstract
Lignin is known to limit the enzyme-mediated hydrolysis of biomass by both restricting substrate swelling and binding to the enzymes. Pretreated mechanical pulp (MP) made from Aspen wood chips was incubated with either 16% sodium sulfite or 32% sodium percarbonate to incorporate similar amounts of sulfonic and carboxylic acid groups onto the lignin (60 mmol/kg substrate) present in the pulp without resulting in significant delignification. When Simon's stain was used to assess potential enzyme accessibility to the cellulose, it was apparent that both post-treatments enhanced accessibility and cellulose hydrolysis. To further elucidate how acid group addition might influence potential enzyme binding to lignin, Protease Treated Lignin (PTL) was isolated from the original and modified mechanical pulps and added to a cellulose rich, delignified Kraft pulp. As anticipated, the PTLs from both the oxidized and sulfonated substrates proved less inhibitory and adsorbed less enzymes than did the PTL derived from the original pulp. Subsequent analyses indicated that both the sulfonated and oxidized lignin samples contained less phenolic hydroxyl groups, resulting in enhanced hydrophilicity and a more negative charge which decreased the non-productive binding of the cellulase enzymes to the lignin., (Copyright © 2020 Wu, Chandra, Takada, Liu, Renneckar, Kim, Kim and Saddler.)
- Published
- 2020
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7. Acidic deep eutectic solvent assisted isolation of lignin containing nanocellulose from thermomechanical pulp.
- Author
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Jiang J, Carrillo-Enríquez NC, Oguzlu H, Han X, Bi R, Saddler JN, Sun RC, and Jiang F
- Subjects
- Chemical Fractionation, Choline chemistry, Hot Temperature, Hydrolysis, Lignin isolation & purification, Lignin chemistry, Nanoparticles chemistry, Oxalic Acid chemistry, Solvents chemistry, Wood chemistry
- Abstract
Nanocellulose is a promising material but its isolation generally requires unrecyclable hazardous chemicals and high energy consumption and its overall yield is low due to the use of high purity cellulose as precursor. In order to overcome these shortcomings, in this study, thermomechanical pulp (TMP) was investigated as a precursor for isolating lignin containing nanocellulose (LNC) using an environmentally friendly acidic deep eutectic solvent (DES) pre-treatment. Flat "ribbon" like LNCs (around 7.1 nm wide, 3.7 nm thick) with uniformly distributed lignin nanoparticles of 20-50 nm in diameter were successfully obtained at 57 % yield under optimum pre-treatment conditions (90 °C, 6 h, 1:1 oxalic acid dihydrate to choline chloride ratio). The LNCs exhibit cellulose Iβ structure, high lignin content (32.6 %), and high thermal stability (T
max of 358 °C). In general, green acidic DES pre-treatment has shown high efficiency in converting high lignin content biomass into value-added LNC, which benefits both lignocellulose utilization and environmental protection., (Copyright © 2020 Elsevier Ltd. All rights reserved.)- Published
- 2020
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8. Enzyme-Mediated Lignocellulose Liquefaction Is Highly Substrate-Specific and Influenced by the Substrate Concentration or Rheological Regime.
- Author
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van der Zwan T, Sigg A, Hu J, Chandra RP, and Saddler JN
- Abstract
The high viscosities/yield stresses of lignocellulose slurries makes their industrial processing a significant challenge. However, little is known regarding the degree to which liquefaction and its enzymatic requirements are specific to a substrate's physicochemical and rheological properties. In the work reported here, the substrate- and rheological regime-specificities of liquefaction of various substrates were assessed using real-time in-rheometer viscometry and offline oscillatory rheometry when hydrolyzed by combinations of cellobiohydrolase ( Trichoderma reesei Cel7A), endoglucanase ( Humicola insolens Cel45A), glycoside hydrolase (GH) family 10 xylanase, and GH family 11 xylanase. In contrast to previous work that has suggested that endoglucanase activity dominates enzymatic liquefaction, all of the enzymes were shown to have at least some liquefaction capacity depending on the substrate and reaction conditions. The contribution of individual enzymes was found to be influenced by the rheological regime; in the concentrated regime, the cellobiohydrolase outperformed the endoglucanase, achieving 2.4-fold higher yield stress reduction over the same timeframe, whereas the endoglucanase performed best in the semi-dilute regime. It was apparent that the significant differences in rheology and liquefaction mechanisms made it difficult to predict the liquefaction capacity of an enzyme or enzyme cocktail at different substrate concentrations., (Copyright © 2020 van der Zwan, Sigg, Hu, Chandra and Saddler.)
- Published
- 2020
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9. The use of fluorescent protein-tagged carbohydrate-binding modules to evaluate the influence of drying on cellulose accessibility and enzymatic hydrolysis.
- Author
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Mboowa D, Khatri V, and Saddler JN
- Abstract
The influence of drying on cellulose accessibility and enzymatic hydrolysis was assessed. Dissolving pulp was differentially dried by freeze-, air- and oven-drying at 50 °C and subsequently hydrolyzed using the commercial CTec 3 cellulase preparation. It was apparent that drying reduced the ease of enzymatic hydrolysis of all of the substrates with a pronounced reduction (48%) exhibited by the oven-dried pulp. To assess if the ease of hydrolysis was due to enzyme accessibility to the substrate, microscopy (SEM), FTIR spectroscopy, water retention value (WRV), fiber aspect ratio analysis, Simons' stain and the selective binding of Fluorescent Protein-tagged Carbohydrate Binding Modules (FP-CBMs): CBM3a (crystalline cellulose) and CBM17 (amorphous cellulose) in combination with confocal laser scanning microscopy (CLSM) were used. The combined methods indicated that, if the gross characteristics of the substrate limited enzyme accessibility, the cellulases, as represented by the FP-CBMs, could not in turn access the finer structural components of the cellulosic substrates., Competing Interests: There are no financial conflicts of interest to declare., (This journal is © The Royal Society of Chemistry.)
- Published
- 2020
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10. Elucidation of Changes in Cellulose Ultrastructure and Accessibility in Hardwood Fractionation Processes with Carbohydrate Binding Modules.
- Author
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Novy V, Nielsen F, Olsson J, Aïssa K, Saddler JN, Wallberg O, and Galbe M
- Abstract
We have recently presented a sequential treatment method, in which steam explosion (STEX) was followed by hydrotropic extraction (HEX), to selectively fractionate cellulose, hemicellulose, and lignin in hardwood into separate process streams. However, above a treatment severity threshold, the structural alterations in the cellulose-enriched fraction appeared to restrict the enzymatic hydrolyzability and delignification efficiency. To better understand the ultrastructural changes in the cellulose, hardwood chips were treated by single (STEX or HEX) and combined treatments (STEX and HEX), and the cellulose accessibility quantified with carbohydrate-binding modules (CBMs) that bind preferentially to crystalline (CBM2a) and paracrystalline cellulose (CBM17). Fluorescent-tagged versions of the CBMs were used to map the spatial distribution of cellulose substructures with confocal laser scanning microscopy. With increasing severities, STEX increased the apparent crystallinity (CBM2a/CBM17-ratio) and overall accessibility (CBM2aH6 + CBM17) of the cellulose, whereas HEX demonstrated the opposite trend. The respective effects could also be discerned in the combined treatments where increasing severities further resulted in higher hemicellulose dissolution and, although initially beneficial, in stagnating accessibility and hydrolyzability. This study suggests that balancing the severities in the two treatments is required to maximize the fractionation and simultaneously achieve a reactive and accessible cellulose that is readily hydrolyzable., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)
- Published
- 2020
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11. Potential of Xylanases to Reduce the Viscosity of Micro/Nanofibrillated Bleached Kraft Pulp.
- Author
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Tian D, Zhong N, Leung J, Shen F, Hu J, and Saddler JN
- Abstract
The generally high viscosity of micro/nanofibrillated cellulose limits its applications in cream and fluid products. A bleached softwood Kraft (BSK) pulp was refined with increasing energy (500-2500 kWh t
-1 ) to produce micro/nanofibrillated cellulose (MNBSK). Subsequent xylanase treatment was shown to influence the viscosity, gel point, aspect ratio, and fiber surface morphology of the MNBSK. It was apparent that the accessibility to xylanases was increased even at low refining energies (500 kWh t-1 ). Depending on the initial degree of cellulose fibrillation, xylanase treatment decreased the viscosity of the MNBSK from 4190-2030 to 681-243 Pa·s at a shear rate of 0.01 s-1 , corresponding to the reduction in the aspect ratio from 183-296 to 163-194. It was likely that the xylanases were predominantly acting on the xylan present on the fiber surfaces, reducing the cross-linking points on the cellulose fibers and consequently resulting in the reduction in MNBSK viscosity.- Published
- 2020
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12. The influence of lignin on the effectiveness of using a chemithermomechanical pulping based process to pretreat softwood chips and pellets prior to enzymatic hydrolysis.
