Sample records for xylan

  1. Evolution of Xylan Substitution Patterns in Gymnosperms and Angiosperms: Implications for Xylan Interaction with Cellulose1[CC-BY

    PubMed Central

    Li, An; Gomes, Thiago C.F.

    2016-01-01

    The interaction between cellulose and xylan is important for the load-bearing secondary cell wall of flowering plants. Based on the precise, evenly spaced pattern of acetyl and glucuronosyl (MeGlcA) xylan substitutions in eudicots, we recently proposed that an unsubstituted face of xylan in a 2-fold helical screw can hydrogen bond to the hydrophilic surfaces of cellulose microfibrils. In gymnosperm cell walls, any role for xylan is unclear, and glucomannan is thought to be the important cellulose-binding polysaccharide. Here, we analyzed xylan from the secondary cell walls of the four gymnosperm lineages (Conifer, Gingko, Cycad, and Gnetophyta). Conifer, Gingko, and Cycad xylan lacks acetylation but is modified by arabinose and MeGlcA. Interestingly, the arabinosyl substitutions are located two xylosyl residues from MeGlcA, which is itself placed precisely on every sixth xylosyl residue. Notably, the Gnetophyta xylan is more akin to early-branching angiosperms and eudicot xylan, lacking arabinose but possessing acetylation on alternate xylosyl residues. All these precise substitution patterns are compatible with gymnosperm xylan binding to hydrophilic surfaces of cellulose. Molecular dynamics simulations support the stable binding of 2-fold screw conifer xylan to the hydrophilic face of cellulose microfibrils. Moreover, the binding of multiple xylan chains to adjacent planes of the cellulose fibril stabilizes the interaction further. Our results show that the type of xylan substitution varies, but an even pattern of xylan substitution is maintained among vascular plants. This suggests that 2-fold screw xylan binds hydrophilic faces of cellulose in eudicots, early-branching angiosperm, and gymnosperm cell walls. PMID:27325663

  2. Reducing the heterogeneity of xylan through processing.

    PubMed

    Zhang, Wei; Johnson, Amanda M; Barone, Justin R; Renneckar, Scott

    2016-10-05

    Glycerol thermal processing (GTP) of hardwood biomass at temperatures between 200 and 240°C facilitated stepwise biopolymer fractionation, while limiting significant degradation of the major hemicellulose, glucuronoxylan, into water-extractable oligosaccharides. After GTP pretreatment and sequential water and organic solvent extraction, up to 80% of the initial xylan remained in the pretreated biomass. The majority of the xylan from GTP pretreated and water/solvent extracted biomass was removed using a mild alkali extraction and the composition was compared to xylan directly isolated from untreated hardwood. The precipitated xylan from the neutralized alkaline filtrate was isolated as a water insoluble xylan portion (WIX). The residual xylan dissolved in the neutralized filtrate was precipitated in cold methanol and recovered as the water soluble xylan portion (WSX). Results showed that xylan in WIX was in a polymeric form with a number average degree of polymerization (DP) over 100, whereas the WSX had a much lower average DP of 27 (ca) and contained more substitution. As the processing severity increased during GTP pretreatment, the proportion of WIX increased and the purity of the xylan within the WIX sample reached 84% based on compositional analysis. FT-IR analysis of WIX revealed that xylan isolated after GTP contained peaks related to a reduced carbonyl signal compared to the control. Furthermore, crude WSX contained less xylan with more lignin contamination at severe GTP conditions. The recovery of the xylan in two portions facilitated a preferential purification strategy resulting in WIX with an extremely narrow polydispersity index between 1.1 and 1.25, dependent upon the GTP severity. This study provided insight into fractionating higher molecular weight xylan that may serve value-added applications such as healthcare materials and advanced packaging. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Effects of Xylan Side-Chain Substitutions on Xylan-Cellulose Interactions and Implications for Thermal Pretreatment of Cellulosic Biomass.

    PubMed

    Pereira, Caroline S; Silveira, Rodrigo L; Dupree, Paul; Skaf, Munir S

    2017-04-10

    Lignocellulosic biomass is mainly constituted by cellulose, hemicellulose, and lignin and represents an important resource for the sustainable production of biofuels and green chemistry materials. Xylans, a common hemicellulose, interact with cellulose and often exhibit various side chain substitutions including acetate, (4-O-methyl) glucuronic acid, and arabinose. Recent studies have shown that the distribution of xylan substitutions is not random, but follows patterns that are dependent on the plant taxonomic family and cell wall type. Here, we use molecular dynamics simulations to investigate the role of substitutions on xylan interactions with the hydrophilic cellulose face, using the recently discovered xylan decoration pattern of the conifer gymnosperms as a model. The results show that α-1,2-linked substitutions stabilize the binding of single xylan chains independently of the nature of the substitution and that Ca 2+ ions can mediate cross-links between glucuronic acid substitutions of two neighboring xylan chains, thus stabilizing binding. At high temperature, xylans move from the hydrophilic to the hydrophobic cellulose surface and are also stabilized by Ca 2+ cross-links. Our results help to explain the role of substitutions on xylan-cellulose interactions, and improve our understanding of the plant cell wall architecture and the fundamentals of biomass pretreatments.

  4. Co-operative actions and degradation analysis of purified xylan-degrading enzymes from Thermomonospora fusca BD25 on oat-spelt xylan.

    PubMed

    Tuncer, M; Ball, A S

    2003-01-01

    To determine and quantify the products from the degradation of xylan by a range of purified xylan-degrading enzymes, endoxylanase, beta-xylosidase and alpha-l-arabinofuranosidase produced extracellularly by Thermomonospora fusca BD25. The amounts of reducing sugars released from oat-spelt xylan by the actions of endoxylanase, beta-xylosidase and alpha-l-arabinofuranosidase were equal to 28.1, 4.6 and 7% hydrolysis (as xylose equivalents) of the substrate used, respectively. However, addition of beta-xylosidase and alpha-l-arabinofuranosidase preparation to endoxylanase significantly enhanced (70 and 20% respectively) the action of endoxylanase on the substrate. The combination of purified endoxylanase, beta-xylosidase and alpha-l-arabinofuranosidase preparations produced a greater sugar yield (58.6% hydrolysis) and enhanced the total reducing sugar yield by around 50%. The main xylooligosaccharide products released using the action of endoxylanase alone on oat-spelt xylan were identified as xylobiose and xylopentose. alpha-l-Arabinofuranosidase was able to release arabinose and xylobiose from oat-spelt xylan. In the presence of all three purified enzymes the hydrolysis products of oat-spelt xylan were mainly xylose, arabinose and substituted xylotetrose with lesser amount of substituted xylotriose. The addition of the beta-xylosidase and alpha-l-arabinofuranosidase enzymes to purified xylanases more than doubled the degradation of xylan from 28 to 58% of the total substrate with xylose and arabinose being the major sugars produced. The results highlight the role of xylan de-branching enzymes in the degradation of xylan and suggest that the use of enzyme cocktails may significantly improve the hydrolysis of xylan in industrial processes.

  5. Biochemical characterization of rice xylan O-acetyltransferases.

    PubMed

    Zhong, Ruiqin; Cui, Dongtao; Dasher, Robert L; Ye, Zheng-Hua

    2018-06-01

    Rice xylan is predominantly monoacetylated at O-2 and O-3, and 14 rice DUF231 proteins were demonstrated to be xylan acetyltransferases. Acetylated xylans are the principal hemicellulose in the cell walls of grass species. Because xylan acetylation impedes the conversion of cellulosic biomass into biofuels, knowledge on acetyltransferases catalyzing xylan acetylation in grass species will be instrumental for a better utilization of grass biomass for biofuel production. Xylan in rice (Oryza sativa) is predominantly monoacetylated at O-2 and O-3 with a total degree of acetylation of 0.19. In this report, we have characterized 14 rice DUF231 proteins (OsXOAT1 to OsXOAT14) that are phylogenetically grouped together with Arabidopsis xylan acetyltransferases ESK1 and its close homologs. Complementation analysis demonstrated that the expression of OsXOAT1 to OsXOAT7 in the Arabidopsis esk1 mutant was able to rescue its defects in 2-O- and 3-O-monoacetylation and 2,3-di-O-acetylation. Activity assay of recombinant proteins revealed that all 14 OsXOATs exhibited acetyltransferase activities capable of transferring acetyl groups from acetyl-CoA to the xylohexaose acceptor with 10 of them having high activities. Structural analysis of the OsXOAT-catalyzed products showed that the acetylated structural units consisted mainly of 2-O- and 3-O-monoacetylated xylosyl residues with a minor amount of 2,3-di-O-acetylated xylosyl units, which is consistent with the acetyl substitution pattern of rice xylan. Further kinetic studies revealed that OsXOAT1, OsXOAT2, OsXOAT5, OsXOAT6 and OsXOAT7 had high affinity toward the xylohexaose acceptor. Our results provide biochemical evidence indicating that OsXOATs are acetyltransferases involved in xylan acetylation in rice.

  6. Maillard reaction products from chitosan-xylan ionic liquid solution.

    PubMed

    Luo, Yuqiong; Ling, Yunzhi; Wang, Xiaoying; Han, Yang; Zeng, Xianjie; Sun, Runcang

    2013-10-15

    A facile method is reported to prepare Maillard reaction products (MRPs) from chitosan and xylan in co-solvent ionic liquid. UV absorbance and fluorescence changes were regarded as indicators of the occurrence of Maillard reaction. FT-IR, NMR, XRD and TG were used to investigate the structure of chitosan-xylan conjugate. The results revealed that when chitosan reacted with xylan in ionic liquid, the hydrogen bonds in chitosan were destroyed, the facts resulted in the formation of chitosan-xylan MRPs. Moreover, when the mass ratio of chitosan to xylan was 1:1, the Maillard reaction proceeded easily. In addition, relatively high antioxidant property was also noted for the chitosan-xylan conjugate with mass ratio 1:1. So the obtained chitosan-xylan MRP is a promising antioxidant agent for food industry. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. 2-Hydroxypropyltrimethylammonium xylan adsorption onto rod-like cellulose nanocrystal.

    PubMed

    Sim, Jae Hyun; Dong, Shuping; Röemhild, Katrin; Kaya, Abdulaziz; Sohn, Daewon; Tanaka, Keiji; Roman, Maren; Heinze, Thomas; Esker, Alan R

    2015-02-15

    Chemical incompatibility and relatively weak interaction between lignocellulosic fibers and synthetic polymers have made studies of wood fiber-thermoplastic composite more challenging. In this study, adsorption of 2-hydroxypropyltrimethylammonium xylans onto rod-like cellulose nanocrystals are investigated by zeta-potential measurements, and polarized and depolarized dynamic light scattering as a factor for better understanding of lignocellulosic fibers and cellulose nanocrystals. Zeta-potential measurements show xylan derivative adsorption onto cellulose nanocrystals. Decay time distributions of the ternary system and binary system from dynamic light scattering show that aggregates exist in the binary system and they disappear in the ternary system. At low 2-hydroxypropyltrimethylammonium xylan concentrations relative to that of cellulose nanocrystal, xylan derivatives adsorbed onto some of the cellulose nanocrystal. Hence, more xylan derivatives adsorbed onto cellulose nanocrystal increased with increasing xylan derivative concentration. Also, the concentration dependence of the ratio of the rotational diffusion coefficient to the translational diffusion coefficient revealed a strong adsorptive interaction between xylan derivatives and the cellulose nanocrystals. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Novel polyurethanes from xylan and TDI: Preparation and characterization

    USDA-ARS?s Scientific Manuscript database

    In this work a novel polyurethane was developed involving xylan and tolylene-2,4-diisocyanate (TDI). Polymer synthesis was achieved via conventional heat or microwave-assisted reaction in dimethylsulfoxide. Because xylan has multiple OH groups on each polymer chain, the TDI/xylan molar ratio neede...

  9. Xylan extraction from pretreated sugarcane bagasse using alkaline and enzymatic approaches.

    PubMed

    Sporck, Daniele; Reinoso, Felipe A M; Rencoret, Jorge; Gutiérrez, Ana; Del Rio, José C; Ferraz, André; Milagres, Adriane M F

    2017-01-01

    New biorefinery concepts are necessary to drive industrial use of lignocellulose biomass components. Xylan recovery before enzymatic hydrolysis of the glucan component is a way to add value to the hemicellulose fraction, which can be used in papermaking, pharmaceutical, and food industries. Hemicellulose removal can also facilitate subsequent cellulolytic glucan hydrolysis. Sugarcane bagasse was pretreated with an alkaline-sulfite chemithermomechanical process to facilitate subsequent extraction of xylan by enzymatic or alkaline procedures. Alkaline extraction methods yielded 53% (w/w) xylan recovery. The enzymatic approach provided a limited yield of 22% (w/w) but produced the xylan with the lowest contamination with lignin and glucan components. All extracted xylans presented arabinosyl side groups and absence of acetylation. 2D-NMR data suggested the presence of O -methyl-glucuronic acid and p -coumarates only in enzymatically extracted xylan. Xylans isolated using the enzymatic approach resulted in products with molecular weights (Mw) lower than 6 kDa. Higher Mw values were detected in the alkali-isolated xylans. Alkaline extraction of xylan provided a glucan-enriched solid readily hydrolysable with low cellulase loads, generating hydrolysates with a high glucose/xylose ratio. Hemicellulose removal before enzymatic hydrolysis of the cellulosic fraction proved to be an efficient manner to add value to sugarcane bagasse biorefining. Xylans with varied yield, purity, and structure can be obtained according to the extraction method. Enzymatic extraction procedures produce high-purity xylans at low yield, whereas alkaline extraction methods provided higher xylan yields with more lignin and glucan contamination. When xylan extraction is performed with alkaline methods, the residual glucan-enriched solid seems suitable for glucose production employing low cellulase loadings.

  10. Ice templated and cross-linked xylan/nanocrystalline cellulose hydrogels

    Treesearch

    Tobias Köhnke; Thomas Elder; Hans Theliander; Arthur J. Ragauskas

    2014-01-01

    Structured xylan-based hydrogels, reinforced with cellulose nanocrystals (CNCs), have successfully been prepared from water suspensions by cross-linking during freeze-casting. In order to induce cross-linking during the solidification/sublimation operation, xylan was first oxidized using sodium periodate to introduce dialdehydes. The oxidized xylan was then mixed with...

  11. Laccase catalysed grafting of phenolic onto xylan to improve its applicability in films

    NASA Astrophysics Data System (ADS)

    Pei, Jicheng; Wang, Bing; Zhang, Fangdong; Li, Zhongyang; Yin, Yunbei; Zhang, Dongxu

    2015-07-01

    Xylan can be tailored for various value-added applications. However, its use in aqueous systems is hampered by its complex structure, and small molecular weight. This research aimed at improving the xylan molecular weight and changing its structure. Laccase-catalysed oxidation of 4-coumaric acid (PCA), ferulic acid (FA), syringaldehyde (SD), and vanillin (VA) onto xylan was grafted to study the changes in its structure, tensile properties, and antibacterial activities. A Fourier transform infrared (FTIR) spectrum analyser was used to observe the changes in functional groups of xylan. The results showed a band at 1635 cm-1 corresponding to the stretching vibration of conjugated carbonyl carboxy hemoglobin and a benzene ring structure were strengthened; the appearance of a new band between 1200 cm-1 and 1270 cm-1 corresponding to alkyl ethers on the aryl C-O stretching vibration was due to the fact that during the grafting process, the number of benzene ring structures increased and covalent connections occurred between phenols and xylan. The reaction mechanism for the laccase-catalysed oxidation of phenol compounds onto xylan was preliminary explored by 13C-NMR. The results showed that PCA-xylan, FA-xylan graft poly onto xylan by Cγ ester bond, SD-xylan graft poly onto xylan by ether bond and an ester bond, and VD-xylan graft poly onto xylan by ether bond. The film strength of xylan derivatives has been significantly increased, especially for the PCA-xylan derivative. The increases in tensile stress at break, tensile strength, tensile yield stress, and Young's modulus were: 24.04%, 31.30%, 55.56%, and 28.21%, respectively. After laccase/phenolics were modified, xylan had a good antibacterial effect to E. coli, Corynebacterium glutamicum, and Bacillus subtilis. The SD-xylan, FA-xylan, and PCA-xylan showed a greater efficacy against E. coli, Corynebacterium glutamicum, and Bacillus subtilis, respectively.

  12. The pattern of xylan acetylation suggests xylan may interact with cellulose microfibrils as a twofold helical screw in the secondary plant cell wall of Arabidopsis thaliana.

    PubMed

    Busse-Wicher, Marta; Gomes, Thiago C F; Tryfona, Theodora; Nikolovski, Nino; Stott, Katherine; Grantham, Nicholas J; Bolam, David N; Skaf, Munir S; Dupree, Paul

    2014-08-01

    The interaction between xylan and cellulose microfibrils is important for secondary cell wall properties in vascular plants; however, the molecular arrangement of xylan in the cell wall and the nature of the molecular bonding between the polysaccharides are unknown. In dicots, the xylan backbone of β-(1,4)-linked xylosyl residues is decorated by occasional glucuronic acid, and approximately one-half of the xylosyl residues are O-acetylated at C-2 or C-3. We recently proposed that the even, periodic spacing of GlcA residues in the major domain of dicot xylan might allow the xylan backbone to fold as a twofold helical screw to facilitate alignment along, and stable interaction with, cellulose fibrils; however, such an interaction might be adversely impacted by random acetylation of the xylan backbone. Here, we investigated the arrangement of acetyl residues in Arabidopsis xylan using mass spectrometry and NMR. Alternate xylosyl residues along the backbone are acetylated. Using molecular dynamics simulation, we found that a twofold helical screw conformation of xylan is stable in interactions with both hydrophilic and hydrophobic cellulose faces. Tight docking of xylan on the hydrophilic faces is feasible only for xylan decorated on alternate residues and folded as a twofold helical screw. The findings suggest an explanation for the importance of acetylation for xylan-cellulose interactions, and also have implications for our understanding of cell wall molecular architecture and properties, and biological degradation by pathogens and fungi. They will also impact strategies to improve lignocellulose processing for biorefining and bioenergy. © 2014 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.

  13. The pattern of xylan acetylation suggests xylan may interact with cellulose microfibrils as a twofold helical screw in the secondary plant cell wall of Arabidopsis thaliana

    PubMed Central

    Busse-Wicher, Marta; Gomes, Thiago C F; Tryfona, Theodora; Nikolovski, Nino; Stott, Katherine; Grantham, Nicholas J; Bolam, David N; Skaf, Munir S; Dupree, Paul

    2014-01-01

    The interaction between xylan and cellulose microfibrils is important for secondary cell wall properties in vascular plants; however, the molecular arrangement of xylan in the cell wall and the nature of the molecular bonding between the polysaccharides are unknown. In dicots, the xylan backbone of β-(1,4)-linked xylosyl residues is decorated by occasional glucuronic acid, and approximately one-half of the xylosyl residues are O-acetylated at C-2 or C-3. We recently proposed that the even, periodic spacing of GlcA residues in the major domain of dicot xylan might allow the xylan backbone to fold as a twofold helical screw to facilitate alignment along, and stable interaction with, cellulose fibrils; however, such an interaction might be adversely impacted by random acetylation of the xylan backbone. Here, we investigated the arrangement of acetyl residues in Arabidopsis xylan using mass spectrometry and NMR. Alternate xylosyl residues along the backbone are acetylated. Using molecular dynamics simulation, we found that a twofold helical screw conformation of xylan is stable in interactions with both hydrophilic and hydrophobic cellulose faces. Tight docking of xylan on the hydrophilic faces is feasible only for xylan decorated on alternate residues and folded as a twofold helical screw. The findings suggest an explanation for the importance of acetylation for xylan–cellulose interactions, and also have implications for our understanding of cell wall molecular architecture and properties, and biological degradation by pathogens and fungi. They will also impact strategies to improve lignocellulose processing for biorefining and bioenergy. PMID:24889696

  14. Effects of cationic xylan from annual plants on the mechanical properties of paper.

    PubMed

    Deutschle, Alexander L; Römhild, Katrin; Meister, Frank; Janzon, Ron; Riegert, Christiane; Saake, Bodo

    2014-02-15

    Xylan from oat spelt and wheat was used as an additive to enhance the dry strength of paper. The absorption of xylan by the cellulose fibers was increased by cationization to different degrees of substitution. Paper hand sheets with different doses of xylan and industrial cationic starch were produced, and the mechanical properties were determined. Absorption measurements of cationic oat spelt xylan on pulp fibers explained the differing influences of low and high cationized xylan addition on paper strength. The addition of cationic oat spelt xylan with a degree of substitution of 0.1 at a 4% dose provided the largest improvement in the tensile-index (67%), burst-index (105%) and tear-index (77%). Compared to cationic starch, cationic oat spelt xylan additives led to similar paper strength values, excepting the tear strength. The structural differences and protein impurities made the wheat xylan unsuitable as a strength additive for paper pulp. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. Hydrolysis kinetics of tulip tree xylan in hot compressed water.

    PubMed

    Yoon, Junho; Lee, Hun Wook; Sim, Seungjae; Myint, Aye Aye; Park, Hee Jeong; Lee, Youn-Woo

    2016-08-01

    Lignocellulosic biomass, a promising renewable resource, can be converted into numerous valuable chemicals post enzymatic saccharification. However, the efficacy of enzymatic saccharification of lignocellulosic biomass is low; therefore, pretreatment is necessary to improve the efficiency. Here, a kinetic analysis was carried out on xylan hydrolysis, after hot compressed water pretreatment of the lignocellulosic biomass conducted at 180-220°C for 5-30min, and on subsequent xylooligosaccharide hydrolysis. The weight ratio of fast-reacting xylan to slow-reacting xylan was 5.25 in tulip tree. Our kinetic results were applied to three different reaction systems to improve the pretreatment efficiency. We found that semi-continuous reactor is promising. Lower reaction temperatures and shorter space times in semi-continuous reactor are recommended for improving xylan conversion and xylooligosaccharide yield. In the theoretical calculation, 95% of xylooligosaccharide yield and xylan conversion were achieved simultaneously with high selectivity (desired product/undesired product) of 100 or more. Copyright © 2016. Published by Elsevier Ltd.

  16. Cellulose- and xylan-degrading thermophilic anaerobic bacteria from biocompost.

    PubMed

    Sizova, M V; Izquierdo, J A; Panikov, N S; Lynd, L R

    2011-04-01

    Nine thermophilic cellulolytic clostridial isolates and four other noncellulolytic bacterial isolates were isolated from self-heated biocompost via preliminary enrichment culture on microcrystalline cellulose. All cellulolytic isolates grew vigorously on cellulose, with the formation of either ethanol and acetate or acetate and formate as principal fermentation products as well as lactate and glycerol as minor products. In addition, two out of nine cellulolytic strains were able to utilize xylan and pretreated wood with roughly the same efficiency as for cellulose. The major products of xylan fermentation were acetate and formate, with minor contributions of lactate and ethanol. Phylogenetic analyses of 16S rRNA and glycosyl hydrolase family 48 (GH48) gene sequences revealed that two xylan-utilizing isolates were related to a Clostridium clariflavum strain and represent a distinct novel branch within the GH48 family. Both isolates possessed high cellulase and xylanase activity induced independently by either cellulose or xylan. Enzymatic activity decayed after growth cessation, with more-rapid disappearance of cellulase activity than of xylanase activity. A mixture of xylan and cellulose was utilized simultaneously, with a significant synergistic effect observed as a reduction of lag phase in cellulose degradation.

  17. Xylan - A potential contaminant for lunar samples and Antarctic meteorites

    NASA Astrophysics Data System (ADS)

    Wright, I. P.; Russell, S. S.; Boyd, S. R.; Meyer, C.; Pillinger, C. T.

    The possibility that lunar samples have been contaminated by the proprietary lubricant paint known as Xylan, which has been applied to screw threads in dry-N sample processing cabinets at NASA JSC, is considered. From a sample analysis using sealed-tube and stepped combustion, it is argued that the unexpectedly high concentration of organic materials found in EET A79001 is not due to Xylan contamination. It is considered unlikely that previous C and N analyses of lunar samples have been affected by the introduction of Xylan.

  18. Chemical depolymerization of switchgrass xylan with oligosaccharide product analysis by HPAEC-PAD and mass spectrometry

    USDA-ARS?s Scientific Manuscript database

    Xylan is a barrier to enzymatic hydrolysis of plant cell walls. It is well accepted that the xylan layer needs to be removed to efficiently hydrolyze cellulose and consequently pretreatment conditions are in part optimized for maximal xylan depolymerization or displacement. Xylan consists of a lon...

  19. Study on the Modification of Bleached Eucalyptus Kraft Pulp Using Birch Xylan

    Treesearch

    Wenjia Han; Chuanshan Zhao; Thomas Elder; Rendang Yang; Dongho Kim; Yunqiao Pu; Jeffery Hsieh; Arthur J. Ragauskas

    2012-01-01

    In this study, birch xylan was deposited onto elementally chlorine free (ECF) bleached eucalyptus kraft pulp, and the corresponding changes in physical properties were determined. An aqueous 5% birch xylan solution at pH 9 was added to 5 wt% slurry of bleached kraft eucalyptus fibers, with stirring at 70 C for 15 min after which the pH was adjusted to 5–6. The xylan...

  20. Deep Eutectic Solvent Aqueous Solutions as Efficient Media for the Solubilization of Hardwood Xylans.

    PubMed

    Morais, Eduarda S; Mendonça, Patrícia V; Coelho, Jorge F J; Freire, Mara G; Freire, Carmen S R; Coutinho, João A P; Silvestre, Armando J D

    2018-02-22

    This work contributes to the development of integrated lignocellulosic-based biorefineries by the pioneering exploitation of hardwood xylans by solubilization and extraction in deep eutectic solvents (DES). DES formed by choline chloride and urea or acetic acid were initially evaluated as solvents for commercial xylan as a model compound. The effects of temperature, molar ratio, and concentration of the DES aqueous solutions were evaluated and optimized by using a response surface methodology. The results obtained demonstrated the potential of these solvents, with 328.23 g L -1 of xylan solubilization using 66.7 wt % DES in water at 80 °C. Furthermore, xylans could be recovered by precipitation from the DES aqueous media in yields above 90 %. The detailed characterization of the xylans recovered after solubilization in aqueous DES demonstrated that 4-O-methyl groups were eliminated from the 4-O-methylglucuronic acids moieties and uronic acids (15 %) were cleaved from the xylan backbone during this process. The similar M w values of both pristine and recovered xylans confirmed the success of the reported procedure. DES recovery in four additional extraction cycles was also demonstrated. Finally, the successful extraction of xylans from Eucalyptus globulus wood by using aqueous solutions of DES was demonstrated. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Properties of polyvinyl alcohol/xylan composite films with citric acid.

    PubMed

    Wang, Shuaiyang; Ren, Junli; Li, Weiying; Sun, Runcang; Liu, Shijie

    2014-03-15

    Composite films of xylan and polyvinyl alcohol were produced with citric acid as a new plasticizer or a cross-linking agent. The effects of citric acid content and polyvinyl alcohol/xylan weight ratio on the mechanical properties, thermal stability, solubility, degree of swelling and water vapor permeability of the composite films were investigated. The intermolecular interactions and morphology of composite films were characterized by FTIR spectroscopy and SEM. The results indicated that polyvinyl alcohol/xylan composite films had good compatibility. With an increase in citric acid content from 10% to 50%, the tensile strength reduced from 35.1 to 11.6 MPa. However, the elongation at break increased sharply from 15.1% to 249.5%. The values of water vapor permeability ranged from 2.35 to 2.95 × 10(-7)g/(mm(2)h). Interactions between xylan and polyvinyl alcohol in the presence of citric acid become stronger, which were caused by hydrogen bond and ester bond formation among the components during film forming. Copyright © 2013. Published by Elsevier Ltd.

  2. Preparation and characterization of safe microparticles based on xylan.

    PubMed

    Cartaxo da Costa Urtiga, Silvana; Aquino Azevedo de Lucena Gabi, Camilla; Rodrigues de Araújo Eleamen, Giovanna; Santos Souza, Bartolomeu; Pessôa, Hilzeth de Luna Freire; Marcelino, Henrique Rodrigues; Afonso de Moura Mendonça, Elisângela; Egito, Eryvaldo Sócrates Tabosa do; Oliveira, Elquio Eleamen

    2017-10-01

    This work describes the preparation and evaluation of safe xylan-based microparticles prepared by cross-linking polymerization using sodium trimetaphosphate. The resulting microparticles were evaluated for morphology, particle size, polymer-cross-link agent interaction, and in vitro toxicity. The microparticles showed narrow monodisperse size distributions with their mean sizes being between 3.5 and 12.5 µm in dried state. FT-IR analyzes confirmed the interaction between sodium trimetaphosphate and xylan during the cross-linking process with formation of phosphate ester bonds. Additionally, the X-ray diffraction patterns and FT-IR analyzes suggested that little or no cross-linking agent remained inside the microparticles. Furthermore, the in-vitro studies using Artemia salina and human erythrocytes revealed that the microparticles are not toxic. Therefore, the overall results suggest that these xylan microparticles can be used as a platform for new drug delivery system.

  3. Glycosyl transferases in family 61 mediate arabinofuranosyl transfer onto xylan in grasses.

    PubMed

    Anders, Nadine; Wilkinson, Mark D; Lovegrove, Alison; Freeman, Jacqueline; Tryfona, Theodora; Pellny, Till K; Weimar, Thilo; Mortimer, Jennifer C; Stott, Katherine; Baker, John M; Defoin-Platel, Michael; Shewry, Peter R; Dupree, Paul; Mitchell, Rowan A C

    2012-01-17

    Xylan, a hemicellulosic component of the plant cell wall, is one of the most abundant polysaccharides in nature. In contrast to dicots, xylan in grasses is extensively modified by α-(1,2)- and α-(1,3)-linked arabinofuranose. Despite the importance of grass arabinoxylan in human and animal nutrition and for bioenergy, the enzymes adding the arabinosyl substitutions are unknown. Here we demonstrate that knocking-down glycosyltransferase (GT) 61 expression in wheat endosperm strongly decreases α-(1,3)-linked arabinosyl substitution of xylan. Moreover, heterologous expression of wheat and rice GT61s in Arabidopsis leads to arabinosylation of the xylan, and therefore provides gain-of-function evidence for α-(1,3)-arabinosyltransferase activity. Thus, GT61 proteins play a key role in arabinoxylan biosynthesis and therefore in the evolutionary divergence of grass cell walls.

  4. Xylan from corn cobs, a promising polymer for drug delivery: production and characterization.

    PubMed

    Oliveira, Elquio Eleamen; Silva, Acarília Eduardo; Júnior, Toshiyuki Nagashima; Gomes, Monique Christine Salgado; Aguiar, Larissa Muratori; Marcelino, Henrique Rodrigues; Araújo, Ivonete Batista; Bayer, Marc P; Ricardo, Nágila M P S; Oliveira, Anselmo Gomes; Egito, Eryvaldo Sócrates Tabosa

    2010-07-01

    Although many authors have reported several beneficial effects ascribed to xylan, such as inhibitory action on mutagenicity activity, antiphlogistic effects, and mitogenic and comitogenic activities, few papers have investigated a systematic study on the technological properties of this polymer. The aim of the present work was to evaluate xylan as a promise raw material for the pharmaceutical industry. The water-insoluble xylan samples were extracted from corn cobs following several steps. The obtained powered sample was analyzed by infrared and RMN spectroscopy, and characterized regarding their particle size, bulk and tap densities, compressibility index, compactability, Hausner ratio, and angle of repose. According to the results, infrared and RMN spectroscopy were shown to be able to evaluate the xylan structural conformation and composition, respectively. In addition, rheological data demonstrated that xylan powder obtained from corn cobs may be characterized as a material with low density and very cohesive flow properties. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  5. Phenylacetic and Phenylpropionic Acids Do Not Affect Xylan Degradation by Ruminococcus albus

    PubMed Central

    Reveneau, Carine; Adams, Sarah E.; Cotta, M. A.; Morrison, M.

    2003-01-01

    Since the addition of either ruminal fluid or a combination of phenylacetic and phenylpropionic acids (PAA/PPA) has previously been shown to dramatically improve cellulose degradation and growth of Ruminococcus albus, it was of interest to determine the effects of these additives on xylan-grown cultures. Although cell-bound xylanase activity increased when either PAA/PPA or ruminal fluid was added to the growth medium, total xylanase did not change, and neither of these supplements affected the growth or xylan-degrading capacity of R. albus 8. Similarly, neither PAA/PPA nor ruminal fluid affected xylan degradation by multiple strains of R. albus when xylan prepared from oat spelts was used as a carbohydrate source. These results show that the xylanolytic potential of R. albus is not conditional on the availability of PAA/PPA or other components of ruminal fluid. PMID:14602663

  6. High oxygen nanocomposite barrier films based on xylan and nanocrystalline cellulose

    Treesearch

    Amit Saxena; Thomas J. Elder; Jeffrey Kenvin; Arthur J. Ragauskas

    2010-01-01

    The goal of this work is to produce nanocomposite film with low oxygen permeability by casting an aqueous solution containing xylan, sorbitol and nanocrystalline cellulose. The morphology of the resulting nanocomposite films was examined by scanning electron microscopy and atomic force microscopy which showed that control films containing xylan and sorbitol had a more...

  7. Selective chemical oxidation and depolymerization of switchgrass (Panicum virgatum L.) xylan with oligosaccharide product analysis by mass spectrometry

    USDA-ARS?s Scientific Manuscript database

    Xylan is a barrier to enzymatic hydrolysis of plant cell walls. It is well accepted that the xylan layer needs to be removed to efficiently hydrolyze cellulose and consequently pretreatment conditions are in part optimized for maximal xylan depolymerization or displacement. Xylan consists of a long ...

  8. Mutations of Arabidopsis TBL32 and TBL33 affect xylan acetylation and secondary wall deposition

    DOE PAGES

    Yuan, Youxi; Teng, Quincy; Zhong, Ruiqin; ...

    2016-01-08

    Xylan is a major acetylated polymer in plant lignocellulosic biomass and it can be monoand di-acetylated at O-2 and O-3 as well as mono-acetylated at O-3 of xylosyl residues that is substituted with glucuronic acid (GlcA) at O-2. Based on the finding that ESK1, an Arabidopsis thaliana DUF231 protein, specifically mediates xylan 2-O- and 3-O-monoacetylation, we previously proposed that different acetyltransferase activities are required for regiospecific acetyl substitutions of xylan. Here, we demonstrate the functional roles of TBL32 and TBL33, two ESK1 close homologs, in acetyl substitutions of xylan. Simultaneous mutations of TBL32 and TBL33 resulted in a significant reductionmore » in xylan acetyl content and endoxylanase digestion of the mutant xylan released GlcA-substituted xylooligomers without acetyl groups. Structural analysis of xylan revealed that the tbl32 tbl33 mutant had a nearly complete loss of 3-O-acetylated, 2-O-GlcA-substituted xylosyl residues. A reduction in 3-Omonoacetylated and 2,3-di-O-acetylated xylosyl residues was also observed. Simultaneous mutations of TBL32, TBL33 and ESK1 resulted in a severe reduction in xylan acetyl level down to 15% of that of the wild type, and concomitantly, severely collapsed vessels and stunted plant growth. In particular, the S2 layer of secondary walls in xylem vessels of tbl33 esk1 and tbl32 tbl33 esk1 exhibited an altered structure, indicating abnormal assembly of secondary wall polymers. Furthermore, these results demonstrate that TBL32 and TBL33 play an important role in xylan acetylation and normal deposition of secondary walls.« less

  9. Mutations of Arabidopsis TBL32 and TBL33 affect xylan acetylation and secondary wall deposition

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yuan, Youxi; Teng, Quincy; Zhong, Ruiqin

    Xylan is a major acetylated polymer in plant lignocellulosic biomass and it can be monoand di-acetylated at O-2 and O-3 as well as mono-acetylated at O-3 of xylosyl residues that is substituted with glucuronic acid (GlcA) at O-2. Based on the finding that ESK1, an Arabidopsis thaliana DUF231 protein, specifically mediates xylan 2-O- and 3-O-monoacetylation, we previously proposed that different acetyltransferase activities are required for regiospecific acetyl substitutions of xylan. Here, we demonstrate the functional roles of TBL32 and TBL33, two ESK1 close homologs, in acetyl substitutions of xylan. Simultaneous mutations of TBL32 and TBL33 resulted in a significant reductionmore » in xylan acetyl content and endoxylanase digestion of the mutant xylan released GlcA-substituted xylooligomers without acetyl groups. Structural analysis of xylan revealed that the tbl32 tbl33 mutant had a nearly complete loss of 3-O-acetylated, 2-O-GlcA-substituted xylosyl residues. A reduction in 3-Omonoacetylated and 2,3-di-O-acetylated xylosyl residues was also observed. Simultaneous mutations of TBL32, TBL33 and ESK1 resulted in a severe reduction in xylan acetyl level down to 15% of that of the wild type, and concomitantly, severely collapsed vessels and stunted plant growth. In particular, the S2 layer of secondary walls in xylem vessels of tbl33 esk1 and tbl32 tbl33 esk1 exhibited an altered structure, indicating abnormal assembly of secondary wall polymers. Furthermore, these results demonstrate that TBL32 and TBL33 play an important role in xylan acetylation and normal deposition of secondary walls.« less

  10. Effects of Impurities in Alkali-Extracted Xylan on Its Enzymatic Hydrolysis to Produce Xylo-Oligosaccharides.

    PubMed

    Shen, Rui; Li, Hong-Qiang; Zhang, Jie; Xu, Jian

    2016-07-01

    As the second abundant natural carbohydrate, xylan is normally prepared through alkaline extraction and then used for xylo-oligosaccharides (XOS) production. However, the extracted xylan inevitably contains salt, ethanol, and pigment. In order to investigate the effects of these impurities on XOS production, the alkaline-extracted xylan with different kinds and concentrations of impurities was made and then hydrolyzed using alkaline xylanase (EC 3.2.1.8) to produce XOS. The results showed that a certain concentration of salt (NaCl) promoted the XOS production, while ethanol and pigment inhibited the enzymatic hydrolysis process significantly. The color value mainly ascribed to the phenolic compounds binding to xylan was a key restriction factor in the enzymatic hydrolysis later stage. Using optimal xylan sample (with 10 mg/mL NaCl, color value of 4.6 × 10(5), without ethanol) as substrate, the highest XOS yield of 58.58 % was obtained. As the substrate of XOS production, prepared xylan should contain colored materials and ethanol as less as possible, however, retains appropriate salt.

  11. Bioconversion of Xylan to triglycerides by oil-rich yeasts. [Cryptococcus albidus; Cryptococcus terricoluus; Trichosporon

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Fall, R.; Phelps, P.; Spindler, D.

    A series of lipid-accumulating yeasts was examined for their potential to saccharify xylan and accumulate triglyceride. Of the genera tested, including Candida, Cryptococcus, Lipomyces, Rhodosporidium, Rhodotorula, and Trichosporon, only Crytococcus and Trichosporon isolates saccharified xylan. All of the strains could assimilate xylose and accumuate triglyceride under nitrogen-limiting conditions. Strains of Cryptococcus albidus were found to be especially useful for a one-step saccharification of xylan coupled to triglyceride synthesis. Crytococcus terricolus, a strain constitutive for lipid accumulation, lacked extracellular xylanase, but did assimilate xylose and xylobiose and was able to continuously convert xylan to triglyceride if the culture medium was supplementedmore » with xylanase. 22 references.« less

  12. Preparation, characterization and in vitro anticoagulant activity of corn stover xylan sulfates.

    PubMed

    Cheng, He-Li; Liu, Hao; Feng, Qing-Hua; Xie, Yi-Min; Zhan, Huai-Yu

    2017-02-01

    A new anticoagulant agent was prepared by introducing sulfate groups into corn stover xylan through homogeneous reactions. Three organic solvents, N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO) and formamide (FA), were adopted as reaction media, with the assistance of LiCl. Structural characterization by FT-IR and 13 CNMR showed that xylan sulfate (XS) could be successfully synthesized with SO 3 ∙Pyridine (SO 3 ∙Py) complexes sulfation reagent in the three media. The effect of sulfation temperature, sulfation time, media type and molar ratio of -SO 3 /-OH on the degree of substitution (DS) and degree of the polymerization (DP) were studied. DMF/LiCl were more effective than DMSO/LiCl and FA/LiCl in preparation of xylan sulfate with high DS. The optimal conditions for sulfation were obtained when SO 3 ∙Py complex was added to DMF/LiCl with -SO 3 /-OH ratio of 1.5:1 and maintained at 50 °C for 3 h. Degree of polymerization of xylan was decreased during the sulfation process and DMF/LiCl offered the least xylan degradation as compared with DMSO/LiCl or FA/LiCl. Anticoagulant activities of the resultant xylan sulfates with different DS were evaluated by using activated partial thromboplastin time (APTT), thrombin time (TT), and prothrombin time (PT). Results indicated that the introducing of sulfate groups into xylan did endow the polysaccharides with anticoagulant activity. The APTT and TT of XS with DS of 1.20 reached 141 and 45.3 s at a dosage of 20 μg/mL, while the APTT and TT values for the blank sample were only 35.5 and 15.6 s. Furthermore, coagulation time was prolonged with the increase of DS and the concentration of XS. Our findings provide new insights into the value-added utilization of agricultural biomass.

  13. A Highly Efficient Xylan-Utilization System in Aspergillus niger An76: A Functional-Proteomics Study.

    PubMed

    Gong, Weili; Dai, Lin; Zhang, Huaiqiang; Zhang, Lili; Wang, Lushan

    2018-01-01

    Xylan constituted with β-1,4-D-xylose linked backbone and diverse substituted side-chains is the most abundant hemicellulose component of biomass, which can be completely and rapidly degraded into fermentable sugars by Aspergillus niger . This is of great value for obtaining renewable biofuels and biochemicals. To clarify the underlying mechanisms associated with highly efficient xylan degradation, assimilation, and metabolism by A. niger , we utilized functional proteomics to analyze the secreted proteins, sugar transporters, and intracellular proteins of A. niger An76 grown on xylan-based substrates. Results demonstrated that the complete xylanolytic enzyme system required for xylan degradation and composed of diverse isozymes was secreted in a sequential order. Xylan-backbone-degrading enzymes were preferentially induced by xylose or other soluble sugars, which efficiently produced large amounts of xylooligosaccharides (XOS) and xylose; however, XOS was more efficient than xylose in triggering the expression of the key transcription activator XlnR, resulting in higher xylanase activity and shortening xylanase-production time. Moreover, the substituted XOS was responsible for improving the abundance of side-chain-degrading enzymes, specific transporters, and key reductases and dehydrogenases in the pentose catabolic pathway. Our findings indicated that industries might be able to improve the species and concentrations of xylan-degrading enzymes and shorten fermentation time by adding abundant intermediate products of natural xylan (XOS) to cultures of filamentous fungi.

  14. A Highly Efficient Xylan-Utilization System in Aspergillus niger An76: A Functional-Proteomics Study

    PubMed Central

    Gong, Weili; Dai, Lin; Zhang, Huaiqiang; Zhang, Lili; Wang, Lushan

    2018-01-01

    Xylan constituted with β-1,4-D-xylose linked backbone and diverse substituted side-chains is the most abundant hemicellulose component of biomass, which can be completely and rapidly degraded into fermentable sugars by Aspergillus niger. This is of great value for obtaining renewable biofuels and biochemicals. To clarify the underlying mechanisms associated with highly efficient xylan degradation, assimilation, and metabolism by A. niger, we utilized functional proteomics to analyze the secreted proteins, sugar transporters, and intracellular proteins of A. niger An76 grown on xylan-based substrates. Results demonstrated that the complete xylanolytic enzyme system required for xylan degradation and composed of diverse isozymes was secreted in a sequential order. Xylan-backbone-degrading enzymes were preferentially induced by xylose or other soluble sugars, which efficiently produced large amounts of xylooligosaccharides (XOS) and xylose; however, XOS was more efficient than xylose in triggering the expression of the key transcription activator XlnR, resulting in higher xylanase activity and shortening xylanase-production time. Moreover, the substituted XOS was responsible for improving the abundance of side-chain-degrading enzymes, specific transporters, and key reductases and dehydrogenases in the pentose catabolic pathway. Our findings indicated that industries might be able to improve the species and concentrations of xylan-degrading enzymes and shorten fermentation time by adding abundant intermediate products of natural xylan (XOS) to cultures of filamentous fungi. PMID:29623069

  15. Degradation and utilization of xylan by the ruminal bacteria Butyrivibrio fibrisolvens and Selenomonas ruminantium.

    PubMed

    Cotta, M A; Zeltwanger, R L

    1995-12-01

    The cross-feeding of xyland hydrolysis products between the xylanolytic bacterium Butyrivibrio fibrisolvens H17c and the xylooligosaccharide-fermenting bacterium Selenomonas ruminantium GA192 was investigated. Cultures were grown anaerobically in complex medium containing oat spelt xylan, and the digestion of xylan and the generation and subsequent utilization of xylooligosaccharide intermediates were monitored over time. Monocultures of B. fibrisolvens rapidly degraded oat spelt xylan, and a pool of extracellular degradation intermediates composed of low-molecular-weight xylooligosaccharides (xylobiose through xylopentaose and larger, unidentified oligomers) accumulated in these cultures. The ability of S. ruminantium to utilize the products of xylanolysis by B. fibrisolvens was demonstrated by its ability to grow on xylan that had first been digested by the extracellular xylanolytic enzymes of B. fibrisolvens. Although enzymatic hydrolysis converted the xylan to soluble products, this alone was not sufficient to assure complete utilization by S. ruminantium, and considerable quantities of oligosaccharides remained following growth. Stable xylan-utilizing cocultures of S. ruminantium and B. fibrisolvens were established, and the utilization of xylan was monitored. Despite the presence of an oligosaccharide-fermenting organism, accumulations of acid-alcohol soluble products were still noted; however, the composition of carbohydrates present in these cultures differed from that seen when B. fibrisolvens was cultivated alone. Residual carbohydrates present at various times during growth were of higher average degree of polymerization in cocultures than in cultures of B. fibrisolvens alone. Structural characterization of these residual products may help define the limitations on the assimilation of xylooligosaccharides by ruminal bacteria.

  16. Candida xylanilytica sp. nov., a xylan-degrading yeast species isolated from Thailand.

    PubMed

    Boonmak, Chanita; Limtong, Savitree; Jindamorakot, Sasitorn; Am-In, Somjit; Yongmanitchai, Wichien; Suzuki, Ken-ichiro; Nakase, Takashi; Kawasaki, Hiroko

    2011-05-01

    Xylan is a major component of hemicellulose, which constitutes about 40 % of plant biomass. Hydrolysis of xylan into simple sugars is one of the important steps in the conversion of lignocellulosic material to value-added products. During an investigation of cellulose- and xylan-degrading yeasts, two yeast strains that were able to use cellulose and xylan as sole carbon source were found to represent a phylogenetically distinct species in the Spathaspora clade. The closest species in terms of pairwise sequence similarity in the D1/D2 domain of the LSU rRNA gene was Candida subhashii. The novel species can be distinguished from the other species in the Spathaspora clade based on the ability to assimilate methanol and raffinose, growth in medium containing 60 % glucose, and growth at 42 °C. It ferments glucose but not other carbohydrates. The name Candida xylanilytica sp. nov. is proposed for this species. The type strain is KU-Xn11(T) ( = NBRC 106499(T)  = BCC 34694(T)  = CBS 11761(T)).

  17. In situ enzyme aided adsorption of soluble xylan biopolymers onto cellulosic material.

    PubMed

    Chimphango, Annie F A; Görgens, J F; van Zyl, W H

    2016-06-05

    The functional properties of cellulose fibers can be modified by adsorption of xylan biopolymers. The adsorption is improved when the degree of biopolymers substitution with arabinose and 4-O-methyl-glucuronic acid (MeGlcA) side groups, is reduced. α-l-Arabinofuranosidase (AbfB) and α-d-glucuronidase (AguA) enzymes were applied for side group removal, to increase adsorption of xylan from sugarcane (Saccharum officinarum L) bagasse (BH), bamboo (Bambusa balcooa) (BM), Pinus patula (PP) and Eucalyptus grandis (EH) onto cotton lint. The AguA treatment increased the adsorption of all xylans by up to 334%, whereas, the AbfB increased the adsorption of the BM and PP by 31% and 44%, respectively. A combination of AguA and AbfB treatment increased the adsorption, but to a lesser extent than achieved with AguA treatment. This indicated that the removal of the glucuronic acid side groups provided the most significant increase in xylan adsorption to cellulose, in particular through enzymatic treatment. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. A novel xylan degrading β-D-xylosidase: purification and biochemical characterization.

    PubMed

    Michelin, Michele; Peixoto-Nogueira, Simone C; Silva, Tony M; Jorge, João A; Terenzi, Héctor F; Teixeira, José A; Polizeli, Maria de Lourdes T M

    2012-11-01

    Aspergillus ochraceus, a thermotolerant fungus isolated in Brazil from decomposing materials, produced an extracellular β-xylosidase that was purified using DEAE-cellulose ion exchange chromatography, Sephadex G-100 and Biogel P-60 gel filtration. β-xylosidase is a glycoprotein (39 % carbohydrate content) and has a molecular mass of 137 kDa by SDS-PAGE, with optimal temperature and pH at 70 °C and 3.0-5.5, respectively. β-xylosidase was stable in acidic pH (3.0-6.0) and 70 °C for 1 h. The enzyme was activated by 5 mM MnCl₂ (28 %) and MgCl₂ (20 %) salts. The β-xylosidase produced by A. ochraceus preferentially hydrolyzed p-nitrophenyl-β-D-xylopyranoside, exhibiting apparent K(m) and V(max) values of 0.66 mM and 39 U (mg protein)⁻¹ respectively, and to a lesser extent p-nitrophenyl-β-D-glucopyranoside. The enzyme was able to hydrolyze xylan from different sources, suggesting a novel β-D-xylosidase that degrades xylan. HPLC analysis revealed xylans of different compositions which allowed explaining the differences in specificity observed by β-xylosidase. TLC confirmed the capacity of the enzyme in hydrolyzing xylan and larger xylo-oligosaccharides, as xylopentaose.

  19. Engineering the Xylan Utilization System in Bacillus subtilis for Production of Acidic Xylooligosaccharides

    Treesearch

    Mun Su Rhee; Lusha Wei; Neha Sawhney; John D. Rice; Franz J. St. John; Jason C. Hurlbert; James F. Preston

    2014-01-01

    Xylans are the predominant polysaccharides in hemicelluloses and an important potential source of biofuels and chemicals. The ability of Bacillus subtilis subsp. subtilis strain 168 to utilize xylans has been ascribed to secreted glycoside hydrolase family 11 (GH11) and GH30 endoxylanases, encoded by the xynA and...

  20. Conversion of xylan by recyclable spores of Bacillus subtilis displaying thermophilic enzymes.

    PubMed

    Mattossovich, Rosanna; Iacono, Roberta; Cangiano, Giuseppina; Cobucci-Ponzano, Beatrice; Isticato, Rachele; Moracci, Marco; Ricca, Ezio

    2017-11-28

    The Bacillus subtilis spore has long been used to display antigens and enzymes. Spore display can be accomplished by a recombinant and a non-recombinant approach, with the latter proved more efficient than the recombinant one. We used the non-recombinant approach to independently adsorb two thermophilic enzymes, GH10-XA, an endo-1,4-β-xylanase (EC 3.2.1.8) from Alicyclobacillus acidocaldarius, and GH3-XT, a β-xylosidase (EC 3.2.1.37) from Thermotoga thermarum. These enzymes catalyze, respectively, the endohydrolysis of (1-4)-β-D-xylosidic linkages of xylans and the hydrolysis of (1-4)-β-D-xylans to remove successive D-xylose residues from the non-reducing termini. We report that both purified enzymes were independently adsorbed on purified spores of B. subtilis. The adsorption was tight and both enzymes retained part of their specific activity. When spores displaying either GH10-XA or GH3-XT were mixed together, xylan was hydrolysed more efficiently than by a mixture of the two free, not spore-adsorbed, enzymes. The high total activity of the spore-bound enzymes is most likely due to a stabilization of the enzymes that, upon adsorption on the spore, remained active at the reaction conditions for longer than the free enzymes. Spore-adsorbed enzymes, collected after the two-step reaction and incubated with fresh substrate, were still active and able to continue xylan degradation. The recycling of the mixed spore-bound enzymes allowed a strong increase of xylan degradation. Our results indicate that the two-step degradation of xylans can be accomplished by mixing spores displaying either one of two required enzymes. The two-step process occurs more efficiently than with the two un-adsorbed, free enzymes and adsorbed spores can be reused for at least one other reaction round. The efficiency of the process, the reusability of the adsorbed enzymes, and the well documented robustness of spores of B. subtilis indicate the spore as a suitable platform to display enzymes

  1. Xylan hydrolysis in Populus trichocarpa × P. deltoides and model substrates during hydrothermal pretreatment.

    PubMed

    Trajano, Heather L; Pattathil, Sivakumar; Tomkins, Bruce A; Tschaplinski, Timothy J; Hahn, Michael G; Van Berkel, Gary J; Wyman, Charles E

    2015-03-01

    Previous studies defined easy and difficult to hydrolyze fractions of hemicellulose that may result from bonds among cellulose, hemicellulose, and lignin. To understand how such bonds affect hydrolysis, Populus trichocarpa × Populus deltoides, holocellulose isolated from P. trichocarpa × P. deltoides and birchwood xylan were subjected to hydrothermal flow-through pretreatment. Samples were characterized by glycome profiling, HPLC, and UPLC-MS. Glycome profiling revealed steady fragmentation and removal of glycans from solids during hydrolysis. The extent of polysaccharide fragmentation, hydrolysis rate, and total xylose yield were lowest for P. trichocarpa × P. deltoides and greatest for birchwood xylan. Comparison of results from P. trichocarpa × P. deltoides and holocellulose suggested that lignin-carbohydrate complexes reduce hydrolysis rates and limit release of large xylooligomers. Smaller differences between results with holocellulose and birchwood xylan suggest xylan-cellulose hydrogen bonds limited hydrolysis, but to a lesser extent. These findings imply cell wall structure strongly influences hydrolysis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Engineering of plants with improved properties as biofuels feedstocks by vessel-specific complementation of xylan biosynthesis mutants

    PubMed Central

    2012-01-01

    Background Cost-efficient generation of second-generation biofuels requires plant biomass that can easily be degraded into sugars and further fermented into fuels. However, lignocellulosic biomass is inherently recalcitrant toward deconstruction technologies due to the abundant lignin and cross-linked hemicelluloses. Furthermore, lignocellulosic biomass has a high content of pentoses, which are more difficult to ferment into fuels than hexoses. Engineered plants with decreased amounts of xylan in their secondary walls have the potential to render plant biomass a more desirable feedstock for biofuel production. Results Xylan is the major non-cellulosic polysaccharide in secondary cell walls, and the xylan deficient irregular xylem (irx) mutants irx7, irx8 and irx9 exhibit severe dwarf growth phenotypes. The main reason for the growth phenotype appears to be xylem vessel collapse and the resulting impaired transport of water and nutrients. We developed a xylan-engineering approach to reintroduce xylan biosynthesis specifically into the xylem vessels in the Arabidopsis irx7, irx8 and irx9 mutant backgrounds by driving the expression of the respective glycosyltransferases with the vessel-specific promoters of the VND6 and VND7 transcription factor genes. The growth phenotype, stem breaking strength, and irx morphology was recovered to varying degrees. Some of the plants even exhibited increased stem strength compared to the wild type. We obtained Arabidopsis plants with up to 23% reduction in xylose levels and 18% reduction in lignin content compared to wild-type plants, while exhibiting wild-type growth patterns and morphology, as well as normal xylem vessels. These plants showed a 42% increase in saccharification yield after hot water pretreatment. The VND7 promoter yielded a more complete complementation of the irx phenotype than the VND6 promoter. Conclusions Spatial and temporal deposition of xylan in the secondary cell wall of Arabidopsis can be manipulated by

  3. In vitro antioxidant, anticoagulant and antimicrobial activity and in inhibition of cancer cell proliferation by xylan extracted from corn cobs.

    PubMed

    Melo-Silveira, Raniere Fagundes; Fidelis, Gabriel Pereira; Costa, Mariana Santana Santos Pereira; Telles, Cinthia Beatrice Silva; Dantas-Santos, Nednaldo; de Oliveira Elias, Susana; Ribeiro, Vanessa Bley; Barth, Afonso Luis; Macedo, Alexandre José; Leite, Edda Lisboa; Rocha, Hugo Alexandre Oliveira

    2012-01-01

    Xylan is one of most abundant polymer after cellulose. However, its potential has yet to be completely recognized. Corn cobs contain a considerable reservoir of xylan. The aim of this work was to study some of the biological activities of xylan obtained from corn cobs after alkaline extraction enhanced by ultrasonication. Physical chemistry and infrared analyses showed 130 kDa heteroxylan containing mainly xylose:arabinose: galactose:glucose (5.0:1.5:2.0:1.2). Xylan obtained exhibited total antioxidant activity corresponding to 48.5 mg of ascorbic acid equivalent/g of xylan. Furthermore, xylan displayed high ferric chelating activity (70%) at 2 mg/mL. Xylan also showed anticoagulant activity in aPTT test. In antimicrobial assay, the polysaccharide significantly inhibited bacterial growth of Klebsiella pneumoniae. In a test with normal and tumor human cells, after 72 h, only HeLa tumor cell proliferation was inhibited (p < 0.05) in a dose-dependent manner by xylan, reaching saturation at around 2 mg/mL, whereas 3T3 normal cell proliferation was not affected. The results suggest that it has potential clinical applications as antioxidant, anticoagulant, antimicrobial and antiproliferative compounds.

  4. In Vitro Antioxidant, Anticoagulant and Antimicrobial Activity and in Inhibition of Cancer Cell Proliferation by Xylan Extracted from Corn Cobs

    PubMed Central

    Melo-Silveira, Raniere Fagundes; Fidelis, Gabriel Pereira; Costa, Mariana Santana Santos Pereira; Telles, Cinthia Beatrice Silva; Dantas-Santos, Nednaldo; de Oliveira Elias, Susana; Ribeiro, Vanessa Bley; Barth, Afonso Luis; Macedo, Alexandre José; Leite, Edda Lisboa; Rocha, Hugo Alexandre Oliveira

    2012-01-01

    Xylan is one of most abundant polymer after cellulose. However, its potential has yet to be completely recognized. Corn cobs contain a considerable reservoir of xylan. The aim of this work was to study some of the biological activities of xylan obtained from corn cobs after alkaline extraction enhanced by ultrasonication. Physical chemistry and infrared analyses showed 130 kDa heteroxylan containing mainly xylose:arabinose: galactose:glucose (5.0:1.5:2.0:1.2). Xylan obtained exhibited total antioxidant activity corresponding to 48.5 mg of ascorbic acid equivalent/g of xylan. Furthermore, xylan displayed high ferric chelating activity (70%) at 2 mg/mL. Xylan also showed anticoagulant activity in aPTT test. In antimicrobial assay, the polysaccharide significantly inhibited bacterial growth of Klebsiella pneumoniae. In a test with normal and tumor human cells, after 72 h, only HeLa tumor cell proliferation was inhibited (p < 0.05) in a dose-dependent manner by xylan, reaching saturation at around 2 mg/mL, whereas 3T3 normal cell proliferation was not affected. The results suggest that it has potential clinical applications as antioxidant, anticoagulant, antimicrobial and antiproliferative compounds. PMID:22312261

  5. Designer xylanosomes: protein nanostructures for enhanced xylan hydrolysis

    USDA-ARS?s Scientific Manuscript database

    This work is the first report of the successful design, construction, and application of multi-functional, self-assembling biocatalysts for targeted xylan hydrolysis, termed xylanosomes. Using the architecture of cellulosomes found in some anaerobic cellulolytic microbes, four different xylanosomes...

  6. An unusual xylan in Arabidopsis primary cell walls is synthesised by GUX3, IRX9L, IRX10L and IRX14

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mortimer, Jenny C.; Faria-Blanc, Nuno; Yu, Xiaolan

    Xylan is a crucial component of many plant primary and secondary cell walls. However, the structure and function of xylan in the dicotyledon primary cell wall is not well understood. Here, we characterized a xylan that is specific to tissues enriched in Arabidopsis primary cell walls. Unlike previously described xylans, this xylan carries a pentose linked 1–2 to the α-1,2-d-glucuronic acid (GlcA) side chains on the β-1,4-Xyl backbone. The frequent and precisely regular spacing of GlcA substitutions every six xylosyl residues along the backbone is also unlike that previously observed in secondary cell wall xylan. Molecular genetics, in vitro assays,more » and expression data suggest that IRX9L, IRX10L and IRX14 are required for xylan backbone synthesis in primary cell wall synthesising tissues. IRX9 and IRX10 are not involved in the primary cell wall xylan synthesis but are functionally exchangeable with IRX9L and IRX10L. GUX3 is the only glucuronyltransferase required for the addition of the GlcA decorations on the xylan. Lastly, the differences in xylan structure in primary versus secondary cell walls might reflect the different roles in cross-linking and interaction with other cell wall components.« less

  7. An unusual xylan in Arabidopsis primary cell walls is synthesised by GUX3, IRX9L, IRX10L and IRX14

    DOE PAGES

    Mortimer, Jenny C.; Faria-Blanc, Nuno; Yu, Xiaolan; ...

    2015-06-04

    Xylan is a crucial component of many plant primary and secondary cell walls. However, the structure and function of xylan in the dicotyledon primary cell wall is not well understood. Here, we characterized a xylan that is specific to tissues enriched in Arabidopsis primary cell walls. Unlike previously described xylans, this xylan carries a pentose linked 1–2 to the α-1,2-d-glucuronic acid (GlcA) side chains on the β-1,4-Xyl backbone. The frequent and precisely regular spacing of GlcA substitutions every six xylosyl residues along the backbone is also unlike that previously observed in secondary cell wall xylan. Molecular genetics, in vitro assays,more » and expression data suggest that IRX9L, IRX10L and IRX14 are required for xylan backbone synthesis in primary cell wall synthesising tissues. IRX9 and IRX10 are not involved in the primary cell wall xylan synthesis but are functionally exchangeable with IRX9L and IRX10L. GUX3 is the only glucuronyltransferase required for the addition of the GlcA decorations on the xylan. Lastly, the differences in xylan structure in primary versus secondary cell walls might reflect the different roles in cross-linking and interaction with other cell wall components.« less

  8. Investigating Mass Transport Limitations on Xylan Hydrolysis During Dilute Acid Pretreatment of Poplar

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mittal, Ashutosh; Pilath, Heid M.; Parent, Yves

    2014-04-28

    Mass transport limitations could be an impediment to achieving high sugar yields during biomass pretreatment and thus be a critical factor in the economics of biofuels production. The objective of this work was to study the mass transfer restrictions imposed by the structure of biomass on the hydrolysis of xylan during dilute acid pretreatment of biomass. Mass transfer effects were studied by pretreating poplar wood at particle sizes ranging from 10 micrometers to 10 mm. This work showed a significant reduction in the rate of xylan hydrolysis in poplar when compared to the intrinsic rate of hydrolysis for isolated xylanmore » that is possible in the absence of mass transfer. In poplar samples we observed no significant difference in the rates of xylan hydrolysis over more than two orders of magnitude in particle size. It appears that no additional mass transport restrictions are introduced by increasing particle size from 10 micrometers to 10 mm. This work suggests that the rates of xylan hydrolysis in biomass particles are limited primarily by the diffusion of hydrolysis products out of plant cell walls. A mathematical description is presented to describe the kinetics of xylan hydrolysis that includes transport of the hydrolysis products through biomass into the bulk solution. The modeling results show that the effective diffusion coefficient of the hydrolysis products in the cell wall is several orders of magnitude smaller than typical values in other applications signifying the role of plant cell walls in offering resistance to diffusion of the hydrolysis products.« less

  9. The complex of xylan and iodine: the induction and detection of nanoscale order

    Treesearch

    Xiaochun Yu; Rajai H. Atalla

    2005-01-01

    The complex of xylan and iodine and its formation in a solution of xylan, CaCl2, and I2 + KI was investigated by UV/Vis, second-derivative UV/Vis, and Raman spectroscopy. The complex forms only at very high concentrations of CaCl2, suggesting that when the water available in the solution is not sufficient to fully hydrate the calcium cation the chelation with the...

  10. Carboxymethylated-, hydroxypropylsulfonated- and quaternized xylan derivative films

    Treesearch

    Ivan Simkovic; Ivan Kelnar; Iveta Uhliarikova; Raniero Mdndichi; Anurag Mandalika; Thomas Elder

    2014-01-01

    Under alkaline/water conditions carboxymethyl, 2-hydroxypropylsulfonate and trimethylammonium-2-hydroxypropyl groups were introduced into xylan in one step with the goal to prepare film specimens. The materials were characterized by NMR, SEC-MALS, TG/DTG/DTA, AFM and mechanical testing. The properties of triple, double and mono-substituted materials were compared. The...

  11. Moisture barrier properties of xylan composite films

    Treesearch

    Amit Saxena; Thomas J. Elder; Arthur J. Ragauskas

    2011-01-01

    Moisture barrier properties of films based on xylan reinforced with several cellulosic resources including nanocrystalline cellulose, acacia bleached kraft pulp fibers and softwood kraft fibers have been evaluated. Measurements of water vapor transmission rate (WVTR) were performed by a modification of the wet cup method described by ASTM E 96-95, indicating that...

  12. Biomimetic Inks Based on Cellulose Nanofibrils and Cross-Linkable Xylans for 3D Printing.

    PubMed

    Markstedt, Kajsa; Escalante, Alfredo; Toriz, Guillermo; Gatenholm, Paul

    2017-11-22

    This paper presents a sustainable all-wood-based ink which can be used for 3D printing of constructs for a large variety of applications such as clothes, furniture, electronics, and health care products with a customized design and versatile gel properties. The 3D printing technologies where the material is dispensed in the form of liquids, so called inks, have proven suitable for 3D printing dispersions of cellulose nanofibrils (CNFs) because of their unique shear thinning properties. In this study, novel inks were developed with a biomimetic approach where the structural properties of cellulose and the cross-linking function of hemicelluloses that are found in the plant cell wall were utilized. The CNF was mixed with xylan, a hemicellulose extracted from spruce, to introduce cross-linking properties which are essential for the final stability of the printed ink. For xylan to be cross-linkable, it was functionalized with tyramine at different degrees. Evaluation of different ink compositions by rheology measurements and 3D printing tests showed that the degree of tyramine substitution and the ratio of CNFs to xylan-tyramine in the prepared inks influenced the printability and cross-linking density. Both two-layered gridded structures and more complex 3D constructs were printed. Similarly to conventional composites, the interactions between the components and their miscibility are important for the stability of the printed and cross-linked ink. Thus, the influence of tyramine on the adsorption of xylan to cellulose was studied with a quartz crystal microbalance to verify that the functionalization had little influence on xylan's adsorption to cellulose. Utilizing xylan-tyramine in the CNF dispersions resulted in all-wood-based inks which after 3D printing can be cross-linked to form freestanding gels while at the same time, the excellent printing properties of CNFs remain intact.

  13. Folding of xylan onto cellulose fibrils in plant cell walls revealed by solid-state NMR

    NASA Astrophysics Data System (ADS)

    Simmons, Thomas J.; Mortimer, Jenny C.; Bernardinelli, Oigres D.; Pöppler, Ann-Christin; Brown, Steven P.; Deazevedo, Eduardo R.; Dupree, Ray; Dupree, Paul

    2016-12-01

    Exploitation of plant lignocellulosic biomass is hampered by our ignorance of the molecular basis for its properties such as strength and digestibility. Xylan, the most prevalent non-cellulosic polysaccharide, binds to cellulose microfibrils. The nature of this interaction remains unclear, despite its importance. Here we show that the majority of xylan, which forms a threefold helical screw in solution, flattens into a twofold helical screw ribbon to bind intimately to cellulose microfibrils in the cell wall. 13C solid-state magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy, supported by in silico predictions of chemical shifts, shows both two- and threefold screw xylan conformations are present in fresh Arabidopsis stems. The twofold screw xylan is spatially close to cellulose, and has similar rigidity to the cellulose microfibrils, but reverts to the threefold screw conformation in the cellulose-deficient irx3 mutant. The discovery that induced polysaccharide conformation underlies cell wall assembly provides new principles to understand biomass properties.

  14. Folding of xylan onto cellulose fibrils in plant cell walls revealed by solid-state NMR.

    PubMed

    Simmons, Thomas J; Mortimer, Jenny C; Bernardinelli, Oigres D; Pöppler, Ann-Christin; Brown, Steven P; deAzevedo, Eduardo R; Dupree, Ray; Dupree, Paul

    2016-12-21

    Exploitation of plant lignocellulosic biomass is hampered by our ignorance of the molecular basis for its properties such as strength and digestibility. Xylan, the most prevalent non-cellulosic polysaccharide, binds to cellulose microfibrils. The nature of this interaction remains unclear, despite its importance. Here we show that the majority of xylan, which forms a threefold helical screw in solution, flattens into a twofold helical screw ribbon to bind intimately to cellulose microfibrils in the cell wall. 13 C solid-state magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy, supported by in silico predictions of chemical shifts, shows both two- and threefold screw xylan conformations are present in fresh Arabidopsis stems. The twofold screw xylan is spatially close to cellulose, and has similar rigidity to the cellulose microfibrils, but reverts to the threefold screw conformation in the cellulose-deficient irx3 mutant. The discovery that induced polysaccharide conformation underlies cell wall assembly provides new principles to understand biomass properties.

  15. Identification of an algal xylan synthase indicates that there is functional orthology between algal and plant cell wall biosynthesis

    DOE PAGES

    Jensen, Jacob Kruger; Busse-Wicher, Marta; Poulsen, Christian Peter; ...

    2018-02-20

    Insights into the evolution of plant cell walls have important implications for comprehending these diverse and abundant biological structures. In order to understand the evolving structure-function relationships of the plant cell wall, it is imperative to trace the origin of its different components. The present study is focused on plant 1,4-β-xylan, tracing its evolutionary origin by genome and transcriptome mining followed by phylogenetic analysis, utilizing a large selection of plants and algae. It substantiates the findings by heterologous expression and biochemical characterization of a charophyte alga xylan synthase. Of the 12 known gene classes involved in 1,4-β-xylan formation, XYS1/IRX10 inmore » plants, IRX7, IRX8, IRX9, IRX14 and GUX occurred for the first time in charophyte algae. An XYS1/IRX10 ortholog from Klebsormidium flaccidum, designated K. flaccidumXYLAN SYNTHASE-1 (KfXYS1), possesses 1,4-β-xylan synthase activity, and 1,4-β-xylan occurs in the K. flaccidum cell wall. Finally, these data suggest that plant 1,4-β-xylan originated in charophytes and shed light on the origin of one of the key cell wall innovations to occur in charophyte algae, facilitating terrestrialization and emergence of polysaccharide-based plant cell walls.« less

  16. Identification of an algal xylan synthase indicates that there is functional orthology between algal and plant cell wall biosynthesis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jensen, Jacob Kruger; Busse-Wicher, Marta; Poulsen, Christian Peter

    Insights into the evolution of plant cell walls have important implications for comprehending these diverse and abundant biological structures. In order to understand the evolving structure-function relationships of the plant cell wall, it is imperative to trace the origin of its different components. The present study is focused on plant 1,4-β-xylan, tracing its evolutionary origin by genome and transcriptome mining followed by phylogenetic analysis, utilizing a large selection of plants and algae. It substantiates the findings by heterologous expression and biochemical characterization of a charophyte alga xylan synthase. Of the 12 known gene classes involved in 1,4-β-xylan formation, XYS1/IRX10 inmore » plants, IRX7, IRX8, IRX9, IRX14 and GUX occurred for the first time in charophyte algae. An XYS1/IRX10 ortholog from Klebsormidium flaccidum, designated K. flaccidumXYLAN SYNTHASE-1 (KfXYS1), possesses 1,4-β-xylan synthase activity, and 1,4-β-xylan occurs in the K. flaccidum cell wall. Finally, these data suggest that plant 1,4-β-xylan originated in charophytes and shed light on the origin of one of the key cell wall innovations to occur in charophyte algae, facilitating terrestrialization and emergence of polysaccharide-based plant cell walls.« less

  17. Dual-component system dimethyl sulfoxide/LiCl as a solvent and catalyst for homogeneous ring-opening grafted polymerization of ε-caprolactone onto xylan.

    PubMed

    Zhang, Xue-Qin; Chen, Ming-Jie; Liu, Chuan-Fu; Sun, Run-Cang

    2014-01-22

    The preparation of xylan-graft-poly(ε-caprolactone) (xylan-g-PCL) copolymers was investigated by homogeneous ring-opening polymerization (ROP) in a dual-component system containing Lewis base LiCl and strong polar aprotic solvent dimethyl sulfoxide (DMSO). DMSO/LiCl acted as solvent, base, and catalyst for the ROP reaction. The effects of the parameters, including the reaction temperature, molar ratio of ε-caprolactone (ε-CL) to anhydroxylose units (AXU) in xylan, and reaction time, on the degree of substitution (DS) and weight percent of PCL side chain (WPCL) were investigated. The results showed that xylan-g-PCL copolymers with low DS in the range of 0.03-0.39 were obtained under the given conditions. The Fourier transform infrared spectroscopy (FTIR), (1)H nuclear magnetic resonance (NMR), (13)C NMR, (1)H-(1)H correlation spectroscopy (COSY), and (1)H-(13)C correlation two-dimensional (2D) NMR [heteronuclear single-quantum coherence (HSQC)] characterization provided more evidence of the attachment of side chains onto xylan. Only one ε-CL was confirmed to be attached onto xylan with each side chain. Integration of resonances assigned to the substituted C2 and C3 in the HSQC spectrum also indicated 69.23 and 30.77% of PCL side chains attached to AXU at C3 and C2 positions, respectively. Although the attachment of PCL onto xylan led to the decreased thermal stability of xylan, the loss of unrecovered xylan fractions with low molecular weight because of the high solubility of xylan in DMSO/LiCl resulted in the increased thermal stability of the samples. This kind of xylan derivative has potential application in environmentally friendly and biodegradable materials considering the good biodegradability of xylan and PCL.

  18. Dilute acid hydrolysis of paper birch : kinetics studies of xylan and acetyl-group hydrolysis

    Treesearch

    Mark T. Maloney; Thomas W. Chapman; Andrew J. Baker

    1985-03-01

    Batch hydrolysis kinetics of paper birch (Betula papyrifera) xylan and its associated acetyl groups in dilute sulfuric acid have been measured for acid concentrations of between 0.04 and 0.18 M and temperatures of between 100 and 170°C. Only 5% of the cellulose was hydrolyzed for up to 85% xylan removal. Rate data were correlated well by a parallel reaction model based...

  19. Transcriptomic Analysis of Xylan Utilization Systems in Paenibacillus sp

    Treesearch

    Neha Sawhney; Casey Crooks; Franz St. John; James F. Preston; R. M. Kelly

    2014-01-01

    Xylans, including methylglucuronoxylans (MeGXn) and methylglucuronoarabinoxylans (MeGAXn), are the predominant polysaccharides in hemicellulose fractions of dicots and monocots available for conversion to biofuels and chemicals. Paenibacillus sp. strain JDR-2 (Pjdr2) efficiently depolymerizes MeGX

  20. Temperature-dependent phase behaviour of tetrahydrofuran–water alters solubilization of xylan to improve co-production of furfurals from lignocellulosic biomass

    DOE PAGES

    Smith, Micholas Dean; Cai, Charles M.; Cheng, Xiaolin; ...

    2018-03-06

    Xylose, Xylan, Hemicellulose, CELF, THF, Co-solvent, Pretreatment, Biomass ABSTRACT: Xylan is an important polysaccharide found in the hemicellulose fraction of lignocellulosic biomass that can be hydrolysed to xylose and further dehydrated to the furfural, an important renewable platform fuel precursor. Here, pairing molecular simulation and experimental evidences, we reveal how the unique temperature-dependent phase behaviour of water-tetrahydrofuran (THF) co-solvent can delay xylan solubilization to synergistically improve catalytic co-processing of biomass to furfural and 5-HMF. Our results indicate, based on polymer correlations between polymer conformational behaviour and solvent quality, that both co-solvent and aqueous environments serve as ‘good’ solvents for xylan.more » Interestingly, the simulations also revealed that unlike other cell-wall components (i.e., lignin and cellulose), the make-up of the solvation shell of xylan in THF-water is dependent on the temperature-phase behaviour. At temperatures between 333K and 418K, THF and water become immiscible, and THF is evacuated from the solvation shell of xylan, while above and below this temperature range, THF and water are both present in the polysaccharide’s solvation shell. This suggested that the solubilization of xylan in THF-water may be similar to aqueous-only solutions at temperatures between 333K and 418K and different outside this range. Experimental reactions on beachwood xylan corroborate this hypothesis by demonstrating 2-fold reduction of xylan solubilization in THF-water within a miscible temperature regime (445K) and unchanged solubilization within an immiscible regime (400K). Translating this phase-dependent behaviour to processing of maple wood chips, we demonstrate how the weaker xylan solvation in THF-water under miscible conditions can delay furfural production from xylose, allowing 5-HMF production from cellulose to “catch-up” such that their high yield production from

  1. Temperature-dependent phase behaviour of tetrahydrofuran–water alters solubilization of xylan to improve co-production of furfurals from lignocellulosic biomass

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Smith, Micholas Dean; Cai, Charles M.; Cheng, Xiaolin

    Xylose, Xylan, Hemicellulose, CELF, THF, Co-solvent, Pretreatment, Biomass ABSTRACT: Xylan is an important polysaccharide found in the hemicellulose fraction of lignocellulosic biomass that can be hydrolysed to xylose and further dehydrated to the furfural, an important renewable platform fuel precursor. Here, pairing molecular simulation and experimental evidences, we reveal how the unique temperature-dependent phase behaviour of water-tetrahydrofuran (THF) co-solvent can delay xylan solubilization to synergistically improve catalytic co-processing of biomass to furfural and 5-HMF. Our results indicate, based on polymer correlations between polymer conformational behaviour and solvent quality, that both co-solvent and aqueous environments serve as ‘good’ solvents for xylan.more » Interestingly, the simulations also revealed that unlike other cell-wall components (i.e., lignin and cellulose), the make-up of the solvation shell of xylan in THF-water is dependent on the temperature-phase behaviour. At temperatures between 333K and 418K, THF and water become immiscible, and THF is evacuated from the solvation shell of xylan, while above and below this temperature range, THF and water are both present in the polysaccharide’s solvation shell. This suggested that the solubilization of xylan in THF-water may be similar to aqueous-only solutions at temperatures between 333K and 418K and different outside this range. Experimental reactions on beachwood xylan corroborate this hypothesis by demonstrating 2-fold reduction of xylan solubilization in THF-water within a miscible temperature regime (445K) and unchanged solubilization within an immiscible regime (400K). Translating this phase-dependent behaviour to processing of maple wood chips, we demonstrate how the weaker xylan solvation in THF-water under miscible conditions can delay furfural production from xylose, allowing 5-HMF production from cellulose to “catch-up” such that their high yield production from

  2. Characterization of xylan in the early stages of secondary cell wall formation in tobacco bright yellow-2 cells.

    PubMed

    Ishii, Tadashi; Matsuoka, Keita; Ono, Hiroshi; Ohnishi-Kameyama, Mayumi; Yaoi, Katsuro; Nakano, Yoshimi; Ohtani, Misato; Demura, Taku; Iwai, Hiroaki; Satoh, Shinobu

    2017-11-15

    The major polysaccharides present in the primary and secondary walls surrounding plant cells have been well characterized. However, our knowledge of the early stages of secondary wall formation is limited. To address this, cell walls were isolated from differentiating xylem vessel elements of tobacco bright yellow-2 (BY-2) cells induced by VASCULAR-RELATED NAC-DOMAIN7 (VND7). The walls of induced VND7-VP16-GR BY-2 cells consisted of cellulose, pectic polysaccharides, hemicelluloses, and lignin, and contained more xylan and cellulose compared with non-transformed BY-2 and uninduced VND7-VP16-GR BY-2 cells. A reducing end sequence of xylan containing rhamnose and galaturonic acid- residues is present in the walls of induced, uninduced, and non-transformed BY-2 cells. Glucuronic acid residues in xylan from walls of induced cells are O-methylated, while those of xylan in non-transformed BY-2 and uninduced cells are not. Our results show that xylan changes in chemical structure and amounts during the early stages of xylem differentiation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Using isolated cell wall xylan to identify recalcitrant oligosaccharides

    USDA-ARS?s Scientific Manuscript database

    Herbaceous biomass is a renewable source of carbohydrates with potential for use in microbial conversion to biofuels. Xylan comprises 20-40% of herbaceous biomass cell wall material and its full depolymerization benefits the economics of bioconversion. To understand the limitations of commercial enz...

  4. Removal of glucuronic acid from xylan is a strategy to improve the conversion of plant biomass to sugars for bioenergy.

    PubMed

    Lyczakowski, Jan J; Wicher, Krzysztof B; Terrett, Oliver M; Faria-Blanc, Nuno; Yu, Xiaolan; Brown, David; Krogh, Kristian B R M; Dupree, Paul; Busse-Wicher, Marta

    2017-01-01

    Plant lignocellulosic biomass can be a source of fermentable sugars for the production of second generation biofuels and biochemicals. The recalcitrance of this plant material is one of the major obstacles in its conversion into sugars. Biomass is primarily composed of secondary cell walls, which is made of cellulose, hemicelluloses and lignin. Xylan, a hemicellulose, binds to the cellulose microfibril and is hypothesised to form an interface between lignin and cellulose. Both softwood and hardwood xylan carry glucuronic acid side branches. As xylan branching may be important for biomass recalcitrance and softwood is an abundant, non-food competing, source of biomass it is important to investigate how conifer xylan is synthesised. Here, we show using Arabidopsis gux mutant biomass that removal of glucuronosyl substitutions of xylan can allow 30% more glucose and over 700% more xylose to be released during saccharification. Ethanol yields obtained through enzymatic saccharification and fermentation of gux biomass were double those obtained for non-mutant material. Our analysis of additional xylan branching mutants demonstrates that absence of GlcA is unique in conferring the reduced recalcitrance phenotype. As in hardwoods, conifer xylan is branched with GlcA. We use transcriptomic analysis to identify conifer enzymes that might be responsible for addition of GlcA branches onto xylan in industrially important softwood. Using a combination of in vitro and in vivo activity assays, we demonstrate that a white spruce ( Picea glauca ) gene, PgGUX , encodes an active glucuronosyl transferase. Glucuronic acid introduced by PgGUX reduces the sugar release of Arabidopsis gux mutant biomass to wild-type levels indicating that it can fulfil the same biological function as native glucuronosylation. Removal of glucuronic acid from xylan results in the largest increase in release of fermentable sugars from Arabidopsis plants that grow to the wild-type size. Additionally, plant

  5. Active Site Mapping of Xylan-Deconstructing Enzymes with Arabinoxylan Oligosaccharides Produced by Automated Glycan Assembly.

    PubMed

    Senf, Deborah; Ruprecht, Colin; de Kruijff, Goswinus H M; Simonetti, Sebastian O; Schuhmacher, Frank; Seeberger, Peter H; Pfrengle, Fabian

    2017-03-02

    Xylan-degrading enzymes are crucial for the deconstruction of hemicellulosic biomass, making the hydrolysis products available for various industrial applications such as the production of biofuel. To determine the substrate specificities of these enzymes, we prepared a collection of complex xylan oligosaccharides by automated glycan assembly. Seven differentially protected building blocks provided the basis for the modular assembly of 2-substituted, 3-substituted, and 2-/3-substituted arabino- and glucuronoxylan oligosaccharides. Elongation of the xylan backbone relied on iterative additions of C4-fluorenylmethoxylcarbonyl (Fmoc) protected xylose building blocks to a linker-functionalized resin. Arabinofuranose and glucuronic acid residues have been selectively attached to the backbone using fully orthogonal 2-(methyl)naphthyl (Nap) and 2-(azidomethyl)benzoyl (Azmb) protecting groups at the C2 and C3 hydroxyls of the xylose building blocks. The arabinoxylan oligosaccharides are excellent tools to map the active site of glycosyl hydrolases involved in xylan deconstruction. The substrate specificities of several xylanases and arabinofuranosidases were determined by analyzing the digestion products after incubation of the oligosaccharides with glycosyl hydrolases. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. A group of Populus trichocarpa DUF231 proteins exhibit differential O-acetyltransferase activities toward xylan.

    PubMed

    Zhong, Ruiqin; Cui, Dongtao; Ye, Zheng-Hua

    2018-01-01

    Wood represents the most abundant biomass produced by plants and one of its major components is acetyl xylan. Acetylation in xylan can occur at O-2 or O-3 of a xylosyl residue, at both O-2 and O-3 of a xylosyl residue, and at O-3 of a xylosyl residue substituted at O-2 with glucuronic acid. Acetyltransferases responsible for the regiospecific acetylation of xylan in tree species have not yet been characterized. Here we report the biochemical characterization of twelve Populus trichocarpa DUF231-containing proteins, named PtrXOATs, for their roles in the regiospecific acetylation of xylan. The PtrXOAT genes were found to be differentially expressed in Populus organs and among them, PtrXOAT1, PtrXOAT2, PtrXOAT9 and PtrXOAT10 exhibited the highest level of expression in stems undergoing wood formation. Activity assays of recombinant proteins demonstrated that all twelve PtrXOAT proteins were able to transfer acetyl groups from acetyl CoA onto a xylohexaose acceptor with PtrXOAT1, PtrXOAT2, PtrXOAT3, PtrXOAT11 and PtrXOAT12 having the highest activity. Structural analysis of the PtrXOAT-catalyzed reaction products using 1H NMR spectroscopy revealed that PtrXOAT1, PtrXAOT2 and PtrXOAT3 mediated 2-O- and 3-O-monoacetylation and 2,3-di-O-acetylation of xylosyl residues and PtrXOAT11 and PtrXOAT12 only catalyzed 2-O- and 3-O-monoacetylation of xylosyl residues. Of the twelve PtrXOATs, only PtrXOAT9 and PtrXOAT10 were capable of transferring acetyl groups onto the O-3 position of 2-O-glucuronic acid-substituted xylosyl residues. Furthermore, when expressed in the Arabidopsis eskimo1 mutant, PtrXOAT1, PtrXAOT2 and PtrXOAT3 were able to rescue the defects in xylan acetylation. Together, these results demonstrate that the twelve PtrXOATs are acetyltransferases with different roles in xylan acetylation in P. trichocarpa.

  7. Simultaneous catalytic conversion of cellulose and corncob xylan under temperature programming for enhanced sorbitol and xylitol production.

    PubMed

    Ribeiro, Lucília Sousa; Órfão, José J de Melo; Pereira, Manuel Fernando Ribeiro

    2017-11-01

    Sorbitol and xylitol yields can be improved by converting cellulose and xylan simultaneously, due to a synergetic effect between both substrates. Furthermore, both yields can be greatly enhanced by simply adjusting the reaction conditions regarding the optimum for the production of each product, since xylitol (from xylan) and sorbitol (from cellulose) yields are maximized when the reaction is carried out at 170 and 205°C, respectively. Therefore, the combination of a simultaneous conversion of cellulose and xylan with a two-step temperature approach, which consists in the variation of the reaction temperature from 170 to 205°C after 2h, showed to be a good strategy for maximizing the production of sorbitol and xylitol directly from mixture of cellulose and xylan. Using this new and environmentally friendly approach, yields of sorbitol and xylitol of 75 and 77%, respectively, were obtained after 6h of reaction. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Designer biomass for next-generation biorefineries: leveraging recent insights into xylan structure and biosynthesis.

    PubMed

    Smith, Peter J; Wang, Hsin-Tzu; York, William S; Peña, Maria J; Urbanowicz, Breeanna R

    2017-01-01

    Xylans are the most abundant noncellulosic polysaccharides in lignified secondary cell walls of woody dicots and in both primary and secondary cell walls of grasses. These polysaccharides, which comprise 20-35% of terrestrial biomass, present major challenges for the efficient microbial bioconversion of lignocellulosic feedstocks to fuels and other value-added products. Xylans play a significant role in the recalcitrance of biomass to degradation, and their bioconversion requires metabolic pathways that are distinct from those used to metabolize cellulose. In this review, we discuss the key differences in the structural features of xylans across diverse plant species, how these features affect their interactions with cellulose and lignin, and recent developments in understanding their biosynthesis. In particular, we focus on how the combined structural and biosynthetic knowledge can be used as a basis for biomass engineering aimed at developing crops that are better suited as feedstocks for the bioconversion industry.