- Author
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Takada M, Chandra R, Wu J, and Saddler JN
- Subjects
- Biomass, Cellulose, Hydrolysis, Lignin, Wood
- Abstract
Over the last century the pulp and paper sector has assessed various technologies to fractionate woody biomass to produce strong, bright fibers. Several of these processes have also been assessed for their potential to pretreat and fractionate biomass to enhance the subsequent enzymatic hydrolysis of the cellulosic component. Although many of these pretreatments are effective on agricultural residues, softwoods have proven more recalcitrant, primarily due to their high lignin content and structure. As delignification is too expensive to be used routinely a more economically attractive approach might be to alter the lignin. Recent work has shown that, using a modified chemithermomechanical pulping (CTMP) "front end", lignin can be modified and relocated. This significantly enhanced hemicellulose recovery and enzyme-mediated cellulose hydrolysis of woody biomass. As well as being effective on wood chips, the modified CTMP pretreatment process also enhanced the bioconversion of densified feedstocks such as pellets., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)
- Published
- 2020
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13. Laccase-mediated hydrophilization of lignin decreases unproductive enzyme binding but limits subsequent enzymatic hydrolysis at high substrate concentrations.
- Author
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van der Zwan T, Chandra RP, and Saddler JN
- Subjects
- Biomass, Hydrolysis, Steam, Laccase, Lignin
- Abstract
One of the predominant mechanisms by which lignin restricts effective enzymatic deconstruction of lignocellulosic materials is the unproductive adsorption of enzymes. Although this inhibition can be partially mitigated through hydrophilization of lignin during thermochemical pretreatment, these types of treatments could potentially worsen slurry rheology, consequently making it more difficult to process the material at high substrate concentrations. In the work reported here, laccases were used to specifically modify lignin hydrophilicity within steam-pretreated substrate via in situ phenolic compound grafting. While lignin hydrophilization reduced unproductive enzyme adsorption, high-solids hydrolysis efficiency decreased significantly due to mass transfer limitations. It was apparent that low-solids hydrolysis experiments were a poor predictor of substrate digestibility at high-solids conditions and that substrate-water interactions impacted both substrate digestibility and slurry rheology., (Copyright © 2019 Elsevier Ltd. All rights reserved.)
- Published
- 2019
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14. The influence of lignin migration and relocation during steam pretreatment on the enzymatic hydrolysis of softwood and corn stover biomass substrates.
- Author
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Takada M, Chandra RP, and Saddler JN
- Subjects
- Hydrolysis, Biomass, Cellulases chemistry, Lignin chemistry, Steam, Zea mays chemistry
- Abstract
To be effective, steam pretreatment is typically carried out at temperatures/pressures above the glass transition point (Tg) of biomass lignin so that it can partly fluidize and relocate. The relocation of Douglas-fir and corn stover derived lignin was compared with the expectation that, with the corn stover lignin's lower hydrophobicity and molecular weight, it would be more readily fluidized. It was apparent that the Tg of lignin decreased as the moisture increased, with the easier access of steam to the corn stover lignin promoting its plasticization. Although the softwood lignin was more recalcitrant, when it was incorporated onto filter paper, it too could be plasticized, with its relocation enhancing enzymatic hydrolysis. When lignin recondensation was minimized, the increased hydrophobicity suppressed lignin relocation. It was apparent that differences in the accessibility of the lignin present in Douglas-fir and corn stover to steam significantly impacted lignin fluidization, relocation, and subsequent cellulose hydrolysis., (© 2019 Wiley Periodicals, Inc.)
- Published
- 2019
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15. Functionalizing Cellulose Nanocrystals with Click Modifiable Carbohydrate-Binding Modules.
- Author
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Aïssa K, Karaaslan MA, Renneckar S, and Saddler JN
- Subjects
- Catalysis, Click Chemistry, Hydrogels chemistry, Polyethylene Glycols chemistry, Carbohydrates chemistry, Cellulose chemistry, Cellulose metabolism, Nanoparticles chemistry, Nanoparticles metabolism
- Abstract
Functionalized cellulose nanocrystals (CNC) have unique properties that make them attractive in various applications such as drug delivery, hydrogels, and emulsions. However, the predominant chemical methods currently used to functionalize cellulose nanocrystals have a large environmental footprint. Although greener methods are desirable, the relatively inert nature of cellulose crystals presents a major challenge to their potential modification in aqueous media. In the work reported here, carbohydrate binding modules (CBMs) were used to introduce new functionality to cellulose surfaces. CBM2a, which has a strong affinity for crystalline cellulose, was functionalized with an alkyne at the terminal amine position. The alkyne group, which was introduced onto the cellulose surface with CBM2a, underwent a Click reaction with polyethylene glycol (PEG) to modify CNC surfaces. This provided a strong, non-covalent modification of cellulose surfaces that was carried out in a one-pot reaction in aqueous media. The CBM-PEG modification of cellulose surfaces increased CNC redispersion after drying and improved suspension stability based on steric interactions. It was apparent that hybrid polysaccharide-protein, self-assembled nanoparticles could be effectively produced, with potential for nanomedicine, immunoassay, and drug delivery applications.
- Published
- 2019
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16. The Potential of Using Immobilized Xylanases to Enhance the Hydrolysis of Soluble, Biomass Derived Xylooligomers.
- Author
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Hu J, Davies J, Mok YK, Arato C, and Saddler JN
- Abstract
Earlier work had indicated that enzyme-mediated hydrolysis of xylooligomer-rich water-soluble streams (derived from steam pre-treated wheat straw) resulted in the effective production of xylose which was subsequently used to produce bio-glycol. In the work reported here, both the thermostability and recyclability of xylanases were significantly improved by covalent immobilizing the enzymes onto alginate beads. The immobilized xylanases showed a lower hydrolytic potential (~55% xylooligomer conversion) compared to the commercial xylanase cocktail HTec3 (~90% xylooligomer conversion) when used at the same protein loading concentration. This was likely due to the less efficient immobilization of key higher molecular weight enzymes (>75 kDa), such as β-xylosidases. However, enzyme immobilization could be improved by lowering the glutaraldehyde loading used to activate the alginate beads, resulting in improved hydrolysis efficacy (~65% xylooligomer conversion). Enzyme immobilization improved enzyme thermostability (endoxylanase and β-xylosidase activities were improved by 80% and 40%, respectively, after 24 h hydrolysis) and this allowed the immobilized enzymes to be reused/recycled for multiple rounds of hydrolysis (up to five times) without any significant reduction in their hydrolytic potential.
- Published
- 2018
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17. Enhancing bacterial cellulose production via adding mesoporous halloysite nanotubes in the culture medium.
- Author
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Tian D, Shen F, Hu J, Renneckar S, and Saddler JN
- Abstract
Although bacterial cellulose (BC) is a fascinating, highly pure cellulose material for various downstream applications, production has been challenged by its low productivity. This work reported a facile route to significantly enhance BC yield without compromising its structural advantages via adding mesoporous halloysite nanotubes (HNTs) in the culture medium at static cultivations. The BC productivity of Gluconacetobacter xylinus was increased from 2.2 to 5.9 g L
-1 after 15 days of cultivation when 2 wt% of HNTs was added into the standard fructose medium. It appeared that the dual functionality of cell immobilization and oxygen release of the HNTs were responsible for enhancing the BC productivity. Moreover, the HNTs-resulted BC pellicle exhibited negligible content of HNTs contamination (∼2 wt%), higher degree of crystallinity (87.7%) and porosity (assessed by water holding capacity, 12.7 g g-1 ), and showed promising applications especially in the bio-adsorption field., (Copyright © 2018 Elsevier Ltd. All rights reserved.)- Published
- 2018
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18. Minimizing cellulase inhibition of whole slurry biomass hydrolysis through the addition of carbocation scavengers during acid-catalyzed pretreatment.
- Author
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Zhai R, Hu J, and Saddler JN
- Subjects
- Cellulases, Cellulose, Hydrolysis, Biomass, Cellulase, Lignin
- Abstract
The aim of this work was to study how to minimize cellulase inhibition of whole slurry biomass hydrolysis through addition of carbocation scavengers during acid-catalyzed pretreatment. Various potential carbocation scavengers were compared and their inhibition mitigating effects towards the hydrolytic performance of cellulase enzymes was assessed. The results indicated that the addition of carbocation scavengers during the pretreatment process could not only alleviate the inhibitory effect of the phenolics on the enzymatic hydrolysis but also increase the accessibility of cellulases to the pretreated substrates. It appeared that lignin-derived compounds such as 4-hydroxybenzoic acid, vanillic acid, syringic acid could all serve as efficient scavengers to alleviate the inhibitory effect of phenolics on cellulose hydrolysis where the syringic acid showed the best mitigating effect. By combining the carbocation scavengers in the pretreatment process, an improved cellulose hydrolysis of the pretreated whole slurry could be achieved without any post detoxification step., (Copyright © 2018 Elsevier Ltd. All rights reserved.)
- Published
- 2018
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19. The inhibition of hemicellulosic sugars on cellulose hydrolysis are highly dependant on the cellulase productive binding, processivity, and substrate surface charges.