  9. The complex physiology of Cellvibrio japonicus xylan degradation relies on a single cytoplasmic β-xylosidase for xylo-oligosaccharide utilization

    DOE PAGES

    Blake, Andrew D.; Beri, Nina R.; Guttman, Hadassa S.; ...

    2017-12-21

    Lignocellulose degradation by microbes plays a central role in global carbon cycling, human gut metabolism, and renewable energy technologies. While considerable effort has been put into understanding the biochemical aspects of lignocellulose degradation, much less work has been done to understand how these enzymes work in an in vivo context. Here in this paper, we report a systems level study of xylan degradation in the saprophytic bacterium Cellvibrio japonicus. Transcriptome analysis indicated seven genes that encode carbohydrate active enzymes were up-regulated during growth with xylan containing media. In-frame deletion analysis of these genes found that only gly43F is critical formore » utilization of xylo-oligosaccharides, xylan, and arabinoxylan. Heterologous expression of gly43F was sufficient for the utilization of xylo-oligosaccharides in Escherichia coli. Additional analysis found that the xyn11A, xyn11B, abf43L, abf43K, and abf51A gene products were critical for utilization of arabinoxylan. Furthermore, a predicted transporter (CJA_1315) was required for effective utilization of xylan substrates, and we propose this unannotated gene be called xntA (xylan transporter A). Our major findings are 1) C. japonicus employs both secreted and surface associated enzymes for xylan degradation, which differs from the strategy used for cellulose degradation, and 2) a single cytoplasmic β-xylosidase is essential for the utilization of xylo-oligosaccharides.« less

  10. The complex physiology of Cellvibrio japonicus xylan degradation relies on a single cytoplasmic β-xylosidase for xylo-oligosaccharide utilization

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Blake, Andrew D.; Beri, Nina R.; Guttman, Hadassa S.

    Lignocellulose degradation by microbes plays a central role in global carbon cycling, human gut metabolism, and renewable energy technologies. While considerable effort has been put into understanding the biochemical aspects of lignocellulose degradation, much less work has been done to understand how these enzymes work in an in vivo context. Here in this paper, we report a systems level study of xylan degradation in the saprophytic bacterium Cellvibrio japonicus. Transcriptome analysis indicated seven genes that encode carbohydrate active enzymes were up-regulated during growth with xylan containing media. In-frame deletion analysis of these genes found that only gly43F is critical formore » utilization of xylo-oligosaccharides, xylan, and arabinoxylan. Heterologous expression of gly43F was sufficient for the utilization of xylo-oligosaccharides in Escherichia coli. Additional analysis found that the xyn11A, xyn11B, abf43L, abf43K, and abf51A gene products were critical for utilization of arabinoxylan. Furthermore, a predicted transporter (CJA_1315) was required for effective utilization of xylan substrates, and we propose this unannotated gene be called xntA (xylan transporter A). Our major findings are 1) C. japonicus employs both secreted and surface associated enzymes for xylan degradation, which differs from the strategy used for cellulose degradation, and 2) a single cytoplasmic β-xylosidase is essential for the utilization of xylo-oligosaccharides.« less

  11. Straw and Xylan Utilization by Pure Cultures of Nitrogen-Fixing Azospirillum spp

    PubMed Central

    Halsall, Dorothy M.; Turner, Graham L.; Gibson, Alan H.

    1985-01-01

    Azospirillum spp. were shown to utilize both straw and xylan, a major component of straw, for growth with an adequate combined N supply and also under N-limiting conditions. For most strains examined, a semisolid agar medium was satisfactory, but several strains appeared to be capable of slow metabolism of the agar. Subsequently, experiments were done with acid-washed sand supplemented with various carbon sources. In these experiments, authenticated laboratory strains, and all 16 recent field isolates from straw-amended soils, of both A. brasilense and A. lipoferum possessed the ability to utilize straw and xylan as energy sources for nitrogen fixation. Neither carboxymethyl cellulose nor cellulose was utilized. The strains and isolates differed in their abilities to utilize xylan and straw and in the efficiency of nitrogenase activity (CO2/C2H2 ratio). Reasonable levels of activity could be maintained for at least 14 days in the sand cultures. Nitrogenase activity (acetylene reduction) was confirmed by 15N2 incorporation. The level of nitrogenase activity observed was dependent on the time of the addition of acetylene to the culture vessels. PMID:16346730

  12. Utilization of xylan by yeasts and its conversion to ethanol by Pichia stipitis strains. [Cryptococcus; Pichia stipitis; Candida shehatae

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lee, H.; Biely, P.; Latta, R.K.

    Yeasts able to grow on D-xylose were screened for the ability to hydrolyze xylan. Xylanase activity was found to be rare; a total of only 19 of more than 250 strains yielded a positive test result. The activity was localized largely in the genus Cryptococcus and in Pichia stipitis and its anamorph Candida shehatae. The ability to hydrolyze xylan was generally uncoupled from that to hydrolyze cellulose; only three of the xylan-positive strains also yielded a positive test for cellulolytic activity. Of the 19 xylanolytic strains. 2. P. stipitis CBS 5773 and CBS 5775, converted xylan into ethanol, with aboutmore » 60% of a theoretical yield computed on the basis of the amount of D-xylose present originally that could be released by acid hydrolysis.« less

  13. Digestion of cellulose and xylan by symbiotic bacteria in the intestine of the Indian flying fox (Pteropus giganteus).

    PubMed

    Prem Anand, A Alwin; Sripathi, K

    2004-09-01

    Bats (Order Chiroptera) are a widely distributed group of mammals. Pteropus giganteus belongs to the Suborder Megachiroptera. This bat consumes fruits and leaves as their major food. Cellulose and xylan are the major composition of leaves. As they consume leaves in their diet, their digestive tract must contain cellulolytic and xylanolytic bacteria which help in the digestion of cellulose and xylan. The cellulolytic and xylanolytic bacteria were isolated and screened on Berg's agar containing cellulose and xylan. The bacteria isolated were characterized biochemically and found to be Proteus vulgaris, Proteus mirabilis, Citrobacter freundii, Serratia liquefaciens and Klebsiella oxytoca. These bacteria help in digestion of cellulose and xylan in the diet of the bat, P. giganteus. Here we show that leaves are also used as a carbohydrate source by these bats. An insectivorous bat, Hipposideros fulvus, was used as a control and does not possess cellulolytic and xylanolytic bacteria.

  14. Isolation and characterization of a xylan with industrial and biomedical applications from edible açaí berries (Euterpe oleraceae).

    PubMed

    Cantu-Jungles, Thaisa Moro; Iacomini, Marcello; Cipriani, Thales R; Cordeiro, Lucimara M C

    2017-04-15

    The chemical features of xylan largely determine its physical and biological properties and its use in the industry. In this work, we describe the occurrence, purification and partial characterization of a xylan in edible açaí berries (Euterpe oleraceae), using a fairly simple and inexpensive method of purification from alkaline açaí extract. A mainly linear (1→4)-β-d-xylan was found as the majority (70%) of alkali extract and 4.2% of the dry matter açaí pulp. This represents the biggest source of xylan found so far in a fruit pulp and could be suitable for applications in the industry and biomedical field. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Green synthesis of silver nanoparticles in xylan solution via Tollens reaction and their detection for Hg(2+).

    PubMed

    Luo, Yuqiong; Shen, Suqin; Luo, Jiwen; Wang, Xiaoying; Sun, Runcang

    2015-01-14

    This work reported a facile and green method to prepare highly stable and uniformly distributed Ag nanoparticles (AgNPs), in which a biopolymer xylan was used as the stabilizing and reducing agent via the Tollens reaction under microwave irradiation. Different variables were evaluated to optimize the reaction conditions. Complete characterization was performed using UV-Vis, XRD, TEM, size distribution analysis and XPS. The results revealed that AgNPs were well dispersed with diameters of 20-35 nm due to the packing of xylan. The optimal conditions were as follows: microwave irradiation temperature was 60-70 °C, microwave power was 800 W, microwave time was 30 min, the ratio of xylan to AgNO3 was 50 mg: 0.13 mmol, and ammonia concentration was 2%. In addition, the AgNPs were collected via high-speed centrifugal separation, and the supernatant was tested by HPAEC, GPC, FT-IR, and NMR. By comparing the structure of xylan before and after the reaction, the reaction mechanism was discussed. It was noted that the xylan-AgNPs composites showed high selectivity and sensitivity for Hg(2+) detection. The other 15 metal ions used had no obvious effect on the detection of Hg(2+), and the limit of detection (LOD) was 4.6 nM, which is lower than the allowed maximum level of 30 nM for drinking water by WHO. In addition, the xylan-AgNPs composites can be applied for Hg(2+) detection in real water samples. This study provides a novel way for the high-value utilization of a rich biomass resource, and a green method for the synthesis of AgNPs for the selective and sensitive detection of harmful heavy metals.

  16. Synthesis and thermal characterization of xylan-graft-polyacrylonitrile.

    PubMed

    Ünlü, Cüneyt H; Öztekin, N Simge; Atıcı, Oya Galioğlu

    2012-10-01

    In this study emulsion polymerization of acrylonitrile using xylan from agricultural waste material (corn cob) and cerium ammonium nitrate was investigated in terms of catalyst acid. Stock ceric solutions were prepared using either nitric or perchloric acid as catalyst. Optimum conditions were determined using different parameters such as reaction time, temperature, and component concentrations. Nitric acid catalyzed reactions resulted in maximum conversion ratio (96%) at 50°C, 1 h where ceric ion, acrylonitrile, xylan, and catalyst concentrations were 21.7 mmol l(-1), 0.5 mol l(-1), 0.2% (w/v), and 0.1 mol l(-1), respectively. However, 83% conversion was obtained with perchloric acid catalysis at 27 °C, 1 h where concentrations were 5.4 mmol l(-1), 0.8 mol l(-1), 0.5% (w/v), and 0.2 mol l(-1), respectively. Copolymer synthesis using perchloric acid was realized at milder conditions than using nitric acid. Thermal analyses of obtained polymers were conducted to characterize copolymers. Results showed that calculated activation energy, maximum degradation temperature, and heat of thermal decomposition changed relying mainly on molecular weight. Copyright © 2012 Elsevier Ltd. All rights reserved.

  17. Molecular Dissection of Xylan Biosynthesis During Wood Formation in Poplar

    EPA Science Inventory

    Xylan, being the second most abundant polysaccharide in dicot wood, is considered to be one of the factors contributing to wood biomass recalcitrance for biofuel production. To better utilize wood as biofuel feedstock, it is crucial to functionally characterize all the genes invo...

  18. Molecular Mechanisms Associated with Xylan Degradation by Xanthomonas Plant Pathogens*

    PubMed Central

    Santos, Camila Ramos; Hoffmam, Zaira Bruna; de Matos Martins, Vanesa Peixoto; Zanphorlin, Leticia Maria; de Paula Assis, Leandro Henrique; Honorato, Rodrigo Vargas; Lopes de Oliveira, Paulo Sérgio; Ruller, Roberto; Murakami, Mario Tyago

    2014-01-01

    Xanthomonas pathogens attack a variety of economically relevant plants, and their xylan CUT system (carbohydrate utilization with TonB-dependent outer membrane transporter system) contains two major xylanase-related genes, xynA and xynB, which influence biofilm formation and virulence by molecular mechanisms that are still elusive. Herein, we demonstrated that XynA is a rare reducing end xylose-releasing exo-oligoxylanase and not an endo-β-1,4-xylanase as predicted. Structural analysis revealed that an insertion in the β7-α7 loop induces dimerization and promotes a physical barrier at the +2 subsite conferring this unique mode of action within the GH10 family. A single mutation that impaired dimerization became XynA active against xylan, and high endolytic activity was achieved when this loop was tailored to match a canonical sequence of endo-β-1,4-xylanases, supporting our mechanistic model. On the other hand, the divergent XynB proved to be a classical endo-β-1,4-xylanase, despite the low sequence similarity to characterized GH10 xylanases. Interestingly, this enzyme contains a calcium ion bound nearby to the glycone-binding region, which is required for catalytic activity and structural stability. These results shed light on the molecular basis for xylan degradation by Xanthomonas and suggest how these enzymes synergistically assist infection and pathogenesis. Our findings indicate that XynB contributes to breach the plant cell wall barrier, providing nutrients and facilitating the translocation of effector molecules, whereas the exo-oligoxylanase XynA possibly participates in the suppression of oligosaccharide-induced immune responses. PMID:25266726

  19. The mechanism by which arabinoxylanases can recognize highly decorated xylans

    USDA-ARS?s Scientific Manuscript database

    The enzymatic degradation of plant cell walls is an important biological process of increasing environmental and industrial significance. Xylan, a major component of the plant cell wall, consists of a backbone of beta 1,4-xylose (Xylp) units that are often decorated with arabinofuranose (Araf) side ...

  20. Enhancing enzymatic hydrolysis of xylan by adding sodium lignosulfonate and long-chain fatty alcohols.

    PubMed

    Lou, Hongming; Yuan, Long; Qiu, Xueqing; Qiu, Kexian; Fu, Jinguo; Pang, Yuxia; Huang, Jinhao

    2016-01-01

    Sodium lignosulfonate (SXSL) and long-chain fatty alcohols (LFAs) could enhance the enzymatic hydrolysis of xylan, and the compound of SXSL and LFAs have synergies on the enzymatic hydrolysis. SXSL shows a strong enhancement in buffer pH range from 4.0 to 6.0. The enhancement increased with the SXSL dosage and the xylanase loading. The cellulose and lignin in corncob substrate could not only adsorb xylanase nonproductively, but also seriously reduce the accessibility of xylanase on xylan to impede the enzymatic hydrolysis of xylan. Cellulase could break the plant cell wall structure of corncob and make additives work better. The xylose yield of corncob at 72h increased from 59.4% to 73.7% by adding the compound of 5g/L SXSL and 0.01% (v/v) n-decanol, which was higher than that without cellulase and additives by 30.7%. Meanwhile, the glucose yield at 72h of corncob increased from 45.8% to 62.3%. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Nanocomposite film prepared by depositing xylan on cellulose nanowhiskers matrix

    Treesearch

    Qining Sun; Anurag Mandalika; Thomas Elder; Sandeep S. Nair; Xianzhi Meng; Fang Huang; Art J. Ragauskas

    2014-01-01

    Novel bionanocomposite films have been prepared by depositing xylan onto cellulose nanowhiskers through a pH adjustment. Analysis of strength properties, water vapour transmission, transparency, surface morphology and thermal decomposition showed the enhancement of film performance. This provides a new green route to the utilization of biomass for sustainable...

  2. Isolation and characterization of unhydrolyzed oligosaccharides from switchgrass (Panicum virgatum, L.) xylan after exhaustive enzymatic treatment with commercial enzyme preparations

    USDA-ARS?s Scientific Manuscript database

    Switchgrass (Panicum virgatum, L.) is a potential renewable source of carbohydrates for use in microbial conversion to biofuels. Xylan comprises approximately 30% of the switchgrass cell wall. To understand the limitations of commercial enzyme mixtures, alkali-extracted, isolated switchgrass xylan w...

  3. The Mechanism by Which Arabinoxylanases Can Recognize Highly Decorated Xylans*

    PubMed Central

    Labourel, Aurore; Crouch, Lucy I.; Brás, Joana L. A.; Jackson, Adam; Rogowski, Artur; Gray, Joseph; Yadav, Madhav P.; Henrissat, Bernard; Fontes, Carlos M. G. A.; Gilbert, Harry J.; Najmudin, Shabir; Baslé, Arnaud; Cuskin, Fiona

    2016-01-01

    The enzymatic degradation of plant cell walls is an important biological process of increasing environmental and industrial significance. Xylan, a major component of the plant cell wall, consists of a backbone of β-1,4-xylose (Xylp) units that are often decorated with arabinofuranose (Araf) side chains. A large penta-modular enzyme, CtXyl5A, was shown previously to specifically target arabinoxylans. The mechanism of substrate recognition displayed by the enzyme, however, remains unclear. Here we report the crystal structure of the arabinoxylanase and the enzyme in complex with ligands. The data showed that four of the protein modules adopt a rigid structure, which stabilizes the catalytic domain. The C-terminal non-catalytic carbohydrate binding module could not be observed in the crystal structure, suggesting positional flexibility. The structure of the enzyme in complex with Xylp-β-1,4-Xylp-β-1,4-Xylp-[α-1,3-Araf]-β-1,4-Xylp showed that the Araf decoration linked O3 to the xylose in the active site is located in the pocket (−2* subsite) that abuts onto the catalytic center. The −2* subsite can also bind to Xylp and Arap, explaining why the enzyme can utilize xylose and arabinose as specificity determinants. Alanine substitution of Glu68, Tyr92, or Asn139, which interact with arabinose and xylose side chains at the −2* subsite, abrogates catalytic activity. Distal to the active site, the xylan backbone makes limited apolar contacts with the enzyme, and the hydroxyls are solvent-exposed. This explains why CtXyl5A is capable of hydrolyzing xylans that are extensively decorated and that are recalcitrant to classic endo-xylanase attack. PMID:27531750

  4. Effect of xylan and lignin removal by batch and flowthrough pretreatment on the enzymatic digestibility of corn stover cellulose.

    PubMed

    Yang, Bin; Wyman, Charles E

    2004-04-05

    Compared with batch systems, flowthrough and countercurrent reactors have important potential advantages for pretreating cellulosic biomass, including higher hemicellulose sugar yields, enhanced cellulose digestibility, and reduced chemical additions. Unfortunately, they suffer from high water and energy use. To better understand these trade-offs, comparative data are reported on xylan and lignin removal and enzymatic digestibility of cellulose for corn stover pretreated in batch and flowthrough reactors over a range of flow rates between 160 degrees and 220 degrees C, with water only and also with 0.1 wt% sulfuric acid. Increasing flow with just water enhanced the xylan dissolution rate, more than doubled total lignin removal, and increased cellulose digestibility. Furthermore, adding dilute sulfuric acid increased the rate of xylan removal for both batch and flowthrough systems. Interestingly, adding acid also increased the lignin removal rate with flow, but less lignin was left in solution when acid was added in batch. Although the enzymatic hydrolysis of pretreated cellulose was related to xylan removal, as others have shown, the digestibility was much better for flowthrough compared with batch systems, for the same degree of xylan removal. Cellulose digestibility for flowthrough reactors was related to lignin removal as well. These results suggest that altering lignin also affects the enzymatic digestibility of corn stover. Copyright 2004 Wiley Periodicals, Inc.

  5. Kinetic study of hydrolysis of xylan and agricultural wastes with hot liquid water.

    PubMed

    Zhuang, Xinshu; Yuan, Zhenhong; Ma, Longlong; Wu, Chuangzhi; Xu, Mingzhong; Xu, Jingliang; Zhu, Shunni; Qi, Wei

    2009-01-01

    We investigated the kinetics of hot liquid water (HLW) hydrolysis over a 60-min period using a self-designed setup. The reaction was performed within the range 160-220 degrees C, under reaction conditions of 4.0 MPa, a 1:20 solid:liquid ratio (g/mL), at 500 rpm stirring speed. Xylan was chosen as a model compound for hemicelluloses, and two kinds of agricultural wastes-rice straw and palm shell-were used as typical feedstocks representative of herbaceous and woody biomasses, respectively. The hydrolysis reactions for the three kinds of materials followed a first-order sequential kinetic model, and the hydrolysis activation energies were 65.58 kJ/mol for xylan, 68.76 kJ/mol for rice straw, and 95.19 kJ/mol for palm shell. The activation energies of sugar degradation were 147.21 kJ/mol for xylan, 47.08 kJ/mol for rice straw and 79.74 kJ/mol for palm shell. These differences may be due to differences in the composition and construction of the three kinds of materials. In order to reduce the decomposition of sugars, the hydrolysis time of biomasses such as rice straw and palm shell should be strictly controlled.

  6. Bioethanol production by a xylan fermenting thermophilic isolate Clostridium strain DBT-IOC-DC21.

    PubMed

    Singh, Nisha; Puri, Munish; Tuli, Deepak K; Gupta, Ravi P; Barrow, Colin J; Mathur, Anshu S

    2018-06-01

    To overcome the challenges associated with combined bioprocessing of lignocellulosic biomass to biofuel, finding good organisms is essential. An ethanol producing bacteria DBT-IOC-DC21 was isolated from a compost site via preliminary enrichment culture on a pure hemicellulosic substrate and identified as a Clostridium strain by 16S rRNA analysis. This strain presented broad substrate spectrum with ethanol, acetate, lactate, and hydrogen as the primary metabolic end products. The optimum conditions for ethanol production were found to be an initial pH of 7.0, a temperature of 70 °C and an L-G ratio of 0.67. Strain presented preferential hemicellulose fermentation when compared to various substrates and maximum ethanol concentration of 26.61 mM and 43.63 mM was produced from xylan and xylose, respectively. During the fermentation of varying concentration of xylan, a substantial amount of ethanol ranging from 25.27 mM to 67.29 mM was produced. An increased ethanol concentration of 40.22 mM was produced from a mixture of cellulose and xylan, with a significant effect observed on metabolic flux distribution. The optimum conditions were used to produce ethanol from 28 g L -1 rice straw biomass (RSB) (equivalent to 5.7 g L -1 of the xylose equivalents) in which 19.48 mM ethanol production was achieved. Thus, Clostridium strain DBT-IOC-DC21 has the potential to perform direct microbial conversion of untreated RSB to ethanol at a yield comparative to xylan fermentation. Copyright © 2018 Elsevier Ltd. All rights reserved.

  7. The Four Arabidopsis Reduced Wall Acetylation Genes are Expressed in Secondary Wall-Containing Cells and Required for the Acetylation of Xylan

    EPA Science Inventory

    Xylan is one of the major polysaccharides in cellulosic biomass, and understanding the mechanisms underlying xylan biosynthesis will potentially help us design strategies to produce cellulosic biomass better suited for biofuel production. Although a number of genes have been show...

  8. Downregulation of RWA genes in hybrid aspen affects xylan acetylation and wood saccharification.

    PubMed

    Pawar, Prashant Mohan-Anupama; Ratke, Christine; Balasubramanian, Vimal K; Chong, Sun-Li; Gandla, Madhavi Latha; Adriasola, Mathilda; Sparrman, Tobias; Hedenström, Mattias; Szwaj, Klaudia; Derba-Maceluch, Marta; Gaertner, Cyril; Mouille, Gregory; Ezcurra, Ines; Tenkanen, Maija; Jönsson, Leif J; Mellerowicz, Ewa J

    2017-06-01

    High acetylation of angiosperm wood hinders its conversion to sugars by glycoside hydrolases, subsequent ethanol fermentation and (hence) its use for biofuel production. We studied the REDUCED WALL ACETYLATION (RWA) gene family of the hardwood model Populus to evaluate its potential for improving saccharification. The family has two clades, AB and CD, containing two genes each. All four genes are expressed in developing wood but only RWA-A and -B are activated by master switches of the secondary cell wall PtNST1 and PtMYB21. Histochemical analysis of promoter::GUS lines in hybrid aspen (Populus tremula × tremuloides) showed activation of RWA-A and -B promoters in the secondary wall formation zone, while RWA-C and -D promoter activity was diffuse. Ectopic downregulation of either clade reduced wood xylan and xyloglucan acetylation. Suppressing both clades simultaneously using the wood-specific promoter reduced wood acetylation by 25% and decreased acetylation at position 2 of Xylp in the dimethyl sulfoxide-extracted xylan. This did not affect plant growth but decreased xylose and increased glucose contents in the noncellulosic monosaccharide fraction, and increased glucose and xylose yields of wood enzymatic hydrolysis without pretreatment. Both RWA clades regulate wood xylan acetylation in aspen and are promising targets to improve wood saccharification. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  9. Direct transformation of xylan-type hemicelluloses to furfural via SnCl₄ catalysts in aqueous and biphasic systems.

    PubMed

    Wang, Wenju; Ren, Junli; Li, Huiling; Deng, Aojie; Sun, Runcang

    2015-05-01

    Direct catalytic transformation of xylan-type hemicelluloses to furfural in the aqueous system and the biphasic system were comparatively investigated under mild conditions. Screening of several promising chlorides for conversion of beech xylan in the aqueous system revealed the Lewis acid SnCl4 was the most effective catalyst. Comparing to the single aqueous system, the bio-based 2-methyltetrahydrofuran (2-MTHF)/H2O biphasic system was more conducive to the synthesis of furfural, in which the highest furfural yield of 78.1% was achieved by using SnCl4 as catalysts under the optimized reaction conditions (150°C, 120 min). Additionally, the influences of xylan-type hemicelluloses with different chemical and structural features from beech, corncob and bagasse on the furfural production were studied. It was found that furfural yield to some extent was determined by the xylose content in hemicelluloses and also had relationships with the molecular weight of hemicelluloses and the degree of crystallization. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. A molecular modeling approach to understand the structure and conformation relationship of (GlcpA)Xylan.

    PubMed

    Guo, Qingbin; Kang, Ji; Wu, Yan; Cui, Steve W; Hu, Xinzhong; Yada, Rickey Y

    2015-12-10

    The structure and conformation relationships of a heteropolysaccharide (GlcpA)Xylan in terms of various molecular weights, Xylp/GlcpA ratio and the distribution of GlcpA along xylan chain were investigated using computer modeling. The adiabatic contour maps of xylobiose, XylpXylp(GlcpA) and (GlcpA)XylpXylp(GlcpA) indicated that the insertion of the side group (GlcpA) influenced the accessible conformational space of xylobiose molecule. RIS-Metropolis Monte Carlo method indicated that insertion of GlcpA side chain induced a lowering effect of the calculated chain extension at low GlcpA:Xylp ratio (GlcpA:Xylp = 1:3). The chain, however, became extended when the ratio of GlcpA:Xylp above 2/3. It was also shown that the spatial extension of the polymer chains was dependent on the distribution of side chain: the random distribution demonstrated the most flexible structure compared to block and alternative distribution. The present studies provide a unique insight into the dependence of both side chain ratio and distribution on the stiffness and flexibility of various (GlcpA)Xylan molecules. Copyright © 2015. Published by Elsevier Ltd.

  11. Three Novel Rice Genes Closely Related to the Arabidopsis IRX9, IRX9L, and IRX14 Genes and Their Roles in Xylan Biosynthesis

    PubMed Central

    Chiniquy, Dawn; Varanasi, Patanjali; Oh, Taeyun; Harholt, Jesper; Katnelson, Jacob; Singh, Seema; Auer, Manfred; Simmons, Blake; Adams, Paul D.; Scheller, Henrik V.; Ronald, Pamela C.

    2013-01-01

    Xylan is the second most abundant polysaccharide on Earth, and represents a major component of both dicot wood and the cell walls of grasses. Much knowledge has been gained from studies of xylan biosynthesis in the model plant, Arabidopsis. In particular, the irregular xylem (irx) mutants, named for their collapsed xylem cells, have been essential in gaining a greater understanding of the genes involved in xylan biosynthesis. In contrast, xylan biosynthesis in grass cell walls is poorly understood. We identified three rice genes Os07g49370 (OsIRX9), Os01g48440 (OsIRX9L), and Os06g47340 (OsIRX14), from glycosyltransferase family 43 as putative orthologs to the putative β-1,4-xylan backbone elongating Arabidopsis IRX9, IRX9L, and IRX14 genes, respectively. We demonstrate that the over-expression of the closely related rice genes, in full or partly complement the two well-characterized Arabidopsis irregular xylem (irx) mutants: irx9 and irx14. Complementation was assessed by measuring dwarfed phenotypes, irregular xylem cells in stem cross sections, xylose content of stems, xylosyltransferase (XylT) activity of stems, and stem strength. The expression of OsIRX9 in the irx9 mutant resulted in XylT activity of stems that was over double that of wild type plants, and the stem strength of this line increased to 124% above that of wild type. Taken together, our results suggest that OsIRX9/OsIRX9L, and OsIRX14, have similar functions to the Arabidopsis IRX9 and IRX14 genes, respectively. Furthermore, our expression data indicate that OsIRX9 and OsIRX9L may function in building the xylan backbone in the secondary and primary cell walls, respectively. Our results provide insight into xylan biosynthesis in rice and how expression of a xylan synthesis gene may be modified to increase stem strength. PMID:23596448

  12. Suppression of xylan endotransglycosylase PtxtXyn10A affects cellulose microfibril angle in secondary wall in aspen wood.

    PubMed

    Derba-Maceluch, Marta; Awano, Tatsuya; Takahashi, Junko; Lucenius, Jessica; Ratke, Christine; Kontro, Inkeri; Busse-Wicher, Marta; Kosik, Ondrej; Tanaka, Ryo; Winzéll, Anders; Kallas, Åsa; Leśniewska, Joanna; Berthold, Fredrik; Immerzeel, Peter; Teeri, Tuula T; Ezcurra, Ines; Dupree, Paul; Serimaa, Ritva; Mellerowicz, Ewa J

    2015-01-01

    Certain xylanases from family GH10 are highly expressed during secondary wall deposition, but their function is unknown. We carried out functional analyses of the secondary-wall specific PtxtXyn10A in hybrid aspen (Populus tremula × tremuloides). PtxtXyn10A function was analysed by expression studies, overexpression in Arabidopsis protoplasts and by downregulation in aspen. PtxtXyn10A overexpression in Arabidopsis protoplasts resulted in increased xylan endotransglycosylation rather than hydrolysis. In aspen, the enzyme was found to be proteolytically processed to a 68 kDa peptide and residing in cell walls. Its downregulation resulted in a corresponding decrease in xylan endotransglycosylase activity and no change in xylanase activity. This did not alter xylan molecular weight or its branching pattern but affected the cellulose-microfibril angle in wood fibres, increased primary growth (stem elongation, leaf formation and enlargement) and reduced the tendency to form tension wood. Transcriptomes of transgenic plants showed downregulation of tension wood related genes and changes in stress-responsive genes. The data indicate that PtxtXyn10A acts as a xylan endotransglycosylase and its main function is to release tensional stresses arising during secondary wall deposition. Furthermore, they suggest that regulation of stresses in secondary walls plays a vital role in plant development. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

  13. Xylan degradation by the human gut Bacteroides xylanisolvens XB1A(T) involves two distinct gene clusters that are linked at the transcriptional level.

    PubMed

    Despres, Jordane; Forano, Evelyne; Lepercq, Pascale; Comtet-Marre, Sophie; Jubelin, Gregory; Chambon, Christophe; Yeoman, Carl J; Berg Miller, Margaret E; Fields, Christopher J; Martens, Eric; Terrapon, Nicolas; Henrissat, Bernard; White, Bryan A; Mosoni, Pascale

    2016-05-04

    Plant cell wall (PCW) polysaccharides and especially xylans constitute an important part of human diet. Xylans are not degraded by human digestive enzymes in the upper digestive tract and therefore reach the colon where they are subjected to extensive degradation by some members of the symbiotic microbiota. Xylanolytic bacteria are the first degraders of these complex polysaccharides and they release breakdown products that can have beneficial effects on human health. In order to understand better how these bacteria metabolize xylans in the colon, this study was undertaken to investigate xylan breakdown by the prominent human gut symbiont Bacteroides xylanisolvens XB1A(T). Transcriptomic analyses of B. xylanisolvens XB1A(T) grown on insoluble oat-spelt xylan (OSX) at mid- and late-log phases highlighted genes in a polysaccharide utilization locus (PUL), hereafter called PUL 43, and genes in a fragmentary remnant of another PUL, hereafter referred to as rPUL 70, which were highly overexpressed on OSX relative to glucose. Proteomic analyses supported the up-regulation of several genes belonging to PUL 43 and showed the important over-production of a CBM4-containing GH10 endo-xylanase. We also show that PUL 43 is organized in two operons and that the knockout of the PUL 43 sensor/regulator HTCS gene blocked the growth of the mutant on insoluble OSX and soluble wheat arabinoxylan (WAX). The mutation not only repressed gene expression in the PUL 43 operons but also repressed gene expression in rPUL 70. This study shows that xylan degradation by B. xylanisolvens XB1A(T) is orchestrated by one PUL and one PUL remnant that are linked at the transcriptional level. Coupled to studies on other xylanolytic Bacteroides species, our data emphasize the importance of one peculiar CBM4-containing GH10 endo-xylanase in xylan breakdown and that this modular enzyme may be used as a functional marker of xylan degradation in the human gut. Our results also suggest that B. xylanisolvens

  14. Facilitating the enzymatic saccharification of pulped bamboo residues by degrading the remained xylan and lignin-carbohydrates complexes.

    PubMed

    Huang, Caoxing; He, Juan; Li, Xin; Min, Douyong; Yong, Qiang

    2015-09-01

    Kraft pulping was performed on bamboo residues and its impact on the chemical compositions and the enzymatic digestibility of the samples were investigated. To improve the digestibility of sample by degrading the xylan and lignin-carbohydrates complexes (LCCs), xylanase and α-L-arabinofuranosidase (AF) were supplemented with cellulase. The results showed more carbohydrates were remained in the samples pulped with low effective alkali (EA) charge, compared to conventional kraft pulping. When 120 IU/g xylanase and 15 IU/g AF were supplemented with 20 FPU/g cellulase, the xylan degradation yield of the sample pulped with 12% EA charge increased from 68.20% to 88.35%, resulting in an increased enzymatic saccharification efficiency from 58.98% to 83.23%. The amount of LCCs in this sample decreased from 8.63/100C9 to 2.99/100C9 after saccharification with these enzymes. The results indicated that degrading the remained xylan and LCCs in the pulp could improve its enzymatic digestibility. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Long-Term Enrichment on Cellulose or Xylan Causes Functional and Taxonomic Convergence of Microbial Communities from Anaerobic Digesters

    PubMed Central

    Jia, Yangyang; Wilkins, David; Lu, Hongyuan; Cai, Mingwei

    2015-01-01

    Cellulose and xylan are two major components of lignocellulosic biomass, which represents a potentially important energy source, as it is abundant and can be converted to methane by microbial action. However, it is recalcitrant to hydrolysis, and the establishment of a complete anaerobic digestion system requires a specific repertoire of microbial functions. In this study, we maintained 2-year enrichment cultures of anaerobic digestion sludge amended with cellulose or xylan to investigate whether a cellulose- or xylan-digesting microbial system could be assembled from sludge previously used to treat neither of them. While efficient methane-producing communities developed under mesophilic (35°C) incubation, they did not under thermophilic (55°C) conditions. Illumina amplicon sequencing results of the archaeal and bacterial 16S rRNA genes revealed that the mature cultures were much lower in richness than the inocula and were dominated by single archaeal (genus Methanobacterium) and bacterial (order Clostridiales) groups, although at finer taxonomic levels the bacteria were differentiated by substrates. Methanogenesis was primarily via the hydrogenotrophic pathway under all conditions, although the identity and growth requirements of syntrophic acetate-oxidizing bacteria were unclear. Incubation conditions (substrate and temperature) had a much greater effect than inoculum source in shaping the mature microbial community, although analysis based on unweighted UniFrac distance found that the inoculum still determined the pool from which microbes could be enriched. Overall, this study confirmed that anaerobic digestion sludge treating nonlignocellulosic material is a potential source of microbial cellulose- and xylan-digesting functions given appropriate enrichment conditions. PMID:26712547

  16. Insight into Aluminum Sulfate-Catalyzed Xylan Conversion into Furfural in a γ-Valerolactone/Water Biphasic Solvent under Microwave Conditions.

    PubMed

    Yang, Tao; Zhou, Yi-Han; Zhu, Sheng-Zhen; Pan, Hui; Huang, Yao-Bing

    2017-10-23

    A simple and efficient biphasic system with an earth-abundant metal salt catalyst was used to produce furfural from xylan with a high yield of up to 87.8 % under microwave conditions. Strikingly, the metal salt Al 2 (SO 4 ) 3 exhibited excellent catalytic activity for xylan conversion, owing to a combination of Lewis and Brønsted acidity and its ability to promote good phase separation. The critical role of the SO 4 2- anion was first analyzed, which resulted in the aforementioned characteristics when combined with the Al 3+ cation. The mixed solvent system with γ-valerolactone (GVL) as the organic phase provided the highest furfural yield, resulting from its good dielectric properties and dissolving capacity, which facilitated the absorption of microwave energy and promoted mass transfer. Mechanistic studies suggested that the xylan-to-furfural conversion proceeded mainly through a hydrolysis-isomerization-dehydration pathway and the hexa-coordinated Lewis acidic [Al(OH) 2 (aq)] + species were the active sites for xylose-xylulose isomerization. Detailed kinetic studies of the subreaction for the xylan conversion revealed that GVL regulates the reaction rates and pathways by promoting the rates of the key steps involved for furfural production and suppressing the side reactions for humin production. Finally, the Al 2 (SO 4 ) 3 catalyst was used for the production of furfural from several lignocellulosic feedstocks, revealing its great potential for other biomass conversions. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Heterologous expression of family 10 xylanases from Acidothermus cellulolyticus enhances the exoproteome of Caldicellulosiruptor bescii and growth on xylan substrates

    DOE PAGES

    Kim, Sun -Ki; Chung, Daehwan; Himmel, Michael E.; ...

    2016-08-22

    The ability to deconstruct plant biomass without conventional pretreatment has made members of the genus Caldicellulosiruptor the target of investigation for the consolidated processing of lignocellulosic biomass to biofuels and bioproducts. These Gram-positive bacteria are hyperthermophilic anaerobes and the most thermophilic cellulolytic organisms so far described. They use both C5 and C6 sugars simultaneously and have the ability to grow well on xylan, a major component of plant cell walls. This is an important advantage for their use to efficiently convert biomass at yields sufficient for an industrial process. For commodity chemicals, yield from substrate is perhaps the most importantmore » economic factor. In an attempt to improve even further the ability of C. bescii to use xylan, we introduced two xylanases from Acidothermus cellulolyticus. Acel_0180 includes tandem carbohydrate-binding modules (CBM2 and CBM3) located at the C-terminus, one of which, CBM2, is not present in C. bescii. Also, the sequences of Xyn10A and Acel_0180 have very little homology with the GH10 domains present in C. bescii. For these reasons, we selected these xylanases as potential candidates for synergistic interaction with those in the C. bescii exoproteome. As a result, heterologous expression of two xylanases from Acidothermus cellulolyticus in Caldicellulosiruptor bescii resulted in a modest, but significant increase in the activity of the exoproteome of C. bescii on xylan substrates. Even though the increase in extracellular activity was modest, the ability of C. bescii to grow on these substrates was dramatically improved suggesting that the xylan substrate/microbe interaction substantially increased deconstruction over the secreted free enzymes alone. In conclusion, we anticipate that the ability to efficiently use xylan, a major component of plant cell walls for conversion of plant biomass to products of interest, will allow the conversion of renewable, sustainable, and

  18. Digestive and physiological effects of a wheat bran extract, arabino-xylan-oligosaccharide, in breakfast cereal

    USDA-ARS?s Scientific Manuscript database

    We assessed whether a wheat bran extract containing arabino-xylan-oligosaccharide (AXOS) elicited a prebiotic effect and influenced other physiologic parameters when consumed in ready-to-eat cereal at two dose levels. This double-blind, randomized, controlled, crossover trial evaluated the effects o...

  19. Synthesis of Acylated Xylan-Based Magnetic Fe3O4 Hydrogels and Their Application for H2O2 Detection

    PubMed Central

    Dai, Qing-Qing; Ren, Jun-Li; Peng, Feng; Chen, Xiao-Feng; Gao, Cun-Dian; Sun, Run-Cang

    2016-01-01

    Acylated xylan-based magnetic Fe3O4 nanocomposite hydrogels (ACX-MNP-gels) were prepared by fabricating Fe3O4 nanoctahedra in situ within a hydrogel matrix which was synthesized by the copolymerization of acylated xylan (ACX) with acrylamide and N-isopropylacrylamide under ultraviolet irradiation. The size of the Fe3O4 fabricated within the hydrogel matrix could be adjusted through controlling the crosslinking concentrations (C). The magnetic hydrogels showed desirable magnetic and mechanical properties, which were confirmed by XRD, Raman spectroscopy, physical property measurement system, SEM, TGA, and compression test. Moreover, the catalytic performance of the magnetic hydrogels was explored. The magnetic hydrogels (C = 7.5 wt %) presented excellent catalytic activity and provided a sensitive response to H2O2 detection even at a concentration level of 5 × 10−6 mol·L−1. This approach to preparing magnetic hydrogels loaded with Fe3O4 nanoparticles endows xylan-based hydrogels with new promising applications in biotechnology and environmental chemistry. PMID:28773811

  20. Supplementation with xylanase and β-xylosidase to reduce xylo-oligomer and xylan inhibition of enzymatic hydrolysis of cellulose and pretreated corn stover

    PubMed Central

    2011-01-01

    Background Hemicellulose is often credited with being one of the important physical barriers to enzymatic hydrolysis of cellulose, and acts by blocking enzyme access to the cellulose surface. In addition, our recent research has suggested that hemicelluloses, particularly in the form of xylan and its oligomers, can more strongly inhibit cellulase activity than do glucose and cellobiose. Removal of hemicelluloses or elimination of their negative effects can therefore become especially pivotal to achieving higher cellulose conversion with lower enzyme doses. Results In this study, cellulase was supplemented with xylanase and β-xylosidase to boost conversion of both cellulose and hemicellulose in pretreated biomass through conversion of xylan and xylo-oligomers to the less inhibitory xylose. Although addition of xylanase and β-xylosidase did not necessarily enhance Avicel hydrolysis, glucan conversions increased by 27% and 8% for corn stover pretreated with ammonia fiber expansion (AFEX) and dilute acid, respectively. In addition, adding hemicellulase several hours before adding cellulase was more beneficial than later addition, possibly as a result of a higher adsorption affinity of cellulase and xylanase to xylan than glucan. Conclusions This key finding elucidates a possible mechanism for cellulase inhibition by xylan and xylo-oligomers and emphasizes the need to optimize the enzyme formulation for each pretreated substrate. More research is needed to identify advanced enzyme systems designed to hydrolyze different substrates with maximum overall enzyme efficacy. PMID:21702938

  1. Compost Grown Agaricus bisporus Lacks the Ability to Degrade and Consume Highly Substituted Xylan Fragments

    PubMed Central

    de Vries, Ronald P.; Gruppen, Harry; Kabel, Mirjam A.

    2015-01-01

    The fungus Agaricus bisporus is commercially grown for the production of edible mushrooms. This cultivation occurs on compost, but not all of this substrate is consumed by the fungus. To determine why certain fractions remain unused, carbohydrate degrading enzymes, water-extracted from mushroom-grown compost at different stages of mycelium growth and fruiting body formation, were analyzed for their ability to degrade a range of polysaccharides. Mainly endo-xylanase, endo-glucanase, β-xylosidase and β-glucanase activities were determined in the compost extracts obtained during mushroom growth. Interestingly, arabinofuranosidase activity able to remove arabinosyl residues from doubly substituted xylose residues and α-glucuronidase activity were not detected in the compost enzyme extracts. This correlates with the observed accumulation of arabinosyl and glucuronic acid substituents on the xylan backbone in the compost towards the end of the cultivation. Hence, it was concluded that compost grown A. bisporus lacks the ability to degrade and consume highly substituted xylan fragments. PMID:26237450

  2. Structural characterization (1->2)-beta-xylose-(1->3)-alpha-arabinose-containing oligosaccharide products of extracted switchgrass (Panicum virgatum, L.) xylan treatment with alpha-arabinofuranosidase and beta-endo-xylanase.

    USDA-ARS?s Scientific Manuscript database

    Switchgrass (Panicum virgatum, L.) is a potential dedicated biomass crop for use in biocatalytic conversion systems to biofuels. Nearly 30% of switchgrass cell wall material is xylan. The complete depolymerization of xylan is desirable both as an additional carbon source for microbial fermentation a...

  3. Complete genome sequence of the xylan-degrading subseafloor bacterium Microcella alkaliphila JAM-AC0309.

    PubMed

    Kurata, Atsushi; Hirose, Yuu; Misawa, Naomi; Wakazuki, Sachiko; Kishimoto, Noriaki; Kobayashi, Tohru

    2016-03-10

    Here we report the complete genome sequence of Microcella alkaliphila JAM-AC0309, which was newly isolated from the deep subseafloor core sediment from offshore of the Shimokita Peninsula of Japan. An array of genes related to utilization of xylan in this bacterium was identified by whole genome analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Xylan-regulated delivery of human keratinocyte growth factor-2 to the inflamed colon by the human anaerobic commensal bacterium Bacteroides ovatus.

    PubMed

    Hamady, Zaed Z R; Scott, Nigel; Farrar, Mark D; Lodge, J Peter A; Holland, Keith T; Whitehead, Terence; Carding, Simon R

    2010-04-01

    Human growth factors are potential therapeutic agents for various inflammatory disorders affecting the gastrointestinal tract. However, they are unstable when administered orally and systemic administration requires high doses increasing the risk of unwanted side effects. Live microorganism-based delivery systems can overcome these problems although they suffer from the inability to control heterologous protein production and there are concerns regarding biosafety and environmental contamination. To overcome these limitations we have developed a new live bacteria drug-delivery system using the human commensal gut bacterium Bacteroides ovatus engineered to secrete human growth factors in response to dietary xylan. The anaerobic nature of B ovatus provides an inherent biosafety feature. B ovatus strains expressing human keratinocyte growth factor-2, which plays a central role in intestinal epithelial homeostasis and repair (BO-KGF), were generated by homologous recombination and evaluated using the dextran sodium sulfate (DSS)-induced model of intestinal epithelial injury and colitis. In response to xylan BO-KGF produced biologically active KGF both in vitro and in vivo. In DSS treated mice administration of xylan and BO-KGF had a significant therapeutic effect in reducing weight loss, improving stool consistency, reducing rectal bleeding, accelerating healing of damaged epithelium, reducing inflammation and neutrophil infiltration, reducing expression of pro-inflammatory cytokines, and accelerating production of goblet cells. BO-KGF and xylan treatment also had a marked prophylactic effect limiting the development of inflammation and disruption of the epithelial barrier. This novel, diet-regulated, live bacterial drug delivery system may be applicable to treating various bowel disorders.

  5. Arabidopsis GUX Proteins Are Glucuronyltransferases Responsible for the Addition of Glucuronic Acid Side Chains onto Xylan

    EPA Science Inventory

    Xylan, the second most abundant cell wall polysaccharide, is composed of a linear backbone of β-(1,4)-linked xylosyl residues that are often substituted with sugar side chains, such as glucuronic acid (GlcA) and methylglucuronic acid (MeGlcA). It has recently been shown that muta...

  6. H2 and acetate transfers during xylan fermentation between a butyrate-producing xylanolytic species and hydrogenotrophic microorganisms from the human gut.

    PubMed

    Chassard, Christophe; Bernalier-Donadille, Annick

    2006-01-01

    The aim of this work was to investigate in vitro interrelationships during xylan fermentation between an H2 and butyrate-producing xylanolytic species recently isolated in our laboratory from human faeces and identified as Roseburia intestinalis and the H2-utilizing acetogen Ruminococcus hydrogenotrophicus or the methanogen Methanobrevibacter smithii. H2 transfer between M. smithii or Ru. hydrogenotrophicus and the xylanolytic species was evidenced, confirming the great potential of these H2-consuming microorganisms to reutilize fermentative H2 during fibre fermentation in the gut. In addition, acetate transfer was demonstrated between the xylanolytic Roseburia sp. and the acetogenic species, both metabolites transfers leading to butyric fermentation of oat xylan without production of H2.

  7. Downregulation of GAUT12 in Populus deltoides by RNA silencing results in reduced recalcitrance, increased growth and reduced xylan and pectin in a woody biofuel feedstock

    DOE PAGES

    Biswal, Ajaya K.; Hao, Zhangying; Pattathil, Sivakumar; ...

    2015-03-12

    The inherent recalcitrance of woody bioenergy feedstocks is a major challenge for their use as a source of second-generation biofuel. Secondary cell walls that constitute the majority of hardwood biomass are rich in cellulose, xylan, and lignin. The interactions among these polymers prevent facile accessibility and deconstruction by enzymes and chemicals. Plant biomass that can with minimal pretreatment be degraded into sugars is required to produce renewable biofuels in a cost-effective manner. The following are the results: GAUT12/IRX8 is a putative glycosyltransferase proposed to be involved in secondary cell wall glucuronoxylan and/or pectin biosynthesis based on concomitant reductions of bothmore » xylan and the pectin homogalacturonan (HG) in Arabidopsis irx8 mutants. Two GAUT12 homologs exist in Populus trichocarpa, PtGAUT12.1 and PtGAUT12.2. Knockdown expression of both genes simultaneously has been shown to reduce xylan content in Populus wood. We tested the proposition that RNA interference (RNAi) downregulation of GAUT12.1 alone would lead to increased sugar release in Populus wood, that is, reduced recalcitrance, based on the hypothesis that GAUT12 synthesizes a wall structure required for deposition of xylan and that cell walls with less xylan and/or modified cell wall architecture would have reduced recalcitrance. Using an RNAi approach, we generated 11 Populus deltoides transgenic lines with 50 to 67% reduced PdGAUT12.1 transcript expression compared to wild type (WT) and vector controls. Ten of the eleven RNAi lines yielded 4 to 8% greater glucose release upon enzymatic saccharification than the controls. The PdGAUT12.1 knockdown (PdGAUT12.1-KD) lines also displayed 12 to 52% and 12 to 44% increased plant height and radial stem diameter, respectively, compared to the controls. Knockdown of PdGAUT12.1 resulted in a 25 to 47% reduction in galacturonic acid and 17 to 30% reduction in xylose without affecting total lignin content, revealing that in

  8. Improving Biofuel Feedstocks by Modifying Xylan Biosynthesis (2013 DOE JGI Genomics of Energy and Environment 8th Annual User Meeting)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lau, Jane

    2013-03-01

    Jane Lau of the Joint BioEnergy Institute on Improving biofuel feedstocks by modifying xylan biosynthesis at the 8th Annual Genomics of Energy Environment Meeting on March 28, 2013 in Walnut Creek, CA.

  9. Treatment of colitis with a commensal gut bacterium engineered to secrete human TGF-β1 under the control of dietary xylan 1.

    PubMed

    Hamady, Zaed Z R; Scott, Nigel; Farrar, Mark D; Wadhwa, Meenu; Dilger, Paula; Whitehead, Terence R; Thorpe, Robin; Holland, Keith T; Lodge, J Peter A; Carding, Simon R

    2011-09-01

    While cytokine therapy and the use of immunosuppressive cytokines such as transforming growth factor-β (TGF-β) offer great potential for the treatment of inflammatory bowel disease (IBD), issues concerning formulation, stability in vivo, delivery to target tissues, and potential toxicity need to be addressed. In consideration of these problems we engineered the human commensal bacterium Bacteroides ovatus for the controlled in situ delivery of TGF-β(1) and treatment of colitis. Sequence encoding the human tgf-β1 gene was cloned downstream of the xylanase promoter in the xylan operon of B. ovatus by homologous recombination. Resulting recombinants (BO-TGF) were tested for TGF-β production in the presence and absence of polysaccharide xylan in vitro and in vivo, and used to treat experimental murine colitis. Clinical and pathological scores were used to assess the effectiveness of therapy. Colonic inflammatory markers including inflammatory cytokine expression were assessed by colorimetric assay and real-time polymerase chain reaction (PCR). BO-TGF secreted high levels of biologically active dimeric TGF-β in vitro and in vivo in a xylan-controlled manner. Administration of xylan in drinking water to BO-TGF-treated mice resulted in a significant clinical improvement of colitis, accelerating healing of damaged colonic epithelium, reducing inflammatory cell infiltration, reducing expression of proinflammatory cytokines, and promoting production of mucin-rich goblet cells in colonic crypts. These beneficial effects are comparable and in most cases superior to that achieved by conventional steroid therapy. This novel drug delivery system has potential for the targeted and controlled delivery of TGF-β(1) and other immunotherapeutic agents for the long-term management of various bowel disorders. Copyright © 2010 Crohn's & Colitis Foundation of America, Inc.

  10. Isolation and Characterization of Bacteria from the Gut of Bombyx mori that Degrade Cellulose, Xylan, Pectin and Starch and Their Impact on Digestion

    PubMed Central

    Anand, A. Alwin Prem; Vennison, S. John; Sankar, S. Gowri; Prabhu, D. Immanual Gilwax; Vasan, P. Thirumalai; Raghuraman, T.; Geoffrey, C. Jerome; Vendan, S. Ezhil

    2010-01-01

    Bombyx mori L. (Lepidoptera: Bombycidae) have been domesticated and widely used for silk production. It feeds on mulberry leaves. Mulberry leaves are mainly composed of pectin, xylan, cellulose and starch. Some of the digestive enzymes that degrade these carbohydrates might be produced by gut bacteria. Eleven isolates were obtained from the digestive tract of B. mori, including the Gram positive Bacillus circulans and Gram negative Proteus vulgaris, Klebsiella pneumoniae, Escherichia coli, Citrobacter freundii, Serratia liquefaciens, Enterobacter sp., Pseudomonas fluorescens, P. aeruginosa, Aeromonas sp., and Erwinia sp.. Three of these isolates, P. vulgaris, K. pneumoniae, C. freundii, were cellulolytic and xylanolytic, P. fluorescens and Erwinia sp., were pectinolytic and K. pneumoniae degraded starch. Aeromonas sp. was able to utilize the CMcellulose and xylan. S. liquefaciens was able to utilize three polysaccharides including CMcellulose, xylan and pectin. B. circulans was able to utilize all four polysaccharides with different efficacy. The gut of B. mori has an alkaline pH and all of the isolated bacterial strains were found to grow and degrade polysaccharides at alkaline pH. The number of cellulolytic bacteria increases with each instar. PMID:20874394

  11. Isolation and characterization of bacteria from the gut of Bombyx mori that degrade cellulose, xylan, pectin and starch and their impact on digestion.

    PubMed

    Anand, A Alwin Prem; Vennison, S John; Sankar, S Gowri; Prabhu, D Immanual Gilwax; Vasan, P Thirumalai; Raghuraman, T; Geoffrey, C Jerome; Vendan, S Ezhil

    2010-01-01

    Bombyx mori L. (Lepidoptera: Bombycidae) have been domesticated and widely used for silk production. It feeds on mulberry leaves. Mulberry leaves are mainly composed of pectin, xylan, cellulose and starch. Some of the digestive enzymes that degrade these carbohydrates might be produced by gut bacteria. Eleven isolates were obtained from the digestive tract of B. mori, including the Gram positive Bacillus circulans and Gram negative Proteus vulgaris, Klebsiella pneumoniae, Escherichia coli, Citrobacter freundii, Serratia liquefaciens, Enterobacter sp., Pseudomonas fluorescens, P. aeruginosa, Aeromonas sp., and Erwinia sp.. Three of these isolates, P. vulgaris, K. pneumoniae, C. freundii, were cellulolytic and xylanolytic, P. fluorescens and Erwinia sp., were pectinolytic and K. pneumoniae degraded starch. Aeromonas sp. was able to utilize the CMcellulose and xylan. S. liquefaciens was able to utilize three polysaccharides including CMcellulose, xylan and pectin. B. circulans was able to utilize all four polysaccharides with different efficacy. The gut of B. mori has an alkaline pH and all of the isolated bacterial strains were found to grow and degrade polysaccharides at alkaline pH. The number of cellulolytic bacteria increases with each instar.

  12. Direct and efficient xylitol production from xylan by Saccharomyces cerevisiae through transcriptional level and fermentation processing optimizations.

    PubMed

    Li, Zhe; Qu, Hongnan; Li, Chun; Zhou, Xiaohong

    2013-12-01

    In this study, four engineered Saccharomyces cerevisiae carrying xylanase, β-xylosidase and xylose reductase genes by different transcriptional regulations were constructed to directly convert xylan to xylitol. According to the results, the high-copy number plasmid required a rigid selection for promoter characteristics, on the contrast, the selection of promoters could be more flexible for low-copy number plasmid. For cell growth and xylitol production, glucose and galactose were found more efficient than other sugars. The semi-aerobic condition and feeding of co-substrates were taken to improve the yield of xylitol. It was found that the strain containing high-copy number plasmid had the highest xylitol yield, but it was sensitive to the change of fermentation. However, the strain carrying low-copy number plasmid was more adaptable to different processes. By optimization of the transcriptional regulation and fermentation processes, the xylitol concentration could be increased of 1.7 folds and the yield was 0.71 g xylitol/g xylan. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Assembly of Xylanases into Designer Cellulosomes Promotes Efficient Hydrolysis of the Xylan Component of a Natural Recalcitrant Cellulosic Substrate

    PubMed Central

    Moraïs, Sarah; Barak, Yoav; Hadar, Yitzhak; Wilson, David B.; Shoham, Yuval; Lamed, Raphael; Bayer, Edward A.

    2011-01-01

    ABSTRACT In nature, the complex composition and structure of the plant cell wall pose a barrier to enzymatic degradation. Nevertheless, some anaerobic bacteria have evolved for this purpose an intriguing, highly efficient multienzyme complex, the cellulosome, which contains numerous cellulases and hemicellulases. The rod-like cellulose component of the plant cell wall is embedded in a colloidal blend of hemicelluloses, a major component of which is xylan. In order to enhance enzymatic degradation of the xylan component of a natural complex substrate (wheat straw) and to study the synergistic action among different xylanases, we have employed a variation of the designer cellulosome approach by fabricating a tetravalent complex that includes the three endoxylanases of Thermobifida fusca (Xyn10A, Xyn10B, and Xyn11A) and an Xyl43A β-xylosidase from the same bacterium. Here, we describe the conversion of Xyn10A and Xyl43A to the cellulosomal mode. The incorporation of the Xyl43A enzyme together with the three endoxylanases into a common designer cellulosome served to enhance the level of reducing sugars produced during wheat straw degradation. The enhanced synergistic action of the four xylanases reflected their immediate juxtaposition in the complex, and these tetravalent xylanolytic designer cellulosomes succeeded in degrading significant (~25%) levels of the total xylan component of the wheat straw substrate. The results suggest that the incorporation of xylanases into cellulosome complexes is advantageous for efficient decomposition of recalcitrant cellulosic substrates—a distinction previously reserved for cellulose-degrading enzymes. PMID:22086489

  14. Complex carbohydrates reduce the frequency of antagonistic interactions among bacteria degrading cellulose and xylan.

    PubMed

    Deng, Yi-Jie; Wang, Shiao Y

    2017-03-01

    Bacterial competition for resources is common in nature but positive interactions among bacteria are also evident. We speculate that the structural complexity of substrate might play a role in mediating bacterial interactions. We tested the hypothesis that the frequency of antagonistic interactions among lignocellulolytic bacteria is reduced when complex polysaccharide is the main carbon source compared to when a simple sugar such as glucose is available. Results using all possible pairwise interactions among 35 bacteria isolated from salt marsh detritus showed that the frequency of antagonistic interactions was significantly lower on carboxymethyl cellulose (CMC)-xylan medium (7.8%) than on glucose medium (15.5%). The two interaction networks were also different in their structures. Although 75 antagonistic interactions occurred on both media, there were 115 that occurred only on glucose and 20 only on CMC-xylan, indicating that some antagonistic interactions were substrate specific. We also found that the frequency of antagonism differed among phylogenetic groups. Gammaproteobacteria and Bacillus sp. were the most antagonistic and they tended to antagonize Bacteroidetes and Actinobacteria, the most susceptible groups. Results from the study suggest that substrate complexity affects how bacteria interact and that bacterial interactions in a community are dynamic as nutrient conditions change. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  15. Furfural Production from d-Xylose and Xylan by Using Stable Nafion NR50 and NaCl in a Microwave-Assisted Biphasic Reaction.

    PubMed

    Le Guenic, Sarah; Gergela, David; Ceballos, Claire; Delbecq, Frederic; Len, Christophe

    2016-08-22

    Pentose dehydration and direct transformation of xylan into furfural were performed in a water-cyclopentyl methyl ether (CPME) biphasic system under microwave irradiation. Heated up between 170 and 190 °C in the presence of Nafion NR50 and NaCl, d-xylose, l-arabinose and xylan gave furfural with maximum yields of 80%, 42% and 55%, respectively. The influence of temperature and reaction time on the reaction kinetics was discussed. This study was also completed by the survey of different reactant ratios, such as organic layer-water or catalyst-inorganic salt ratios. The exchange between proton and cation induced by an excess of NaCl was monitored, and a synergetic effect between the remaining protons and the released HCl was also discovered.

  16. Asparagus IRX9, IRX10, and IRX14A Are Components of an Active Xylan Backbone Synthase Complex that Forms in the Golgi Apparatus1[OPEN

    PubMed Central

    Zeng, Wei; Picard, Kelsey L.; Song, Lili; Wu, Ai-Min; Farion, Isabela M.; Zhao, Jia; Ford, Kris; Bacic, Antony

    2016-01-01

    Heteroxylans are abundant components of plant cell walls and provide important raw materials for the food, pharmaceutical, and biofuel industries. A number of studies in Arabidopsis (Arabidopsis thaliana) have suggested that the IRREGULAR XYLEM9 (IRX9), IRX10, and IRX14 proteins, as well as their homologs, are involved in xylan synthesis via a Golgi-localized complex termed the xylan synthase complex (XSC). However, both the biochemical and cell biological research lags the genetic and molecular evidence. In this study, we characterized garden asparagus (Asparagus officinalis) stem xylan biosynthesis genes (AoIRX9, AoIRX9L, AoIRX10, AoIRX14A, and AoIRX14B) by heterologous expression in Nicotiana benthamiana. We reconstituted and partially purified an active XSC and showed that three proteins, AoIRX9, AoIRX10, and AoIRX14A, are necessary for xylan xylosyltranferase activity in planta. To better understand the XSC structure and its composition, we carried out coimmunoprecipitation and bimolecular fluorescence complementation analysis to show the molecular interactions between these three IRX proteins. Using a site-directed mutagenesis approach, we showed that the DxD motifs of AoIRX10 and AoIRX14A are crucial for the catalytic activity. These data provide, to our knowledge, the first lines of biochemical and cell biological evidence that AoIRX9, AoIRX10, and AoIRX14A are core components of a Golgi-localized XSC, each with distinct roles for effective heteroxylan biosynthesis. PMID:26951434

  17. Effect of yeast extract addition to a mineral salts medium containing hydrolyzed plant xylan on fungal pullulan production.

    PubMed

    Kennedy Ii, Daniel E; West, Thomas P

    2018-05-16

    The ability of the fungus Aureobasidium pullulans ATCC 42023 to produce pullulan from yeast extract-supplemented xylan hydrolysates of the prairie grass prairie cordgrass was examined relative to polysaccharide and cell biomass production, yield, and pullulan content of the polysaccharide. A pullulan concentration of 11.2 g L-1 and yield of 0.79 g g-1 was produced by ATCC 42023 when grown for 168 h at 30°C on the phosphate-buffered hydrolysate supplemented with yeast extract. The highest biomass level being 8.8 g L-1 was produced by ATCC 42023 after 168 h on a yeast extract-supplemented, hydrolysate-containing complete medium lacking sodium chloride. The highest pullulan content of the polysaccharide produced by ATCC 42023 after 168 h on the hydrolysate medium supplemented with yeast extract and ammonium sulfate was 70%. The findings indicate that a polysaccharide with a high pullulan content can be produced at a relatively high yield by the fungus grown on a yeast extract-supplemented xylan hydrolysate, suggesting that pullulan could be produced using a biomass-based process.

  18. Carbon Nanotubes Reinforced Maleic Anhydride-Modified Xylan-g-Poly(N-isopropylacrylamide) Hydrogel with Multifunctional Properties

    PubMed Central

    Liu, Xinxin; Song, Tao; Chang, Minmin; Meng, Ling; Wang, Xiaohui; Sun, Runcang; Ren, Junli

    2018-01-01

    Introducing multifunctional groups and inorganic material imparts xylan-based hydrogels with excellent properties, such as responsiveness to pH, temperature, light, and external magnetic field. In this work, a composite hydrogel was synthesized by introducing acid treated carbon nanotubes (AT-CNTs) into the maleic anhydride modified xylan grafted with poly(N-isopropylacrylamide) (MAX-g-PNIPAM) hydrogels network. It was found that the addition of AT-CNTs affected the MAX-g-PNIPAM hydrogel structure, the swelling ratio and mechanical properties, and imparted the hydrogel with new properties of electrical conductivity and near infrared region (NIR) photothermal conversion. AT-CNTs could reinforce the mechanical properties of MAX-g-PNIPAM hydrogels, being up to 83 kPa for the compressive strength when the amount was 11 wt %, which was eight times than that of PNIPAM hydrogel and four times than that of MAX-g-PNIPAM hydrogel. The electroconductibility was enhanced by the increase of AT-CNTs amounts. Meanwhile, the composite hydrogel also exhibited multiple shape memory and NIR photothermal conversion properties, and water temperature was increased from 26 °C to 56 °C within 8 min under the NIR irradiation. Thus, the AT-CNTs reinforced MAX-g-PNIPAM hydrogel possessed promising multifunctional properties, which offered many potential applications in the fields of biosensors, thermal-arrest technology, and drug-controlled release. PMID:29495611

  19. Carbon Nanotubes Reinforced Maleic Anhydride-Modified Xylan-g-Poly(N-isopropylacrylamide) Hydrogel with Multifunctional Properties.

    PubMed

    Liu, Xinxin; Song, Tao; Chang, Minmin; Meng, Ling; Wang, Xiaohui; Sun, Runcang; Ren, Junli

    2018-02-28

    Introducing multifunctional groups and inorganic material imparts xylan-based hydrogels with excellent properties, such as responsiveness to pH, temperature, light, and external magnetic field. In this work, a composite hydrogel was synthesized by introducing acid treated carbon nanotubes (AT-CNTs) into the maleic anhydride modified xylan grafted with poly(N-isopropylacrylamide) (MAX-g-PNIPAM) hydrogels network. It was found that the addition of AT-CNTs affected the MAX-g-PNIPAM hydrogel structure, the swelling ratio and mechanical properties, and imparted the hydrogel with new properties of electrical conductivity and near infrared region (NIR) photothermal conversion. AT-CNTs could reinforce the mechanical properties of MAX-g-PNIPAM hydrogels, being up to 83 kPa for the compressive strength when the amount was 11 wt %, which was eight times than that of PNIPAM hydrogel and four times than that of MAX-g-PNIPAM hydrogel. The electroconductibility was enhanced by the increase of AT-CNTs amounts. Meanwhile, the composite hydrogel also exhibited multiple shape memory and NIR photothermal conversion properties, and water temperature was increased from 26 °C to 56 °C within 8 min under the NIR irradiation. Thus, the AT-CNTs reinforced MAX-g-PNIPAM hydrogel possessed promising multifunctional properties, which offered many potential applications in the fields of biosensors, thermal-arrest technology, and drug-controlled release.

  20. Development, validation and application of a hydrophilic interaction liquid chromatography-evaporative light scattering detection based method for process control of hydrolysis of xylans obtained from different agricultural wastes.

    PubMed

    Li, Fangbing; Wang, Hui; Xin, Huaxia; Cai, Jianfeng; Fu, Qing; Jin, Yu

    2016-12-01

    Purified standards of xylooligosaccharides (XOSs) (DP2-6) were first prepared from a mixture of XOSs using solid phase extraction (SPE), followed by semi-preparative liquid chromatography both under hydrophilic interaction liquid chromatography (HILIC) modes. Then, an accurate quantitative analysis method based on hydrophilic interaction liquid chromatography-evaporative light scattering detection (HILIC-ELSD) was developed and validated for simultaneous determination of xylose (X1), xylobiose (X2), xylotriose (X3), xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6). This developed HILIC-ELSD method was applied to the comparison of different hydrolysis methods for xylans and assessment of XOSs contents from different agricultural wastes. The result indicated that enzymatic hydrolysis was preferable with fewer by-products and high XOSs yield. The XOSs yield (48.40%) from sugarcane bagasse xylan was the highest, showing conversions of 11.21g X2, 12.75g X3, 4.54g X4, 13.31g X5, and 6.78g X6 from 100g xylan. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Chemical Pretreatment-Independent Saccharifications of Xylan and Cellulose of Rice Straw by Bacterial Weak Lignin-Binding Xylanolytic and Cellulolytic Enzymes

    PubMed Central

    Teeravivattanakit, Thitiporn; Baramee, Sirilak; Phitsuwan, Paripok; Sornyotha, Somphit; Waeonukul, Rattiya; Pason, Patthra; Tachaapaikoon, Chakrit; Poomputsa, Kanokwan; Kosugi, Akihiko; Sakka, Kazuo

    2017-01-01

    ABSTRACT Complete utilization of carbohydrate fractions is one of the prerequisites for obtaining economically favorable lignocellulosic biomass conversion. This study shows that xylan in untreated rice straw was saccharified to xylose in one step without chemical pretreatment, yielding 58.2% of the theoretically maximum value by Paenibacillus curdlanolyticus B-6 PcAxy43A, a weak lignin-binding trifunctional xylanolytic enzyme, endoxylanase/β-xylosidase/arabinoxylan arabinofuranohydrolase. Moreover, xylose yield from untreated rice straw was enhanced to 78.9% by adding endoxylanases PcXyn10C and PcXyn11A from the same bacterium, resulting in improvement of cellulose accessibility to cellulolytic enzyme. After autoclaving the xylanolytic enzyme-treated rice straw, it was subjected to subsequent saccharification by a combination of the Clostridium thermocellum endoglucanase CtCel9R and Thermoanaerobacter brockii β-glucosidase TbCglT, yielding 88.5% of the maximum glucose yield, which was higher than the glucose yield obtained from ammonia-treated rice straw saccharification (59.6%). Moreover, this work presents a new environment-friendly xylanolytic enzyme pretreatment for beneficial hydrolysis of xylan in various agricultural residues, such as rice straw and corn hull. It not only could improve cellulose saccharification but also produced xylose, leading to an improvement of the overall fermentable sugar yields without chemical pretreatment. IMPORTANCE Ongoing research is focused on improving “green” pretreatment technologies in order to reduce energy demands and environmental impact and to develop an economically feasible biorefinery. The present study showed that PcAxy43A, a weak lignin-binding trifunctional xylanolytic enzyme, endoxylanase/β-xylosidase/arabinoxylan arabinofuranohydrolase from P. curdlanolyticus B-6, was capable of conversion of xylan in lignocellulosic biomass such as untreated rice straw to xylose in one step without chemical pretreatment

  2. Chemical Pretreatment-Independent Saccharifications of Xylan and Cellulose of Rice Straw by Bacterial Weak Lignin-Binding Xylanolytic and Cellulolytic Enzymes.

    PubMed

    Teeravivattanakit, Thitiporn; Baramee, Sirilak; Phitsuwan, Paripok; Sornyotha, Somphit; Waeonukul, Rattiya; Pason, Patthra; Tachaapaikoon, Chakrit; Poomputsa, Kanokwan; Kosugi, Akihiko; Sakka, Kazuo; Ratanakhanokchai, Khanok

    2017-11-15

    Complete utilization of carbohydrate fractions is one of the prerequisites for obtaining economically favorable lignocellulosic biomass conversion. This study shows that xylan in untreated rice straw was saccharified to xylose in one step without chemical pretreatment, yielding 58.2% of the theoretically maximum value by Paenibacillus curdlanolyticus B-6 PcAxy43A, a weak lignin-binding trifunctional xylanolytic enzyme, endoxylanase/β-xylosidase/arabinoxylan arabinofuranohydrolase. Moreover, xylose yield from untreated rice straw was enhanced to 78.9% by adding endoxylanases PcXyn10C and PcXyn11A from the same bacterium, resulting in improvement of cellulose accessibility to cellulolytic enzyme. After autoclaving the xylanolytic enzyme-treated rice straw, it was subjected to subsequent saccharification by a combination of the Clostridium thermocellum endoglucanase CtCel9R and Thermoanaerobacter brockii β-glucosidase TbCglT, yielding 88.5% of the maximum glucose yield, which was higher than the glucose yield obtained from ammonia-treated rice straw saccharification (59.6%). Moreover, this work presents a new environment-friendly xylanolytic enzyme pretreatment for beneficial hydrolysis of xylan in various agricultural residues, such as rice straw and corn hull. It not only could improve cellulose saccharification but also produced xylose, leading to an improvement of the overall fermentable sugar yields without chemical pretreatment. IMPORTANCE Ongoing research is focused on improving "green" pretreatment technologies in order to reduce energy demands and environmental impact and to develop an economically feasible biorefinery. The present study showed that PcAxy43A, a weak lignin-binding trifunctional xylanolytic enzyme, endoxylanase/β-xylosidase/arabinoxylan arabinofuranohydrolase from P. curdlanolyticus B-6, was capable of conversion of xylan in lignocellulosic biomass such as untreated rice straw to xylose in one step without chemical pretreatment. It

  3. Modification of Bacterial Cellulose Biofilms with Xylan Polyelectrolytes.

    PubMed

    Santos, Sara M; Carbajo, José M; Gómez, Nuria; Ladero, Miguel; Villar, Juan C

    2017-11-28

    The effect of the addition of two [4-butyltrimethylammonium]-xylan chloride polyelectrolytes (BTMAXs) on bacterial cellulose (BC) was evaluated. The first strategy was to add the polyelectrolytes to the culture medium together with a cell suspension of the bacterium. After one week of cultivation, the films were collected and purified. The second approach consisted of obtaining a purified and homogenized BC, to which the polyelectrolytes were added subsequently. The films were characterized in terms of tear and burst indexes, optical properties, surface free energy, static contact angle, Gurley porosity, SEM, X-ray diffraction and AFM. Although there are small differences in mechanical and optical properties between the nanocomposites and control films, the films obtained by BC synthesis in the presence of BTMAXs were remarkably less opaque, rougher, and had a much lower specular gloss. The surface free energy depends on the BTMAXs addition method. The crystallinity of the composites is lower than that of the control material, with a higher reduction of this parameter in the composites obtained by adding the BTMAXs to the culture medium. In view of these results, it can be concluded that BC-BTMAX composites are a promising new material, for example, for paper restoration.

  4. Structure-function relations of heparin-mimetic sulfated xylan oligosaccharides: inhibition of human immunodeficiency virus-1 infectivity in vitro.

    PubMed

    Stone, A L; Melton, D J; Lewis, M S

    1998-07-01

    Heparins/heparan sulfates modulate the function of proteins and cell membranes in numerous biological systems including normal and disease processes in humans. Heparin has been used for many years as an anticoagulant, and anticoagulant heparin-mimetics were developed several decades ago by chemical sulfation of non-mammalian polysaccharides, e.g., an antithrombotic sulfated xylan. This pharmaceutical, which comprises a mixture of sulfated oligoxylans, also mimics most other biological actions of natural heparins in vitro, including inhibition of the human immunodeficiency virus, but the molecular basis for these actions has been unclear. Here, numerous Components of the sulfated oligoxylan mixture were isolated and when bioassayed in the case of anti-HIV-1 infectivity revealed that a structural specificity underlines the capacity of sulfated xylan to inhibit HIV-1, rather than a non-specific mechanism. Components were isolated by chromatographic fractionation through Bio-Gel P10 in 0.5 M ammonium bicarbonate. This fractionation revealed an elution range associated with apparent molecular weights of approximately 22000 to <1500 relative to standard heparin and heparan sulfates and newly prepared sulfated oligosaccharide standards. Components were characterized by metachromatic absorption spectroscopy, ultracentrifugation, GlcA analysis, and potency against HIV-1 infectivity, both in the tetrazolium cytotoxicity assay and in syncytium-forming assays, in CD4-lymphocytes. Structural specificity was indicated by the differential potencies exhibited by the Components: Highest activity (cytotoxicity) was exhibited by Components in the chromatographic region > or = approximately 5500 in mass (50% effective (inhibitory) concentration = 0.5-0.7 microg ml(-1) in the first fractionation series, and 0.1-0.5 microg ml(-1) in a second series). The potency declined sharply below approximately 5400 in mass, but with an exception; a second structure exhibiting relatively high

  5. Effects of aromatic amino acids, phenylacetate and phenylpropionate on fermentation of xylan by the rumen anaerobic fungi, Neocallimastix frontalis and Piromyces communis.

    PubMed

    Guliye, A Y; Wallace, R J

    2007-10-01

    Anaerobic fungi are important members of the fibrolytic community of the rumen. The aim of this study was to study their requirement for aromatic amino acids (AA) and related phenyl acids (phenylpropionic and phenylacetic acids) for optimal xylan fermentation. Neocallimastix frontalis RE1 and Piromyces communis P were grown in a defined medium containing oat spelts xylan as the sole energy source, plus one of the following N sources: ammonia; ammonia plus a complete mixture of 20 AA commonly found in protein; ammonia plus complete AA mixture minus aromatic AA; ammonia plus phenyl acids; ammonia plus complete AA mixture without aromatic AA plus phenyl acids. Both species grew in all the media, indicating no absolute requirement for AA. The complete AA mixture increased (P<0.05) acetate concentration by 18% and 15%, sugar utilization by 33% and 22% and microbial yield by about 22% and 15% in N. frontalis and P. communis, respectively, in comparison with the treatments that had ammonia as the only N source. Neither the supply of aromatic AA or phenol acids, nor their deletion from the complete AA mixture, affected the fermentation rate, products or yield of either species. AA were not essential for N. frontalis and P. communis, but their growth on xylan was stimulated. The effects could not be explained in terms of aromatic AA alone. Ruminant diets should contain sufficient protein to sustain optimal fibre digestion by ruminal fungi. Aromatic AA or phenyl acids alone cannot replace the complete AA mixture.

  6. A new method for the quantification of monosaccharides, uronic acids and oligosaccharides in partially hydrolyzed xylans by HPAEC-UV/VIS.

    PubMed

    Lorenz, Dominic; Erasmy, Nicole; Akil, Youssef; Saake, Bodo

    2016-04-20

    A new method for the chemical characterization of xylans is presented, to overcome the difficulties in quantification of 4-O-methyl-α-D-glucuronic acid (meGlcA). In this regard, the hydrolysis behavior of xylans from beech and birch wood was investigated to obtain the optimum conditions for hydrolysis, using sulfuric acid. Due to varying linkage strengths and degradation, no general method for complete hydrolysis can be designed. Therefore, partial hydrolysis was applied, yielding monosaccharides and small meGlcA containing oligosaccharides. For a new method by HPAEC-UV/VIS, these samples were reductively aminated by 2-aminobenzoic acid. By quantification of monosaccharides and oligosaccharides, as well as comparison with borate-HPAEC and (13)C NMR-spectroscopy, we revealed that the concentrations meGlcA are significantly underestimated compared to conventional methods. The detected concentrations are 85.4% (beech) and 76.3% (birch) higher with the new procedure. Furthermore, the quantified concentrations of xylose were 9.3% (beech) and 6.5% (birch) higher by considering the unhydrolyzed oligosaccharides as well. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Production of furfural from xylose, xylan and corncob in gamma-valerolactone using FeCl3·6H2O as catalyst.

    PubMed

    Zhang, Luxin; Yu, Hongbing; Wang, Pan; Li, Yong

    2014-01-01

    An efficient and simple one-pot monophasic reaction system with small carbon footprint for converting xylose, xylan and corncob into furfural was developed in gamma-valerolactone (GVL, an ideal sustainable solvent derived from lignocelluloses) by using FeCl3·6H2O as catalyst. Good yields of furfural from xylose were obtained, and the system was shown to work for xylan and corncob as well. A surprisingly high furfural yield of 79.6% from untreated corncob was achieved at 458 K for 100 min. Contrary to what was generally believed, the addition of water, reduced the rate of the reactions, but showed positive effect on preventing the furfural from degradation in GVL. Besides, the C6 sugars (glucose and cellulose) afforded 11.4-24.5% furfural yields when employing this catalytic approach. The reaction system proposed in this manuscript showed great potential for optimizing the catalytic process in furfural production. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. Xylan-Modified-Based Hydrogels with Temperature/pH Dual Sensitivity and Controllable Drug Delivery Behavior

    PubMed Central

    Kong, Wei-Qing; Gao, Cun-Dian; Hu, Shu-Feng; Ren, Jun-Li; Zhao, Li-Hong; Sun, Run-Cang

    2017-01-01

    Among the natural macromolecules potentially used as the scaffold material in hydrogels, xylan has aroused great interest in many fields because of its biocompatibility, low toxicity, and biodegradability. In this work, new pH and thermoresponsive hydrogels were prepared by the cross-linking polymerization of maleic anhydride-modified xylan (MAHX) with N-isopropylacrylamide (NIPAm) and acrylic acid (AA) under UV irradiation to form MAHX-g-P(NIPAm-co-AA) hydrogels. The pore volume, the mechanical properties, and the release rate for drugs of hydrogels could be controlled by the degree of substitution of MAHX. These hydrogels were characterized by swelling ability, lower critical solution temperature (LCST), Fourier-transform infrared (FTIR), and SEM. Furthermore, the cumulative release rate was investigated for acetylsalicylic acid and theophylline, as well as the cytocompatibility MAHX-based hydrogels. Results showed that MAHX-based hydrogels exhibited excellent swelling–deswelling properties, uniform porous structure, and the temperature/pH dual sensitivity. In vitro, the cumulative release rate of acetylsalicylic acid for MAHX-based hydrogels was higher than that for theophylline, and in the gastrointestinal sustained drug release study, the acetylsalicylic acid release rate was extremely slow during the initial 3 h in the gastric fluid (24.26%), and then the cumulative release rate reached to 90.5% after sustained release for 5 h in simulated intestinal fluid. The cytotoxicity experiment demonstrated that MAHX-based hydrogels could promote cell proliferation and had satisfactory biocompatibility with NIH3T3 cells. These results indicated that MAHX-based hydrogels, as new drug carriers, had favorable behavior for intestinal-targeted drug delivery. PMID:28772664

  9. An Aspergillus oryzae acetyl xylan esterase: molecular cloning and characteristics of recombinant enzyme expressed in Pichia pastoris.

    PubMed

    Koseki, Takuya; Miwa, Yozo; Akao, Takeshi; Akita, Osamu; Hashizume, Katsumi

    2006-02-10

    We screened 20,000 clones of an expressed sequence tag (EST) library from Aspergillus oryzae (http://www.nrib.go.jp/ken/EST/db/index.html) and obtained one cDNA clone encoding a protein with similarity to fungal acetyl xylan esterase. We also cloned the corresponding gene, designated as Aoaxe, from the genomic DNA. The deduced amino acid sequence consisted of a putative signal peptide of 31-amino acids and a mature protein of 276-amino acids. We engineered Aoaxe for heterologous expression in P. pastoris. Recombinant AoAXE (rAoAXE) was secreted by the aid of fused alpha-factor secretion signal peptide and accumulated as an active enzyme in the culture medium to a final level of 190 mg/l after 5 days. Purified rAoAXEA before and after treatment with endoglycosidase H migrated by SDS-PAGE with a molecular mass of 31 and 30 kDa, respectively. Purified rAoAXE displayed the greatest hydrolytic activity toward alpha-naphthylacetate (C2), lower activity toward alpha-naphthylpropionate (C3) and no detectable activity toward acyl-chain substrates containing four or more carbon atoms. The recombinant enzyme catalyzed the release of acetic acid from birchwood xylan. No activity was detectable using methyl esters of ferulic, caffeic or sinapic acids. rAoAXE was thermolabile in comparison to other AXEs from Aspergillus.

  10. Extrusion of xylans extracted from corn cobs into biodegradable polymeric materials.

    PubMed

    Bahcegul, Erinc; Akinalan, Busra; Toraman, Hilal E; Erdemir, Duygu; Ozkan, Necati; Bakir, Ufuk

    2013-12-01

    Solvent casting technique, which comprises multiple energy demanding steps including the dissolution of a polymer in a solvent followed by the evaporation of the solvent from the polymer solution, is currently the main technique for the production of xylan based polymeric materials. The present study shows that sufficient water content renders arabinoglucuronoxylan (AGX) polymers extrudable, enabling the production of AGX based polymeric materials in a single step via extrusion, which is economically advantageous to solvent casting process for mass production. AGX polymers with water content of 27% were found to yield extrudates at an extrusion temperature of 90°C. The extruded strips showed very good mechanical properties with an ultimate tensile strength of 76 ± 6 MPa and elongation at break value of 35 ± 8%, which were superior to the mechanical properties of the strips obtained from polylactic acid. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. An extremely thermophilic anaerobic bacterium Caldicellulosiruptor sp. F32 exhibits distinctive properties in growth and xylanases during xylan hydrolysis.

    PubMed

    Ying, Yu; Meng, Dongdong; Chen, Xiaohua; Li, Fuli

    2013-08-15

    An anaerobic, extremely thermophilic, and cellulose- and xylan-degrading bacterium F32 was isolated from biocompost. Sequence analysis of the 16S rRNA gene of this strain showed that it was closely related to Caldicellulosiruptor saccharolyticus DSM 8903 (99.0% identity). Physiological and biochemical data also supported that identification of strain F32 as a Caldicellulosiruptor species. The proteins secreted by Caldicellulosiruptor sp. F32 grown on xylan showed a xylanase activity of 7.74U/mg, which was 2.5 times higher than that of C. saccharolyticus DSM 8903. Based on the genomic sequencing data, 2 xylanase genes, JX030400 and JX030401, were identified in Caldicellulosiruptor sp. F32. The xylanase encoded by JX030401 shared 97% identity with Csac_0696 of C. saccharolyticus DSM 8903, while that encoded by JX030400 shared 94% identity with Athe_0089 of C. bescii DSM 6725, which was not found in the genome of strain DSM 8903. Xylanse encoded by JX030400 had 9-fold higher specific activity than JX030401. Our results indicated that although the 2 strains shared high identity, the xylanase system in Caldicellulosiruptor sp. F32 was more efficient than that in C. saccharolyticus DSM 8903. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. Demonstration of Aflatoxin Inhibitory Activity in a Cotton Seed Coat Xylan

    PubMed Central

    Mellon, J. E.; Cotty, P. J.; Godshall, M. A.; Roberts, E.

    1995-01-01

    An inhibitor of aflatoxin biosynthesis localized in the seed coats of developing cotton was partially purified and characterized. Aqueous extracts from 25-day postanthesis seed coat tissue inhibited aflatoxin (B(inf1)) production in liquid cultures of Aspergillus flavus AF13. Inhibition was concentration dependent, with a 50% effective dose of 173 (mu)g of crude extract per ml of medium. The inhibitor was neutral in charge. Two active fractions were obtained from crude preparations by gel filtration chromatography (BioGel P-100). The purest fraction eluted in the void volume. Carbohydrate composition analysis of this void volume inhibitor indicated a composition of xylose (>90%) and mannose. Aflatoxin production in vitro was inversely related to inhibitor concentration in the fermentation medium (log of aflatoxin versus log of [inhibitor]; r(sup2) = 0.82; P < 0.002). The void volume inhibitor had a 50% effective dose of 6.2 (mu)g/ml, a 28-fold purification of the inhibitor material. These data support the hypothesis that seed coat inhibitory activity is associated with a cottonseed-specific xylan. PMID:16535194

  13. Analysis of functional xylanases in xylan degradation by Aspergillus niger E-1 and characterization of the GH family 10 xylanase XynVII.

    PubMed

    Takahashi, Yui; Kawabata, Hiroaki; Murakami, Shuichiro

    2013-01-01

    Xylanases produced by Aspergillus niger are industrially important and many types of xylanases have been reported. Individual xylanases have been well studied for their enzymatic properties, gene cloning, and heterologous expression. However, less attention has been paid to the relationship between xylanase genes carried on the A. niger genome and xylanases produced by A. niger strains. Therefore, we examined xylanase genes encoded on the genome of A. niger E-1 and xylanases produced in culture. Seven putative xylanase genes, xynI-VII (named in ascending order of the molecular masses of the deduced amino acid sequences), were amplified from the strain E-1 genome using primers designed from the genome sequence of A. niger CBS 513.88 by PCR and phylogenetically classified into three clusters. Additionally, culture supernatant analysis by DE52 anion-exchange column chromatography revealed that this strain produced three xylanases, XynII, XynIII, and XynVII, which were identified by N-terminal amino acid sequencing and MALDI-TOF-MS analyses, in culture when gown in 0.5% xylan medium supplemented with 50 mM succinate. Furthermore, XynVII, the only GH family 10 xylanase in A. niger E-1, was purified and characterized. The purified enzyme showed a single band with a molecular mass of 35 kDa by SDS-PAGE. The highest activity of purified XynVII was observed at 55°C and pH 5.5. The enzyme was stable in the broad pH range of 3-10 and up to 60°C and was resistant to most metal ions and modifying regents. XynVII showed high specificity against beechwood xylan with K m and V max values of 2.8 mg mL(-1) and 127 μmol min(-1)mg(-1), respectively. TLC and MALDI-TOF-MS analyses showed that the final hydrolyzed products of the enzyme from beechwood xylan were xylose, xylobiose, and xylotriose substituted with a 4-o-metylglucuronic acid residue.