- Author
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Zhai R, Hu J, and Saddler JN
- Subjects
- Adsorption, Cellulose, Hydrolysis, Cellulase, Sugars
- Abstract
In this study, the influence of major hemicellulosic sugars (mannose and xylose) on cellulose hydrolysis and major enzyme activities were evaluated by using both commercial enzyme cocktail and purified cellulase monocomponents over a "library" of cellulosic substrates. Surprisingly, the results showed that unlike glucose, mannose/xylose did not inhibit individual cellulase activities but significantly decreased their hydrolytic performance on cellulose substrates. When various enzyme-substrate interactions (e.g. adsorption/desorption, productive binding, and processive moving) were evaluated, it appeared that these hemicellulosic sugars significantly reduced the productive binding and processivity of Cel7A, which in turn limited cellulase hydrolytic efficacy. Among a range of major cellulose characteristics (e.g. crystallinity, degree of polymerization, accessibility, and surface charges), the acid group content of the cellulosic substrates seemed to be the main driver that determined the extent of hemicellulosic sugar inhibition. Our results provided new insights for better understanding the sugar inhibition mechanisms of cellulose hydrolysis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)
- Published
- 2018
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20. Enzyme mediated nanofibrillation of cellulose by the synergistic actions of an endoglucanase, lytic polysaccharide monooxygenase (LPMO) and xylanase.
- Author
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Hu J, Tian D, Renneckar S, and Saddler JN
- Abstract
Physiochemical methods have generally been used to "open-up" biomass substrates/pulps and have been the main method used to fibrillate cellulose. However, recent work has shown that canonical cellulase enzymes such as endoglucanases, in combination with "amorphogenesis inducing" proteins such as lytic polysaccharide monooxygenases (LPMO), swollenin and hemicellulases, are able to increase cellulose accessibility. In the work reported here different combinations of endoglucanase, LPMO and xylanase were applied to Kraft pulps to assess their potential to induce fibrillation at low enzyme loading over a short time period. Although gross fiber properties (fiber length, width and morphology) were relatively unchanged, over a short period of time, the intrinsic physicochemical characteristics of the pulp fibers (e.g. cellulose accessibility/DP/crystallinity/charge) were positively enhanced by the synergistic cooperation of the enzymes. LPMO addition resulted in the oxidative cleavage of the pulps, increasing the negative charge (~100 mmol kg
-1 ) on the cellulose fibers. This improved cellulose nanofibrilliation while stabilizing the nanofibril suspension (zeta potential ζ = ~60 mV), without sacrificing nanocellulose thermostability. The combination of endoglucanase, LPMO and xylanases was shown to facilitate nanofibrillation, potentially reducing the need for mechanical refining while resulting in a pulp with a more uniform nanofibril composition.- Published
- 2018
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21. The potential of tailoring the conditions of steam explosion to produce xylo-oligosaccharides from sugarcane bagasse.
- Author
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Carvalho AFA, Marcondes WF, de Oliva Neto P, Pastore GM, Saddler JN, and Arantes V
- Subjects
- Explosions, Hydrolysis, Oligosaccharides, Steam, Cellulose, Saccharum
- Abstract
In this study, the potential of the steam explosion (SE) method to produce high levels XOS from sugarcane bagasse, a xylan-rich hemicellulosic feedstock, was assessed. The effect of different operating conditions on XOS production yield and selectivity were investigated using a mini-pilot scale SE unit. The results show that even under a non-optimized condition (190 °C, 5 min and 0.5% H
2 SO4 as catalyst), SE led to about 40% xylan recovery as XOS, which was comparable to the well-known, multi-step, enzymatic production of XOS from alkaline-extracted xylan, and other commonly employed chemical methods. In addition, the XOS-rich hydrolysate from SE constituted of greater diversity in the degree of polymerization, which has been shown to be desirable for prebiotic application., (Copyright © 2017 Elsevier Ltd. All rights reserved.)- Published
- 2018
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22. Lignin Sulfonation and SO 2 Addition Enhance the Hydrolyzability of Deacetylated and Then Steam-Pretreated Poplar with Reduced Inhibitor Formation.
- Author
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Tang Y, Dou X, Hu J, Jiang J, and Saddler JN
- Subjects
- Acetylation, Carbonates chemistry, Hydrolysis, Sodium Hydroxide chemistry, Steam, Lignin chemistry, Populus, Sulfones chemistry, Sulfur Dioxide chemistry
- Abstract
The merit of deacetylation of corn stover prior to pretreatment is decreasing the formation of inhibitors and improving enzyme hydrolysis, proved in dilute acid pretreatment. However, few studies are done on how deacetylation would affect bioconversion process containing steam explosion. In this study, the effect of deacetylation on steam explosion was conducted using poplar as substrate. About 57 to 90% of acetyl group in poplar, depending on alkaline types and concentration, was removed by dilute alkaline deacetylation in 6 h. Deacetylation eliminated over 85% of inhibitor formation during downstream steam explosion. However, deacetylation prior to steam explosion decreased the dissolution of hemicellulose, thus reducing the cellulose accessibility of pretreated poplar, finally resulting in 5-20% decrease in glucose yield and 20-35% decrease in xylose yield. The addition of 5% SO
2 during steam explosion significantly improved the hydrolysis of deacetylated and pretreated poplar without significantly increasing the concentration of inhibitors. Incorporating 45 mmol/kg sulfoacid group in lignin fraction of deacetylated and then pretreated poplar dramatically improved the xylose yield to about 100% and increased the glucose yield by 30%.- Published
- 2018
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23. Formulation of an optimized synergistic enzyme cocktail, HoloMix, for effective degradation of various pre-treated hardwoods.
- Author
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Malgas S, Chandra R, Van Dyk JS, Saddler JN, and Pletschke BI
- Subjects
- Hydrolysis, Lignin, Steam, Biomass, Cellulase
- Abstract
In this study, two selected hardwoods were subjected to sodium chlorite delignification and steam explosion, and the impact of pre-treatments on synergistic enzymatic saccharification evaluated. A cellulolytic core-set, CelMix, and a xylanolytic core-set, XynMix, optimised for glucose and xylose release, respectively, were used to formulate HoloMix cocktail for optimal saccharification of various pre-treated hardwoods. For delignified biomass, the optimized HoloMix consisted of 75%:25% protein dosage, CelMix: XynMix, while for untreated and steam exploded biomass the HoloMix consisted of 93.75%:6.25% protein dosage. Saccharification by HoloMix (27.5mgprotein/gbiomass) for 24h achieved 70-100% sugar yields. Pre-treatment of the hardwoods (especially those with a higher proportion of lignin) with a laccase, improved saccharification by HoloMix. This study provided insights into enzymatic hydrolysis of various pre-treated hardwood substrates and showed the same lignocellulolytic cocktail comparable to/if not better than commercial enzyme preparations can be used to efficiently hydrolyse different hardwood species., (Copyright © 2017 Elsevier Ltd. All rights reserved.)
- Published
- 2017
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24. Mechanistic insights into the liquefaction stage of enzyme-mediated biomass deconstruction.
- Author
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van der Zwan T, Hu J, and Saddler JN
- Subjects
- Absorption, Physicochemical, Biomass, Enzyme Activation, Lipase chemistry, Substrate Specificity, Viscosity, Lignin chemistry, Models, Chemical, Populus chemistry, Solutions chemistry, Water chemistry
- Abstract
Effective enzyme-mediated viscosity reduction, disaggregation, or "liquefaction," is required to overcome the rheological challenges resulting from the fibrous, hygroscopic nature of lignocellulosic biomass, particularly at the high solids loadings that will be required for an economically viable process. However, the actual mechanisms involved in enzyme-mediated liquefaction, as determined by viscosity or yield stress reduction, have yet to be fully resolved. Particle fragmentation, interparticle interaction, material dilution, and water-retention capacity were compared for their ability to quantify enzyme-mediated liquefaction of model and more realistic pretreated biomass substrates. It was apparent that material dilution and particle fragmentation occurred simultaneously and that both mechanisms contributed to viscosity/yield stress reduction. However, their relative importance was dependent on the nature of the biomass substrate. Interparticle interaction and enzyme-mediated changes to these interactions was shown to have a significant effect on slurry rheology. Liquefaction was shown to result from the combined action of material dilution, particle fragmentation, and alteration of interactions at particle surfaces. However, the observed changes in water retention capacity did not correlate with yield stress reduction. The relative importance of each mechanism was significantly influenced by the nature of the biomass substrate and its physicochemical properties. An ongoing challenge is that mechanisms, such as refining, which enhance enzyme accessibility to the cellulosic component of the substrate, are detrimental to slurry rheology and will likely impede enzyme-mediated liquefaction when high substrate concentrations are used., (© 2017 Wiley Periodicals, Inc.)
- Published
- 2017
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25. Lignin valorization: lignin nanoparticles as high-value bio-additive for multifunctional nanocomposites.
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Tian D, Hu J, Bao J, Chandra RP, Saddler JN, and Lu C
- Abstract
Background: Although conversion of low value but high-volume lignin by-product to its usable form is one of the determinant factors for building an economically feasible integrated lignocellulose biorefinery, it has been challenged by its structural complexity and inhomogeneity. We and others have shown that uniform lignin nanoparticles can be produced from a wide range of technical lignins, despite the varied lignocellulosic biomass and the pretreatment methods/conditions applied. This value-added nanostructure lignin enriched with multifunctional groups can be a promising versatile material platform for various downstream utilizations especially in the emerging nanocomposite fields., Results: Inspired by the story of successful production and application of nanocellulose biopolymer, two types of uniform lignin nanoparticles (LNPs) were prepared through self-assembling of deep eutectic solvent (DES) and ethanol-organosolv extracted technical lignins derived from a two-stage fractionation pretreatment approach, respectively. Both LPNs exhibited sphere morphology with unique core-shell nanostructure, where the DES-LNPs showed a more uniform particle size distribution. When incorporated into the traditional polymeric matrix such as poly(vinyl alcohol), these LPN products displayed great potential to formulate a transparent nanocomposite film with additional UV-shielding efficacy (reached ~80% at 400 nm with 4 wt% of LNPs) and antioxidant functionalities (reached ~160 μm mol Trolox g
-1 with 4 wt% of LNPs). At the same time, the abundant phenolic hydroxyl groups on the shell of LNPs also provided good interfacial adhesion with PVA matrix through the formation of hydrogen bonding network, which further improved the mechanical and thermal performances of the fabricated LNPs/PVA nanocomposite films., Conclusions: Both LNPs are excellent candidates for producing multifunctional polymer nanocomposites using facile technical route. The prepared transparent and flexible LNPs/PVA composite films with high UV-shielding efficacy, antioxidant activity, and biocompatibility are promising in the advanced packaging field, which potentially provides an additional high-value lignin product stream to the lignocellulose biorefinery. This study could open the door for the production and application of novel LNPs in the nascent bioeconomy.Graphical abstractLignin nanoparticle for transparent nanocomposite film with UV-shielding efficacy.- Published
- 2017
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26. Limitation of cellulose accessibility and unproductive binding of cellulases by pretreated sugarcane bagasse lignin.
- Author
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Siqueira G, Arantes V, Saddler JN, Ferraz A, and Milagres AMF
- Abstract
Background: The effectiveness of the enzymatic hydrolysis of cellulose in plant cell wall is strongly influenced by the access of enzymes to cellulose, which is at least in part limited by the presence of lignin. Although physicochemical treatments preceding the enzymatic catalysis significantly overcome this recalcitrance, the residual lignin can still play a role in the process. Lignin is suggested to act as a barrier, hindering cellulose and limiting the access of the enzymes. It can also unspecifically bind cellulases, reducing the amount of enzymes available to act on cellulose. However, the limiting role of the lignin present in pretreated sugarcane bagasses has not been fully understood yet., Results: A set of sugarcane bagasses pretreated by five leading pretreatment technologies was created and used to assess their accessibility and the unproductive binding capacity of the resulting lignins. Steam explosion and alkaline sulfite pretreatments resulted in more accessible substrates, with approximately 90% of the cellulose hydrolyzed using high enzyme loadings. Enzymatic hydrolysis of alkaline-treated (NaOH) and steam-exploded sugarcane bagasses were strongly affected by unproductive binding at the lowest enzyme loading tested. Analysis of the extracted lignins confirmed the superior binding capacity of these lignins. Sulfite-based pretreatments (alkaline sulfite and acid sulfite) resulted in lignins with lower binding capacities compared to the analogue pretreatments without sulfite (alkaline and acidic). Strong acid groups present in sulfite-based pretreated substrates, attributed to sulfonated lignins, corroborated the lower binding capacities of the lignin present in these substrates. A more advanced enzyme preparation (Cellic CTec3) was shown to be less affected by unproductive binding at low enzyme loading., Conclusions: Pretreatments that increase the accessibility and modify the lignin are necessary in order to decrease the protein binding capacity. The search for the called weak lignin-binding enzymes is of major importance if hydrolysis with low enzyme loadings is the goal for economically viable processes.
- Published
- 2017
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27. A comparison of various lignin-extraction methods to enhance the accessibility and ease of enzymatic hydrolysis of the cellulosic component of steam-pretreated poplar.
- Author
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Tian D, Chandra RP, Lee JS, Lu C, and Saddler JN
- Abstract
Background: Current single-stage delignification-pretreatment technologies to overcome lignocellulosic biomass recalcitrance are usually achieved at the expense of compromising the recovery of the polysaccharide components, particularly the hemicellulose fraction. One way to enhance overall sugar recovery is to tailor an efficient two-stage pretreatment that can pre-extract the more labile hemicellulose component before subjecting the cellulose-rich residual material to a second-stage delignification process. Previous work had shown that a mild steam pretreatment could recover >65% of the hemicellulose from poplar while limiting the acid-catalysed condensation of lignin. This potentially allowed for subsequent lignin extraction using various lignin solvents to produce a more accessible cellulosic substrate., Results: A two-stage approach using steam and/or solvent pretreatment was assessed for its ability to separate hemicellulose and lignin from poplar wood chips while providing a cellulose-rich fraction that could be readily hydrolysed by cellulase enzymes. An initial steam-pretreatment stage was performed over a range of temperatures (160-200 °C) using an equivalent severity factor of 3.6. A higher steam temperature of 190 °C applied over a shorter residence time of 10 min effectively solubilized and recovered 75% of the hemicellulose while enhancing the ability of various solvents [deep eutectic solvent (DES), ethanol organosolv, soda/anthraquinone (soda/AQ) or a hydrotrope] to extract lignin in a second stage. When the second-stage treatments were compared, the mild DES treatment (lactic acid and betaine) at 130 °C, removed comparable amounts of lignin with higher selectivity than did the soda/AQ and organosolv pretreatments at 170 °C. However, the cellulose-rich substrates obtained after the second-stage organosolv and soda/AQ pretreatments showed the highest cellulose accessibility, as measured by the Simon's staining technique. They were also the most susceptible to subsequent enzymatic hydrolysis., Conclusions: The second-stage pretreatments varied in their ability to solubilize and extract the lignin component of steam-pretreated poplar while enhancing the enzymatic hydrolysis of the resulting cellulose-rich residual fractions. Although DES extraction was more selective in extracting lignin from the steam-pretreated substrates, the organosolv and soda/AQ post treatments disrupted the cellulose structure to a greater extent while enhancing the ease of enzymatic hydrolysis. Graphical abstractEffective hemicellulose removal via steam pretreatment followed by subsequent lignin extraction under acidic, alkaline or solvolytic conditions results in a highly accessible, more readily hydrolysed cellulose fraction.
- Published
- 2017
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28. Enhanced delignification of steam-pretreated poplar by a bacterial laccase.
- Author
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Singh R, Hu J, Regner MR, Round JW, Ralph J, Saddler JN, and Eltis LD
- Subjects
- Actinobacteria chemistry, Actinobacteria enzymology, Bacterial Proteins isolation & purification, Biocatalysis, Biofuels supply & distribution, Biomass, Humans, Kinetics, Laccase isolation & purification, Steam, Substrate Specificity, Bacterial Proteins chemistry, Cellulase chemistry, Laccase chemistry, Lignin chemistry, Populus chemistry
- Abstract
The recalcitrance of woody biomass, particularly its lignin component, hinders its sustainable transformation to fuels and biomaterials. Although the recent discovery of several bacterial ligninases promises the development of novel biocatalysts, these enzymes have largely been characterized using model substrates: direct evidence for their action on biomass is lacking. Herein, we report the delignification of woody biomass by a small laccase (sLac) from Amycolatopsis sp. 75iv3. Incubation of steam-pretreated poplar (SPP) with sLac enhanced the release of acid-precipitable polymeric lignin (APPL) by ~6-fold, and reduced the amount of acid-soluble lignin by ~15%. NMR spectrometry revealed that the APPL was significantly syringyl-enriched relative to the original material (~16:1 vs. ~3:1), and that sLac preferentially oxidized syringyl units and altered interunit linkage distributions. sLac's substrate preference among monoaryls was also consistent with this observation. In addition, sLac treatment reduced the molar mass of the APPL by over 50%, as determined by gel-permeation chromatography coupled with multi-angle light scattering. Finally, sLac acted synergistically with a commercial cellulase cocktail to increase glucose production from SPP ~8%. Overall, this study establishes the lignolytic activity of sLac on woody biomass and highlights the biocatalytic potential of bacterial enzymes., Competing Interests: The authors declare no competing financial interests.
- Published
- 2017
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29. Adaptive evolution and metabolic engineering of a cellobiose- and xylose- negative Corynebacterium glutamicum that co-utilizes cellobiose and xylose.
- Author
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Lee J, Saddler JN, Um Y, and Woo HM
- Subjects
- Metabolic Engineering methods, Cellobiose metabolism, Corynebacterium glutamicum metabolism, Xylose metabolism
- Abstract
Background: An efficient microbial cell factory requires a microorganism that can utilize a broad range of substrates to economically produce value-added chemicals and fuels. The industrially important bacterium Corynebacterium glutamicum has been studied to broaden substrate utilizations for lignocellulose-derived sugars. However, C. glutamicum ATCC 13032 is incapable of PTS-dependent utilization of cellobiose because it has missing genes annotated to β-glucosidases (bG) and cellobiose-specific PTS permease., Results: We have engineered and evolved a cellobiose-negative and xylose-negative C. glutamicum that utilizes cellobiose as sole carbon and co-ferments cellobiose and xylose. NGS-genomic and DNA microarray-transcriptomic analysis revealed the multiple genetic mutations for the evolved cellobiose-utilizing strains. As a result, a consortium of mutated transporters and metabolic and auxiliary proteins was responsible for the efficient cellobiose uptake. Evolved and engineered strains expressing an intracellular bG showed a better rate of growth rate on cellobiose as sole carbon source than did other bG-secreting or bG-displaying C. glutamicum strains under aerobic culture. Our strain was also capable of co-fermenting cellobiose and xylose without a biphasic growth, although additional pentose transporter expression did not enhance the xylose uptake rate. We subsequently assessed the strains for simultaneous saccharification and fermentation of cellulosic substrates derived from Canadian Ponderosa Pine., Conclusions: The combinatorial strategies of metabolic engineering and adaptive evolution enabled to construct C. glutamicum strains that were able to co-ferment cellobiose and xylose. This work could be useful in development of recombinant C. glutamicum strains for efficient lignocellulosic-biomass conversion to produce value-added chemicals and fuels.