  14. The integration of dilute acid hydrolysis of xylan and fast pyrolysis of glucan to obtain fermentable sugars.

    PubMed

    Jiang, Liqun; Wu, Nannan; Zheng, Anqing; Zhao, Zengli; He, Fang; Li, Haibin

    2016-01-01

    Fermentable sugars are important intermediates in the biological conversion of biomass. Hemicellulose and amorphous cellulose are easily hydrolyzed to fermentable sugars in dilute acid, whereas crystalline cellulose is more difficult to be hydrolyzed. Cellulose fast pyrolysis is an alternative method to liberate valuable fermentable sugars from biomass. The amount of levoglucosan generated from lignocellulose by fast pyrolysis is usually lower than the theoretical yield based on the cellulose fraction. Pretreatment is a promising route to improve the yield of levoglucosan from lignocellulose. The integration of dilute sulfuric acid hydrolysis and fast pyrolysis to obtain fermentable sugars was evaluated in this study. Dilute sulfuric acid hydrolysis could remove more than 95.1 and 93.4 % of xylan (the main component of hemicellulose) from sugarcane bagasse and corncob with high yield of xylose. On the other hand, dilute sulfuric acid hydrolysis was also an effective pretreatment to enhance levoglucosan yield from lignocellulose. Dilute acid hydrolysis could accumulate glucan (the component of cellulose) and remove most of the alkali and alkaline earth metals which were powerful catalysts during fast pyrolysis. Further increase in dilute acid concentration (from 0 to 2 %) in pretreatment could promote the yield of levoglucosan in fast pyrolysis. The acid pretreated sugarcane bagasse and corncob gave levoglucosan yields of 43.8 and 35.2 % which were obvious higher than those of raw sugarcane bagasse (12.0 %) and corncob (7.0 %). Obtaining fermentable sugars by combination dilute acid hydrolysis of xylan and fast pyrolysis of glucan could make full utilization of biomass, and get fermentable sugars economically from biomass for bio-refinery.

  15. Kinetic Modelling and Experimental Studies for the Effects of Fe 2+ Ions on Xylan Hydrolysis with Dilute-Acid Pretreatment and Subsequent Enzymatic Hydrolysis

    DOE PAGES

    Wei, Hui; Chen, Xiaowen; Shekiro, Joseph; ...

    2018-01-20

    High-temperature (150-170 degrees C) pretreatment of lignocellulosic biomass with mineral acids is well established for xylan breakdown. Fe 2+ is known to be a cocatalyst of this process although kinetics of its action remains unknown. The present work addresses the effect of ferrous ion concentration on sugar yield and degradation product formation from corn stover for the entire two-step treatment, including the subsequent enzymatic cellulose hydrolysis. The feedstock was impregnated with 0.5% acid and 0.75 mM iron cocatalyst, which was found to be optimal in preliminary experiments. The detailed kinetic data of acid pretreatment, with and without iron, was satisfactorilymore » modelled with a four-step linear sequence of first-order irreversible reactions accounting for the formation of xylooligomers, xylose and furfural as intermediates to provide the values of Arrhenius activation energy. Based on this kinetic modelling, Fe 2+ turned out to accelerate all four reactions, with a significant alteration of the last two steps, that is, xylose degradation. Consistent with this model, the greatest xylan conversion occurred at the highest severity tested under 170 ⁰C/30 min with 0.75 mM Fe 2+, with a total of 8% xylan remaining in the pretreated solids, whereas the operational conditions leading to the highest xylose monomer yield, 63%, were milder, 150 degrees C with 0.75 mM Fe 2+ for 20 min. Furthermore, the subsequent enzymatic hydrolysis with the prior addition of 0.75 mM of iron(II) increased the glucose production to 56.3% from 46.3% in the control (iron-free acid). The detailed analysis indicated that conducting the process at lower temperatures yet long residence times benefits the yield of sugars. The above kinetic modelling results of Fe 2+ accelerating all four reactions are in line with our previous mechanistic research showing that the pretreatment likely targets multiple chemistries in plant cell wall polymer networks, including those represented by

  16. Kinetic Modelling and Experimental Studies for the Effects of Fe 2+ Ions on Xylan Hydrolysis with Dilute-Acid Pretreatment and Subsequent Enzymatic Hydrolysis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wei, Hui; Chen, Xiaowen; Shekiro, Joseph

    High-temperature (150-170 degrees C) pretreatment of lignocellulosic biomass with mineral acids is well established for xylan breakdown. Fe 2+ is known to be a cocatalyst of this process although kinetics of its action remains unknown. The present work addresses the effect of ferrous ion concentration on sugar yield and degradation product formation from corn stover for the entire two-step treatment, including the subsequent enzymatic cellulose hydrolysis. The feedstock was impregnated with 0.5% acid and 0.75 mM iron cocatalyst, which was found to be optimal in preliminary experiments. The detailed kinetic data of acid pretreatment, with and without iron, was satisfactorilymore » modelled with a four-step linear sequence of first-order irreversible reactions accounting for the formation of xylooligomers, xylose and furfural as intermediates to provide the values of Arrhenius activation energy. Based on this kinetic modelling, Fe 2+ turned out to accelerate all four reactions, with a significant alteration of the last two steps, that is, xylose degradation. Consistent with this model, the greatest xylan conversion occurred at the highest severity tested under 170 ⁰C/30 min with 0.75 mM Fe 2+, with a total of 8% xylan remaining in the pretreated solids, whereas the operational conditions leading to the highest xylose monomer yield, 63%, were milder, 150 degrees C with 0.75 mM Fe 2+ for 20 min. Furthermore, the subsequent enzymatic hydrolysis with the prior addition of 0.75 mM of iron(II) increased the glucose production to 56.3% from 46.3% in the control (iron-free acid). The detailed analysis indicated that conducting the process at lower temperatures yet long residence times benefits the yield of sugars. The above kinetic modelling results of Fe 2+ accelerating all four reactions are in line with our previous mechanistic research showing that the pretreatment likely targets multiple chemistries in plant cell wall polymer networks, including those represented by

  17. Xylan utilization in human gut commensal bacteria is orchestrated by unique modular organization of polysaccharide-degrading enzymes.

    PubMed

    Zhang, Meiling; Chekan, Jonathan R; Dodd, Dylan; Hong, Pei-Ying; Radlinski, Lauren; Revindran, Vanessa; Nair, Satish K; Mackie, Roderick I; Cann, Isaac

    2014-09-02

    Enzymes that degrade dietary and host-derived glycans represent the most abundant functional activities encoded by genes unique to the human gut microbiome. However, the biochemical activities of a vast majority of the glycan-degrading enzymes are poorly understood. Here, we use transcriptome sequencing to understand the diversity of genes expressed by the human gut bacteria Bacteroides intestinalis and Bacteroides ovatus grown in monoculture with the abundant dietary polysaccharide xylan. The most highly induced carbohydrate active genes encode a unique glycoside hydrolase (GH) family 10 endoxylanase (BiXyn10A or BACINT_04215 and BACOVA_04390) that is highly conserved in the Bacteroidetes xylan utilization system. The BiXyn10A modular architecture consists of a GH10 catalytic module disrupted by a 250 amino acid sequence of unknown function. Biochemical analysis of BiXyn10A demonstrated that such insertion sequences encode a new family of carbohydrate-binding modules (CBMs) that binds to xylose-configured oligosaccharide/polysaccharide ligands, the substrate of the BiXyn10A enzymatic activity. The crystal structures of CBM1 from BiXyn10A (1.8 Å), a cocomplex of BiXyn10A CBM1 with xylohexaose (1.14 Å), and the CBM from its homolog in the Prevotella bryantii B14 Xyn10C (1.68 Å) reveal an unanticipated mode for ligand binding. A minimal enzyme mix, composed of the gene products of four of the most highly up-regulated genes during growth on wheat arabinoxylan, depolymerizes the polysaccharide into its component sugars. The combined biochemical and biophysical studies presented here provide a framework for understanding fiber metabolism by an important group within the commensal bacterial population known to influence human health.

  18. Obtaining cellulose binding and hydrolyzing activity of a family 11 hybrid xylanase by fusion with xylan binding domain.

    PubMed

    Liu, Ming-Qi; Dai, Xian-Jun; Liu, Guang-Fu; Wang, Qian

    2013-03-01

    The xylan binding domain (XBD) and linker sequences (LS) from thermostable and thermophilic Thermomonospora fusca xylanase A (TfxA) was fused to the carboxyl-terminus of a family 11 hybrid xylanase ATx. The constructed chimera (ATxX) was successfully expressed in Pichia pastoris, partially purified to homogeneity, and then characterized in detail. After 96-h 0.25% methanol induction, the xylanase and cellulose activity of ATxX from pPATxX1 transformant culture medium supernatant were 452.1 U/mg and 19.3 U/mg, respectively. SDS-PAGE analysis revealed that the molecular mass of ATxX was about 33.01 kDa. 3.7% ATxX was bound after incubation with 1% microcrystal cellulose at 25 °C for 3 h, while the ATx did not show cellulose binding-hydrolyzing ability. These results suggested that ATx obtained cellulose binding and hydrolyzing ability by fusing with XBD and LS. Enzymatic studies showed that the temperature and pH optimum of the ATxX xylanase activity were 60 °C and pH 5.0, respectively, which were the same as that of ATx. The temperature and pH optimum of the ATxX cellulase activity were 60 °C and pH 6.0, respectively. The major hydrolytic products released by ATxX from birchwood xylan were xylotriose and xylohexaose. Xylooligosaccharides from xylobiose to xylohexaose could be hydrolyzed by ATxX. Mode of action analysis showed that the chimeric ATxX was an endo-acting enzyme. The XBD and LS plays an important role in the binding and hydrolyzing of xylanase to insoluble substrates. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Working towards recalcitrance mechanisms: increased xylan and homogalacturonan production by overexpression of GAlactUronosylTransferase12 (GAUT12) causes increased recalcitrance and decreased growth in Populus

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Biswal, Ajaya K.; Atmodjo, Melani A.; Pattathil, Sivakumar

    The development of fast-growing hardwood trees as a source of lignocellulosic biomass for biofuel and biomaterial production requires a thorough understanding of the plant cell wall structure and function that underlie the inherent recalcitrance properties of woody biomass. Downregulation of GAUT12.1 in Populus deltoides was recently reported to result in improved biomass saccharification, plant growth, and biomass yield. To further understand GAUT12.1 function in biomass recalcitrance and plant growth, here we report the effects of P. trichocarpa GAUT12.1 overexpression in P. deltoides. Increasing GAUT12.1 transcript expression by 7-49% in P. deltoides PtGAUT12.1-overexpression (OE) lines resulted in a nearly complete oppositemore » biomass saccharification and plant growth phenotype to that observed previously in PdGAUT12.1-knockdown (KD) lines. This also included significantly reduced glucose, xylose, and total sugar release (12-13%), plant height (6-54%), stem diameter (8-40%), and overall total aerial biomass yield (48-61%) in 3-month-old, greenhouse-grown PtGAUT12.1-OE lines compared to controls. Total lignin content was unaffected by the gene overexpression. Importantly, selected PtGAUT12.1-OE lines retained the recalcitrance and growth phenotypes upon growth for 9 months in the greenhouse and 2.8 years in the field. PtGAUT12.1-OE plants had significantly smaller leaves with lower relative water content, and significantly reduced stem wood xylem cell numbers and size. At the cell wall level, xylose and galacturonic acid contents increased markedly in total cell walls as well as in soluble and insoluble cell wall extracts, consistent with increased amounts of xylan and homogalacturonan in the PtGAUT12.1-OE lines. This led to increased cell wall recalcitrance, as manifested by the 9-15% reduced amounts of recovered extractable wall materials and 8-15% greater amounts of final insoluble pellet in the PtGAUT12.1-OE lines compared to controls. The combined

  20. Working towards recalcitrance mechanisms: increased xylan and homogalacturonan production by overexpression of GAlactUronosylTransferase12 (GAUT12) causes increased recalcitrance and decreased growth in Populus

    DOE PAGES

    Biswal, Ajaya K.; Atmodjo, Melani A.; Pattathil, Sivakumar; ...

    2018-01-17

    The development of fast-growing hardwood trees as a source of lignocellulosic biomass for biofuel and biomaterial production requires a thorough understanding of the plant cell wall structure and function that underlie the inherent recalcitrance properties of woody biomass. Downregulation of GAUT12.1 in Populus deltoides was recently reported to result in improved biomass saccharification, plant growth, and biomass yield. To further understand GAUT12.1 function in biomass recalcitrance and plant growth, here we report the effects of P. trichocarpa GAUT12.1 overexpression in P. deltoides. Increasing GAUT12.1 transcript expression by 7-49% in P. deltoides PtGAUT12.1-overexpression (OE) lines resulted in a nearly complete oppositemore » biomass saccharification and plant growth phenotype to that observed previously in PdGAUT12.1-knockdown (KD) lines. This also included significantly reduced glucose, xylose, and total sugar release (12-13%), plant height (6-54%), stem diameter (8-40%), and overall total aerial biomass yield (48-61%) in 3-month-old, greenhouse-grown PtGAUT12.1-OE lines compared to controls. Total lignin content was unaffected by the gene overexpression. Importantly, selected PtGAUT12.1-OE lines retained the recalcitrance and growth phenotypes upon growth for 9 months in the greenhouse and 2.8 years in the field. PtGAUT12.1-OE plants had significantly smaller leaves with lower relative water content, and significantly reduced stem wood xylem cell numbers and size. At the cell wall level, xylose and galacturonic acid contents increased markedly in total cell walls as well as in soluble and insoluble cell wall extracts, consistent with increased amounts of xylan and homogalacturonan in the PtGAUT12.1-OE lines. This led to increased cell wall recalcitrance, as manifested by the 9-15% reduced amounts of recovered extractable wall materials and 8-15% greater amounts of final insoluble pellet in the PtGAUT12.1-OE lines compared to controls. The combined

  1. Enhanced agarose and xylan degradation for production of polyhydroxyalkanoates by co-culture of marine bacterium, Saccharophagus degradans and its contaminant, Bacillus cereus

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sawant, Shailesh S.; Salunke, Bipinchandra K.; Taylor, II, Larry E.

    Over reliance on energy or petroleum products has raised concerns both in regards to the depletion of their associated natural resources as well as their increasing costs. Bioplastics derived from microbes are emerging as promising alternatives to fossil fuel derived petroleum plastics. The development of a simple and eco-friendly strategy for bioplastic production with high productivity and yield, which is produced in a cost effective manner utilising abundantly available renewable carbon sources, would have the potential to result in an inexhaustible global energy source. Here we report the biosynthesis of bioplastic polyhydroxyalkanoates (PHAs) in pure cultures of marine bacterium, Saccharophagusmore » degradans 2-40 ( Sde 2-40), its contaminant, Bacillus cereus, and a co-culture of these bacteria ( Sde 2-40 and B. cereus) degrading plant and algae derived complex polysaccharides. Sde 2-40 degraded the complex polysaccharides agarose and xylan as sole carbon sources for biosynthesis of PHAs. The ability of Sde 2-40 to degrade agarose increased after co-culturing with B. cereus. The association of Sde 2-40 with B. cereus resulted in increased cell growth and higher PHA production (34.5% of dry cell weight) from xylan as a carbon source in comparison to Sde 2-40 alone (22.7% of dry cell weight). Lastly, the present study offers an innovative prototype for production of PHA through consolidated bioprocessing of complex carbon sources by pure and co-culture of microorganisms.« less

  2. Enhanced agarose and xylan degradation for production of polyhydroxyalkanoates by co-culture of marine bacterium, Saccharophagus degradans and its contaminant, Bacillus cereus

    DOE PAGES

    Sawant, Shailesh S.; Salunke, Bipinchandra K.; Taylor, II, Larry E.; ...

    2017-02-28

    Over reliance on energy or petroleum products has raised concerns both in regards to the depletion of their associated natural resources as well as their increasing costs. Bioplastics derived from microbes are emerging as promising alternatives to fossil fuel derived petroleum plastics. The development of a simple and eco-friendly strategy for bioplastic production with high productivity and yield, which is produced in a cost effective manner utilising abundantly available renewable carbon sources, would have the potential to result in an inexhaustible global energy source. Here we report the biosynthesis of bioplastic polyhydroxyalkanoates (PHAs) in pure cultures of marine bacterium, Saccharophagusmore » degradans 2-40 ( Sde 2-40), its contaminant, Bacillus cereus, and a co-culture of these bacteria ( Sde 2-40 and B. cereus) degrading plant and algae derived complex polysaccharides. Sde 2-40 degraded the complex polysaccharides agarose and xylan as sole carbon sources for biosynthesis of PHAs. The ability of Sde 2-40 to degrade agarose increased after co-culturing with B. cereus. The association of Sde 2-40 with B. cereus resulted in increased cell growth and higher PHA production (34.5% of dry cell weight) from xylan as a carbon source in comparison to Sde 2-40 alone (22.7% of dry cell weight). Lastly, the present study offers an innovative prototype for production of PHA through consolidated bioprocessing of complex carbon sources by pure and co-culture of microorganisms.« less

  3. Xylanase production with xylan rich lignocellulosic wastes by a local soil isolate of Trichoderma viride

    PubMed Central

    Goyal, Meenakshi; Kalra, K.L.; Sareen, V.K.; Soni, G.

    2008-01-01

    In the present study, cultural and nutritional conditions for enhanced production of xylanase by a local soil isolate of Trichoderma viride, using various lignocellulosic substrates in submerged culture fermentation have been optimized. Of the lignocellulosics used, maize straw was the best inducer followed by jowar straw for xylanase production. The highest activity achieved was between 14 to 17 days of fermentation. A continuous increase in xylanase production was observed with increasing level of lignocellulosics in the medium and highest activity was observed with maize straw at 5% level. Xylanase production with higher levels of lignocellulosics (3 to 5%) of maize, jowar and barseem was found to be higher as compared to that with commercial xylan as carbon source. Sodium nitrate was the best nitrogen source among the six sources used. Maximum xylanase production was achieved with initial medium pH of 3.5–4.0 and incubation temperature of 25ºC.The enzyme preparation was effective in bringing about saccharification of different lignocellulosics. The xylanase production could be further improved by using alkali treated straw as carbon source. PMID:24031262

  4. Altered Expression of Genes Implicated in Xylan Biosynthesis Affects Penetration Resistance against Powdery Mildew.

    PubMed

    Chowdhury, Jamil; Lück, Stefanie; Rajaraman, Jeyaraman; Douchkov, Dimitar; Shirley, Neil J; Schwerdt, Julian G; Schweizer, Patrick; Fincher, Geoffrey B; Burton, Rachel A; Little, Alan

    2017-01-01

    Heteroxylan has recently been identified as an important component of papillae, which are formed during powdery mildew infection of barley leaves. Deposition of heteroxylan near the sites of attempted fungal penetration in the epidermal cell wall is believed to enhance the physical resistance to the fungal penetration peg and hence to improve pre-invasion resistance. Several glycosyltransferase (GT) families are implicated in the assembly of heteroxylan in the plant cell wall, and are likely to work together in a multi-enzyme complex. Members of key GT families reported to be involved in heteroxylan biosynthesis are up-regulated in the epidermal layer of barley leaves during powdery mildew infection. Modulation of their expression leads to altered susceptibility levels, suggesting that these genes are important for penetration resistance. The highest level of resistance was achieved when a GT43 gene was co-expressed with a GT47 candidate gene, both of which have been predicted to be involved in xylan backbone biosynthesis. Altering the expression level of several candidate heteroxylan synthesis genes can significantly alter disease susceptibility. This is predicted to occur through changes in the amount and structure of heteroxylan in barley papillae.

  5. Prospect for Developing a Consolidated Bioprocessing (CBP) Strain Using Xylan as the Substrate: the Case Study of Yarrowia lipolytica

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Wei; Wei, Hui; Alahuhta, Markus

    2016-07-08

    To achieve the goal of developing a direct microbial sugar conversion platform for the production of lipids and drop-in fuels from cellulosic biomass substrate, Yarrowia lipolytica was used to investigate its potential for being developed as CBP strain by expressing cellulase and xylanase enzymes. Y. lipolytica is known to accumulate lipids intracellularly and is capable of metabolizing glucose and xylose to produce lipids; however, due to the lack of the biomass degrading enzymes, it cannot directly utilize lignocellulosic substrates as carbon sources. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in thismore » study, we present successful expression of several xylanases in Y. lipolytica. To the best of our knowledge, this is the first study introducing heterologous hemicellulose genes into the genome of Y. lipolytica. SDS-PAGE and western blotting analysis showed that the endo-xylanase gene XynII and exo-xylosidase gene XlnD were successfully expressed and secreted, and the expressed xylanases were likely either not or sparsely glycosylated, which is advantageous for expression of heterologous proteins from any species. Enzymatic activity tests further demonstrated active expression of XynII and XlnD in Y. lipolytica. Furthermore, synergistic action on converting xylan to xylose was observed when XlnD worked in concert with XynII. XlnD was able to work on the xylo-oligomers generated by XynII, enhancing the xylan conversion to monomeric xylose. The successful expression of these xylanases in Yarrowia further advances us towards our goal to develop a direct microbial conversion process using this organism. and xylose to produce lipids; however, due to the lack of the biomass degrading enzymes, it cannot directly utilize lignocellulosic substrates as carbon sources. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful

  6. β-D-(1→4), β-D-(1→3) 'mixed linkage' xylans from red seaweeds of the order Nemaliales and Palmariales.

    PubMed

    Viana, Adriano G; Noseda, Miguel D; Gonçalves, Alan G; Duarte, Maria Eugênia R; Yokoya, Nair; Matulewicz, Maria C; Cerezo, Alberto S

    2011-06-01

    Xylans from five seaweeds belonging to the order Nemaliales (Galaxaura marginata, Galaxaura obtusata, Tricleocarpacylindrica, Tricleocarpa fragilis, and Scinaia halliae) and one of the order Palmariales (Palmaria palmata) collected on the Brazilian coasts were extracted with hot water and purified from acid xylomannans and/or xylogalactans through Cetavlon precipitation of the acid polysaccharides. The β-D-(1→4), β-D-(1→3) 'mixed linkage' structures were determined using methylation analysis and 1D and 2D NMR spectroscopy. The presence of large sequences of β-(1→4)-linked units suggests transient aggregates of ribbon- or helical-ordered structures that would explain the low optical rotations. Copyright © 2011 Elsevier Ltd. All rights reserved.

  7. Matrix-assisted ultraviolet laser desorption/ionization time-of-flight mass spectrometry of beta-(1 --> 3), beta-(1 --> 4)-xylans from Nothogenia fastigiata using nor-harmane as matrix.

    PubMed

    Fukuyama, Yuko; Kolender, Adriana A; Nishioka, Masae; Nonami, Hiroshi; Matulewicz, María C; Erra-Balsells, Rosa; Cerezo, Alberto S

    2005-01-01

    Three xylan fractions isolated from the red seaweed Nothogenia fastigiata (Nemaliales) were analyzed by ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (UV-MALDI-TOFMS). UV-MALDI-TOFMS was carried out in the linear and reflectron modes, and as routine in the positive and negative ion modes. Of the several matrices tested, nor-harmane was the only effective one giving good spectra in the positive ion mode. The number-average molar masses of two of the fractions, calculated from the distribution profiles, were lower than those determined previously by (1)H NMR analysis, suggesting a decrease in the ionization efficiency with increasing molecular weight; weight-average molar mass and polydispersity index were also determined. As the xylans retained small but significant quantities of calcium salts, the influence of added Ca(2+) as CaCl(2) on UV-MALDI-MS was investigated. The simultaneous addition of sodium chloride and calcium chloride was also analyzed. Addition of sodium chloride did not change the distribution profile of the native sample showing that the inhibitory effect is due to Ca(2+) and not to Cl(-). Addition of calcium chloride with 1:1 analyte/salt molar ratio gave spectra with less efficient desorption/ionization of oligomers; the signals of these oligomers were completely suppressed when the addition of the salt became massive (1:100 analyte/salt molar ratio). Copyright (c) 2005 John Wiley & Sons, Ltd.

  8. A Transcriptomic Analysis of Xylan Mutants Does Not Support the Existence of a Secondary Cell Wall Integrity System in Arabidopsis

    PubMed Central

    Faria-Blanc, Nuno; Mortimer, Jenny C.; Dupree, Paul

    2018-01-01

    Yeast have long been known to possess a cell wall integrity (CWI) system, and recently an analogous system has been described for the primary walls of plants (PCWI) that leads to changes in plant growth and cell wall composition. A similar system has been proposed to exist for secondary cell walls (SCWI). However, there is little data to support this. Here, we analyzed the stem transcriptome of a set of cell wall biosynthetic mutants in order to investigate whether cell wall damage, in this case caused by aberrant xylan synthesis, activates a signaling cascade or changes in cell wall synthesis gene expression. Our data revealed remarkably few changes to the transcriptome. We hypothesize that this is because cells undergoing secondary cell wall thickening have entered a committed programme leading to cell death, and therefore a SCWI system would have limited impact. The absence of transcriptomic responses to secondary cell wall alterations may facilitate engineering of the secondary cell wall of plants. PMID:29636762

  9. A Transcriptomic Analysis of Xylan Mutants Does Not Support the Existence of a Secondary Cell Wall Integrity System in Arabidopsis.

    PubMed

    Faria-Blanc, Nuno; Mortimer, Jenny C; Dupree, Paul

    2018-01-01

    Yeast have long been known to possess a cell wall integrity (CWI) system, and recently an analogous system has been described for the primary walls of plants (PCWI) that leads to changes in plant growth and cell wall composition. A similar system has been proposed to exist for secondary cell walls (SCWI). However, there is little data to support this. Here, we analyzed the stem transcriptome of a set of cell wall biosynthetic mutants in order to investigate whether cell wall damage, in this case caused by aberrant xylan synthesis, activates a signaling cascade or changes in cell wall synthesis gene expression. Our data revealed remarkably few changes to the transcriptome. We hypothesize that this is because cells undergoing secondary cell wall thickening have entered a committed programme leading to cell death, and therefore a SCWI system would have limited impact. The absence of transcriptomic responses to secondary cell wall alterations may facilitate engineering of the secondary cell wall of plants.

  10. Draft genome sequence of Sugiyamaella xylanicola UFMG-CM-Y1884T, a xylan-degrading yeast species isolated from rotting wood samples in Brazil.

    PubMed

    Batista, Thiago M; Moreira, Rennan G; Hilário, Heron O; Morais, Camila G; Franco, Glória R; Rosa, Luiz H; Rosa, Carlos A

    2017-03-01

    We present the draft genome sequence of the type strain of the yeast Sugiyamaella xylanicola UFMG-CM-Y1884 T (= UFMG-CA-32.1 T  = CBS 12683 T ), a xylan-degrading species capable of fermenting d-xylose to ethanol. The assembled genome has a size of ~ 13.7 Mb and a GC content of 33.8% and contains 5971 protein-coding genes. We identified 15 genes with significant similarity to the d-xylose reductase gene from several other fungal species. The draft genome assembled from whole-genome shotgun sequencing of the yeast Sugiyamaella xylanicola UFMG-CM-Y1884 T (= UFMG-CA-32.1 T  = CBS 12683 T ) has been deposited at DDBJ/ENA/GenBank under the accession number MQSX00000000 under version MQSX01000000.

  11. Alteration of cell wall xylan acetylation triggers defense responses that counterbalance the immune deficiencies of plants impaired in the β-subunit of the heterotrimeric G-protein.

    PubMed

    Escudero, Viviana; Jordá, Lucía; Sopeña-Torres, Sara; Mélida, Hugo; Miedes, Eva; Muñoz-Barrios, Antonio; Swami, Sanjay; Alexander, Danny; McKee, Lauren S; Sánchez-Vallet, Andrea; Bulone, Vincent; Jones, Alan M; Molina, Antonio

    2017-11-01

    Arabidopsis heterotrimeric G-protein complex modulates pathogen-associated molecular pattern-triggered immunity (PTI) and disease resistance responses to different types of pathogens. It also plays a role in plant cell wall integrity as mutants impaired in the Gβ- (agb1-2) or Gγ-subunits have an altered wall composition compared with wild-type plants. Here we performed a mutant screen to identify suppressors of agb1-2 (sgb) that restore susceptibility to pathogens to wild-type levels. Out of the four sgb mutants (sgb10-sgb13) identified, sgb11 is a new mutant allele of ESKIMO1 (ESK1), which encodes a plant-specific polysaccharide O-acetyltransferase involved in xylan acetylation. Null alleles (sgb11/esk1-7) of ESK1 restore to wild-type levels the enhanced susceptibility of agb1-2 to the necrotrophic fungus Plectosphaerella cucumerina BMM (PcBMM), but not to the bacterium Pseudomonas syringae pv. tomato DC3000 or to the oomycete Hyaloperonospora arabidopsidis. The enhanced resistance to PcBMM of the agb1-2 esk1-7 double mutant was not the result of the re-activation of deficient PTI responses in agb1-2. Alteration of cell wall xylan acetylation caused by ESK1 impairment was accompanied by an enhanced accumulation of abscisic acid, the constitutive expression of genes encoding antibiotic peptides and enzymes involved in the biosynthesis of tryptophan-derived metabolites, and the accumulation of disease resistance-related secondary metabolites and different osmolites. These esk1-mediated responses counterbalance the defective PTI and PcBMM susceptibility of agb1-2 plants, and explain the enhanced drought resistance of esk1 plants. These results suggest that a deficient PTI-mediated resistance is partially compensated by the activation of specific cell-wall-triggered immune responses. © 2017 The Authors The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.

  12. Crystal Structure and Substrate Specificity Modification of Acetyl Xylan Esterase from Aspergillus luchuensis.

    PubMed

    Komiya, Dai; Hori, Akane; Ishida, Takuya; Igarashi, Kiyohiko; Samejima, Masahiro; Koseki, Takuya; Fushinobu, Shinya

    2017-10-15

    Acetyl xylan esterase (AXE) catalyzes the hydrolysis of the acetyl bonds present in plant cell wall polysaccharides. Here, we determined the crystal structure of AXE from Aspergillus luchuensis ( Al AXEA), providing the three-dimensional structure of an enzyme in the Esterase_phb family. Al AXEA shares its core α/β-hydrolase fold structure with esterases in other families, but it has an extended central β-sheet at both its ends and an extra loop. Structural comparison with a ferulic acid esterase (FAE) from Aspergillus niger indicated that Al AXEA has a conserved catalytic machinery: a catalytic triad (Ser119, His259, and Asp202) and an oxyanion hole (Cys40 and Ser120). Near the catalytic triad of A lAXEA, two aromatic residues (Tyr39 and Trp160) form small pockets at both sides. Homology models of fungal FAEs in the same Esterase_phb family have wide pockets at the corresponding sites because they have residues with smaller side chains (Pro, Ser, and Gly). Mutants with site-directed mutations at Tyr39 showed a substrate specificity similar to that of the wild-type enzyme, whereas those with mutations at Trp160 acquired an expanded substrate specificity. Interestingly, the Trp160 mutants acquired weak but significant type B-like FAE activity. Moreover, the engineered enzymes exhibited ferulic acid-releasing activity from wheat arabinoxylan. IMPORTANCE Hemicelluloses in the plant cell wall are often decorated by acetyl and ferulic acid groups. Therefore, complete and efficient degradation of plant polysaccharides requires the enzymes for cleaving the side chains of the polymer. Since the Esterase_phb family contains a wide array of fungal FAEs and AXEs from fungi and bacteria, our study will provide a structural basis for the molecular mechanism of these industrially relevant enzymes in biopolymer degradation. The structure of the Esterase_phb family also provides information for bacterial polyhydroxyalkanoate depolymerases that are involved in biodegradation of

  13. A glycosyl transferase family 43 protein involved in xylan biosynthesis is associated with straw digestibility in Brachypodium distachyon.

    PubMed

    Whitehead, Caragh; Ostos Garrido, Francisco J; Reymond, Matthieu; Simister, Rachael; Distelfeld, Assaf; Atienza, Sergio G; Piston, Fernando; Gomez, Leonardo D; McQueen-Mason, Simon J

    2018-05-01

    The recalcitrance of secondary plant cell walls to digestion constrains biomass use for the production of sustainable bioproducts and for animal feed. We screened a population of Brachypodium recombinant inbred lines (RILs) for cell wall digestibility using commercial cellulases and detected a quantitative trait locus (QTL) associated with this trait. Examination of the chromosomal region associated with this QTL revealed a candidate gene that encodes a putative glycosyl transferase family (GT) 43 protein, orthologue of IRX14 in Arabidopsis, and hence predicted to be involved in the biosynthesis of xylan. Arabinoxylans form the major matrix polysaccharides in cell walls of grasses, such as Brachypodium. The parental lines of the RIL population carry alternative nonsynonymous polymorphisms in the BdGT43A gene, which were inherited in the RIL progeny in a manner compatible with a causative role in the variation in straw digestibility. In order to validate the implied role of our candidate gene in affecting straw digestibility, we used RNA interference to lower the expression levels of the BdGT43A gene in Brachypodium. The biomass of the silenced lines showed higher digestibility supporting a causative role of the BdGT43A gene, suggesting that it might form a good target for improving straw digestibility in crops. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

  14. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zeng, Yining; Yarbrough, John M.; Mittal, Ashutosh

    Xylan constitutes a significant portion of biomass (e.g. 22% in corn stover used in this study). Xylan is also an important source of carbohydrates, besides cellulose, for renewable and sustainable energy applications. Currently used method for the localization of xylan in biomass is to use fluorescence confocal microscope to image the fluorescent dye labeled monoclonal antibody that specifically binds to xylan. With the rapid adoption of the Raman-based label-free chemical imaging techniques in biology, identifying Raman bands that are unique to xylan would be critical for the implementation of the above label-free techniques for in situ xylan imaging. Unlike ligninmore » and cellulose that have long be assigned fingerprint Raman bands, specific Raman bands for xylan remain unclear. The major challenge is the cellulose in plant cell wall, which has chemical units highly similar to that of xylan. Here we report using xylanase to specifically remove xylan from feedstock. Under various degree of xylan removal, with minimum impact to other major cell wall components, i.e. lignin and cellulose, we have identified Raman bands that could be further tested for chemical imaging of xylan in biomass in situ.« less

  15. Two-stage dilute acid prehydrolysis of biomass

    DOEpatents

    Grohmann, Karel; Torget, Robert W.

    1992-01-01

    A two-stage dilute acid prehydrolysis process on xylan containing hemicellulose in biomass is effected by: treating feedstock of hemicellulosic material comprising xylan that is slow hydrolyzable and xylan that is fast hydrolyzable under predetermined low temperature conditions with a dilute acid for a residence time sufficient to hydrolyze the fast hydrolyzable xylan to xylose; removing said xylose from said fast hydrolyzable xylan and leaving a residue; and treating said residue having a slow hydrolyzable xylan with a dilute acid under predetermined high temperature conditions for a residence time required to hydrolyze said slow hydrolyzable xylan to xylose.

  16. In situ label-free imaging of hemicellulose in plant cell walls using stimulated Raman scattering microscopy

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zeng, Yining; Yarbrough, John M.; Mittal, Ashutosh

    Plant hemicellulose (largely xylan) is an excellent feedstock for renewable energy production and second only to cellulose in abundance. Beyond a source of fermentable sugars, xylan constitutes a critical polymer in the plant cell wall, where its precise role in wall assembly, maturation, and deconstruction remains primarily hypothetical. Effective detection of xylan, particularly by in situ imaging of xylan in the presence of other biopolymers, would provide critical information for tackling the challenges of understanding the assembly and enhancing the liberation of xylan from plant materials. Raman-based imaging techniques, especially the highly sensitive stimulated Raman scattering (SRS) microscopy, have provenmore » to be valuable tools for label-free imaging. However, due to the complex nature of plant materials, especially those same chemical groups shared between xylan and cellulose, the utility of specific Raman vibrational modes that are unique to xylan have been debated. Here, we report a novel approach based on combining spectroscopic analysis and chemical/enzymatic xylan removal from corn stover cell walls, to make progress in meeting this analytical challenge. We have identified several Raman peaks associated with xylan content in cell walls for label-free in situ imaging xylan in plant cell wall. We demonstrated that xylan can be resolved from cellulose and lignin in situ using enzymatic digestion and label-free SRS microscopy in both 2D and 3D. As a result, we believe that this novel approach can be used to map xylan in plant cell walls and that this ability will enhance our understanding of the role played by xylan in cell wall biosynthesis and deconstruction.« less

  17. In situ label-free imaging of hemicellulose in plant cell walls using stimulated Raman scattering microscopy

    DOE PAGES

    Zeng, Yining; Yarbrough, John M.; Mittal, Ashutosh; ...

    2016-11-22

    Plant hemicellulose (largely xylan) is an excellent feedstock for renewable energy production and second only to cellulose in abundance. Beyond a source of fermentable sugars, xylan constitutes a critical polymer in the plant cell wall, where its precise role in wall assembly, maturation, and deconstruction remains primarily hypothetical. Effective detection of xylan, particularly by in situ imaging of xylan in the presence of other biopolymers, would provide critical information for tackling the challenges of understanding the assembly and enhancing the liberation of xylan from plant materials. Raman-based imaging techniques, especially the highly sensitive stimulated Raman scattering (SRS) microscopy, have provenmore » to be valuable tools for label-free imaging. However, due to the complex nature of plant materials, especially those same chemical groups shared between xylan and cellulose, the utility of specific Raman vibrational modes that are unique to xylan have been debated. Here, we report a novel approach based on combining spectroscopic analysis and chemical/enzymatic xylan removal from corn stover cell walls, to make progress in meeting this analytical challenge. We have identified several Raman peaks associated with xylan content in cell walls for label-free in situ imaging xylan in plant cell wall. We demonstrated that xylan can be resolved from cellulose and lignin in situ using enzymatic digestion and label-free SRS microscopy in both 2D and 3D. As a result, we believe that this novel approach can be used to map xylan in plant cell walls and that this ability will enhance our understanding of the role played by xylan in cell wall biosynthesis and deconstruction.« less

  18. Determination of some significant batch culture conditions affecting acetyl-xylan esterase production by Penicillium notatum NRRL-1249

    PubMed Central

    2011-01-01

    Background Acetyl-xylan esterase (AXE, EC 3.1.1.72) hydrolyses acetate group from the linear chain of xylopyranose residues bound by β-1,4-linkage. The enzyme finds commercial applications in bio-bleaching of wood pulp, treating animal feed to increase digestibility, processing food to increase clarification and converting lignocellulosics to feedstock and fuel. In the present study, we report on the production of an extracellular AXE from Penicillium notatum NRRL-1249 by solid state fermentation (SSF). Results Wheat bran at a level of 10 g (with 4 cm bed height) was optimized as the basal substrate for AXE production. An increase in enzyme activity was observed when 7.5 ml of mineral salt solution (MSS) containing 0.1% KH2PO4, 0.05% KCl, 0.05% MgSO4.7H2O, 0.3% NaNO3, 0.001% FeSO4.2H2O and 0.1% (v/w) Tween-80 as an initial moisture content was used. Various nitrogen sources including ammonium sulphate, urea, peptone and yeast extract were compared for enzyme production. Maximal enzyme activity of 760 U/g was accomplished which was found to be highly significant (p ≤ 0.05). A noticeable enhancement in enzyme activity was observed when the process parameters including incubation period (48 h), initial pH (5), 0.2% (w/w) urea as nitrogen source and 0.5% (v/w) Tween-80 as a stimulator were further optimized using a 2-factorial Plackett-Burman design. Conclusion From the results it is clear that an overall improvement of more than 35% in terms of net enzyme activity was achieved compared to previously reported studies. This is perhaps the first report dealing with the use of P. notatum for AXE production under batch culture SSF. The Plackett-Burman model terms were found highly significant (HS), suggesting the potential commercial utility of the culture used (df = 3, LSD = 0.126). PMID:21575210

  19. Enzymatic production of xylooligosaccharides from corn stover and corn cobs treated with aqueous ammonia.

    PubMed

    Zhu, Yongming; Kim, Tae Hyun; Lee, Y Y; Chen, Rongfu; Elander, Richard T

    2006-01-01

    A novel method of producing food-grade xylooligosaccharides from corn stover and corn cobs was investigated. The process starts with pretreatment of feedstock in aqueous ammonia, which results delignified and xylan-rich substrate. The pretreated substrates are subjected to enzymatic hydrolysis of xylan using endoxylanase for production of xylooligosaccharides. The conventional enzyme-based method involves extraction of xylan with a strong alkaline solution to form a liquid intermediate containing soluble xylan. This intermediate is heavily contaminated with various extraneous components. A costly purification step is therefore required before enzymatic hydrolysis. In the present method, xylan is obtained in solid form after pretreatment. Water-washing is all that is required for enzymatic hydrolysis of this material. The complex step of purifying soluble xylan from contaminant is essentially eliminated. Refining of xylooligosaccharides to food-grade is accomplished by charcoal adsorption followed by ethanol elution. Xylanlytic hydrolysis of the pretreated corn stover yielded glucan-rich residue that is easily digestible by cellulase enzyme. The digestibility of the residue reached 86% with enzyme loading of 10 filter paper units/g-glucan. As a feedstock for xylooligosaccharides production, corn cobs are superior to corn stover because of high xylan content and high packing density. The high packing density of corn cobs reduces water input and eventually raises the product concentration.

  20. Hemicellulose block copolymers made from woods for wide-range directed self-assembly lithography enabling wider range of applicable patterning size

    NASA Astrophysics Data System (ADS)

    Morita, Kazuyo; Yamamoto, Kimiko

    2017-03-01

    Xylan, one of hemicellulose family, block copolymer was newly developed for wide-range directed self-assembly lithography (DSA). Xylan is higher hydrophilic material because of having many hydroxy groups in one molecule. It means that xylan block copolymer has a possibility of high-chi block copolymer. Generally, DSA is focused on microphase separation for smaller size with high-chi block copolymer and not well known for larger size. In this study, xylan block copolymer was confirmed enabling wider range of patterning size, from smaller size to larger size. The key of xylan block copolymer is a new molecular structure of block copolymer and sugar chain control technology. Sugar content is the important parameter for not only micro-phase separation property but also line edge roughness (LER) and defects. Based on the sugar control technology, wide-range (hp 8.3nm to 26nm L/S and CD 10nm to 51nm hole) DSA patterning was demonstrated. Additionally it was confirmed that xylan block copolymer is suitable for sequential infiltration synthesis (SIS) process.

  1. Comparative analysis of the ability of Clostridium clariflavum strains and Clostridium thermocellumto utilize hemicellulose and unpretreated plant material

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Izquierdo, Javier A.; Pattathil, Sivakumar; Guseva, Anna

    2014-11-18

    Among themophilic consolidated bioprocessing (CBP) candidate organisms, environmental isolates of Clostridium clariflavum have demonstrated the ability to grow on xylan, and the genome of C. clariflavum DSM 19732 has revealed a number of mechanisms that foster solubilization of hemicellulose that are distinctive relative to the model cellulolytic thermophile Clostridium thermocellum. Growth experiments on xylan, xylooligosaccharides, and xylose reveal that C. clariflavum strains are able to completely break down xylan to xylose and that the environmental strain C. clariflavum sp. 4-2a is able to grow on monomeric xylose. C. clariflavum strains were able to utilize a larger proportion of unpretreated switchgrass,more » and solubilize a higher proportion of glucan, xylan, and arabinan, with strain 4-2a reaching the highest extent of solubilization of these components (64.7 to 69.4%) compared to C. thermocellum (29.5 to 42.5%). In addition, glycome immunoanalyses of residual plant biomass reveal differences in the extent of degradation of easily accessible xylans, with C. clariflavum strains having increased solubilization of this fraction of xylans relative to C. thermocellum. In conclusion, C. clariflavum strains exhibit higher activity than C. thermocellum in the breakdown of hemicellulose and are capable of degrading xylan to xylooligomers and xylose. This capability seems to also play a role in the higher levels of utilization of unpretreated plant material.« less

  2. Novel Endoxylanases of the Moderately Thermophilic Polysaccharide-Degrading Bacterium Melioribacter roseus.

    PubMed

    Rakitin, Andrey L; Ermakova, Alexandra Y; Ravin, Nikolai V

    2015-09-01

    Three endoxylanase-encoding genes from the moderately themophilic chemoorganotrophic bacterium Melioribacter roseus were cloned and expressed in Escherichia coli. Genes xyl2091 (Mros_2091) and xyl2495 (Mros_2495) encode GH10 family hydrolases, whereas xyl2090 (Mros_2090) represents the GH30 family. In addition to catalytic domains, Xyl2090 and Xyl2091 contain carbohydrate-binding modules that could facilitate their binding to xylans and Por sorting domains associated with the sorting of proteins from the periplasm to the outer membrane, where they are covalently attached. Recombinant endoxylanase Xyl2495 exhibited a high specific activity of 1,920 U/mg on birchwood xylan at 40°C. It is active at low temperatures, exhibiting more than 30% of the maximal activity even at 0°C. Endoxylanases Xyl2090 and Xyl2091 have lower specific activities but higher temperature optima at 80°C and 65°C, respectively. Analysis of xylan hydrolysis products revealed that Xyl2090 generates xylo-oligosaccharides longer than xylopentaose. Xylose and xylobiose are the major products of xylan hydrolysis by the recombinant Xyl2091 and Xyl2495. No activity against cellulose was observed for all enzymes. The presence of three xylanases ensures efficient xylan hydrolysis by M. roseus. The highly processive "free" endoxylanase Xyl2495 could hydrolyze xylan under moderate temperatures. Xylan hydrolysis at elevated temperatures could be accomplished by concerted action of two cell-bound xylanases; Xyl2090 that probably degrades xylans to long xylo-oligosaccharides, and Xyl2091 hydrolyzing them to xylose and xylobiose. The new endoxylanases could be useful for saccharification of lignocellulosic biomass in biofuels production, bleaching of paper pulp, and obtaining low molecular weight xylooligosaccharides.