- Published
- 2016
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30. Accessory enzymes influence cellulase hydrolysis of the model substrate and the realistic lignocellulosic biomass.
- Author
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Sun FF, Hong J, Hu J, Saddler JN, Fang X, Zhang Z, and Shen S
- Subjects
- Biomass, Biotechnology, Cellulase isolation & purification, Endo-1,4-beta Xylanases isolation & purification, Endo-1,4-beta Xylanases metabolism, Hydrolysis, Kinetics, Models, Biological, Paper, Substrate Specificity, beta-Glucosidase isolation & purification, beta-Glucosidase metabolism, Cellulase metabolism, Lignin metabolism
- Abstract
The potential of cellulase enzymes in the developing and ongoing "biorefinery" industry has provided a great motivation to develop an efficient cellulase mixture. Recent work has shown how important the role that the so-called accessory enzymes can play in an effective enzymatic hydrolysis. In this study, three newest Novozymes Cellic CTec cellulase preparations (CTec 1/2/3) were compared to hydrolyze steam pretreated lignocellulosic substrates and model substances at an identical FPA loading. These cellulase preparations were found to display significantly different hydrolytic performances irrelevant with the FPA. And this difference was even observed on the filter paper itself when the FPA based assay was revisited. The analysis of specific enzyme activity in cellulase preparations demonstrated that different accessory enzymes were mainly responsible for the discrepancy of enzymatic hydrolysis between diversified substrates and various cellulases. Such the active role of accessory enzymes present in cellulase preparations was finally verified by supplementation with β-glucosidase, xylanase and lytic polysaccharide monooxygenases AA9. This paper provides new insights into the role of accessory enzymes, which can further provide a useful reference for the rational customization of cellulase cocktails in order to realize an efficient conversion of natural lignocellulosic substrates., (Copyright © 2015 Elsevier Inc. All rights reserved.)
- Published
- 2015
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31. A NaBH₄ Coupled Ninhydrin-Based Assay for the Quantification of Protein/Enzymes During the Enzymatic Hydrolysis of Pretreated Lignocellulosic Biomass.
- Author
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Mok YK, Arantes V, and Saddler JN
- Subjects
- Hot Temperature, Biomass, Borohydrides chemistry, Cellulases analysis, Lignin chemistry, Ninhydrin chemistry
- Abstract
Accurate protein quantification is necessary in many of the steps during the enzymatic hydrolysis of pretreated lignocellulosic biomass, from the fundamental determination of enzyme kinetics to techno-economic assessments, such as the use of enzyme recycling strategies, evaluation of enzyme costs, and the optimization of various process steps. In the work described here, a modified, more rapid ninhydrin-based protein quantification assay was developed to better quantify enzyme levels in the presence of lignocellulosic biomass derived compounds. The addition of sodium borohydride followed by acid hydrolysis at 130 °C greatly reduced interference from monosaccharides and oligosaccharides and decreased the assay time 6-fold. The modified ninhydrin assay was shown to be more accurate as compared to various traditional colorimetric protein assays when commercial cellulase enzyme mixtures were quantified under typical pretreated lignocellulosic biomass enzymatic hydrolysis conditions. The relatively short assay time and microplate-reading capability of the modified assay indicated that the method could likely be used for high-throughput protein determination.
- Published
- 2015
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32. The addition of accessory enzymes enhances the hydrolytic performance of cellulase enzymes at high solid loadings.
- Author
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Hu J, Chandra R, Arantes V, Gourlay K, Susan van Dyk J, and Saddler JN
- Subjects
- Biomass, Glycoside Hydrolases metabolism, Hydrolysis, Proteins metabolism, Steam, Xylans metabolism, Zea mays metabolism, Cellulase metabolism
- Abstract
The pretreatment process used and the nature of the biomass feedstock will influence the role that accessory enzymes can play in synergistically interacting with cellulases to effectively deconstruct the substrate. The work reported here assessed the possible boosting effects of the xylanase and lytic polysaccharide monooxygenase (AA9, formerly known as GH61) on the hydrolytic potential of cellulase enzyme mixtures during hydrolysis of steam pretreated poplar and corn stover at high (10-20% w/v) substrate concentrations. A higher proportion of xylanase was required when the substrate had a relatively high xylan content and at high substrate concentrations. In contrast, a relatively small amount of AA9 (about 2 mg/g cellulose) was enough, regardless of the nature or concentration of the substrate. The overall protein loading required to achieve effective hydrolysis of high concentrations of pretreated biomass substrates could be substantially reduced by optimizing the ratio of enzymes in the "cellulase" mixture., (Copyright © 2015 Elsevier Ltd. All rights reserved.)
- Published
- 2015
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33. Horizontal gene transfer and gene dosage drives adaptation to wood colonization in a tree pathogen.
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Dhillon B, Feau N, Aerts AL, Beauseigle S, Bernier L, Copeland A, Foster A, Gill N, Henrissat B, Herath P, LaButti KM, Levasseur A, Lindquist EA, Majoor E, Ohm RA, Pangilinan JL, Pribowo A, Saddler JN, Sakalidis ML, de Vries RP, Grigoriev IV, Goodwin SB, Tanguay P, and Hamelin RC
- Subjects
- Ascomycota pathogenicity, Base Sequence, Colony Count, Microbial, Gene Expression Regulation, Fungal, Genetic Speciation, Genome, Fungal genetics, Host-Pathogen Interactions genetics, Indole Alkaloids metabolism, Molecular Sequence Data, Nitrogen metabolism, Phylogeny, Populus microbiology, Proteolysis, Synteny genetics, Time Factors, Adaptation, Physiological genetics, Ascomycota genetics, Ascomycota growth & development, Gene Dosage, Gene Transfer, Horizontal, Trees microbiology, Wood microbiology
- Abstract
Some of the most damaging tree pathogens can attack woody stems, causing lesions (cankers) that may be lethal. To identify the genomic determinants of wood colonization leading to canker formation, we sequenced the genomes of the poplar canker pathogen, Mycosphaerella populorum, and the closely related poplar leaf pathogen, M. populicola. A secondary metabolite cluster unique to M. populorum is fully activated following induction by poplar wood and leaves. In addition, genes encoding hemicellulose-degrading enzymes, peptidases, and metabolite transporters were more abundant and were up-regulated in M. populorum growing on poplar wood-chip medium compared with M. populicola. The secondary gene cluster and several of the carbohydrate degradation genes have the signature of horizontal transfer from ascomycete fungi associated with wood decay and from prokaryotes. Acquisition and maintenance of the gene battery necessary for growth in woody tissues and gene dosage resulting in gene expression reconfiguration appear to be responsible for the adaptation of M. populorum to infect, colonize, and cause mortality on poplar woody stems.
- Published
- 2015
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34. The accessible cellulose surface influences cellulase synergism during the hydrolysis of lignocellulosic substrates.
- Author
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Hu J, Gourlay K, Arantes V, Van Dyk JS, Pribowo A, and Saddler JN
- Subjects
- Aspergillus niger enzymology, Hydrolysis, Surface Properties, Trichoderma enzymology, Cellulases metabolism, Lignin chemistry
- Abstract
Effective enzymatic hydrolysis of insoluble cellulose requires the synergistic action of a suite of cellulase components. Most previous studies have only assessed cellulase synergism on model cellulosic substrates. When the actions of individual and combinations of cellulases (Cel7A, Cel6A, Cel7B, Cel5A) were assessed on various pretreated lignocellulosic substrates, Cel7A was shown to be the major contributor to overall cellulose hydrolysis, with the other enzymes synergistically enhancing its hydrolytic efficiency, at least partially, by facilitating Cel7A desorption (assessed by a double-sandwich enzyme-linked immunosorbent assay). When the influences of various substrate physicochemical characteristics on the effectiveness of enzyme synergism were assessed, a strong relationship was observed between cellulose accessibility (as determined by the cellulose binding module technique) and the degree of synergism, with greater synergy observed on the more disorganized/accessible cellulose surface., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2015
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35. The use of carbohydrate binding modules (CBMs) to monitor changes in fragmentation and cellulose fiber surface morphology during cellulase- and Swollenin-induced deconstruction of lignocellulosic substrates.