  3. Developing Pericarp of Maize: A Model to Study Arabinoxylan Synthesis and Feruloylation

    PubMed Central

    Chateigner-Boutin, Anne-Laure; Ordaz-Ortiz, José J.; Alvarado, Camille; Bouchet, Brigitte; Durand, Sylvie; Verhertbruggen, Yves; Barrière, Yves; Saulnier, Luc

    2016-01-01

    Cell walls are comprised of networks of entangled polymers that differ considerably between species, tissues and developmental stages. The cell walls of grasses, a family that encompasses major crops, contain specific polysaccharide structures such as xylans substituted with feruloylated arabinose residues. Ferulic acid is involved in the grass cell wall assembly by mediating linkages between xylan chains and between xylans and lignins. Ferulic acid contributes to the physical properties of cell walls, it is a hindrance to cell wall degradability (thus biomass conversion and silage digestibility) and may contribute to pest resistance. Many steps leading to the formation of grass xylans and their cross-linkages remain elusive. One explanation might originate from the fact that many studies were performed on lignified stem tissues. Pathways leading to lignins and feruloylated xylans share several steps, and lignin may impede the release and thus the quantification of ferulic acid. To overcome these difficulties, we used the pericarp of the maize B73 line as a model to study feruloylated xylan synthesis and crosslinking. Using Fourier-transform infra-red spectroscopy and biochemical analyses, we show that this tissue has a low lignin content and is composed of approximately 50% heteroxylans and approximately 5% ferulic acid. Our study shows that, to date, maize pericarp contains the highest level of ferulic acid reported in plant tissue. The detection of feruloylated xylans with a polyclonal antibody shows that the occurrence of these polysaccharides is developmentally regulated in maize grain. We used the genomic tools publicly available for the B73 line to study the expression of genes within families involved or suggested to be involved in the phenylpropanoid pathway, xylan formation, feruloylation and their oxidative crosslinking. Our analysis supports the hypothesis that the feruloylated moiety of xylans originated from feruloylCoA and is transferred by a member

  4. Low acid hydrothermal fractionation of Giant Miscanthus for production of xylose-rich hydrolysate and furfural.

    PubMed

    Kim, Tae Hyun; Ryu, Hyun Jin; Oh, Kyeong Keun

    2016-10-01

    Low acid hydrothermal (LAH) fractionation was developed for the effective recovery of hemicellulosic sugar (mainly xylose) from Miscanthus sacchariflorus Goedae-Uksae 1 (M. GU-1). The xylose yield was maximized at 74.75% when the M. GU-1 was fractionated at 180°C and 0.3wt.% of sulfuric acid for 10min. At this condition, the hemicellulose (mainly xylan) degradation was 86.41%. The difference between xylan degradation and xylose recovery yield, i.e., xylan loss, was 11.66%, as indicated by the formation of decomposed products. The furfural, the value added biochemical product, was also obtained by 0.42g/L at this condition, which was 53.82% of furfural production yield based on the xylan loss. After then, the furfural production continued to increase to a maximum concentration of 1.87g/L, at which point the xylan loss corresponded to 25.87%. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Carbohydrate composition of compost during composting and mycelium growth of Agaricus bisporus.

    PubMed

    Jurak, Edita; Kabel, Mirjam A; Gruppen, Harry

    2014-01-30

    Changes of plant cell wall carbohydrate structures occurring during the process to make suitable compost for growth of Agaricus bisporus are unknown. In this paper, composition and carbohydrate structures in compost samples collected during composting and mycelium growth were analyzed. Furthermore, different extracts of compost samples were prepared with water, 1M and 4M alkali and analyzed. At the beginning of composting, 34% and after 16 days of mycelium growth 27% of dry matter was carbohydrates. Carbohydrate composition analysis showed that mainly cellulose and poorly substituted xylan chains with similar amounts and ratios of xylan building blocks were present in all phases studied. Nevertheless, xylan solubility increased 20% over the period of mycelium growth indicating partial degradation of xylan backbone. Apparently, degradation of carbohydrates occurred over the process studied by both bacteria and fungi, mainly having an effect on xylan-chain length and solubility. Copyright © 2013 Elsevier Ltd. All rights reserved.

  6. Differences in glycosyltransferase family 61 accompany variation in seed coat mucilage composition in Plantago spp.

    PubMed

    Phan, Jana L; Tucker, Matthew R; Khor, Shi Fang; Shirley, Neil; Lahnstein, Jelle; Beahan, Cherie; Bacic, Antony; Burton, Rachel A

    2016-12-01

    Xylans are the most abundant non-cellulosic polysaccharide found in plant cell walls. A diverse range of xylan structures influence tissue function during growth and development. Despite the abundance of xylans in nature, details of the genes and biochemical pathways controlling their biosynthesis are lacking. In this study we have utilized natural variation within the Plantago genus to examine variation in heteroxylan composition and structure in seed coat mucilage. Compositional assays were combined with analysis of the glycosyltransferase family 61 (GT61) family during seed coat development, with the aim of identifying GT61 sequences participating in xylan backbone substitution. The results reveal natural variation in heteroxylan content and structure, particularly in P. ovata and P. cunninghamii, species which show a similar amount of heteroxylan but different backbone substitution profiles. Analysis of the GT61 family identified specific sequences co-expressed with IRREGULAR XYLEM 10 genes, which encode putative xylan synthases, revealing a close temporal association between xylan synthesis and substitution. Moreover, in P. ovata, several abundant GT61 sequences appear to lack orthologues in P. cunninghamii. Our results indicate that natural variation in Plantago species can be exploited to reveal novel details of seed coat development and polysaccharide biosynthetic pathways. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  7. The effect of rabbit age on in vitro caecal fermentation of starch, pectin, xylan, cellulose, compound feed and its fibre.

    PubMed

    Lavrenčič, A

    2007-03-01

    In vitro gas production kinetics of six different substrates, pectin (PEC), xylan (XYL), starch (STA), cellulose (CEL), commercial compound feed (FEED; 201 g crude protein per kg, 155 g crude fibre per kg, 334 g neutral-detergent fibre (NDF) per kg and 190 g acid-detergent fibre (ADF) per kg) and an NDF prepared from commercial compound feed (NDFFEED) were determined using the caecum contents of weaned rabbits (36 days of age) and of rabbits at slaughter age (78 days of age) as inoculums. The cumulated gas production over 96 h of incubation was modelled with Gompertz model, and the kinetic parameters compared. The total potential gas production (parameter 'B' of the Gompertz model) was not affected (P>0.05) by the inoculum source, except with STA, where rabbits at slaughter weight had significantly higher total potential fermentability (314 ml/g dry matter (DM)) than those at weaning age (189 ml/g DM). Intensities of fermentation (maximum fermentation rate; MFR) of PEC (32.2 ml/h) and XYL (24.4 ml/h) were significantly greater in rabbits at weaning, while that of STA (45 ml/h) was significantly lower than at slaughter age (23.0, 14.3 and 14.0 ml/h for PEC, XYL and STA, respectively). The MFRs of CEL and NDFFEED were very similar between inoculum sources. In the first 10 h of fermentation which correspond to the normal retention time of the substrates in the caecum, the highest amount of gas was produced from PEC, followed by FEED and XYL. These substrates had a time of maximum fermentation rate (TMFR) at both rabbit ages short enough (8.0 and 9.5 h for PEC, 9.5 and 6.6 h for FEED, 13.7 and 14.2 h for XYL at weaning and at slaughter age, respectively) to be almost completely fermented in vivo.

  8. GH10 XynA is the main xylanase identified in the crude enzymatic extract of Paenibacillus sp. A59 when grown on xylan or lignocellulosic biomass.

    PubMed

    Ghio, Silvina; Insani, Ester M; Piccinni, Florencia E; Talia, Paola M; Grasso, Daniel H; Campos, Eleonora

    2016-01-01

    A novel bacterial isolate with polysaccharides degrading activity was identified as Paenibacillus sp., and named Paenibacillus sp. A59. Even though it is a strict mesophile, optimal xylanase activity of the crude enzymatic extract was achieved between 50°C and 70°C and more than 60% of the activity was retained after incubation for 48h at 50°C, indicating thermotolerance of the enzymes involved. The extract was also active on pre-treated sugarcane residue (SCR) and wheat straw, releasing xylobiose and xylose as the main products, therefore confirming its predominantly xylanolytic activity. By zymograms and mass spectrometry of crude enzymatic extracts of xylan or SCR cultures, a 32kDa GH10 beta- 1,4- endoxylanase with xylanase and no CMCase activity was identified. We named this enzyme XynA and it was the only xylanase identified under both conditions assayed, suggesting that it is a good candidate for recombinant expression and evaluation in hemicelluloses deconstruction applications. Also, a protein with two S-layer homology domains (SLH) and a large uncharacterized C-terminal domain as well as an ABC substrate binding protein were identified in crude extracts of SCR cultures. We propose that Paenibacillus sp. A59 uses a system similar to anaerobic and other Gram positive bacteria, with SLH-domain proteins anchoring polysaccharide-degrading enzymes close to the membrane and the substrate binding protein assisting translocation of simple sugars to the cell interior. Copyright © 2016 Elsevier GmbH. All rights reserved.

  9. Synthesis of furfural from xylose, xylan, and biomass using AlCl3·6H2O in biphasic media via xylose isomerization to xylulose.

    PubMed

    Yang, Yu; Hu, Chang-Wei; Abu-Omar, Mahdi M

    2012-02-13

    Furfural was prepared in high yields (75 %) from the reaction of xylose in a water-tetrahydrofuran biphasic medium containing AlCl(3)·6H2O and NaCl under microwave heating at 140 °C. The reaction profile revealed the formation of xylulose as an intermediate en route to the dehydration product (furfural). The reaction under these conditions reached completion in 45 min. The aqueous phase containing AlCl(3)·6H(2)O and NaCl could be recycled multiple times (>5) without any loss of activity or selectivity for furfural. Extension of this biphasic reaction system to include xylan as the starting material afforded furfural in 64 % yield. The use of corn stover, pinewood, switchgrass, and poplar gave furfural in 55, 38, 56, and 64 % yield, respectively, at 160 °C. Even though AlCl(3)·6H(2)O did not affect the conversion of crystalline cellulose, moderate yields of the by-product 5-hydroxymethylfurfural (HMF) were noted. The highest HMF yield of 42 % was obtained from pinewood. The coproduction of HMF and furfural from biomass was attributed to the weakening of the cellulose network in the biomass, as a result of hemicellulose hydrolysis. The multifunctional capacity of AlCl(3)·6H(2)O (hemicellulose hydrolysis, xylose isomerization, and xylulose dehydration) in combination with its ease of recyclability make it an attractive candidate/catalyst for the selective synthesis of furfural from various biomass feedstocks. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Down-Regulation of PoGT47C Expression in Poplar Results in a Reduced Glucuronoxylan Content and an Increased Wood Digestibility by Cellulase

    EPA Science Inventory

    Xylan is the second most abundant polysaccharide in dicot wood. Unraveling the biosynthetic pathway of xylan is important not only for our understanding of the process of wood formation but also for our rational engineering of wood for biofuel production. Although several glycosy...

  11. Diversity in production of xyaln-degrading enzymes among species belonging to the Trichoderma section Longibrachiatum

    USDA-ARS?s Scientific Manuscript database

    Xylan is an important part of plant biomass and represents a renewable raw material for biorefineries. Contrary to cellulose, the structure of hemicellulose is quite complex. Therefore, the biodegradation of xylan needs the cooperation of many enzymes. For industrial production of xylanase multienzy...

  12. On the specificity and mode of action of a xylanase from Trametes hirsuta (Wulf.) Pilát.

    PubMed

    Kubacková, M; Karácsonyi, S; Bilisics, L; Toman, R

    1979-11-01

    The mode of action of the extracellular endo-(1 leads to 4)-beta-D-xylanase produced by Trametes hirsuta on a (4-0-methyl-D-glucurono)-D-xylan and a modified, essentially neutral D-xylan from white willow (Salix alba L.) has been studied. Xylotetraose and xylohexaose, together with aldotetraouronic and aldohexaouronic acids, were the main products. The acidic oligosaccharides had a 4-O-methyl-D-glucopyranosyluronic acid group attached to the non-reducing D-xylosyl end-group. The action pattern of the xylanase corresponds to that of a typical endo-enzyme that acts more readily in the middle of chain, and the specific region of its action appears to involve five D-xylosyl residues. The products of the enzymic treatment of the D-xylan have revealed a regular distribution of the 4-O-methyl-D-glucopyranosyluronic acid groups attached to the D-xylan backbone.

  13. Action of a GH115 α-glucuronidase from Amphibacillus xylanus at alkaline condition promotes release of 4-O-methylglucopyranosyluronic acid from glucuronoxylan and arabinoglucuronoxylan.

    PubMed

    Yan, Ruoyu; Vuong, Thu V; Wang, Weijun; Master, Emma R

    2017-09-01

    Glucuronic acid and/or 4-O-methyl-glucuronic acid (GlcA/MeGlcA) are substituents of the main xylans present in hardwoods, conifers, and many cereal grains. α-Glucuronidases from glycoside hydrolase family GH115 can target GlcA/MeGlcA from both internally and terminally substituted regions of xylans. The current study describes the first GH115 α-glucuronidase, AxyAgu115A, from the alkaliphilic organism Amphilbacillus xylanus. AxyAgu115A was active in a wide pH range, and demonstrated better performance in alkaline condition compared to other characterized GH115 α-glucuronidases, which generally show optimal activity in acidic conditions. Specifically, its relative activity between pH 5.0 and pH 8.5 was above 80%, and was 35% of maximum at pH 10.5; although the enzyme lost 30% and 80% relative residual activity after 24-h pre-incubation at pH 9 and pH 10, respectively. AxyAgu115A was also similarly active towards glucuronoxylan as well as comparatively complex xylans such as spruce arabinoglucurunoxylan. Accommodation of complex xylans was supported by docking analyses that predicted accessibility of AxyAgu115A to branched xylo-oligosaccharides. MeGlcA release by AxyAgu115A from each xylan sample was increased by up to 30% by performing the reaction at pH 11.0 rather than pH 4.0, revealing applied benefits of AxyAgu115A for xylan recovery and processing. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Xylanase supplementation on enzymatic saccharification of dilute acid pretreated poplars at different severities

    Treesearch

    Chao Zhang; Xinshu Zhuang; Zhao Jiang Wang; Fred Matt; Franz St. John; J.Y. Zhu

    2013-01-01

    Three pairs of solid substrates from dilute acid pretreatment of two poplar wood samples were enzymatically hydrolyzed by cellulase preparations supplemented with xylanase. Supplementation of xylanase improved cellulose saccharification perhaps due to improved cellulose accessibility by xylan hydrolysis. Total xylan removal directly affected enzymatic cellulose...

  15. Single-step microwave-assisted hot water extraction of hemicelluloses from selected lignocellulosic materials - A biorefinery approach.

    PubMed

    Mihiretu, Gezahegn T; Brodin, Malin; Chimphango, Annie F; Øyaas, Karin; Hoff, Bård H; Görgens, Johann F

    2017-10-01

    The viability of single-step microwave-induced pressurized hot water conditions for co-production of xylan-based biopolymers and bioethanol from aspenwood sawdust and sugarcane trash was investigated. Extraction of hemicelluloses was conducted using microwave-assisted pressurized hot water system. The effects of temperature and time on extraction yield and enzymatic digestibility of resulting solids were determined. Temperatures between 170-200°C for aspenwood and 165-195°C for sugarcane trash; retention times between 8-22min for both feedstocks, were selected for optimization purpose. Maximum xylan extraction yields of 66 and 50%, and highest cellulose digestibilities of 78 and 74%, were attained for aspenwood and sugarcane trash respectively. Monomeric xylose yields for both feedstocks were below 7%, showing that the xylan extracts were predominantly in non-monomeric form. Thus, single-step microwave-assisted hot water method is viable biorefinery approach to extract xylan from lignocelluloses while rendering the solid residues sufficiently digestible for ethanol production. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Hemicelluloses prior to aspen chemithermomechanical pulping: pre-extraction, separation, and characterization.

    PubMed

    Liu, Wei; Hou, Qingxi; Mao, Changbin; Yuan, Zhirun; Li, Kecheng

    2012-05-16

    A portion of hemicelluloses and acetic acid can be pre-extracted with dilute sulfuric acid prior to the aspen chemithermomechanical pulp process. The streams collected from the second press-impregnation stage after acid pre-extraction contain a significant amount of acid pre-extracted hemicelluloses. Most of the total sugars obtained from the pressate were xylan, in which xylan was further hydrolyzed to sugar monomers under the acid pre-extraction condition. To fully understand the characteristics of hemicelluloses yielded prior to pulping, the pre-extracted hemicelluloses were separated and characterized by FT-IR, (1)H NMR, and thermogravimetric analysis in this study. Most of the FT-IR bonds from the hemicelluloses agreed well with the other two spectra of birch xylan and CA0050 xylan, except a new absorption at 1734 cm(-1) contributed to acetyl groups. The hemicelluloses obtained from acid pre-extraction began to decompose significantly at about 225 °C, slightly lower in comparison with organosolv and alkaline hemicelluloses reported in the literature.

  17. A xylanase gene directly cloned from the genomic DNA of alkaline wastewater sludge showing application potential in the paper industry.

    PubMed

    Zhao, Yanyu; Luo, Huiying; Meng, Kun; Shi, Pengjun; Wang, Guozeng; Yang, Peilong; Yuan, Tiezheng; Yao, Bin

    2011-09-01

    A xylanase gene, aws-2x, was directly cloned from the genomic DNA of the alkaline wastewater sludge using degenerated PCR and modified TAIL-PCR. The deduced amino acid sequence of AWS-2x shared the highest identity (60%) with the xylanase from Chryseobacterium gleum belonging to the glycosyl hydrolase GH family 10. Recombinant AWS-2x was expressed in Escherichia coli BL21 (DE3) and purified to electrophoretic homogeneity. The enzyme showed maximal activity at pH 7.5 and 55 °C, maintained more than 50% of maximal activity when assayed at pH 9.0, and was stable over a wide pH range from 4.0 to 11.0. The specific activity of AWS-2x towards hardwood xylan (beechwood and birchwood xylan) was significantly higher than that to cereal xylan (oat spelt xylan and wheat arabinoxylan). These properties make AWS-2x a potential candidate for application in the pulp and paper industry.

  18. Cloning and characterization of alpha-glucuronidase enzyme

    USDA-ARS?s Scientific Manuscript database

    Hemicellulose is the second largest source of biomass on Earth. Xylan, a polymer of beta-1,4-linked xylose residues, is a common component of hemicellulose. The enzymes xylanase and beta-xylosidase hydrolyze the xylan into xylose which can then be fermented into value-added products. However, the...

  19. Loss of Arabidopsis GAUT12/IRX8 causes anther indehiscence and leads to reduced G lignin associated with altered matrix polysaccharide deposition

    PubMed Central

    Hao, Zhangying; Avci, Utku; Tan, Li; Zhu, Xiang; Glushka, John; Pattathil, Sivakumar; Eberhard, Stefan; Sholes, Tipton; Rothstein, Grace E.; Lukowitz, Wolfgang; Orlando, Ron; Hahn, Michael G.; Mohnen, Debra

    2014-01-01

    GAlactUronosylTransferase12 (GAUT12)/IRregular Xylem8 (IRX8) is a putative glycosyltransferase involved in Arabidopsis secondary cell wall biosynthesis. Previous work showed that Arabidopsis irregular xylem8 (irx8) mutants have collapsed xylem due to a reduction in xylan and a lesser reduction in a subfraction of homogalacturonan (HG). We now show that male sterility in the irx8 mutant is due to indehiscent anthers caused by reduced deposition of xylan and lignin in the endothecium cell layer. The reduced lignin content was demonstrated by histochemical lignin staining and pyrolysis Molecular Beam Mass Spectrometry (pyMBMS) and is associated with reduced lignin biosynthesis in irx8 stems. Examination of sequential chemical extracts of stem walls using 2D 13C-1H Heteronuclear Single-Quantum Correlation (HSQC) NMR spectroscopy and antibody-based glycome profiling revealed a reduction in G lignin in the 1 M KOH extract and a concomitant loss of xylan, arabinogalactan and pectin epitopes in the ammonium oxalate, sodium carbonate, and 1 M KOH extracts from the irx8 walls compared with wild-type walls. Immunolabeling of stem sections using the monoclonal antibody CCRC-M138 reactive against an unsubstituted xylopentaose epitope revealed a bi-lamellate pattern in wild-type fiber cells and a collapsed bi-layer in irx8 cells, suggesting that at least in fiber cells, GAUT12 participates in the synthesis of a specific layer or type of xylan or helps to provide an architecture framework required for the native xylan deposition pattern. The results support the hypothesis that GAUT12 functions in the synthesis of a structure required for xylan and lignin deposition during secondary cell wall formation. PMID:25120548

  20. Isolation, screening and characterization of a novel extracellular xylanase from Aspergillus niger (KP874102.1) and its application in orange peel hydrolysis.

    PubMed

    Uday, Uma Shankar Prasad; Majumdar, Ria; Tiwari, Onkar Nath; Mishra, Umesh; Mondal, Abhijit; Bandyopadhyay, Tarun Kanti; Bhunia, Biswanath

    2017-12-01

    In the present work, a potent xylanase producing fungal strain Aspergillus niger (KP874102.1) was isolated through cultural and morphological observations from soil sample of Baramura forest, Tripura west, India. 28S rDNA technique was applied for genomic identification of this fungal strain. The isolated strain was found to be phylogenetically closely related to Aspergillus niger. Kinetic constants such as K m and V max for extracellular xylanase were determined using various substrate such as beech wood xylan, oat spelt xylan and CM cellulose through Lineweaver-Burk plot. K m , V max and K cat for beech wood xylan are found to be 2.89mg/ml, 2442U and 426178Umlmg -1 respectively. Crude enzyme did not show also CM cellulose activity. The relative efficiency of oat spelt xylan was found to be 0.819 with respect to beech wood xylan. After acid hydrolysis, enzyme was able to produce reducing sugar with 17.7, 35.5, 50.8 and 65% (w/w) from orange peel after 15, 30, 45 and 60min incubation with cellulase free xylanase and maximum reducing sugar formation rate was found to be 55.96μg/ml/min. Therefore, the Aspergillus niger (KP874102.1) is considered as a potential candidate for enzymatic hydrolysis of orange peel. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Asparagus Spears as a Model to Study Heteroxylan Biosynthesis during Secondary Wall Development

    PubMed Central

    Wu, Aimin; Picard, Kelsey; Lampugnani, Edwin R.; Cheetamun, Roshan; Beahan, Cherie; Cassin, Andrew; Lonsdale, Andrew; Doblin, Monika S.; Bacic, Antony

    2015-01-01

    Garden asparagus (Asparagus officinalis L.) is a commercially important crop species utilized for its excellent source of vitamins, minerals and dietary fiber. However, after harvest the tissue hardens and its quality rapidly deteriorates because spear cell walls become rigidified due to lignification and substantial increases in heteroxylan content. This latter observation prompted us to investigate the in vitro xylan xylosyltransferase (XylT) activity in asparagus. The current model system for studying heteroxylan biosynthesis, Arabidopsis, whilst a powerful genetic system, displays relatively low xylan XylT activity in in vitro microsomal preparations compared with garden asparagus therefore hampering our ability to study the molecular mechanism(s) of heteroxylan assembly. Here, we analyzed physiological and biochemical changes of garden asparagus spears stored at 4 °C after harvest and detected a high level of xylan XylT activity that accounts for this increased heteroxylan. The xylan XylT catalytic activity is at least thirteen-fold higher than that reported for previously published species, including Arabidopsis and grasses. A biochemical assay was optimized and up to seven successive Xyl residues were incorporated to extend the xylotetraose (Xyl4) acceptor backbone. To further elucidate the xylan biosynthesis mechanism, we used RNA-seq to generate an Asparagus reference transcriptome and identified five putative xylan biosynthetic genes (AoIRX9, AoIRX9-L, AoIRX10, AoIRX14_A, AoIRX14_B) with AoIRX9 having an expression profile that is distinct from the other genes. We propose that Asparagus provides an ideal biochemical system to investigate the biochemical aspects of heteroxylan biosynthesis and also offers the additional benefit of being able to study the lignification process during plant stem maturation. PMID:25894575

  2. Biopolymer nanocomposite films reinforced with nanocellulose whiskers

    Treesearch

    Amit Saxena; Marcus Foston; Mohamad Kassaee; Thomas J. Elder; Arthur J. Ragauskas

    2011-01-01

    A xylan nanocomposite film with improved strength and barrier properties was prepared by a solution casting using nanocellulose whiskers as a reinforcing agent. The 13C cross-polarization magic angle spinning (CP/MAS) nuclear magnetic resonance (NMR) analysis of the spectral data obtained for the NCW/xylan nanocomposite films indicated the signal intensity originating...

  3. Xylose production from corn stover biomass by steam explosion combined with enzymatic digestibility.

    PubMed

    Liu, Zhi-Hua; Chen, Hong-Zhang

    2015-10-01

    A novel conversion process using steam explosion combined with enzymatic digestibility was exploited to increase sugar yield. Results showed that glucan and xylan recovery decreased with the increase of holding temperature and residence time in SE, respectively, while glucan and xylan conversion exhibited an opposite trend. The optimal conditions of steam explosion were 160 °C and 48 min, under which glucan and xylan recovery was 93.4% and 71.6%, respectively. Glucan and xylan conversion at 18% solid loading by periodic peristalsis increased by 3.4-5.8% and 4.5-6.2%, respectively, compared with that by water baths shaker. In the whole process, glucose, xylose and total sugar yield reached to 77.3%, 62.8% and 72.3%, respectively. The yield of hydroxymethyl furfural, furfural and lignin-derived products was 6.3 × 10(-2), 7.5 × 10(-2) and less than 3.7 × 10(-2) g/100 g feedstock, respectively. This novel conversion process increased sugar recovery, reduced degradation products formation, improved digestibility efficiency, and hence increased sugar yield. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Identification and characterisation of xylanolytic yeasts isolated from decaying wood and sugarcane bagasse in Brazil.

    PubMed

    Lara, Carla A; Santos, Renata O; Cadete, Raquel M; Ferreira, Carla; Marques, Susana; Gírio, Francisco; Oliveira, Evelyn S; Rosa, Carlos A; Fonseca, César

    2014-06-01

    In this study, yeasts associated with lignocellulosic materials in Brazil, including decaying wood and sugarcane bagasse, were isolated, and their ability to produce xylanolytic enzymes was investigated. A total of 358 yeast isolates were obtained, with 198 strains isolated from decaying wood and 160 strains isolated from decaying sugarcane bagasse samples. Seventy-five isolates possessed xylanase activity in solid medium and were identified as belonging to nine species: Candida intermedia, C. tropicalis, Meyerozyma guilliermondii, Scheffersomyces shehatae, Sugiyamaella smithiae, Cryptococcus diffluens, Cr. heveanensis, Cr. laurentii and Trichosporon mycotoxinivorans. Twenty-one isolates were further screened for total xylanase activity in liquid medium with xylan, and five xylanolytic yeasts were selected for further characterization, which included quantitative analysis of growth in xylan and xylose and xylanase and β-D-xylosidase activities. The yeasts showing the highest growth rate and cell density in xylan, Cr. laurentii UFMG-HB-48, Su. smithiae UFMG-HM-80.1 and Sc. shehatae UFMG-HM-9.1a, were, simultaneously, those exhibiting higher xylanase activity. Xylan induced the highest level of (extracellular) xylanase activity in Cr. laurentii UFMG-HB-48 and the highest level of (intracellular, extracellular and membrane-associated) β-D-xylosidase activity in Su. smithiae UFMG-HM-80.1. Also, significant β-D-xylosidase levels were detected in xylan-induced cultures of Cr. laurentii UFMG-HB-48 and Sc. shehatae UFMG-HM-9.1a, mainly in extracellular and intracellular spaces, respectively. Under xylose induction, Cr. laurentii UFMG-HB-48 showed the highest intracellular β-D-xylosidase activity among all the yeast tested. C. tropicalis UFMG-HB 93a showed its higher (intracellular) β-D-xylosidase activity under xylose induction and higher at 30 °C than at 50 °C. This study revealed different xylanolytic abilities and strategies in yeasts to metabolise xylan and

  5. Adsorption of lignocelluloses of model pre-hydrolysis liquor on activated carbon.

    PubMed

    Fatehi, Pedram; Ryan, Jennifer; Ni, Yonghao

    2013-03-01

    The main objective of this work was to study the adsorption behavior of various components dissolved in the pre-hydrolysis of kraft process on activated carbon. In this work, model prehydrolysis liquor (PHL) solutions (MPHL)s were prepared via mixing various commercially available monosugars, xylan, lignin and furfural; and their adsorption performance on activated carbon (AC) was investigated. In singular (one component) MPHL/AC systems, furfural had the maximum and xylose had the minimum adsorption, and the adsorption of monosugars was basically similar on AC. Also, polydiallyldimethylammonium chloride (PDADMAC) was added (0.5 g/l) to singular xylan or lignin MPHL/AC system, which increased the lignin and xylan adsorptions to 350 and 190 mg/g on AC, respectively. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. Enzymatic pulp upgrade for producing high-value cellulose out of a Kraft paper pulp.

    PubMed

    Hutterer, Christian; Kliba, Gerhard; Punz, Manuel; Fackler, Karin; Potthast, Antje

    2017-07-01

    The high-yield separation of polymeric parts from wood-derived lignocellulosic material is indispensable in biorefinery concepts. For the separation of cellulose and xylan from hardwood paper pulps to obtain pulps of high cellulose contents, simple alkaline extractions were found to be the most suitable technology, although having certain limitations. These are embodied by residual alkali resistant xylan incorporated in the pulp matrix. Further purification in order to produce pure cellulose with a low uniformity could be achieved selectively degrading residual xylan and depolymerizing the cellulose macromolecules by xylanase and cellulase. The latter help to adjust cellulose chain lengths for certain dissolving pulp grades while reducing the demand for ozone in subsequent TCF bleaching. Experiments applying different commercially available enzyme preparations revealed the dependency of xylanase performance on the residual xylan content in pulps being stimulated by additional cellulase usage. The action of the latter strongly depends on the cellulose allomorphy confirming the impact of the pulp morphology. Hence, the combined application of both types of enzymes offers a high potential for upgrading pulps in order to produce a pure and high-value cellulose product. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Production of prebiotic-xylooligosaccharides from alkali pretreated mahogany and mango wood sawdust by using purified xylanase of Clostridium strain BOH3.

    PubMed

    Rajagopalan, Gobinath; Shanmugavelu, Kavitha; Yang, Kun-Lin

    2017-07-01

    Xylooligosaccharides (XOS) are emerging prebiotics which can be produced from lignocellulosic biomass including agro-residues and hardwood. In this study, we report the production of XOS from thermal-alkali pretreated hardwood such as mahogany and mango by using a purified xylanase from Clostridium strain BOH3. In the first approach, pure xylan is extracted from mahogany and mango hardwood and then the pure xylan is hydrolyzed by using the xylanase. In this case, 572 and 504mg XOS/g pure xylan were obtained from mahogany and mango woods, respectively. In the second approach, the same xylanase is employed to hydrolyze sawdust of hardwood after different types of pretreatments. After a thermal (121°C for 15min) pretreatment under a mild alkaline (0.05N NaOH) condition, the pretreated mahogany and mango sawdust can be utilized directly to produce 89.5 and 67.6mg XOS/g pretreated sawdust, respectively. XOS produced from the pretreated sawdust show strong prebiotic effects on Bifidobacteria and Lactobacilli. This report shows the possibility of producing XOS from pretreated woody wastes without using pure xylan as a substrate. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Biochemical characterization of two thermostable xylanolytic enzymes encoded by a gene cluster of Caldicellulosiruptor owensensis.

    PubMed

    Mi, Shuofu; Jia, Xiaojing; Wang, Jinzhi; Qiao, Weibo; Peng, Xiaowei; Han, Yejun

    2014-01-01

    The xylanolytic extremely thermophilic bacterium Caldicellulosiruptor owensensis provides a promising platform for xylan utilization. In the present study, two novel xylanolytic enzymes, GH10 endo-β-1,4-xylanase (Coxyn A) and GH39 β-1,4-xylosidase (Coxyl A) encoded in one gene cluster of C.owensensis were heterogeneously expressed and biochemically characterized. The optimum temperature of the two xylanlytic enzymes was 75°C, and the respective optimum pH for Coxyn A and Coxyl A was 7.0 and 5.0. The difference of Coxyn A and Coxyl A in solution was existing as monomer and homodimer respectively, it was also observed in predicted secondary structure. Under optimum condition, the catalytic efficiency (kcat/Km) of Coxyn A was 366 mg ml(-1) s(-1) on beechwood xylan, and the catalytic efficiency (kcat/Km) of Coxyl A was 2253 mM(-1) s(-1) on pNP-β-D-xylopyranoside. Coxyn A degraded xylan to oligosaccharides, which were converted to monomer by Coxyl A. The two intracellular enzymes might be responsible for xylooligosaccharides utilization in C.owensensis, also provide a potential way for xylan degradation in vitro.

  9. Identification and use of the putative Bacteroides ovatus xylanase promoter for the inducible production of recombinant human proteins.

    PubMed

    Hamady, Zaed Z R; Farrar, Mark D; Whitehead, Terence R; Holland, Keith T; Lodge, J Peter A; Carding, Simon R

    2008-10-01

    The use of genetically modified bacteria to deliver biologically active molecules directly to the gut has become an increasingly attractive area of investigation. The challenge of regulation of production of the therapeutic molecule and colonization of the bowel led us to investigate Bacteroides ovatus for the production of these molecules, due to its ability to colonize the colon and xylan utilization properties. Here we have identified the putative xylanase promoter. The 5' region of the corresponding mRNA was determined by 5'RACE analysis and the transcription initiation site was identified 216 bp upstream of the ATG start codon. The putative xylanase promoter was regulated by xylan in a dose- and time-dependent manner, and repressed by glucose. This promoter was subsequently used to direct the controlled expression of a gene encoding the human intestinal trefoil factor (TFF-3) after integration as a single copy into the chromosome of B. ovatus. The resulting strain produced biologically active TFF-3 in the presence of xylan. These findings identify the B. ovatus xylanase operon promoter and show that it can be utilized to direct xylan-inducible expression of heterologous eukaryotic genes in B. ovatus.

  10. Characterization of Potential Polysaccharide Utilization Systems in the Marine Bacteroidetes Gramella Flava JLT2011 Using a Multi-Omics Approach

    PubMed Central

    Tang, Kai; Lin, Yingfan; Han, Yu; Jiao, Nianzhi

    2017-01-01

    Members of phylum Bacteroidetes are distributed across diverse marine niches and Flavobacteria is often the predominant bacterial class decomposing algae-derived polysaccharides. Here, we report the complete genome of Gramella flava JLT2011 (Flavobacteria) isolated from surface water of the southeastern Pacific. A remarkable genomic feature is that the number of glycoside hydrolase (GH) genes in the genome of G. flava JLT2011 is more than 2-fold higher than that of other Gramella species. The functional profiles of the GHs suggest extensive variation in Gramella species. Growth experiments revealed that G. flava JLT2011 has the ability to utilize a wide range of polysaccharides for growth such as xylan and homogalacturonan in pectin. Nearly half of all GH genes were located on the multi-gene polysaccharide utilization loci (PUL) or PUL-like systems in G. flava JLT2011. This species was also found to harbor the two xylan PULs and a pectin PUL, respectively. Gene expression data indicated that more GHs and sugar-specific outer-membrane susC-susD systems were found in the presence of xylan than in the presence of pectin, suggesting a different strategy for heteropolymeric xylan and homoglacturonan utilization. Multi-omics data (transcriptomics, proteomics, and metabolomics) indicated that xylan PULs and pectin PUL are respectively involved in the catabolism of their corresponding polysaccharides. This work presents a comparison of polysaccharide decomposition within a genus and expands current knowledge on the diversity and function of PULs in marine Bacteroidetes, thereby deepening our understanding of their ecological role in polysaccharide remineralization in the marine system. PMID:28261179

  11. Dissecting the Genetic Basis for Seed Coat Mucilage Heteroxylan Biosynthesis in Plantago ovata Using Gamma Irradiation and Infrared Spectroscopy

    PubMed Central

    Tucker, Matthew R.; Ma, Chao; Phan, Jana; Neumann, Kylie; Shirley, Neil J.; Hahn, Michael G.; Cozzolino, Daniel; Burton, Rachel A.

    2017-01-01

    Seeds from the myxospermous species Plantago ovata release a polysaccharide-rich mucilage upon contact with water. This seed coat derived mucilage is composed predominantly of heteroxylan (HX) and is utilized as a gluten-free dietary fiber supplement to promote human colorectal health. In this study, a gamma-irradiated P. ovata population was generated and screened using histological stains and Fourier Transform Mid Infrared (FTMIR) spectroscopy to identify putative mutants showing defects in seed coat mucilage HX composition and/or structure. FTMIR analysis of dry seed revealed variation in regions of the IR spectra previously linked to xylan structure in Secale cereale (rye). Subsequent absorbance ratio and PCA multivariate analysis identified 22 putative mutant families with differences in the HX IR fingerprint region. Many of these showed distinct changes in the amount and subtle changes in structure of HX after mucilage extrusion, while 20% of the putative HX mutants identified by FTMIR showed no difference in staining patterns of extruded mucilage compared to wild-type. Transcriptional screening analysis of two putative reduced xylan in mucilage (rxm) mutants, rxm1 and rxm3, revealed that changes in HX levels in rxm1 correlate with reduced transcription of known and novel genes associated with xylan synthesis, possibly indicative of specific co-regulatory units within the xylan biosynthetic pathway. These results confirm that FTMIR is a suitable method for identifying putative mutants with altered mucilage HX composition in P. ovata, and therefore forms a resource to identify novel genes involved in xylan biosynthesis. PMID:28377777

  12. Characterization and identification of the xylanolytic enzymes from Aspergillus fumigatus Z5.

    PubMed

    Miao, Youzhi; Li, Juan; Xiao, Zhizhuang; Shen, Qirong; Zhang, Ruifu

    2015-06-23

    Plant biomass, the most abundant natural material on earth, represents a vast source of food and energy in nature. As the main component of plant biomass, xylan is a complex polysaccharide comprising a linear β(1,4)-linked backbone of xylosyl residues substituted by acetyl, arabinosyl, glucuronysyl and 4-O-methylglucuronycyl residues. Aspergillus fumigatus Z5 is an efficient plant biomass depolymerization fungus. In this study, its crude xylanolytic enzymes were characterized and identified by two-dimensional gel electrophoresis (2-DE). The optimal temperature for the crude xylanases was close to 60 °C, the highest xylanase activity was achieved at pH ranged from 3 to 6, and the crude xylanases also showed a very broad region of pH (3-11) stability. The maximal xylanase activity of 21.45 U · ml(-1) was observed in the fourth day of cultivation at 50 °C and 150 rpm with 2 % xylan as the sole carbon source. Zymogram analysis indicated that there were more than seven secreted proteins with xylanase activity. In the crude enzyme, two major endoxylanases, five cellulases and several associated enzymes were identified to be involved in the hydrolysis of polysaccharides. Of the total 13 xylanase genes in the Z5 genome, 11 were observed using q-PCR to be induced by xylan, one of which, An endo-1,4-β-xylanase with a low secretion level, was also expressed and characterized. The final hydrolysis products of xylan by crude enzyme mainly consisted of xylobiose. This study provides a comprehensive understanding of the depolymerization of xylan by Z5 and will help to design enzymatic strategies for plant biomass utilization.

  13. Comparative evaluation of acid and alkaline sulfite pretreatments for enzymatic saccharification of bagasses from three different sugarcane hybrids.

    PubMed

    Monte, Joseana R; Laurito-Friend, Debora F; Ferraz, André; Milagres, Adriane M F

    2018-04-26

    Sugarcane bagasses from three experimental sugarcane hybrids and a mill-reference sample were used to compare the efficiency and mode of action of acid and alkaline sulfite pretreatment processes. Varied chemical loads and reaction temperatures were used to prepare samples with distinguished characteristics regarding xylan and lignin removals, as well as sulfonation levels of residual lignins. The pretreatment with low sulfite loads (5%) under acidic conditions (pH 2) provided maximum glucose yield of 70% during enzymatic hydrolysis with cellulases (10 FPU/g) and β-glucosidases (20 UI/g bagasse). In this case, glucan enzymatic conversion from pretreated materials was mostly associated with extensive xylan removal (70-100%) and partial delignification occurred during the pretreatment. The use of low sulfite loads under acidic conditions required pretreatment temperatures of 160°C. In contrast, at a lower pretreatment temperature (120°C), alkaline sulfite process achieved similar glucan digestibility, but required a higher sulfite load (7.5%). Residual xylans from acid pretreated materials were almost completely hydrolysed by commercial enzymes, contrasting with relatively lower xylan to xylose conversions observed in alkaline pretreated samples. Efficient xylan removal during acid sulfite pretreatment and also during enzymatic digestion can be useful to enhance glucan accessibility and digestibility by cellulases. Alkaline sulfite process also provided substrates with high glucan digestibility, mainly associated with delignification and sulfonation of residual lignins. The results demonstrate that temperature, pH and sulfite can be combined for reducing lignocellulose recalcitrance and achieve similar glucan conversion rates in the alkaline and acid sulfite pretreated bagasses. This article is protected by copyright. All rights reserved. © 2018 American Institute of Chemical Engineers.

  14. Determination of the modes of action and synergies of xylanases by analysis of xylooligosaccharide profiles over time using fluorescence-assisted carbohydrate electrophoresis.

    PubMed

    Gong, Weili; Zhang, Huaiqiang; Tian, Li; Liu, Shijia; Wu, Xiuyun; Li, Fuli; Wang, Lushan

    2016-07-01

    The structure of xylan, which has a 1,4-linked β-xylose backbone with various substituents, is much more heterogeneous and complex than that of cellulose. Because of this, complete degradation of xylan needs a large number of enzymes that includes GH10, GH11, and GH3 family xylanases together with auxiliary enzymes. Fluorescence-assisted carbohydrate electrophoresis (FACE) is able to accurately differentiate unsubstituted and substituted xylooligosaccharides (XOS) in the heterogeneous products generated by different xylanases and allows changes in concentrations of specific XOS to be analyzed quantitatively. Based on a quantitative analysis of XOS profiles over time using FACE, we have demonstrated that GH10 and GH11 family xylanases immediately degrade xylan into sizeable XOS, which are converted into smaller XOS in a much lower speed. The shortest substituted XOS produced by hydrolysis of the substituted xylan backbone by GH10 and GH11 family xylanases were MeGlcA(2) Xyl3 and MeGlcA(2) Xyl4 , respectively. The unsubstituted xylan backbone was degraded into xylose, xylobiose, and xylotriose by both GH10 and GH11 family xylanases; the product profiles are not family-specific but, instead, depend on different subsite binding affinities in the active sites of individual enzymes. Synergystic action between xylanases and β-xylosidase degraded MeGlcA(2) Xyl4 into xylose and MeGlcA(2) Xyl3 but further degradation of MeGlcA(2) Xyl3 required additional enzymes. Synergy between xylanases and β-xylosidase was also found to significantly accelerate the conversion of XOS into xylose. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. The effect of lignin removal by alkaline peroxide pretreatment on the susceptibility of corn stover to purified cellulolytic and xylanolytic enzymes.