- Author
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Gourlay K, Hu J, Arantes V, Penttilä M, and Saddler JN
- Subjects
- Microscopy, Confocal, Protein Binding, X-Ray Diffraction, Cellulase metabolism, Cellulose chemistry, Cellulose metabolism, Lignin chemistry, Lignin metabolism
- Abstract
Although the actions of many of the hydrolytic enzymes involved in cellulose hydrolysis are relatively well understood, the contributions that amorphogenesis-inducing proteins might contribute to cellulose deconstruction are still relatively undefined. Earlier work has shown that disruptive proteins, such as the non-hydrolytic non-oxidative protein Swollenin, can open up and disaggregate the less-ordered regions of lignocellulosic substrates. Within the cellulosic fraction, relatively disordered, amorphous regions known as dislocations are known to occur along the length of the fibers. It was postulated that Swollenin might act synergistically with hydrolytic enzymes to initiate biomass deconstruction within these dislocation regions. Carbohydrate binding modules (CBMs) that preferentially bind to cellulosic substructures were fluorescently labeled. They were imaged, using confocal microscopy, to assess the distribution of crystalline and amorphous cellulose at the fiber surface, as well as to track changes in surface morphology over the course of enzymatic hydrolysis and fiber fragmentation. Swollenin was shown to promote targeted disruption of the cellulosic structure at fiber dislocations., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2015
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36. The enzymatic hydrolysis of pretreated pulp fibers predominantly involves "peeling/erosion" modes of action.
- Author
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Arantes V, Gourlay K, and Saddler JN
- Abstract
Background: There is still considerable debate regarding the actual mechanism by which a "cellulase mixture" deconstructs cellulosic materials, with accessibility to the substrate at the microscopic level being one of the major restrictions that limits fast, complete cellulose hydrolysis. In the work reported here we tried to determine the predominant mode of action, at the fiber level, of how a cellulase mixture deconstructs pretreated softwood and hardwood pulp fibers. Quantitative changes in the pulp fibers derived from different pretreated biomass substrates were monitored throughout the course of enzymatic hydrolysis to see if the dominant mechanisms involved either the fragmentation/cutting of longer fibers to shorter fibers or their "peeling/delamination/erosion," or if both cutting and peeling mechanisms occurred simultaneously., Results: Regardless of the source of biomass, the type of pretreatment and the chemical composition of the substrate, under typical hydrolysis conditions (50°C, pH 4.8, mixing) longer pulp fibers (fiber length >200 μm) were rapidly broken down until a relatively constant fiber length of 130 to 160 μm was reached. In contrast, shorter fibers with an initial average fiber length of 130 to 160 μm showed no significant change in length despite their substantial hydrolysis. The fragmentation/cutting mode of deconstruction was only observed on longer fibers at early stages of hydrolysis. Although the fiber fragmentation mode of deconstruction was not greatly influenced by enzyme loading, it was significantly inhibited by glucose and was mainly observed during initial mixing of the enzyme and substrate. In contrast, significant changes in the fiber width occurred throughout the course of hydrolysis for all of the substrates, suggesting that fiber width may limit the rate and extent of cellulose hydrolysis., Conclusion: It appears that, at the fiber level, pretreated pulp fibers are hydrolyzed through a two-step mode of action involving an initial rapid fragmentation followed by simultaneous swelling and peeling/erosion of the fragmented fibers. This latter mechanism is the predominant mode of action involved in effectively hydrolyzing the cellulose present in pretreated wood substrates.
- Published
- 2014
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37. Lignin valorization: improving lignin processing in the biorefinery.
- Author
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Ragauskas AJ, Beckham GT, Biddy MJ, Chandra R, Chen F, Davis MF, Davison BH, Dixon RA, Gilna P, Keller M, Langan P, Naskar AK, Saddler JN, Tschaplinski TJ, Tuskan GA, and Wyman CE
- Subjects
- Biofuels, Carbon, Carbon Fiber, Crops, Agricultural chemistry, Crops, Agricultural genetics, Crops, Agricultural metabolism, Elastomers, Lignin chemistry, Lignin genetics, Bioengineering methods, Cellulose chemistry, Lignin biosynthesis
- Abstract
Research and development activities directed toward commercial production of cellulosic ethanol have created the opportunity to dramatically increase the transformation of lignin to value-added products. Here, we highlight recent advances in this lignin valorization effort. Discovery of genetic variants in native populations of bioenergy crops and direct manipulation of biosynthesis pathways have produced lignin feedstocks with favorable properties for recovery and downstream conversion. Advances in analytical chemistry and computational modeling detail the structure of the modified lignin and direct bioengineering strategies for future targeted properties. Refinement of biomass pretreatment technologies has further facilitated lignin recovery, and this coupled with genetic engineering will enable new uses for this biopolymer, including low-cost carbon fibers, engineered plastics and thermoplastic elastomers, polymeric foams, fungible fuels, and commodity chemicals., (Copyright © 2014, American Association for the Advancement of Science.)
- Published
- 2014
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38. The synergistic action of accessory enzymes enhances the hydrolytic potential of a "cellulase mixture" but is highly substrate specific.
- Author
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Hu J, Arantes V, Pribowo A, and Saddler JN
- Abstract
Background: Currently, the amount of protein/enzyme required to achieve effective cellulose hydrolysis is still too high. One way to reduce the amount of protein/enzyme required is to formulate a more efficient enzyme cocktail by adding so-called accessory enzymes such as xylanase, lytic polysaccharide monooxygenase (AA9, formerly known as GH61), etc., to the cellulase mixture. Previous work has shown the strong synergism that can occur between cellulase and xylanase mixtures during the hydrolysis of steam pretreated corn stover, requiring lower protein loading to achieve effective hydrolysis. However, relatively high loadings of xylanases were required. When family 10 and 11 endo-xylanases and family 5 xyloglucanase were supplemented to a commercial cellulase mixture varying degrees of improved hydrolysis over a range of pretreated, lignocellulosic substrates were observed., Results: The potential synergistic interactions between cellulase monocomponents and hemicellulases from family 10 and 11 endo-xylanases (GH10 EX and GH11 EX) and family 5 xyloglucanase (GH5 XG), during hydrolysis of various steam pretreated lignocellulosic substrates, were assessed. It was apparent that the hydrolytic activity of cellulase monocomponents was enhanced by the addition of accessory enzymes although the "boosting" effect was highly substrate specific. The GH10 EX and GH5 XG both exhibited broad substrate specificity and showed strong synergistic interaction with the cellulases when added individually. The GH10 EX was more effective on steam pretreated agriculture residues and hardwood substrates whereas GH5 XG addition was more effective on softwood substrates. The synergistic interaction between GH10 EX and GH5 XG when added together further enhanced the hydrolytic activity of the cellulase enzymes over a range of pretreated lignocellulosic substrates. GH10 EX addition could also stimulate further cellulose hydrolysis when added to the hydrolysis reactions when the rate of hydrolysis had levelled off., Conclusions: Endo-xylanases and xyloglucanases interacted synergistically with cellulases to improve the hydrolysis of a range of pretreated lignocellulosic substrates. However, the extent of improved hydrolysis was highly substrate dependent. It appears that those accessory enzymes, such as GH10 EX and GH5 XG, with broader substrate specificities promoted the greatest improvements in the hydrolytic performance of the cellulase mixture on all of the pretreated biomass substrates.
- Published
- 2013
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39. The development and use of an ELISA-based method to follow the distribution of cellulase monocomponents during the hydrolysis of pretreated corn stover.
- Author
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Pribowo AY, Hu J, Arantes V, and Saddler JN
- Abstract
Background: It is widely recognised that fast, effective hydrolysis of pretreated lignocellulosic substrates requires the synergistic action of multiple types of hydrolytic and some non-hydrolytic proteins. However, due to the complexity of the enzyme mixture, the enzymes interaction with and interference from the substrate and a lack of specific methods to follow the distribution of individual enzymes during hydrolysis, most of enzyme-substrate interaction studies have used purified enzymes and pure cellulose model substrates. As the enzymes present in a typical "cellulase mixture" need to work cooperatively to achieve effective hydrolysis, the action of one enzyme is likely to influence the behaviour of others. The action of the enzymes will be further influenced by the nature of the lignocellulosic substrate. Therefore, it would be beneficial if a method could be developed that allowed us to follow some of the individual enzymes present in a cellulase mixture during hydrolysis of more commercially realistic biomass substrates., Results: A high throughput immunoassay that could quantitatively and specifically follow individual cellulase enzymes during hydrolysis was developed. Using monoclonal and polyclonal antibodies (MAb and PAb, respectively), a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed to specifically quantify cellulase enzymes from Trichoderma reesei: cellobiohydrolase I (Cel7A), cellobiohydrolase II (Cel6A), and endoglucanase I (Cel7B). The interference from substrate materials present in lignocellulosic supernatants could be minimized by dilution., Conclusion: A double-antibody sandwich ELISA was able to detect and quantify individual enzymes when present in cellulase mixtures. The assay was sensitive over a range of relatively low enzyme concentration (0 - 1 μg/ml), provided the enzymes were first pH adjusted and heat treated to increase their antigenicity. The immunoassay was employed to quantitatively monitor the adsorption of cellulase monocomponents, Cel7A, Cel6A, and Cel7B, that were present in both Celluclast and Accellerase 1000, during the hydrolysis of steam-pretreated corn stover (SPCS). All three enzymes exhibited different individual adsorption profiles. The specific and quantitative adsorption profiles observed with the ELISA method were in agreement with earlier work where more labour intensive enzyme assay techniques were used.
- Published
- 2013
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40. A two-stage pretreatment approach to maximise sugar yield and enhance reactive lignin recovery from poplar wood chips.
- Author
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Panagiotopoulos IA, Chandra RP, and Saddler JN
- Subjects
- Carbohydrates analysis, Ethanol, Hydrolysis, Steam, Sulfuric Acids, Lignin isolation & purification, Polysaccharides isolation & purification, Populus chemistry, Wood chemistry
- Abstract
A two-stage pretreatment approach, employing steam followed by organosolv treatment, was assessed for its ability to fractionate and recover most of the hemicellulose, lignin and cellulose components of poplar wood chips. A mild steaming stage was initially used to maximise hemicellulose sugar recovery, with 63% of the original xylan solubilised and recovered after this stage and close to 90% recovered in total. Rather than hindering subsequent organosolv delignification, the prior steam treatment enhanced lignin solubilisation with more than 66% of the original lignin removed after the two-stage pretreatment. The extracted lignin contained at least equal or greater amounts of functional groups as compared to the lignin solubilised after a single-stage organosolv pretreatment. More than 98% of the original cellulose was recovered after the two-stage pretreatment and 88% of the cellulose could be hydrolysed to glucose at enzyme loading of 5FPU/g cellulose after 72h., (Copyright © 2012 Elsevier Ltd. All rights reserved.)
- Published
- 2013
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41. Effect of replacing polyol by organosolv and kraft lignin on the property and structure of rigid polyurethane foam.
- Author
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Pan X and Saddler JN
- Abstract
Background: Lignin is one of the three major components in plant cell walls, and it can be isolated (dissolved) from the cell wall in pretreatment or chemical pulping. However, there is a lack of high-value applications for lignin, and the commonest proposal for lignin is power and steam generation through combustion. Organosolv ethanol process is one of the effective pretreatment methods for woody biomass for cellulosic ethanol production, and kraft process is a dominant chemical pulping method in paper industry. In the present research, the lignins from organosolv pretreatment and kraft pulping were evaluated to replace polyol for producing rigid polyurethane foams (RPFs)., Results: Petroleum-based polyol was replaced with hardwood ethanol organosolv lignin (HEL) or hardwood kraft lignin (HKL) from 25% to 70% (molar percentage) in preparing rigid polyurethane foam. The prepared foams contained 12-36% (w/w) HEL or 9-28% (w/w) HKL. The density, compressive strength, and cellular structure of the prepared foams were investigated and compared. Chain extenders were used to improve the properties of the RPFs., Conclusions: It was found that lignin was chemically crosslinked not just physically trapped in the rigid polyurethane foams. The lignin-containing foams had comparable structure and strength up to 25-30% (w/w) HEL or 19-23% (w/w) HKL addition. The results indicated that HEL performed much better in RPFs and could replace more polyol at the same strength than HKL because the former had a better miscibility with the polyol than the latter. Chain extender such as butanediol could improve the strength of lignin-containing RPFs.
- Published
- 2013
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42. Does densification influence the steam pretreatment and enzymatic hydrolysis of softwoods to sugars?
- Author
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Kumar L, Tooyserkani Z, Sokhansanj S, and Saddler JN
- Subjects
- Cellulose chemistry, Chromatography, High Pressure Liquid, Hydrolysis, Particle Size, Biofuels, Cellulose analysis, Ethanol chemistry, Glucose biosynthesis, Polysaccharides analysis, Steam, Wood chemistry
- Abstract
The global trade in wood pellets continues to grow. However, their potential as a feedstock for large scale cellulosic ethanol production has not been evaluated. We anticipated that the reduced moisture content and pressure exerted on the wood biomass during the pelletisation process would result in some carbohydrate loss as well as making the biomass more recalcitrant to pretreatment and subsequent hydrolysis. However, when softwood chips and pellets were steam pretreated at medium severity, little hemicellulose loss occurred while more than two-thirds of the cellulose present in the cellulose rich water insoluble fractions were hydrolysed (at 20 FPU cellulase/g cellulose). In addition, prior steaming substantially reduced the particle size of the wood chips enabling direct pelletisation without the need for grinding. Surprisingly, it was also possible to apply a single steam pretreatment to facilitate both pelletisation and subsequent enzymatic hydrolysis without the need for a further pretreatment step., (Copyright © 2012 Elsevier Ltd. All rights reserved.)
- Published
- 2012
- Full Text
- View/download PDF
43. Use of substructure-specific carbohydrate binding modules to track changes in cellulose accessibility and surface morphology during the amorphogenesis step of enzymatic hydrolysis.
- Author
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Gourlay K, Arantes V, and Saddler JN
- Abstract
Background: Cellulose amorphogenesis, described as the non-hydrolytic "opening up" or disruption of a cellulosic substrate, is becoming increasingly recognized as one of the key steps in the enzymatic deconstruction of cellulosic biomass when used as a feedstock for fuels and chemicals production. Although this process is thought to play a major role in facilitating hydrolysis, the lack of quantitative techniques capable of accurately describing the molecular-level changes occurring in the substrate during amorphogenesis has hindered our understanding of this process., Results: In this work, techniques for measuring changes in cellulose accessibility are reviewed and a new quantitative assay method is described. Carbohydrate binding modules (CBMs) with specific affinities for crystalline (CBM2a) or amorphous (CBM44) cellulose were used to track specific changes in the surface morphology of cotton fibres during amorphogenesis. The extents of phosphoric acid-induced and Swollenin-induced changes to cellulose accessibility were successfully quantified using this technique., Conclusions: The adsorption of substructure-specific CBMs can be used to accurately quantify the extent of changes to cellulose accessibility induced by non-hydrolytic disruptive proteins. The technique provided a quick, accurate and quantitative measure of the accessibility of cellulosic substrates. Expanding the range of CBMs used for adsorption studies to include those specific for such compounds as xylan or mannan should also allow for the accurate quantitative tracking of the accessibility of these and other polymers within the lignocellulosic biomass matrix.
- Published
- 2012
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44. The adsorption and enzyme activity profiles of specific Trichoderma reesei cellulase/xylanase components when hydrolyzing steam pretreated corn stover.
- Author
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Pribowo A, Arantes V, and Saddler JN
- Subjects
- Adsorption, Chromatography, Liquid, Hydrolysis, Lignin metabolism, Mass Spectrometry, Substrate Specificity, Zea mays chemistry, beta-Glucosidase, Biotechnology methods, Cellulases metabolism, Endo-1,4-beta Xylanases metabolism, Steam, Trichoderma enzymology, Zea mays metabolism
- Abstract
Recycling of enzymes during biomass conversion is one potential strategy to reduce the cost of the hydrolysis step of cellulosic ethanol production. Devising an efficient enzyme recycling strategy requires a good understanding of how the enzymes adsorb, distribute, and interact with the substrate during hydrolysis. We investigated the interaction of individual Trichoderma reesei enzymes present in a commercial cellulase mixture during the hydrolysis of steam-pretreated corn stover (SPCS). The enzyme profiles were followed using zymograms, gel electrophoresis, enzyme activity assays and mass spectrometry. The adsorption and activity profiles of 6 specific enzymes Cel7A (CBH I), Cel7B (EG I), Cel5A (EG II), Xyn 10 (endo-1,4-β-xylanase III), Xyn 11 (endo-xylanase II), and β-glucosidase were characterized. Initially, each of the enzymes rapidly adsorbed onto the SPCS. However, this was followed by partial desorption to an adsorption equilibrium where the Cel7A, Cel7B, Xyn 10, and β-glucosidase were partially adsorbed to the SPCS and also found free in solution throughout the course of hydrolysis. In contrast, the Cel5A and Xyn 11 components remained primarily free in the supernatant. The Cel7A component also exhibited a partial desorption when the rate of hydrolysis leveled off as evidenced by MUC zymogram and SDS-PAGE. Those cellulase components that did not bind to the substrate were generally less stable and lost their activities within the first 24h when compared to enzymes that were distributed in both the liquid and solid phases. Therefore, to ensure maximum enzyme activity recovery, enzyme recycling seems to be most effective when short-term rounds of hydrolysis are combined with the recovery of enzymes from both the liquid and the solid phases and potentially enzyme supplementation to replenish lost activity., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2012
- Full Text
- View/download PDF
45. Fibre size does not appear to influence the ease of enzymatic hydrolysis of organosolv-pretreated softwoods.
- Author
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Del Rio LF, Chandra RP, and Saddler JN
- Subjects
- Crystallization, Hydrolysis, Polymers chemistry, Substrate Specificity, Enzymes metabolism, Wood
- Abstract
To determine the effect of fibre size on enzymatic hydrolysis, organosolv-pretreated lodgepole pine was size-fractionated into six substrates ranging in average size from 0.20 to 3.4mm. Other than the fines fraction (<0.2mm) which contained most of the lignin, the fractionated substrates were more readily hydrolyzed than the original substrate with nearly complete hydrolysis after 72 h at 5 FPU g(-1) cellulose. Surprisingly, fibre size was found to have little influence on enzymatic hydrolysis likely due to similarities in the substrates' chemical composition, accessible surface area, cellulose crystallinity and degree of polymerization. To determine the influence of the fines on enzymatic hydrolysis, their content was artificially increased (from 8.9% to 55.4%) however; this did not have a noticeable effect. These results show that within the range of fibre sizes tested, other substrate characteristics likely play a more significant role in the ease of hydrolysis of pretreated substrates., (Copyright © 2011 Elsevier Ltd. All rights reserved.)