    PubMed

    Selig, Michael J; Vinzant, Todd B; Himmel, Michael E; Decker, Stephen R

    2009-05-01

    Pretreatment of corn stover with alkaline peroxide (AP) at pH 11.5 resulted in reduction of lignin content in the residual solids as a function of increasing batch temperature. Scanning electron microscopy of these materials revealed notably more textured surfaces on the plant cell walls as a result of the delignifying pretreatment. As expected, digestion of the delignified samples with commercial cellulase preparations showed an inverse relationship between the content of lignin present in the residual solids after pretreatment and the extent of both glucan and xylan conversion achievable. Digestions with purified enzymes revealed that decreased lignin content in the pretreated solids did not significantly impact the extent of glucan conversion achievable by cellulases alone. Not until purified xylanolytic activities were included with the cellulases were significant improvements in glucan conversion realized. In addition, an inverse relationship was observed between lignin content after pretreatment and the extent of xylan conversion achievable in a 24-h period with the xylanolytic enzymes in the absence of the cellulases. This observation, coupled with the direct relationship between enzymatic xylan and glucan conversion observed in a number of cases, suggests that the presence of lignins may not directly occlude cellulose present in lignocelluloses but rather impact cellulase action indirectly by its association with xylan.

  16. A Comparison of Polysaccharide Substrates and Reducing Sugar Methods for the Measurement of endo-1,4-β-Xylanase.

    PubMed

    McCleary, Barry V; McGeough, Paraic

    2015-11-01

    The most commonly used method for the measurement of the level of endo-xylanase in commercial enzyme preparations is the 3,5-dinitrosalicylic acid (DNS) reducing sugar method with birchwood xylan as substrate. It is well known that with the DNS method, much higher enzyme activity values are obtained than with the Nelson-Somogyi (NS) reducing sugar method. In this paper, we have compared the DNS and NS reducing sugar assays using a range of xylan-type substrates and accurately compared the molar response factors for xylose and a range of xylo-oligosaccharides. Purified beechwood xylan or wheat arabinoxylan is shown to be a suitable replacement for birchwood xylan which is no longer commercially available, and it is clearly demonstrated that the DNS method grossly overestimates endo-xylanase activity. Unlike the DNS assay, the NS assay gave the equivalent colour response with equimolar amounts of xylose, xylobiose, xylotriose and xylotetraose demonstrating that it accurately measures the quantity of glycosidic bonds cleaved by the endo-xylanase. The authors strongly recommend cessation of the use of the DNS assay for measurement of endo-xylanase due to the fact that the values obtained are grossly overestimated due to secondary reactions in colour development.

  17. Degradable Bone Graft Substitute for Effective Delivery of Multiple Growth Factors in the Treatment of Nonunion Fractures

    DTIC Science & Technology

    2012-08-01

    growth factors directly to the bone defect site can enhance repair of non-union fractures. In this study, a new chitosan /xylan composite hydrogel was...delivery aspect of this study did not succeed, treatment with the new xylan/ chitosan hydrogel alone was enough to heal serious fractures that did not...characteristics of the hydrogel in question could be tested as well as performing in vitro cell work. The previous supplier of chitosan , the main polymer

  18. The thermophilic biomass-degrading fungus Thielavia terrestris Co3Bag1 produces a hyperthermophilic and thermostable β-1,4-xylanase with exo- and endo-activity.

    PubMed

    García-Huante, Yolanda; Cayetano-Cruz, Maribel; Santiago-Hernández, Alejandro; Cano-Ramírez, Claudia; Marsch-Moreno, Rodolfo; Campos, Jorge E; Aguilar-Osorio, Guillermo; Benitez-Cardoza, Claudia G; Trejo-Estrada, Sergio; Hidalgo-Lara, María Eugenia

    2017-01-01

    A hyperthermophilic and thermostable xylanase of 82 kDa (TtXynA) was purified from the culture supernatant of T. terrestris Co3Bag1, grown on carboxymethyl cellulose (CMC), and characterized biochemically. TtXynA showed optimal xylanolytic activity at pH 5.5 and at 85 °C, and retained more than 90% of its activity at a broad pH range (4.5-10). The enzyme is highly thermostable with a half-life of 23.1 days at 65 °C, and active in the presence of several metal ions. Circular dichroism spectra strongly suggest the enzyme gains secondary structures when temperature increases. TtXynA displayed higher substrate affinity and higher catalytic efficiency towards beechwood xylan than towards birchwood xylan, oat-spelt xylan, and CMC. According to its final hydrolysis products, TtXynA displays endo-/exo-activity, yielded xylobiose, an unknown oligosaccharide containing about five residues of xylose and a small amount of xylose on beechwood xylan. Finally, this report represents the description of the first fungal hyperthermophilic xylanase which is produced by T. terrestris Co3Bag1. Since TtXynA displays relevant biochemical properties, it may be a suitable candidate for biotechnological applications carried out at high temperatures, like the enzymatic pretreatment of plant biomass for the production of bioethanol.

  19. A family 30 glucurono-xylanase from Bacillus subtilis LC9: Expression, characterization and its application in Chinese bread making.

    PubMed

    Guo, Yalan; Gao, Zhen; Xu, Jiaxing; Chang, Siyuan; Wu, Bin; He, Bingfang

    2018-05-22

    A GH30-8 endoxylanase was identified from an environmental Bacillus subtilis isolate following growth selection on aspen wood glucuronoxylan. The putative endoxylanase was cloned for protein expression and characterization in the Gram-positive protease deficient protein expression host B. subtilis WB800. The extracellular activity obtained was 55 U/mL, which was 14.5-fold higher than that obtained with the native species. The apparent molecular mass of BsXyn30 was estimated as 43 kDa by SDS-PAGE. BsXyn30 showed an optimal activity at pH 7.0 and 60 °C. Recombinant BsXyn30 displayed maximum activity against aspen wood xylan, followed by beechwood xylan but showed no catalytic activity on arabinose-substituted xylans. Analysis of hydrolyzed products of beechwood xylan by thin-layer chromatography and mass spectroscopy revealed the presence of xylooligosaccharides with a single methyl-glucuronic acid residue. BsXyn30 exhibited very low activity for hydrolysis xylotetraose and xylopentaose, but had no detectable activity against xylobiose and xylotriose. Using BsXyn30 as an additive in breadmaking, a decrease in water-holding capacity, an increase in dough expansion as well as improvements in volume and specific volume of the bread were recorded. Thus, the present study provided the basis for the application of GH30 xylanase in breadmaking. Copyright © 2018 Elsevier B.V. All rights reserved.

  20. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pérez-Pimienta, Jose A.; Vargas-Tah, Alejandra; López-Ortega, Karla M.

    Agave bagasse (AGB) has gained recognition as a drought-tolerant biofuel feedstock with high productivity in semiarid regions. A comparative analysis of ionic liquid (IL) and organosolv (OV) pretreatment technologies in AGB was performed using a sequential enzymatic saccharification and fermentation (SESF) strategy with cellulolytic enzymes and the ethanologenic Escherichia coli strain MS04. After pretreatment, 86% of xylan and 45% of lignin were removed from OV-AGB, whereas IL-AGB reduced lignin content by 28% and xylan by 50% when compared to the untreated biomass. High glucan ( > 90%) and xylan ( > 83%) conversion was obtained with both pretreated samples. Duringmore » the fermentation stage (48 h), 12.1 and 12.7 kg of ethanol were produced per 100 kg of untreated AGB for IL and OV, respectively. These comparative analyses showed the advantages of SESF using IL and OV in a biorefinery configuration where a better understanding of AGB recalcitrance is key for future applications.« less

  1. Investigation of enzyme formulation on pretreated switchgrass.

    PubMed

    Falls, Matthew; Shi, Jian; Ebrik, Mirvat A; Redmond, Tim; Yang, Bin; Wyman, Charles E; Garlock, Rebecca; Balan, Venkatesh; Dale, Bruce E; Pallapolu, V Ramesh; Lee, Y Y; Kim, Youngmi; Mosier, Nathan S; Ladisch, Michael R; Hames, Bonnie; Thomas, Steve; Donohoe, Bryon S; Vinzant, Todd B; Elander, Richard T; Warner, Ryan E; Sierra-Ramirez, Rocio; Holtzapple, Mark T

    2011-12-01

    This work studied the benefits of adding different enzyme cocktails (cellulase, xylanase, β-glucosidase) to pretreated switchgrass. Pretreatment methods included ammonia fiber expansion (AFEX), dilute-acid (DA), liquid hot water (LHW), lime, lime+ball-milling, soaking in aqueous ammonia (SAA), and sulfur dioxide (SO(2)). The compositions of the pretreated materials were analyzed and showed a strong correlation between initial xylan composition and the benefits of xylanase addition. Adding xylanase dramatically improved xylan yields for SAA (+8.4%) and AFEX (+6.3%), and showed negligible improvement (0-2%) for the pretreatments with low xylan content (dilute-acid, SO(2)). Xylanase addition also improved overall yields with lime+ball-milling and SO(2) achieving the highest overall yields from pretreated biomass (98.3% and 93.2%, respectively). Lime+ball-milling obtained an enzymatic yield of 92.3kg of sugar digested/kg of protein loaded. Copyright © 2011 Elsevier Ltd. All rights reserved.

  2. The biosynthesis and wall-binding of hemicelluloses in cellulose-deficient maize cells: an example of metabolic plasticity.

    PubMed

    de Castro, María; Miller, Janice G; Acebes, José Luis; Encina, Antonio; García-Angulo, Penélope; Fry, Stephen C

    2015-04-01

    Cell-suspension cultures (Zea mays L., Black Mexican sweet corn) habituated to 2,6-dichlorobenzonitrile (DCB) survive with reduced cellulose owing to hemicellulose network modification. We aimed to define the hemicellulose metabolism modifications in DCB-habituated maize cells showing a mild reduction in cellulose at different stages in the culture cycle. Using pulse-chase radiolabeling, we fed habituated and non-habituated cultures with [(3)H]arabinose, and traced the distribution of (3)H-pentose residues between xylans, xyloglucans and other polymers in several cellular compartments for 5 h. Habituated cells were slower taking up exogenous [(3)H]arabinose. Tritium was incorporated into polysaccharide-bound arabinose and xylose residues, but habituated cells diverted a higher proportion of their new [(3)H]xylose residues into (hetero) xylans at the expense of xyloglucan synthesis. During logarithmic growth, habituated cells showed slower vesicular trafficking of polymers, especially xylans. Moreover, habituated cells showed a decrease in the strong wall-binding of all pentose-containing polysaccharides studied; correspondingly, especially in log-phase cultures, habituation increased the proportion of (3)H-hemicelluloses ([(3)H]xylans and [(3)H]xyloglucan) sloughed into the medium. These findings could be related to the cell walls' cellulose-deficiency, and consequent reduction in binding sites for hemicelluloses; the data could also reflect the habituated cells' reduced capacity to integrate arabinoxylans by extra-protoplasmic phenolic cross-linking, as well as xyloglucans, during wall assembly. © 2015 Institute of Botany, Chinese Academy of Sciences.

  3. C-Terminal carbohydrate-binding module 9_2 fused to the N-terminus of GH11 xylanase from Aspergillus niger.

    PubMed

    Xu, Wenxuan; Liu, Yajuan; Ye, Yanxin; Liu, Meng; Han, Laichuang; Song, Andong; Liu, Liangwei

    2016-10-01

    The 9_2 carbohydrate-binding module (C2) locates natively at the C-terminus of the GH10 thermophilic xylanase from Thermotoga marimita. When fused to the C-terminus, C2 improved thermostability of a GH11 xylanase (Xyn) from Aspergillus niger. However, a question is whether the C-terminal C2 would have a thermostabilizing effect when fused to the N-terminus of a catalytic module. A chimeric enzyme, C2-Xyn, was created by step-extension PCR, cloned in pET21a(+), and expressed in E. coli BL21(DE3). The C2-Xyn exhibited a 2 °C higher optimal temperature, a 2.8-fold longer thermostability, and a 4.5-fold higher catalytic efficiency on beechwood xylan than the Xyn. The C2-Xyn exhibited a similar affinity for binding to beechwood xylan and a higher affinity for oat-spelt xylan than Xyn. C2 is a thermostabilizing carbohydrate-binding module and provides a model of fusion at an enzymatic terminus inconsistent with the modular natural terminal location.

  4. Structural Insights into the Thermophilic Adaption Mechanism of Endo-1,4-β-Xylanase from Caldicellulosiruptor owensensis.

    PubMed

    Liu, Xin; Liu, Tengfei; Zhang, Yuebin; Xin, Fengjiao; Mi, Shuofu; Wen, Boting; Gu, Tianyi; Shi, Xinyuan; Wang, Fengzhong; Sun, Lichao

    2018-01-10

    Xylanases (EC 3.2.1.8) are a kind of enzymes degrading xylan to xylooligosaccharides (XOS) and have been widely used in a variety of industrial applications. Among them, xylanases from thermophilic microorganisms have distinct advantages in industries that require high temperature conditions. The CoXynA gene, encoding a glycoside hydrolase (GH) family 10 xylanase, was identified from thermophilic Caldicellulosiruptor owensensis and was overexpressed in Escherichia coli. Recombinant CoXynA showed optimal activity at 90 °C with a half-life of about 1 h at 80 °C and exhibited highest activity at pH 7.0. The activity of CoXynA activity was affected by a variety of cations. CoXynA showed distinct substrate specificities for beechwood xylan and birchwood xylan. The crystal structure of CoXynA was solved and a molecular dynamics simulation of CoXynA was performed. The relatively high thermostability of CoXynA was proposed to be due to the increased overall protein rigidity resulting from the reduced length and fluctuation of Loop 7.

  5. Rapid analysis of composition and reactivity in cellulosic biomass feedstocks with near-infrared spectroscopy.

    PubMed

    Payne, Courtney E; Wolfrum, Edward J

    2015-01-01

    Obtaining accurate chemical composition and reactivity (measures of carbohydrate release and yield) information for biomass feedstocks in a timely manner is necessary for the commercialization of biofuels. Our objective was to use near-infrared (NIR) spectroscopy and partial least squares (PLS) multivariate analysis to develop calibration models to predict the feedstock composition and the release and yield of soluble carbohydrates generated by a bench-scale dilute acid pretreatment and enzymatic hydrolysis assay. Major feedstocks included in the calibration models are corn stover, sorghum, switchgrass, perennial cool season grasses, rice straw, and miscanthus. We present individual model statistics to demonstrate model performance and validation samples to more accurately measure predictive quality of the models. The PLS-2 model for composition predicts glucan, xylan, lignin, and ash (wt%) with uncertainties similar to primary measurement methods. A PLS-2 model was developed to predict glucose and xylose release following pretreatment and enzymatic hydrolysis. An additional PLS-2 model was developed to predict glucan and xylan yield. PLS-1 models were developed to predict the sum of glucose/glucan and xylose/xylan for release and yield (grams per gram). The release and yield models have higher uncertainties than the primary methods used to develop the models. It is possible to build effective multispecies feedstock models for composition, as well as carbohydrate release and yield. The model for composition is useful for predicting glucan, xylan, lignin, and ash with good uncertainties. The release and yield models have higher uncertainties; however, these models are useful for rapidly screening sample populations to identify unusual samples.

  6. Rapid analysis of composition and reactivity in cellulosic biomass feedstocks with near-infrared spectroscopy

    DOE PAGES

    Payne, Courtney E.; Wolfrum, Edward J.

    2015-03-12

    Obtaining accurate chemical composition and reactivity (measures of carbohydrate release and yield) information for biomass feedstocks in a timely manner is necessary for the commercialization of biofuels. Our objective was to use near-infrared (NIR) spectroscopy and partial least squares (PLS) multivariate analysis to develop calibration models to predict the feedstock composition and the release and yield of soluble carbohydrates generated by a bench-scale dilute acid pretreatment and enzymatic hydrolysis assay. Major feedstocks included in the calibration models are corn stover, sorghum, switchgrass, perennial cool season grasses, rice straw, and miscanthus. Here are the results: We present individual model statistics tomore » demonstrate model performance and validation samples to more accurately measure predictive quality of the models. The PLS-2 model for composition predicts glucan, xylan, lignin, and ash (wt%) with uncertainties similar to primary measurement methods. A PLS-2 model was developed to predict glucose and xylose release following pretreatment and enzymatic hydrolysis. An additional PLS-2 model was developed to predict glucan and xylan yield. PLS-1 models were developed to predict the sum of glucose/glucan and xylose/xylan for release and yield (grams per gram). The release and yield models have higher uncertainties than the primary methods used to develop the models. In conclusion, it is possible to build effective multispecies feedstock models for composition, as well as carbohydrate release and yield. The model for composition is useful for predicting glucan, xylan, lignin, and ash with good uncertainties. The release and yield models have higher uncertainties; however, these models are useful for rapidly screening sample populations to identify unusual samples.« less

  7. Localization of Cell Wall Polysaccharides in Normal and Compression Wood of Radiata Pine: Relationships with Lignification and Microfibril Orientation1

    PubMed Central

    Donaldson, Lloyd A.; Knox, J. Paul

    2012-01-01

    The distribution of noncellulosic polysaccharides in cell walls of tracheids and xylem parenchyma cells in normal and compression wood of Pinus radiata, was examined to determine the relationships with lignification and cellulose microfibril orientation. Using fluorescence microscopy combined with immunocytochemistry, monoclonal antibodies were used to detect xyloglucan (LM15), β(1,4)-galactan (LM5), heteroxylan (LM10 and LM11), and galactoglucomannan (LM21 and LM22). Lignin and crystalline cellulose were localized on the same sections used for immunocytochemistry by autofluorescence and polarized light microscopy, respectively. Changes in the distribution of noncellulosic polysaccharides between normal and compression wood were associated with changes in lignin distribution. Increased lignification of compression wood secondary walls was associated with novel deposition of β(1,4)-galactan and with reduced amounts of xylan and mannan in the outer S2 (S2L) region of tracheids. Xylan and mannan were detected in all lignified xylem cell types (tracheids, ray tracheids, and thick-walled ray parenchyma) but were not detected in unlignified cell types (thin-walled ray parenchyma and resin canal parenchyma). Mannan was absent from the highly lignified compound middle lamella, but xylan occurred throughout the cell walls of tracheids. Using colocalization measurements, we confirmed that polysaccharides containing galactose, mannose, and xylose have consistent correlations with lignification. Low or unsubstituted xylans were localized in cell wall layers characterized by transverse cellulose microfibril orientation in both normal and compression wood tracheids. Our results support the theory that the assembly of wood cell walls, including lignification and microfibril orientation, may be mediated by changes in the amount and distribution of noncellulosic polysaccharides. PMID:22147521

  8. Modeling changes in biomass composition during microwave-based alkali pretreatment of switchgrass.

    PubMed

    Keshwani, Deepak R; Cheng, Jay J

    2010-01-01

    This study used two different approaches to model changes in biomass composition during microwave-based pretreatment of switchgrass: kinetic modeling using a time-dependent rate coefficient, and a Mamdani-type fuzzy inference system. In both modeling approaches, the dielectric loss tangent of the alkali reagent and pretreatment time were used as predictors for changes in amounts of lignin, cellulose, and xylan during the pretreatment. Training and testing data sets for development and validation of the models were obtained from pretreatment experiments conducted using 1-3% w/v NaOH (sodium hydroxide) and pretreatment times ranging from 5 to 20 min. The kinetic modeling approach for lignin and xylan gave comparable results for training and testing data sets, and the differences between the predictions and experimental values were within 2%. The kinetic modeling approach for cellulose was not as effective, and the differences were within 5-7%. The time-dependent rate coefficients of the kinetic models estimated from experimental data were consistent with the heterogeneity of individual biomass components. The Mamdani-type fuzzy inference was shown to be an effective approach to model the pretreatment process and yielded predictions with less than 2% deviation from the experimental values for lignin and with less than 3% deviation from the experimental values for cellulose and xylan. The entropies of the fuzzy outputs from the Mamdani-type fuzzy inference system were calculated to quantify the uncertainty associated with the predictions. Results indicate that there is no significant difference between the entropies associated with the predictions for lignin, cellulose, and xylan. It is anticipated that these models could be used in process simulations of bioethanol production from lignocellulosic materials.

  9. Rapid analysis of composition and reactivity in cellulosic biomass feedstocks with near-infrared spectroscopy

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Payne, Courtney E.; Wolfrum, Edward J.

    Obtaining accurate chemical composition and reactivity (measures of carbohydrate release and yield) information for biomass feedstocks in a timely manner is necessary for the commercialization of biofuels. Our objective was to use near-infrared (NIR) spectroscopy and partial least squares (PLS) multivariate analysis to develop calibration models to predict the feedstock composition and the release and yield of soluble carbohydrates generated by a bench-scale dilute acid pretreatment and enzymatic hydrolysis assay. Major feedstocks included in the calibration models are corn stover, sorghum, switchgrass, perennial cool season grasses, rice straw, and miscanthus. Here are the results: We present individual model statistics tomore » demonstrate model performance and validation samples to more accurately measure predictive quality of the models. The PLS-2 model for composition predicts glucan, xylan, lignin, and ash (wt%) with uncertainties similar to primary measurement methods. A PLS-2 model was developed to predict glucose and xylose release following pretreatment and enzymatic hydrolysis. An additional PLS-2 model was developed to predict glucan and xylan yield. PLS-1 models were developed to predict the sum of glucose/glucan and xylose/xylan for release and yield (grams per gram). The release and yield models have higher uncertainties than the primary methods used to develop the models. In conclusion, it is possible to build effective multispecies feedstock models for composition, as well as carbohydrate release and yield. The model for composition is useful for predicting glucan, xylan, lignin, and ash with good uncertainties. The release and yield models have higher uncertainties; however, these models are useful for rapidly screening sample populations to identify unusual samples.« less

  10. One-step pretreatment of yellow poplar biomass using peracetic acid to enhance enzymatic digestibility.

    PubMed

    Lee, Hyeong Rae; Kazlauskas, Romas J; Park, Tai Hyun

    2017-09-22

    Pretreatment of biomass with dilute acid requires high temperatures of >160 °C to remove xylan and does not remove lignin. Here we report that the addition of peracetic acid, a strong oxidant, to mild dilute acid pretreatment reduces the temperature requirement to only 120 °C. Pretreatment of yellow poplar with peracetic acid (300 mM, 2.3 wt%) and dilute sulfuric acid (100 mM, 1.0 wt%) at 120 °C for 5 min removed 85.7% of the xylan and 90.4% of the lignin leaving a solid consisting of 75.6% glucan, 6.0% xylan and 4.7% lignin. Low enzyme loadings of 5 FPU/g glucan and 10 pNPGU/g glucan converted this solid to glucose with an 84.0% yield. This amount of glucose was 2.5 times higher than with dilute acid-pretreated solid and 13.8 times higher than with untreated yellow poplar. Thus, the addition of peracetic acid, easily generated from acetic acid and hydrogen peroxide, dramatically increases the effectiveness of dilute acid pretreatment of biomass.

  11. Heterologous expression of xylanase enzymes in lipogenic yeast Yarrowia lipolytica

    DOE PAGES

    Wang, Wei; Wei, Hui; Alahuhta, Markus; ...

    2014-12-02

    In order to develop a direct microbial sugar conversion platform for the production of lipids, drop-in fuels and chemicals from cellulosic biomass substrate, we chose Yarrowia lipolytica as a viable demonstration strain. Y. lipolytica is known to accumulate lipids intracellularly and is capable of metabolizing sugars to produce lipids; however, it lacks the lignocellulose-degrading enzymes needed to break down biomass directly. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful expression of several xylanases in Y. lipolytica. The XynII and XlnD expressing Yarrowia strains exhibited an abilitymore » to grow on xylan mineral plates. This was shown by Congo Red staining of halo zones on xylan mineral plates. Enzymatic activity tests further demonstrated active expression of XynII and XlnD in Y. lipolytica. Furthermore, synergistic action in converting xylan to xylose was observed when XlnD acted in concert with XynII. Finally, the successful expression of these xylanases in Yarrowia further advances us toward our goal to develop a direct microbial conversion process using this organism.« less

  12. Application of pigeon pea (Cajanus cajan) stalks as raw material for xylooligosaccharides production.

    PubMed

    Samanta, A K; Jayapal, Natasha; Kolte, A P; Senani, S; Sridhar, Manpal; Mishra, Sukriti; Prasad, C S; Suresh, K P

    2013-04-01

    Pigeon pea (Cajanus cajan) is a perennial plant widely cultivated in tropical and subtropical regions of many countries. The present studies aimed to produce xylooligosaccharides (XOS) from pigeon pea stalks in order to do value addition. The chemical analysis of stalks revealed 18.33 ± 1.40 % hemicelluloses in addition to cellulose, protein, and lignin. Sodium hydroxide coupled with steam application enabled almost 96 % recovery of original xylan, present in the pigeon pea stalks. Enzymatic hydrolysis of xylan led to production of XOS namely, xylobiose and xylotriose. Response surface model indicated a maximum yield of xylobiose (0.502 mg/ml) under the hydrolysis conditions of pH 4.91, temperature at 48.11 °C, enzyme dose at 11.01 U, and incubation time at 15.65 h. The ideal conditions for higher xylotriose yield (0.204 mg/ml) were pH 5.44, temperature at 39.29 °C, enzyme dose at 3.23 U, and incubation time at 15.26 h. The present investigation was successful in assessing the prospect of using pigeon pea stalks as a raw material for xylan extraction vis-à-vis XOS production.

  13. Xylanase and β-xylosidase production by Aspergillus ochraceus: new perspectives for the application of wheat straw autohydrolysis liquor.

    PubMed

    Michelin, Michele; Polizeli, Maria de Lourdes T M; Ruzene, Denise S; Silva, Daniel P; Vicente, António A; Jorge, João A; Terenzi, Héctor F; Teixeira, José A

    2012-01-01

    The xylanase biosynthesis is induced by its substrate-xylan. The high xylan content in some wastes such as wheat residues (wheat bran and wheat straw) makes them accessible and cheap sources of inducers to be mainly applied in great volumes of fermentation, such as those of industrial bioreactors. Thus, in this work, the main proposal was incorporated in the nutrient medium wheat straw particles decomposed to soluble compounds (liquor) through treatment of lignocellulosic materials in autohydrolysis process, as a strategy to increase and undervalue xylanase production by Aspergillus ochraceus. The wheat straw autohydrolysis liquor produced in several conditions was used as a sole carbon source or with wheat bran. The best conditions for xylanase and β-xylosidase production were observed when A. ochraceus was cultivated with 1% wheat bran added of 10% wheat straw liquor (produced after 15 min of hydrothermal treatment) as carbon source. This substrate was more favorable when compared with xylan, wheat bran, and wheat straw autohydrolysis liquor used separately. The application of this substrate mixture in a stirred tank bioreactor indicated the possibility of scaling up the process to commercial production.

  14. Improvement of bread making quality by supplementation with a recombinant xylanase produced by Pichia pastoris.

    PubMed

    de Queiroz Brito Cunha, Carolina Cândida; Gama, Aline Rodrigues; Cintra, Lorena Cardoso; Bataus, Luiz Artur Mendes; Ulhoa, Cirano José

    2018-01-01

    Xylanases (EC 3.2.1.8) are hydrolytic enzymes, which randomly cleave the β-1,4-linked xylose residues from xylan. The synthetic gene xynBS27 from Streptomyces sp. S27 was successfully cloned and expressed in Pichia pastoris. The full-length gene consists of 729 bp and encodes 243 amino acids including 51 residues of a putative signal peptide. This enzyme was purified in two steps and was shown to have a molecular weight of 20 kDa. The purified r-XynBS27 was active against beechwood xylan and oat spelt xylan as expected for GH 11 family. The optimum pH and temperature values for the enzyme were 6.0 and 75 °C, respectively. The Km and Vmax were 12.38 mg/mL and 13.68 μmol min/mg, respectively. The r-XynBS27 showed high xylose tolerance and was inhibited by some metal ions and by SDS. r-XynBS27 was employed as an additive in the bread making process. A decrease in firmness, stiffness and consistency, and improvements in specific volume and reducing sugar content were recorded.

  15. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Wei; Wei, Hui; Alahuhta, Markus

    In order to develop a direct microbial sugar conversion platform for the production of lipids, drop-in fuels and chemicals from cellulosic biomass substrate, we chose Yarrowia lipolytica as a viable demonstration strain. Y. lipolytica is known to accumulate lipids intracellularly and is capable of metabolizing sugars to produce lipids; however, it lacks the lignocellulose-degrading enzymes needed to break down biomass directly. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful expression of several xylanases in Y. lipolytica. The XynII and XlnD expressing Yarrowia strains exhibited an abilitymore » to grow on xylan mineral plates. This was shown by Congo Red staining of halo zones on xylan mineral plates. Enzymatic activity tests further demonstrated active expression of XynII and XlnD in Y. lipolytica. Furthermore, synergistic action in converting xylan to xylose was observed when XlnD acted in concert with XynII. Finally, the successful expression of these xylanases in Yarrowia further advances us toward our goal to develop a direct microbial conversion process using this organism.« less

  16. Gene stacking of multiple traits for high yield of fermentable sugars in plant biomass

    DOE PAGES

    Aznar, Aude; Chalvin, Camille; Shih, Patrick M.; ...

    2018-01-09

    Second-generation biofuels produced from biomass can help to decrease dependency on fossil fuels, bringing about many economic and environmental benefits. To make biomass more suitable for biorefinery use, we need a better understanding of plant cell wall biosynthesis. Increasing the ratio of C6 to C5 sugars in the cell wall and decreasing the lignin content are two important targets in engineering of plants that are more suitable for downstream processing for second-generation biofuel production. Here, we have studied the basic mechanisms of cell wall biosynthesis and identified genes involved in biosynthesis of pectic galactan, including the GALS1 galactan synthase andmore » the UDP-galactose/UDP-rhamnose transporter URGT1. We have engineered plants with a more suitable biomass composition by applying these findings, in conjunction with synthetic biology and gene stacking tools. Plants were engineered to have up to fourfold more pectic galactan in stems by overexpressing GALS1, URGT1, and UGE2, a UDP-glucose epimerase. Furthermore, the increased galactan trait was engineered into plants that were already engineered to have low xylan content by restricting xylan biosynthesis to vessels where this polysaccharide is essential. Finally, the high galactan and low xylan traits were stacked with the low lignin trait obtained by expressing the QsuB gene encoding dehydroshikimate dehydratase in lignifying cells. In conclusion, the results show that approaches to increasing C6 sugar content, decreasing xylan, and reducing lignin content can be combined in an additive manner. Thus, the engineered lines obtained by this trait-stacking approach have substantially improved properties from the perspective of biofuel production, and they do not show any obvious negative growth effects. The approach used in this study can be readily transferred to bioenergy crop plants.« less

  17. Gene stacking of multiple traits for high yield of fermentable sugars in plant biomass

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Aznar, Aude; Chalvin, Camille; Shih, Patrick M.

    Second-generation biofuels produced from biomass can help to decrease dependency on fossil fuels, bringing about many economic and environmental benefits. To make biomass more suitable for biorefinery use, we need a better understanding of plant cell wall biosynthesis. Increasing the ratio of C6 to C5 sugars in the cell wall and decreasing the lignin content are two important targets in engineering of plants that are more suitable for downstream processing for second-generation biofuel production. Here, we have studied the basic mechanisms of cell wall biosynthesis and identified genes involved in biosynthesis of pectic galactan, including the GALS1 galactan synthase andmore » the UDP-galactose/UDP-rhamnose transporter URGT1. We have engineered plants with a more suitable biomass composition by applying these findings, in conjunction with synthetic biology and gene stacking tools. Plants were engineered to have up to fourfold more pectic galactan in stems by overexpressing GALS1, URGT1, and UGE2, a UDP-glucose epimerase. Furthermore, the increased galactan trait was engineered into plants that were already engineered to have low xylan content by restricting xylan biosynthesis to vessels where this polysaccharide is essential. Finally, the high galactan and low xylan traits were stacked with the low lignin trait obtained by expressing the QsuB gene encoding dehydroshikimate dehydratase in lignifying cells. In conclusion, the results show that approaches to increasing C6 sugar content, decreasing xylan, and reducing lignin content can be combined in an additive manner. Thus, the engineered lines obtained by this trait-stacking approach have substantially improved properties from the perspective of biofuel production, and they do not show any obvious negative growth effects. The approach used in this study can be readily transferred to bioenergy crop plants.« less

  18. Elucidation of the Molecular Basis for Arabinoxylan-Debranching Activity of a Thermostable Family GH62 α-l-Arabinofuranosidase from Streptomyces thermoviolaceus

    PubMed Central

    Wang, Weijun; Mai-Gisondi, Galina; Stogios, Peter J.; Kaur, Amrit; Xu, Xiaohui; Cui, Hong; Turunen, Ossi; Savchenko, Alexei

    2014-01-01

    Xylan-debranching enzymes facilitate the complete hydrolysis of xylan and can be used to alter xylan chemistry. Here, the family GH62 α-l-arabinofuranosidase from Streptomyces thermoviolaceus (SthAbf62A) was shown to have a half-life of 60 min at 60°C and the ability to cleave α-1,3 l-arabinofuranose (l-Araf) from singly substituted xylopyranosyl (Xylp) backbone residues in wheat arabinoxylan; low levels of activity on arabinan as well as 4-nitrophenyl α-l-arabinofuranoside were also detected. After selective removal of α-1,3 l-Araf substituents from disubstituted Xylp residues present in wheat arabinoxylan, SthAbf62A could also cleave the remaining α-1,2 l-Araf substituents, confirming the ability of SthAbf62A to remove α-l-Araf residues that are (1→2) and (1→3) linked to monosubstituted β-d-Xylp sugars. Three-dimensional structures of SthAbf62A and its complex with xylotetraose and l-arabinose confirmed a five-bladed β-propeller fold and revealed a molecular Velcro in blade V between the β1 and β21 strands, a disulfide bond between Cys27 and Cys297, and a calcium ion coordinated in the central channel of the fold. The enzyme-arabinose complex structure further revealed a narrow and seemingly rigid l-arabinose binding pocket situated at the center of one side of the β propeller, which stabilized the arabinofuranosyl substituent through several hydrogen-bonding and hydrophobic interactions. The predicted catalytic amino acids were oriented toward this binding pocket, and the catalytic essentiality of Asp53 and Glu213 was confirmed by site-specific mutagenesis. Complex structures with xylotetraose revealed a shallow cleft for xylan backbone binding that is open at both ends and comprises multiple binding subsites above and flanking the l-arabinose binding pocket. PMID:24951792

  19. aguA, the gene encoding an extracellular alpha-glucuronidase from Aspergillus tubingensis, is specifically induced on xylose and not on glucuronic acid.

    PubMed

    de Vries, R P; Poulsen, C H; Madrid, S; Visser, J

    1998-01-01

    An extracellular alpha-glucuronidase was purified and characterized from a commercial Aspergillus preparation and from culture filtrate of Aspergillus tubingensis. The enzyme has a molecular mass of 107 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 112 kDa as determined by mass spectrometry, has a determined pI just below 5.2, and is stable at pH 6.0 for prolonged times. The pH optimum for the enzyme is between 4.5 and 6.0, and the temperature optimum is 70 degrees C. The alpha-glucuronidase is active mainly on small substituted xylo-oligomers but is also able to release a small amount of 4-O-methylglucuronic acid from birchwood xylan. The enzyme acts synergistically with endoxylanases and beta-xylosidase in the hydrolysis of xylan. The enzyme is N glycosylated and contains 14 putative N-glycosylation sites. The gene encoding this alpha-glucuronidase (aguA) was cloned from A. tubingensis. It consists of an open reading frame of 2,523 bp and contains no introns. The gene codes for a protein of 841 amino acids, containing a eukaryotic signal sequence of 20 amino acids. The mature protein has a predicted molecular mass of 91,790 Da and a calculated pI of 5.13. Multiple copies of the gene were introduced in A. tubingensis, and expression was studied in a highly overproducing transformant. The aguA gene was expressed on xylose, xylobiose, and xylan, similarly to genes encoding endoxylanases, suggesting a coordinate regulation of expression of xylanases and alpha-glucuronidase. Glucuronic acid did not induce the expression of aguA and also did not modulate the expression on xylose. Addition of glucose prevented expression of aguA on xylan but only reduced the expression on xylose.

  20. Lignin-carbohydrate complexes from sisal (Agave sisalana) and abaca (Musa textilis): chemical composition and structural modifications during the isolation process.

    PubMed

    Del Río, José C; Prinsen, Pepijn; Cadena, Edith M; Martínez, Ángel T; Gutiérrez, Ana; Rencoret, Jorge

    2016-05-01

    Two types of lignins occurred in different lignin-carbohydrate fractions, a lignin enriched in syringyl units, less condensed, preferentially associated with xylans, and a lignin with more guaiacyl units, more condensed, associated with glucans. Lignin-carbohydrate complexes (LCC) were isolated from the fibers of sisal (Agave sisalana) and abaca (Musa textilis) according to a plant biomass fractionation procedure recently developed and which was termed as "universally" applicable to any type of lignocellulosic material. Two LCC fractions, namely glucan-lignin (GL) and xylan-lignin (XL), were isolated and differed in the content and composition of carbohydrates and lignin. In both cases, GL fractions were enriched in glucans and comparatively depleted in lignin, whereas XL fractions were depleted in glucans, but enriched in xylans and lignin. Analysis by two-dimensional Nuclear Magnetic Resonance (2D-NMR) and Derivatization Followed by Reductive Cleavage (DFRC) indicated that the XL fractions were enriched in syringyl (S)-lignin units and β-O-4' alkyl-aryl ether linkages, whereas GL fractions have more guaiacyl (G)-lignin units and less β-O-4' alkyl-aryl ether linkages per lignin unit. The data suggest that the structural characteristics of the lignin polymers are not homogeneously distributed within the same plant and that two different lignin polymers with different composition and structure might be present. The analyses also suggested that acetates from hemicelluloses and the acyl groups (acetates and p-coumarates) attached to the γ-OH of the lignin side chains were extensively hydrolyzed and removed during the LCC fractionation process. Therefore, caution must be paid when using this fractionation approach for the structural characterization of plants with acylated hemicelluloses and lignins. Finally, several chemical linkages (phenylglycosides and benzyl ethers) could be observed to occur between lignin and xylans in these plants.

  1. Biochemical and thermodynamic characteristics of thermo-alkali-stable xylanase from a novel polyextremophilic Bacillus halodurans TSEV1.

    PubMed

    Kumar, Vikash; Satyanarayana, T

    2013-09-01

    The purified extracellular xylanase of polyextremophilic Bacillus halodurans TSEV1 has been visualized as a single band on SDS-PAGE and eluted as single peak by gel filtration, with a molecular mass of 40 kDa. The peptide finger print and cloned xylanase gene sequence analyses indicate that this enzyme belongs to GH family 10. The active site carboxyl residues are mainly involved in catalysis, while tryptophan residues are involved in substrate binding. The enzyme is optimally active at 80 °C and pH 9.0, and stable in the pH range of 7.0-12.0 with T 1/2 of 35 min at 80 °C (pH 9.0). Activation energy for birch wood xylan hydrolysis is 30.51 kJ mol(-1). The K m, V max and k cat (birchwood xylan) are 2.05 mg ml(-1), 333.33 μmol mg(-1 )min(-1) and 3.33 × 10(4) min(-1), respectively. The pKa1 and pKa2 of ionizable groups of the active site that influence V max are 8.51 and 11.0. The analysis of thermodynamic parameters for xylan hydrolysis suggests this as a spontaneous process. The enzyme is resistant to chemical denaturants like urea and guanidinium-HCl. The site-directed mutagenesis of catalytic glutamic acid residues (E196 and E301) resulted in a complete loss of activity. The birch wood xylan hydrolyzate contained xylobiose and xylotriose as the main products without any trace of xylose, and the enzyme hydrolyzes xylotetraose and xylopentaose rapidly to xylobiose. Thermo-alkali-stability, resistance to various chemical denaturants and mode of action make it a useful biocatalyst for generating xylo-oligosaccharides from agro-residues and bleaching of pulp in paper industries.

  2. aguA, the Gene Encoding an Extracellular α-Glucuronidase from Aspergillus tubingensis, Is Specifically Induced on Xylose and Not on Glucuronic Acid

    PubMed Central

    de Vries, Ronald P.; Poulsen, Charlotte H.; Madrid, Susan; Visser, Jaap

    1998-01-01

    An extracellular α-glucuronidase was purified and characterized from a commercial Aspergillus preparation and from culture filtrate of Aspergillus tubingensis. The enzyme has a molecular mass of 107 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 112 kDa as determined by mass spectrometry, has a determined pI just below 5.2, and is stable at pH 6.0 for prolonged times. The pH optimum for the enzyme is between 4.5 and 6.0, and the temperature optimum is 70°C. The α-glucuronidase is active mainly on small substituted xylo-oligomers but is also able to release a small amount of 4-O-methylglucuronic acid from birchwood xylan. The enzyme acts synergistically with endoxylanases and β-xylosidase in the hydrolysis of xylan. The enzyme is N glycosylated and contains 14 putative N-glycosylation sites. The gene encoding this α-glucuronidase (aguA) was cloned from A. tubingensis. It consists of an open reading frame of 2,523 bp and contains no introns. The gene codes for a protein of 841 amino acids, containing a eukaryotic signal sequence of 20 amino acids. The mature protein has a predicted molecular mass of 91,790 Da and a calculated pI of 5.13. Multiple copies of the gene were introduced in A. tubingensis, and expression was studied in a highly overproducing transformant. The aguA gene was expressed on xylose, xylobiose, and xylan, similarly to genes encoding endoxylanases, suggesting a coordinate regulation of expression of xylanases and α-glucuronidase. Glucuronic acid did not induce the expression of aguA and also did not modulate the expression on xylose. Addition of glucose prevented expression of aguA on xylan but only reduced the expression on xylose. PMID:9440512

  3. Influence of dietary fiber on xylanolytic and cellulolytic bacteria of adult pigs.

    PubMed Central

    Varel, V H; Robinson, I M; Jung, H J

    1987-01-01

    Xylanolytic and cellulolytic bacteria were enumerated over an 86-day period from fecal samples of 10 8-month-old gilts that were fed either a control or a 40% alfalfa meal (high-fiber) diet. Fecal samples were collected from all pigs on days 0, 3, 5, 12, 25, 37, 58, and 86. Overall, the numbers of xylanolytic bacteria producing greater than 5-mm-diameter zones of clearing on 0.24% xylan roll tube medium after 24 to 36 h of incubation were 1.6 X 10(8) and 4.2 X 10(8)/g (dry weight) of feces for the control pigs and those fed the high-fiber diet, respectively. After 1 week of incubation, a large number of smaller zones of clearing (1 to 2 mm) appeared. Besides Bacteroides succinogenes and Ruminococcus flavefaciens, which produced faint zones of clearing in xylan roll tubes, three strains which closely resembled B. ruminicola hydrolyzed and used xylan for growth. The overall numbers of cellulolytic bacteria producing zones of clearing in 0.5% agar roll tube medium were 0.36 X 10(8) and 4.1 X 10(8)/g for the control pigs and those fed the high-fiber diet, respectively. B. succinogenes was the predominant cellulolytic isolate from both groups of pigs, and R. flavefaciens was found in a ratio of approximately 1 to 15 with B. succinogenes. Degradation of xylan and cellulose, measured by in vitro dry matter disappearance after inoculation with fecal samples, was significantly greater for pigs fed the high-fiber diet than that for the controls. These data suggest that the number of fibrolytic microorganisms and their activity in the large intestine of the adult pig can be increased by feeding pigs high-alfalfa-fiber diets and that these organisms are similar to those found in the rumen. PMID:3030194

  4. Elucidation of Factors Effecting Enzymatic Saccharification using Transgenic Hardwoods

    NASA Astrophysics Data System (ADS)

    Min, Douyong

    Three groups of transgenic wood samples were used as starting materials to elucidate the recalcitrance of enzymatic saccharification with/without pretreatments. The first group of transgenic wood samples is low lignin P. trichocarpa. The second group is low xylan P. trichocarpa. The third one is 12 hybrid poplars which have different levels of S/V ratio and lignin content. Four pretreatments were carried out in this research including dilute sulfuric acid, green liquor, auto hydrolysis and ozone delignification. The behavior among pretreatments as a function of removal of lignin appears to be different. Lignin is the major factor of recalcitrance of the lignocellulosic material to ethanol conversion process. Xylan also plays key role in this process. In addition, the crude milled wood lignin was isolated from these three groups of transgenic samples. Lignin carbohydrate complexes was characterized by 1H-13C HMQC and 13C NMR. Thus the effect of LCCs on enzymatic saccharification was elucidated. High S/V ratio propels the lignin removal during pretreatments however; high S/V ratio retards the enzymatic saccharification on the lignocellulosic material without pretreatments. The level of LCCs linkages accounts for additional recalcitrance of the lignocellulosic material to ethanol conversion process. The amount of LCCs linkages is affected by xylan content, lignin content and S/V ratio.