- Published
- 2012
- Full Text
- View/download PDF
46. The enhancement of enzymatic hydrolysis of lignocellulosic substrates by the addition of accessory enzymes such as xylanase: is it an additive or synergistic effect?
- Author
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Hu J, Arantes V, and Saddler JN
- Abstract
Background: We and other workers have shown that accessory enzymes, such as β-glucosidase, xylanase, and cellulase cofactors, such as GH61, can considerably enhance the hydrolysis effectiveness of cellulase cocktails when added to pretreated lignocellulosic substrates. It is generally acknowledged that, among the several factors that hamper our current ability to attain efficient lignocellulosic biomass conversion yields at low enzyme loadings, a major problem lies in our incomplete understanding of the cooperative action of the different enzymes acting on pretreated lignocellulosic substrates., Results: The reported work assessed the interaction between cellulase and xylanase enzymes and their potential to improve the hydrolysis efficiency of various pretreated lignocellulosic substrates when added at low protein loadings. When xylanases were added to the minimum amount of cellulase enzymes required to achieve 70% cellulose hydrolysis of steam pretreated corn stover (SPCS), or used to partially replace the equivalent cellulase dose, both approaches resulted in enhanced enzymatic hydrolysis. However, the xylanase supplementation approach increased the total protein loading required to achieve significant improvements in hydrolysis (an additive effect), whereas the partial replacement of cellulases with xylanase resulted in similar improvements in hydrolysis without increasing enzyme loading (a synergistic effect). The enhancement resulting from xylanase-aided synergism was higher when enzymes were added simultaneously at the beginning of hydrolysis. This co-hydrolysis of the xylan also influenced the gross fiber characteristics (for example, fiber swelling) resulting in increased accessibility of the cellulose to the cellulase enzymes. These apparent increases in accessibility enhanced the steam pretreated corn stover digestibility, resulting in three times faster cellulose and xylan hydrolysis, a seven-fold decrease in cellulase loading and a significant increase in the hydrolysis performance of the optimized enzyme mixture. When a similar xylanase-aided enhancement strategy was assessed on other pretreated lignocellulosic substrates, equivalent increases in hydrolysis efficiency were also observed., Conclusions: It was apparent that the 'blocking effect' of xylan was one of the major mechanisms that limited the accessibility of the cellulase enzymes to the cellulose. However, the synergistic interaction of the xylanase and cellulase enzymes was also shown to significantly improve cellulose accessibility through increasing fiber swelling and fiber porosity and also plays a major role in enhancing enzyme accessibility.
- Published
- 2011
- Full Text
- View/download PDF
47. Relatively high-substrate consistency hydrolysis of steam-pretreated sweet sorghum bagasse at relatively low cellulase loading.
- Author
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Shen F, Zhong Y, Saddler JN, and Liu R
- Subjects
- Biotransformation, Cellulase metabolism, Cellulose metabolism, Endo-1,4-beta Xylanases metabolism, Hydrolysis, Steam, Sulfur Dioxide metabolism, beta-Glucosidase metabolism, Biotechnology methods, Glucans metabolism, Glucose biosynthesis, Sorghum metabolism, Xylans metabolism
- Abstract
Sweet sorghum bagasse (SSB) was steam pretreated in the conditions of 190 °C for 5 min to assess its amenability to the pretreatment and enzymatic hydrolysis. Results showed that pretreatment conditions were robust enough to pretreat SSB with maximum of 87% glucan and 72% xylan recovery. Subsequent enzymatic hydrolysis showed that the pretreated SSB at 2% substrate consistency resulted in maximum of 70% glucan-glucose conversion. Increasing substrate consistency from 2% to 16% led to a significant reduction in glucan conversion. However, the decrease ratio of glucan-glucose conversion was the minimum when the consistency increased from 2% to 12%. When the pretreated SSB consistency of 12% was applied for hydrolysis, increase in cellulase loading from 7.5 up to 20 filter paper units (FPU)/g glucan resulted only in 14% increase in glucan-glucose conversion compared to 20% increase with cellulase loading varying from 2.5 to 7.5 FPU/g glucan. More than 10 cellobiase units (CBU)/g glucan β-glucosidase supplementation had no noticeable improvement on glucan-glucose conversion. Additionally, supplementation of xylanase was found to significantly increase glucan-glucose conversion from 50% to 80% with the substrate consistency of 12%, when the cellulase and β-glucosidase loadings were at relatively low enzyme loadings (7.5 FPU/g and 10 CBU/g glucan). It appeared that residual xylan played a critical role in hindering the ease of hydrolysis of SSB. A proper xylanase addition was suggested to achieve a high hydrolysis yield at relatively high substrate consistency with relatively low enzyme loadings.
- Published
- 2011
- Full Text
- View/download PDF
48. Evaluation of hemicellulose removal by xylanase and delignification on SHF and SSF for bioethanol production with steam-pretreated substrates.
- Author
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Shen F, Kumar L, Hu J, and Saddler JN
- Subjects
- Chromatography, Gas, Chromatography, High Pressure Liquid, Fermentation, Hydrolysis, Polysaccharides isolation & purification, Steam, Biofuels, Cellulose chemistry, Endo-1,4-beta Xylanases metabolism, Ethanol metabolism, Polysaccharides metabolism, Pseudotsuga chemistry
- Abstract
Steam-pretreated sweet sorghum bagasse (SSB) and Douglas-fir (DF) were employed for SHF and SSF to evaluate the effects of xylanase supplementation and delignification on ethanol production. Results indicated final ethanol concentration in SHF could reach 28.4 g/L (SSB) and 20.4 g/L (DF) by xylanase supplementation with the increase of 46% and 61% in comparison with controls. The delignification could significantly enhance final ethanol concentration to 31.2g/L (SSB) and 30.2 g/L (DF) with the increase of 61% and 138%. In SSF, final ethanol concentration in the delignified SSB and DF arrived at 27.6 g/L and 34.3 g/L with the increase of 26% and 157% compared with controls. However, only 2.2 g/L (SSB) and 6.9 g/L (DF) ethanol were obtained with xylanase supplementation. According to these results, it could be concluded that delignification was beneficial to improve ethanol production of SHF and SSF. The xylanase supplementation (0.12 g protein/g glucan) was only positive to SHF while retarded SSF seriously., (Copyright © 2011 Elsevier Ltd. All rights reserved.)
- Published
- 2011
- Full Text
- View/download PDF
49. The effects of increasing swelling and anionic charges on the enzymatic hydrolysis of organosolv-pretreated softwoods at low enzyme loadings.
- Author
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Del Rio LF, Chandra RP, and Saddler JN
- Subjects
- Cellulose metabolism, Hydrolysis, Pinus chemistry, Pinus metabolism, Static Electricity, Wood chemistry, Enzymes metabolism, Wood metabolism
- Abstract
Organosolv-pretreated Lodgepole pine substrates were physically and chemically treated to increase their hydrophilicity and swelling as these are two substrate attributes which have been shown to improve cellulolytic hydrolysis. Surprisingly, mechanical treatment of the organosolv-treated substrates by PFI-mill refining did not significantly increase hydrolysis yields despite decreases in particle size and crystallinity and increases in swelling. However, sulfonation of the substrate did, significantly, increase enzymatic hydrolysis at loadings of both 5 and 2.5 FPU g(-1) cellulose (from 80% to 95% and from 35% to 80%, respectively). In addition, sulfonation resulted in an increase in the amount of free enzymes detected during the course of hydrolysis to a maximum of 80% after 72 h. This suggested that the beneficial effects of sulfonation were primarily due to a decrease in the non-specific binding of the cellulases to the lignin., (Copyright © 2011 Wiley Periodicals, Inc.)
- Published
- 2011
- Full Text
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50. The isolation, characterization and effect of lignin isolated from steam pretreated Douglas-fir on the enzymatic hydrolysis of cellulose.
- Author
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Nakagame S, Chandra RP, Kadla JF, and Saddler JN
- Subjects
- Adsorption, Hydrolysis, Lignin chemistry, Magnetic Resonance Spectroscopy, Peptide Hydrolases, Solubility, Spectrum Analysis, Temperature, Cellulase metabolism, Cellulose metabolism, Lignin isolation & purification, Pseudotsuga chemistry, Steam
- Abstract
Douglas-fir was SO(2)-steam pretreated at different severities (190, 200, and 210°C) to assess the possible negative effect of the residual and isolated lignins on the enzymatic hydrolysis of the steam pretreated substrates. When various isolated lignins were added to the Avicel hydrolysis reactions, the decrease in glucose yields ranged from 15.2% to 29.0% after 72 h. It was apparent that the better hydrolysis yields obtained at higher pretreatment severities were more a result of the greater accessibly of the cellulose rather than any specific change in the non-productive binding of the lignin to the enzymes. FTIR and (13)C NMR characterization indicated that the lignin in the steam pretreated substrates became more condensed with increasing severity, suggesting that the cellulases were adsorbed to the lignin by hydrophobic interactions. Electrostatic interactions were also involved as the positively charged cellulase components were preferentially adsorbed to the lignins., (Copyright © 2010 Elsevier Ltd. All rights reserved.)
- Published
- 2011
- Full Text
- View/download PDF
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