  5. Insights into the plant polysaccharide degradation potential of the xylanolytic yeast Pseudozyma brasiliensis.

    PubMed

    Kaupert Neto, Antonio Adalberto; Borin, Gustavo Pagotto; Goldman, Gustavo Henrique; Damásio, André Ricardo de Lima; Oliveira, Juliana Velasco de Castro

    2016-03-01

    In second-generation (2G) bioethanol production, plant cell-wall polysaccharides are broken down to release fermentable sugars. The enzymes of this process are classified as carbohydrate-active enzymes (CAZymes) and contribute substantially to the cost of biofuel production. A novel basidiomycete yeast species, Pseudozyma brasiliensis, was recently discovered. It produces an endo-β-1,4-xylanase with a higher specific activity than other xylanases. This enzyme is essential for the hydrolysis of biomass-derived xylan and has an important role in 2G bioethanol production. In spite of the P. brasiliensis biotechnological potential, there is no information about how it breaks down polysaccharides. For the first time, we characterized the secretome of P. brasiliensis grown on different carbon sources (xylose, xylan, cellobiose and glucose) and also under starvation conditions. The growth and consumption of each carbohydrate and the activity of the CAZymes of culture supernatants were analyzed. The CAZymes found in its secretomes, validated by enzymatic assays, have the potential to hydrolyze xylan, mannan, cellobiose and other polysaccharides. The data show that this yeast is a potential source of hydrolases, which can be used for biomass saccharification. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  6. Purification and biochemical properties of multiple xylanases from Aspergillus ochraceus tolerant to Hg2+ ion and a wide range of pH.

    PubMed

    Michelin, Michele; Silva, Tony M; Jorge, João A; Polizeli, Maria de Lourdes T M

    2014-09-01

    Production of multiple xylanases, in which each enzyme has a specific characteristic, can be one strategy to achieve the effective hydrolysis of xylan. Three xylanases (xyl 1, xyl 2, and xyl 3) from Aspergillus ochraceus were purified by chromatography using diethylaminoethyl (DEAE) cellulose, Biogel P-60, and Sephadex G-100 columns. These enzymes are glycoproteins of low molecular weight with an optimum temperature at 60 °C. The glycosylation presented is apparently not related to thermostability, since xyl 3 (20 % carbohydrate) was more thermostable than xyl 2 (67 % carbohydrate). Xyl 3 was able to retain most of its activity in a wide range of pH (3.5-8.0), while xyl 1 and xyl 2 presented optimum pH of 6.0. Xyl 1 and xyl 2 were activated by 5 and 10 mM MnCl2 and CoCl2, while xyl 3 was activated by 1 mM of the same compounds. Interestingly, xyl 2 presented high tolerance toward mercury ion. Xylanases from A. ochraceus hydrolyzed xylans of different origins, such as birchwood, oat spelt, larchwood, and eucalyptus (around 90 % or more), except xyl 2 and xyl 3 that hydrolyzed with lesser efficiency eucalyptus (66.7 %) and oat spelt (44.8 %) xylans.

  7. Nucleic acid compositions and the encoding proteins

    DOEpatents

    Preston, III, James F.; Chow, Virginia; Nong, Guang; Rice, John D.; St. John, Franz J.

    2014-09-02

    The subject invention provides at least one nucleic acid sequence encoding an aldouronate-utilization regulon isolated from Paenibacillus sp. strain JDR-2, a bacterium which efficiently utilizes xylan and metabolizes aldouronates (methylglucuronoxylosaccharides). The subject invention also provides a means for providing a coordinately regulated process in which xylan depolymerization and product assimilation are coupled in Paenibacillus sp. strain JDR-2 to provide a favorable system for the conversion of lignocellulosic biomass to biobased products. Additionally, the nucleic acid sequences encoding the aldouronate-utilization regulon can be used to transform other bacteria to form organisms capable of producing a desired product (e.g., ethanol, 1-butanol, acetoin, 2,3-butanediol, 1,3-propanediol, succinate, lactate, acetate, malate or alanine) from lignocellulosic biomass.

  8. Nucleic acids, compositions and uses thereof

    DOEpatents

    Preston, III, James F.; Chow, Virginia [Gainesville, FL; Nong, Guang [Gainesville, FL; Rice, John D [Gainesville, FL; John, Franz J [Baltimore, MD

    2012-02-21

    The subject invention provides at least one nucleic acid sequence encoding an aldouronate-utilization regulon isolated from Paenibacillus sp. strain JDR-2, a bacterium which efficiently utilizes xylan and metabolizes aldouronates (methylglucuronoxylosaccharides). The subject invention also provides a means for providing a coordinately regulated process in which xylan depolymerization and product assimilation are coupled in Paenibacillus sp. strain JDR-2 to provide a favorable system for the conversion of lignocellulosic biomass to biobased products. Additionally, the nucleic acid sequences encoding the aldouronate-utilization regulon can be used to transform other bacteria to form organisms capable of producing a desired product (e.g., ethanol, 1-butanol, acetoin, 2,3-butanediol, 1,3-propanediol, succinate, lactate, acetate, malate or alanine) from lignocellulosic biomass.

  9. Interactions of fungi from fermented sausage with regenerated cellulose casings.

    PubMed

    Sreenath, Hassan K; Jeffries, Thomas W

    2011-11-01

    This research examined cellulolytic effects of fungi and other microbes present in cured sausages on the strength and stability of regenerated cellulose casings (RCC) used in the sausage industry. Occasionally during the curing process, RCC would split or fail, thereby leading to loss of product. The fungus Penicillium sp. BT-F-1, which was isolated from fermented sausages, and other fungi, which were introduced to enable the curing process, produced small amounts of cellulases on RCC in both liquid and solid cultivations. During continued incubation for 15-60 days in solid substrate cultivation (SSC) on RCC support, the fungus Penicillium sp isolate BT-F-1 degraded the casings' dry weights by 15-50% and decreased their tensile strengths by ~75%. Similarly commercial cellulase(s) resulted in 20-50% degradation of RCC in 48 h. During incubation with Penicillium sp BT-F-1, the surface structure of RCC collapsed, resulting in loss of strength and stability of casings. The matrix of industrial RCC comprised 88-93% glucose polymer residues with 0.8-4% xylan impurities. Premature casing failure appeared to result from operating conditions in the manufacturing process that allowed xylan to build up in the extrusion bath. The sausage fungus Penicillium sp BT-F-1 produced xylanases to break down soft xylan pockets prior to slow cellulosic dissolution of RCC.

  10. Low-molecular-weight lignin-rich fraction in the extract of cultured Lentinula edodes mycelia attenuates carbon tetrachloride-induced toxicity in primary cultures of rat hepatocytes.

    PubMed

    Yoshioka, Yasuko; Kojima, H; Tamura, A; Tsuji, K; Tamesada, M; Yagi, K; Murakami, N

    2012-01-01

    The extract of cultured Lentinula edodes mycelia (LEM) is a medicinal food ingredient that has hepatoprotective effects. In this study, we fractionated the LEM extract to explore novel active compounds related to hepatoprotection by using primary cultures of rat hepatocytes exposed to carbon tetrachloride (CCl(4)). The LEM extract and the fractions markedly inhibited the release of alanine aminotransferase (ALT) from hepatocytes damaged by CCl(4) into the culture medium. The strongest hepatocyte-protective activity was seen in a fraction (Fr. 2) in which a 50% ethanol extract was further eluted with 50% methanol and separated using reverse-phase HPLC. Fr. 2 had an average molecular weight of 2753, and the main components are lignin (49%) and saccharides (36%, of which xylose comprises 41%). Therefore, Fr. 2 was presumed to be a low-molecular-weight compound consisting mainly of lignin and xylan-like polysaccharides. The hepatocyte-protective activity was observed even after digestion of xylan-like polysaccharides in Fr.2 and confirmed with low-molecular-weight lignin (LM-lignin) alone. In addition, Fr. 2, the xylan-digested Fr. 2 and LM-lignin showed higher superoxide dismutase (SOD)-like activity than the LEM extract. These results suggested that the effective fraction in the LEM extract related to hepatocyte protection consisted mainly of LM-lignin, and its antioxidant activity partially contributes to the hepatocyte-protective activity of the LEM extract.

  11. A highly thermostable alkaline cellulase-free xylanase from thermoalkalophilic Bacillus sp. JB 99 suitable for paper and pulp industry: purification and characterization.

    PubMed

    Shrinivas, Dengeti; Savitha, Gunashekaran; Raviranjan, Kumar; Naik, Gajanan Ramchandra

    2010-11-01

    A highly thermostable alkaline xylanase was purified to homogeneity from culture supernatant of Bacillus sp. JB 99 using DEAE-Sepharose and Sephadex G-100 gel filtration with 25.7-fold increase in activity and 43.5% recovery. The molecular weight of the purified xylanase was found to be 20 kDA by SDS-PAGE and zymogram analysis. The enzyme was optimally active at 70 °C, pH 8.0 and stable over pH range of 6.0-10.0.The relative activity at 9.0 and 10.0 were 90% and 85% of that of pH 8.0, respectively. The enzyme showed high thermal stability at 60 °C with 95% of its activity after 5 h. The K (m) and V (max) of enzyme for oat spelt xylan were 4.8 mg/ml and 218.6 µM min(-1) mg(-1), respectively. Analysis of N-terminal amino acid sequence revealed that the xylanase belongs to glycosyl hydrolase family 11 from thermoalkalophilic Bacillus sp. with basic pI. Substrate specificity showed a high activity on xylan-containing substrate and cellulase-free nature. The hydrolyzed product pattern of oat spelt xylan on thin-layer chromatography suggested xylanase as an endoxylanase. Due to these properties, xylanase from Bacillus sp. JB 99 was found to be highly compatible for paper and pulp industry.

  12. Characterization of a uronate dehydrogenase from Thermobispora bispora for production of glucaric acid from hemicellulose substrate.

    PubMed

    Li, Yaxian; Xue, Yemin; Cao, Zhigang; Zhou, Tao; Alnadari, Fawze

    2018-06-23

    A thermostable uronate dehydrogenase Tb-UDH from Thermobispora bispora was over-expressed in Escherichia coli using the T7 polymerase expression system. The Tb-UDH was purified by metal affinity chromatography, and gave a single band on SDS-PAGE. The maximum activity on glucuronic acid was found at 60 °C and pH 7.0. The purified enzyme retained over 58% of its activity after holding a pH ranging from 7.0 to 7.5 for 1 h at 60 °C. The K m and V max values of the purified Tb-UDH for Glucuronic acid (GluUA) were 0.165 mM and 117.7 U mg -1 , respectively, those for galacturonic acid (GalUA) were 0.115 mM and 104.2 U mg -1 , respectively, and those for NAD + were 0.120 mM and 133.3 U mg -1 , respectively; the turnover number (k cat ) with GluUA as a substrate was higher than that with GalUA; however, the Michaelis constant (K m ) for GalUA was lower than that for GluUA. After 60 min of incubation at 50 °C, Tb-UDH exhibited a conversion ratio for glucuronic acid to the glucaric acid of 84% on chemical reagent and 81.3% on hydrolysates from breech xylans formed by xylanase and α-glucuronidase. This work shows that biocatalytic routes have great potential for the conversion of hemicellulose substrate into value-added products derived from renewable biomass. TOC GRAPHIC: (A) The structure of the xylan is described and the site of action of the xylan degrading enzyme is indicated. (B) The effect of substrate concentration on recombinant Tb-UDH activity when galacturonic acid was used as substrate. (C) SDS-PAGE analysis of E. coli BL21 (DE3) harboring pET-20b(+) and pET-20b-Tb-UDH. (D) Oxidative conversion of glucuronic acid from a beechwood xylan to glucaric acid.

  13. Investigation of Plant Cell Wall Properties: A Study of Contributions from the Nanoscale to the Macroscale Impacting Cell Wall Recalcitrance

    NASA Astrophysics Data System (ADS)

    Crowe, Jacob Dillon

    Biochemical conversion of lignocellulosic biomass to fuel ethanol is one of a few challenging, yet opportune technologies that can reduce the consumption of petroleum-derived transportation fuels, while providing parallel reductions in greenhouse gas emissions. Biomass recalcitrance, or resistance to deconstruction, is a major technical challenge that limits effective conversion of biomass to fermentable sugars, often requiring a costly thermochemical pretreatment step to improve biomass deconstruction. Biomass recalcitrance is imparted largely by the secondary cell wall, a complex polymeric matrix of cell wall polysaccharides and aromatic heteropolymers, that provides structural stability to cells and enables plant upright growth. Polymers within the cell wall can vary both compositionally and structurally depending upon plant species and anatomical fraction, and have varied responses to thermochemical pretreatments. Cell wall properties impacting recalcitrance are still not well understood, and as a result, the goal of this dissertation is to investigate structural features of the cell wall contributing to recalcitrance (1) in diverse anatomical fractions of a single species, (2) in response to diverse pretreatments, and (3) resulting from genetic modification. In the first study, feedstock cell wall heterogeneity was investigated in anatomical (stem, leaf sheaths, and leaf blades) and internode fractions of switchgrass at varying tissue maturities. Lignin content was observed as the key contributor to recalcitrance in maturing stem tissues only, with non-cellulosic substituted glucuronoarabinoxylans and pectic polysaccharides contributing to cell wall recalcitrance in leaf sheath and leaf blades. Hydroxycinnamate (i.e., saponifiable p-coumarate and ferulate) content along with xylan and pectin extractability decreased with tissue maturity, suggesting lignification is only one component imparting maturity specific cell wall recalcitrance. In the second study

  14. Evidence of a Critical Role for Cellodextrin Transporte 2 (CDT-2) in Both Cellulose and Hemicellulose Degradation and Utilization in Neurospora crassa

    PubMed Central

    Cai, Pengli; Gu, Ruimeng; Wang, Bang; Li, Jingen; Wan, Li; Tian, Chaoguang; Ma, Yanhe

    2014-01-01

    CDT-1 and CDT-2 are two cellodextrin transporters discovered in the filamentous fungus Neurospora crassa. Previous studies focused on characterizing the role of these transporters in only a few conditions, including cellulose degradation, and the function of these two transporters is not yet completely understood. In this study, we show that deletion of cdt-2, but not cdt-1, results in growth defects not only on Avicel but also on xylan. cdt-2 can be highly induced by xylan, and this mutant has a xylodextrin consumption defect. Transcriptomic analysis of the cdt-2 deletion strain on Avicel and xylan showed that major cellulase and hemicellulase genes were significantly down-regulated in the cdt-2 deletion strain and artificial over expression of cdt-2 in N. crassa increased cellulase and hemicellulase production. Together, these data clearly show that CDT-2 plays a critical role in hemicellulose sensing and utilization. This is the first time a sugar transporter has been assigned a function in the hemicellulose degradation pathway. Furthermore, we found that the transcription factor XLR-1 is the major regulator of cdt-2, while cdt-1 is primarily regulated by CLR-1. These results deepen our understanding of the functions of both cellodextrin transporters, particularly for CDT-2. Our study also provides novel insight into the mechanisms for hemicellulose sensing and utilization in N. crassa, and may be applicable to other cellulolytic filamentous fungi. PMID:24586693

  15. Feasibility test of utilizing Saccharophagus degradans 2-40(T) as the source of crude enzyme for the saccharification of lignocellulose.

    PubMed

    Jung, Young Hoon; Kim, Hyun Kyung; Song, Du-Sup; Choi, In-Geol; Yang, Taek Ho; Lee, Hee Jong; Seung, Doyoung; Kim, Kyoung Heon

    2014-04-01

    In the conversion of lignocellulose into high-value products, including fuels and chemicals, the production of cellulase and the enzymatic hydrolysis for producing fermentable sugar are the largest contributors to the cost of production of the final products. The marine bacterium Saccharophagus degradans 2-40(T) can degrade more than ten different complex polysaccharides found in the ocean, including cellulose and xylan. Accordingly, S. degradans has been actively considered as a practical source of crude enzymes needed for the saccharification of lignocellulose to produce ethanol by others including a leading commercial company. However, the overall enzyme system of S. degradans for hydrolyzing cellulose and hemicellulose has not been quantitatively evaluated yet in comparison with commercial enzymes. In this study, the inductions and activities of cellulase and xylanase of cell-free lysate of S. degradans were investigated. The growth of S. degradans cells and the activities of cellulase and xylanase were promoted by adding 2 % of cellulose and xylan mixture (cellulose:xylan = 4:3 in mass ratio) to the aquarium salt medium supplemented with 0.2 % glucose. The specific cellulase activity of the cell-free lysate of S. degradans, as determined by the filter paper activity assay, was approximately 70 times lower than those of commercial cellulases, including Celluclast 1.5 L and Accellerase 1000. These results imply that significant improvement in the cellulase activity of S. degradans is needed for the industrial uses of S. degradans as the enzyme source.

  16. Cloning, expression and characterization of β-xylosidase from Aspergillus niger ASKU28.

    PubMed

    Choengpanya, Khuanjarat; Arthornthurasuk, Siriphan; Wattana-amorn, Pakorn; Huang, Wan-Ting; Plengmuankhae, Wandee; Li, Yaw-Kuen; Kongsaeree, Prachumporn T

    2015-11-01

    β-Xylosidases catalyze the breakdown of β-1,4-xylooligosaccharides, which are produced from degradation of xylan by xylanases, to fermentable xylose. Due to their important role in xylan degradation, there is an interest in using these enzymes in biofuel production from lignocellulosic biomass. In this study, the coding sequence of a glycoside hydrolase family 3 β-xylosidase from Aspergillus niger ASKU28 (AnBX) was cloned and expressed in Pichia pastoris as an N-terminal fusion protein with the α-mating factor signal sequence (α-MF) and a poly-histidine tag. The expression level was increased to 5.7 g/l in a fermenter system as a result of optimization of only five codons near the 5' end of the α-MF sequence. The recombinant AnBX was purified to homogeneity through a single-step Phenyl Sepharose chromatography. The enzyme exhibited an optimal activity at 70°C and at pH 4.0-4.5, and a very high kinetic efficiency toward a xyloside substrate. AnBX demonstrated an exo-type activity with retention of the β-configuration, and a synergistic action with xylanase in hydrolysis of beechwood xylan. This study provides comprehensive data on characterization of a glycoside hydrolase family 3 β-xylosidase that have not been determined in any prior investigations. Our results suggested that AnBX may be useful for degradation of lignocellulosic biomass in bioethanol production, pulp bleaching process and beverage industry. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Production of xylanase and β-xylosidase from autohydrolysis liquor of corncob using two fungal strains.

    PubMed

    Michelin, Michele; Polizeli, Maria de Lourdes T M; Ruzene, Denise S; Silva, Daniel P; Ruiz, Héctor A; Vicente, António A; Jorge, João A; Terenzi, Héctor F; Teixeira, José A

    2012-09-01

    Agroindustrial residues are materials often rich in cellulose and hemicellulose. The use of these substrates for the microbial production of enzymes of industrial interest is mainly due to their high availability associated with their low cost. In this work, corncob (CCs) particles decomposed to soluble compounds (liquor) were incorporated in the microbial growth medium through autohydrolysis, as a strategy to increase and undervalue xylanase and β-xylosidase production by Aspergillus terricola and Aspergillus ochraceus. The CCs autohydrolysis liquor produced at 200 °C for 5, 15, 30 or 50 min was used as the sole carbon source or associated with untreated CC. The best condition for enzyme synthesis was observed with CCs submitted to 30 min of autohydrolysis. The enzymatic production with untreated CCs plus CC liquor was higher than with birchwood xylan for both microorganisms. A. terricola produced 750 total U of xylanase (144 h cultivation) and 30 total U of β-xylosidase (96-168 h) with 0.75% untreated CCs and 6% CCs liquor, against 650 total U of xylanase and 2 total U of β-xylosidase in xylan; A. ochraceus produced 605 total U of xylanase and 56 total U of β-xylosidase (168 h cultivation) with 1% untreated CCs and 10% CCs liquor against 400 total U of xylanase and 38 total U of β-xylosidase in xylan. These results indicate that the treatment of agroindustrial wastes through autohydrolysis can be a viable strategy in the production of high levels of xylanolytic enzymes.

  18. Fate of Carbohydrates and Lignin during Composting and Mycelium Growth of Agaricus bisporus on Wheat Straw Based Compost

    PubMed Central

    Jurak, Edita; Punt, Arjen M.; Arts, Wim; Kabel, Mirjam A.; Gruppen, Harry

    2015-01-01

    In wheat straw based composting, enabling growth of Agaricus bisporus mushrooms, it is unknown to which extent the carbohydrate-lignin matrix changes and how much is metabolized. In this paper we report yields and remaining structures of the major components. During the Phase II of composting 50% of both xylan and cellulose were metabolized by microbial activity, while lignin structures were unaltered. During A. bisporus’ mycelium growth (Phase III) carbohydrates were only slightly consumed and xylan was found to be partially degraded. At the same time, lignin was metabolized for 45% based on pyrolysis GC/MS. Remaining lignin was found to be modified by an increase in the ratio of syringyl (S) to guaiacyl (G) units from 0.5 to 0.7 during mycelium growth, while fewer decorations on the phenolic skeleton of both S and G units remained. PMID:26436656

  19. Screening, identification, and characterization of a GH43 family β-xylosidase/α-arabinofuranosidase from a compost microbial metagenome.

    PubMed

    Matsuzawa, Tomohiko; Kaneko, Satoshi; Yaoi, Katsuro

    2015-11-01

    A putative glycoside hydrolase family 43 β-xylosidase/α-arabinofuranosidase (CoXyl43) that promotes plant biomass saccharification was isolated via functional screening of a compost microbial metagenomic library and characterized. CoXyl43 promoted the saccharification of plant biomasses, including xylans (xylan and arabinoxylan), rice straw, and Erianthus, by degrading xylooligosaccharide residues to monosaccharide residues. The recombinant CoXyl43 protein exhibited both β-xylosidase and α-arabinofuranosidase activities for chromogenic substrates, with optimal activity at pH 7.5 and 55 °C. Both of these activities were inactivated by ethanol, dimethylsulfoxide, and zinc and copper ions but were activated by manganese ions. Only the β-xylosidase activity of recombinant CoXyl43 was enhanced in the presence of calcium ions. These results indicate that CoXyl43 exhibits unique enzymatic properties useful for biomass saccharification.

  20. Alkaline organosolv pretreatment of corn stover for enhancing the enzymatic digestibility.

    PubMed

    Yuan, Wei; Gong, Zhiwei; Wang, Guanghui; Zhou, Wenting; Liu, Yi; Wang, Xuemin; Zhao, Mi

    2018-06-14

    In the present study, a sodium hydroxide-methanol solution (SMs) pretreatment of corn stover was described to overcome biomass recalcitrance for the first time. Effects of sodium hydroxide loading, solid-to-liquid ratio, processing time and temperature on enzymatic saccharification were studied in detail. The SMs pretreatment could significantly enhance the enzyme accessibility of corn stover, minimize the degradation of sugar polymers, and decrease the energy consumption. 97.5% glucan and 83.5% xylan were preserved in the regenerated corn stover under the optimal condition. Subsequent enzymatic digestibilities of glucan and xylan reached 97.2% and 80.3%, respectively. The enzyme susceptibility of the regenerated samples was explained by their physical and chemical characteristics. This strategy provides a promising alternative for better techno-economic of the lignocelluloses-to-sugars routes. Copyright © 2018 Elsevier Ltd. All rights reserved.

  1. Bioabatement with hemicellulase supplementation to reduce enzymatic hydrolysis inhibitors

    USDA-ARS?s Scientific Manuscript database

    Removal of inhibitory compounds by bioabatement, combined with xylan hydrolysis, enables effective cellulose hydrolysis of pretreated corn stover, for fermentation of the sugars to fuel ethanol or other products. The fungus Coniochaeta ligniaria NRRL30616 eliminates most enzyme and fermentation inhi...

  2. The diversity and specificity of the extracellular proteome in the cellulolytic bacterium Caldicellulosiruptor bescii is driven by the nature of the cellulosic growth substrate

    DOE PAGES

    Poudel, Suresh; Giannone, Richard J.; Basen, Mirko; ...

    2018-03-23

    Background: Caldicellulosiruptor bescii is a thermophilic cellulolytic bacterium that efficiently deconstructs lignocellulosic biomass into sugars, which subsequently can be fermented into alcohols, such as ethanol, and other products. Deconstruction of complex substrates by C. bescii involves a myriad of highly abundant, substrate-specific extracellular solute binding proteins (ESBPs) and carbohydrate-active enzymes (CAZymes) containing carbohydrate-binding modules (CBMs). Mass spectrometry-based proteomics was employed to investigate how these substrate recognition proteins and enzymes vary as a function of lignocellulosic substrates.Results:Proteomic analysis revealed several key extracellular proteins that respond specifically to either C5 or C6 mono- and polysaccharides. These include proteins of unknown functions (PUFs),more » ESBPs, and CAZymes. ESBPs that were previously shown to interact more efficiently with hemicellulose and pectin were detected in high abundance during growth on complex C5 substrates, such as switchgrass and xylan. Some proteins, such as Athe_0614 and Athe_2368, whose functions are not well defined were predicted to be involved in xylan utilization and ABC transport and were significantly more abundant in complex and C5 substrates, respectively. The proteins encoded by the entire glucan degradation locus (GDL; Athe_1857, 1859, 1860, 1865, 1867, and 1866) were highly abundant under all growth conditions, particularly when C. bescii was grown on cellobiose, switchgrass, or xylan. In contrast, the glycoside hydrolases Athe_0609 (Pullulanase) and 0610, which both possess CBM20 and a starch binding domain, appear preferential to C5/complex substrate deconstruction. Some PUFs, such as Athe_2463 and 2464, were detected as highly abundant when grown on C5 substrates (xylan and xylose), also suggesting C5-substrate specificity. In conclusion, this study reveals the protein membership of the C. bescii secretome and demonstrates its plasticity

  3. The diversity and specificity of the extracellular proteome in the cellulolytic bacterium Caldicellulosiruptor bescii is driven by the nature of the cellulosic growth substrate

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Poudel, Suresh; Giannone, Richard J.; Basen, Mirko

    Background: Caldicellulosiruptor bescii is a thermophilic cellulolytic bacterium that efficiently deconstructs lignocellulosic biomass into sugars, which subsequently can be fermented into alcohols, such as ethanol, and other products. Deconstruction of complex substrates by C. bescii involves a myriad of highly abundant, substrate-specific extracellular solute binding proteins (ESBPs) and carbohydrate-active enzymes (CAZymes) containing carbohydrate-binding modules (CBMs). Mass spectrometry-based proteomics was employed to investigate how these substrate recognition proteins and enzymes vary as a function of lignocellulosic substrates.Results:Proteomic analysis revealed several key extracellular proteins that respond specifically to either C5 or C6 mono- and polysaccharides. These include proteins of unknown functions (PUFs),more » ESBPs, and CAZymes. ESBPs that were previously shown to interact more efficiently with hemicellulose and pectin were detected in high abundance during growth on complex C5 substrates, such as switchgrass and xylan. Some proteins, such as Athe_0614 and Athe_2368, whose functions are not well defined were predicted to be involved in xylan utilization and ABC transport and were significantly more abundant in complex and C5 substrates, respectively. The proteins encoded by the entire glucan degradation locus (GDL; Athe_1857, 1859, 1860, 1865, 1867, and 1866) were highly abundant under all growth conditions, particularly when C. bescii was grown on cellobiose, switchgrass, or xylan. In contrast, the glycoside hydrolases Athe_0609 (Pullulanase) and 0610, which both possess CBM20 and a starch binding domain, appear preferential to C5/complex substrate deconstruction. Some PUFs, such as Athe_2463 and 2464, were detected as highly abundant when grown on C5 substrates (xylan and xylose), also suggesting C5-substrate specificity. In conclusion, this study reveals the protein membership of the C. bescii secretome and demonstrates its plasticity

  4. The diversity and specificity of the extracellular proteome in the cellulolytic bacterium Caldicellulosiruptor bescii is driven by the nature of the cellulosic growth substrate.

    PubMed

    Poudel, Suresh; Giannone, Richard J; Basen, Mirko; Nookaew, Intawat; Poole, Farris L; Kelly, Robert M; Adams, Michael W W; Hettich, Robert L

    2018-01-01

    Caldicellulosiruptor bescii is a thermophilic cellulolytic bacterium that efficiently deconstructs lignocellulosic biomass into sugars, which subsequently can be fermented into alcohols, such as ethanol, and other products. Deconstruction of complex substrates by C. bescii involves a myriad of highly abundant, substrate-specific extracellular solute binding proteins (ESBPs) and carbohydrate-active enzymes (CAZymes) containing carbohydrate-binding modules (CBMs). Mass spectrometry-based proteomics was employed to investigate how these substrate recognition proteins and enzymes vary as a function of lignocellulosic substrates. Proteomic analysis revealed several key extracellular proteins that respond specifically to either C5 or C6 mono- and polysaccharides. These include proteins of unknown functions (PUFs), ESBPs, and CAZymes. ESBPs that were previously shown to interact more efficiently with hemicellulose and pectin were detected in high abundance during growth on complex C5 substrates, such as switchgrass and xylan. Some proteins, such as Athe_0614 and Athe_2368, whose functions are not well defined were predicted to be involved in xylan utilization and ABC transport and were significantly more abundant in complex and C5 substrates, respectively. The proteins encoded by the entire glucan degradation locus (GDL; Athe_1857, 1859, 1860, 1865, 1867, and 1866) were highly abundant under all growth conditions, particularly when C. bescii was grown on cellobiose, switchgrass, or xylan. In contrast, the glycoside hydrolases Athe_0609 (Pullulanase) and 0610, which both possess CBM20 and a starch binding domain, appear preferential to C5/complex substrate deconstruction. Some PUFs, such as Athe_2463 and 2464, were detected as highly abundant when grown on C5 substrates (xylan and xylose), also suggesting C5-substrate specificity. This study reveals the protein membership of the C. bescii secretome and demonstrates its plasticity based on the complexity (mono

  5. Quantitative proteomic study of Aspergillus Fumigatus secretome revealed deamidation of secretory enzymes.

    PubMed

    Adav, Sunil S; Ravindran, Anita; Sze, Siu Kwan

    2015-04-24

    Aspergillus sp. plays an essential role in lignocellulosic biomass recycling and is also exploited as cell factories for the production of industrial enzymes. This study profiled the secretome of Aspergillus fumigatus when grown with cellulose, xylan and starch by high throughput quantitative proteomics using isobaric tags for relative and absolute quantification (iTRAQ). Post translational modifications (PTMs) of proteins play a critical role in protein functions. However, our understanding of the PTMs in secretory proteins is limited. Here, we present the identification of PTMs such as deamidation of secreted proteins of A. fumigatus. This study quantified diverse groups of extracellular secreted enzymes and their functional classification revealed cellulases and glycoside hydrolases (32.9%), amylases (0.9%), hemicellulases (16.2%), lignin degrading enzymes (8.1%), peptidases and proteases (11.7%), chitinases, lipases and phosphatases (7.6%), and proteins with unknown function (22.5%). The comparison of quantitative iTRAQ results revealed that cellulose and xylan stimulates expression of specific cellulases and hemicellulases, and their abundance level as a function of substrate. In-depth data analysis revealed deamidation as a major PTM of key cellulose hydrolyzing enzymes like endoglucanases, cellobiohydrolases and glucosidases. Hemicellulose degrading endo-1,4-beta-xylanase, monosidases, xylosidases, lignin degrading laccase, isoamyl alcohol oxidase and oxidoreductases were also found to be deamidated. The filamentous fungi play an essential role in lignocellulosic biomass recycling and fungal strains belonging to Aspergillus were also exploited as cell factories for the production of organic acids, pharmaceuticals, and industrially important enzymes. In this study, extracellular proteins secreted by thermophilic A. fumigatus when grown with cellulose, xylan and starch were profiled using isobaric tags for relative and absolute quantification (iTRAQ) by

  6. A Simple Method for the Determination of Xylanase Activity on Insoluble Substrates

    USDA-ARS?s Scientific Manuscript database

    The propensity for a xylanase to convert insoluble (arabino)xylan into soluble oligosaccharides is an important parameter in the baking, pulp and paper, prebiotics, and biofuel industries. Current methods for determining xylanase activity on insoluble substrates are labor intensive, non-specific, or...

  7. Maleic acid treatment of biologically detoxified corn stover liquor

    USDA-ARS?s Scientific Manuscript database

    Elimination of microbial and/or enzyme inhibitors from pretreated lignocellulose is critical for effective cellulose conversion and yeast fermentation of liquid hot-water (LHW) pretreated corn stover. In this study, xylan oligomers were hydrolyzed using either maleic acid or hemicellulases. Other so...

  8. H-ZSM5 Catalyzed co-pyrolysis of biomass and plastics

    USDA-ARS?s Scientific Manuscript database

    This study aims at addressing two important problems vital to agriculture, disposal of agricultural plastics and production of drop-in fuels from biomass via co-pyrolysis of both feedstocks. Mixtures of biomass (switchgrass, cellulose, xylan and lignin) and plastic (polyethylene terephthalate (PET),...

  9. Production of Sporotrichum thermophile xylanase by solid state fermentation utilizing deoiled Jatropha curcas seed cake and its application in xylooligosachharide synthesis.

    PubMed

    Sadaf, Ayesha; Khare, S K

    2014-02-01

    De-oiled Jatropha curcas seed cake, a plentiful by-product of biodiesel industry was used as substrate for the production of a useful xylanase from Sporotrichum thermophile in solid state fermentation. Under the optimized conditions, 1025U xylanase/g (deoiled seed cake) was produced. The xylanase exhibited half life of 4h at 45°C and 71.44min at 50°C respectively. It was stable in a broad pH range of 7.0-11.0. Km and Vmax were 12.54mg/ml and 454.5U/ml/min respectively. S. thermophile xylanase is an endoxylanase free of exoxylanase activity, hence advantageous for xylan hydrolysis to produce xylooligosachharides. Hydrolysis of oat spelt xylan by S. thermophile xylanase yielded 73% xylotetraose, 15.4% xylotriose and 10% xylobiose. The S. thermophile endoxylanase thus seem potentially useful in the food industries. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. Corn fiber, cobs and stover: enzyme-aided saccharification and co-fermentation after dilute acid pretreatment.

    PubMed

    Van Eylen, David; van Dongen, Femke; Kabel, Mirjam; de Bont, Jan

    2011-05-01

    Three corn feedstocks (fibers, cobs and stover) available for sustainable second generation bioethanol production were subjected to pretreatments with the aim of preventing formation of yeast-inhibiting sugar-degradation products. After pretreatment, monosaccharides, soluble oligosaccharides and residual sugars were quantified. The size of the soluble xylans was estimated by size exclusion chromatography. The pretreatments resulted in relatively low monosaccharide release, but conditions were reached to obtain most of the xylan-structures in the soluble part. A state of the art commercial enzyme preparation, Cellic CTec2, was tested in hydrolyzing these dilute acid-pretreated feedstocks. The xylose and glucose liberated were fermented by a recombinant Saccharomyces cerevisiae strain. In the simultaneous enzymatic saccharification and fermentation system employed, a concentration of more than 5% (v/v) (0.2g per g of dry matter) of ethanol was reached. Copyright © 2011 Elsevier Ltd. All rights reserved.

  11. Purification and characterization of a thermostable hypothetical xylanase from Aspergillus oryzae HML366.

    PubMed

    He, Haiyan; Qin, Yongling; Li, Nan; Chen, Guiguang; Liang, Zhiqun

    2015-03-01

    In the current study, fermentation broth of Aspergillus oryzae HML366 in sugar cane bagasse was subjected to ultrafiltration and ion exchange chromatography, and two xylanases, XynH1 and XynH2, were purified. Time-of-flight mass spectrometry coupled with SDS-PAGE analysis revealed that XynH1 is identical to the hypothetical A. oryzae RIB40 protein XP_001826985.1, with a molecular weight of 33.671 kDa. Likewise, XynH2 was identified as xylanase XynF1 with a molecular weight of 35.402 kDa. Sequence analysis indicated that XynH1 belongs to glycosyl hydrolases family 10. The specific activity of XynH1 was measured at 476.9 U/mg. Optimal xylanase activity was observed at pH 6.0, and enzyme remained active within pH 4.0-10.0 and at a temperature below 70 °C. Mg(2+), Mn(2+), Ca(2+), and K(+) enhanced the XynH1 xylanase activity to 146, 122, 114, and 108%, respectively. XynH1 hydrolyzed Birchwood xylan and Larchwood xylan effectively. The K m and V max of XynH1 values determined were 1.16 mM and 336 μmol/min/mg with Birchwood xylan as the substrate. A. oryzae HML366 xylanase XynH1 showed superior heat and pH tolerance, therefore may have significant applications in paper and biofuel industries. These studies constitute the first investigation of the xylanase activities of the hypothetical protein XP_001826985.1 form A. oryzae.

  12. Cloning, Expression and Characteristics of a Novel Alkalistable and Thermostable Xylanase Encoding Gene (Mxyl) Retrieved from Compost-Soil Metagenome

    PubMed Central

    Verma, Digvijay; Kawarabayasi, Yutaka; Miyazaki, Kentaro; Satyanarayana, Tulasi

    2013-01-01

    Background The alkalistable and thermostable xylanases are in high demand for pulp bleaching in paper industry and generating xylooligosaccharides by hydrolyzing xylan component of agro-residues. The compost-soil samples, one of the hot environments, are expected to be a rich source of microbes with thermostable enzymes. Methodology/Principal Findings Metagenomic DNA from hot environmental samples could be a rich source of novel biocatalysts. While screening metagenomic library constructed from DNA extracted from the compost-soil in the p18GFP vector, a clone (TSDV-MX1) was detected that exhibited clear zone of xylan hydrolysis on RBB xylan plate. The sequencing of 6.321 kb DNA insert and its BLAST analysis detected the presence of xylanase gene that comprised 1077 bp. The deduced protein sequence (358 amino acids) displayed homology with glycosyl hydrolase (GH) family 11 xylanases. The gene was subcloned into pET28a vector and expressed in E. coli BL21 (DE3). The recombinant xylanase (rMxyl) exhibited activity over a broad range of pH and temperature with optima at pH 9.0 and 80°C. The recombinant xylanase is highly thermostable having T1/2 of 2 h at 80°C and 15 min at 90°C. Conclusion/Significance This is the first report on the retrieval of xylanase gene through metagenomic approach that encodes an enzyme with alkalistability and thermostability. The recombinant xylanase has a potential application in paper and pulp industry in pulp bleaching and generating xylooligosaccharides from the abundantly available agro-residues. PMID:23382818

  13. The Botrytis cinerea xylanase Xyn11A contributes to virulence with its necrotizing activity, not with its catalytic activity

    PubMed Central

    2010-01-01

    Background The Botrytis cinerea xylanase Xyn11A has been previously shown to be required for full virulence of this organism despite its poor contribution to the secreted xylanase activity and the low xylan content of B. cinerea hosts. Intriguingly, xylanases from other fungi have been shown to have the property, independent of the xylan degrading activity, to induce necrosis when applied to plant tissues, so we decided to test the hypothesis that secreted Xyn11A contributes to virulence by promoting the necrosis of the plant tissue surrounding the infection, therefore facilitating the growth of this necrotroph. Results We show here that Xyn11A has necrotizing activity on plants and that this capacity is conserved in site-directed mutants of the protein lacking the catalytic activity. Besides, Xyn11A contributes to the infection process with the necrotizing and not with the xylan hydrolyzing activity, as the catalytically-impaired Xyn11A variants were able to complement the lower virulence of the xyn11A mutant. The necrotizing activity was mapped to a 30-amino acids peptide in the protein surface, and this region was also shown to mediate binding to tobacco spheroplasts by itself. Conclusions The main contribution of the xylanase Xyn11A to the infection process of B. cinerea is to induce necrosis of the infected plant tissue. A conserved 30-amino acids region on the enzyme surface, away from the xylanase active site, is responsible for this effect and mediates binding to plant cells. PMID:20184750

  14. Contribution of acetate to butyrate formation by human faecal bacteria.

    PubMed

    Duncan, Sylvia H; Holtrop, Grietje; Lobley, Gerald E; Calder, A Graham; Stewart, Colin S; Flint, Harry J

    2004-06-01

    Acetate is normally regarded as an endproduct of anaerobic fermentation, but butyrate-producing bacteria found in the human colon can be net utilisers of acetate. The butyrate formed provides a fuel for epithelial cells of the large intestine and influences colonic health. [1-(13)C]Acetate was used to investigate the contribution of exogenous acetate to butyrate formation. Faecalibacterium prausnitzii and Roseburia spp. grown in the presence of 60 mm-acetate and 10 mm-glucose derived 85-90 % butyrate-C from external acetate. This was due to rapid interchange between extracellular acetate and intracellular acetyl-CoA, plus net acetate uptake. In contrast, a Coprococcus-related strain that is a net acetate producer derived only 28 % butyrate-C from external acetate. Different carbohydrate-derived energy sources affected butyrate formation by mixed human faecal bacteria growing in continuous or batch cultures. The ranking order of butyrate production rates was amylopectin > oat xylan > shredded wheat > inulin > pectin (continuous cultures), and inulin > amylopectin > oat xylan > shredded wheat > pectin (batch cultures). The contribution of external acetate to butyrate formation in these experiments ranged from 56 (pectin) to 90 % (xylan) in continuous cultures, and from 72 to 91 % in the batch cultures. This is consistent with a major role for bacteria related to F. prausnitzii and Roseburia spp. in butyrate formation from a range of substrates that are fermented in the large intestine. Variations in the dominant metabolic type of butyrate producer between individuals or with variations in diet are not ruled out, however, and could influence butyrate supply in the large intestine.

  15. Highly thermostable GH39 ß-xylosidase from a Geobacillus sp. strain WSUCF1

    USDA-ARS?s Scientific Manuscript database

    Background Complete enzymatic hydrolysis of xylan to xylose requires the action of endoxylanase and ß-xylosidase. ß-xylosidases play an important part in hydrolyzing xylo-oligosaccharides to xylose. Thermostable ß-xylosidases have been a focus of attention as industrially important enzymes due to th...

  16. Plant cell wall: Never too much acetate

    DOE PAGES

    Scheller, Henrik V.

    2017-03-03

    Here, plant cell walls incorporate a variety of acetylated polysaccharides. In addition to enzymes catalysing acetylation (acetyltransferases), plants could produce enzymes to remove acetyl groups (acetylesterases). Previously, pectin acetylesterases were known and now a xylan acetylesterase has been identified — and it has many surprises.

  17. Genomic and transcriptomic analysis of carbohydrate utilization by Paenibacillus sp. JDR-2: systems for bioprocessing plant polysaccharides

    Treesearch

    Neha Sawhney; Casey Crooks; Virginia Chow; James F. Preston; Franz St. John

    2016-01-01

    Background: Polysaccharides comprising plant biomass are potential resources for conversion to fuels and chemicals. These polysaccharides include xylans derived from the hemicellulose of hardwoods and grasses, soluble beta-glucans from cereals and starch as the primary form of energy storage in plants. Paenibacillus sp...

  18. and applications of microbial ß-D-xylosidases featuring the catalytically efficient enzyme from Selenomonas ruminantium

    USDA-ARS?s Scientific Manuscript database

    Xylan 1,4-beta-D xylosidase catalyzes hydrolysis of nonreducing end xylose residues from xylooligosaccharides. The enzyme is currently used in several industrial-scale processes for food and materials, and on a grander scale, the enzyme could find employment along side cellulases and other hemicell...

  19. Bioethanol production from corn stover using aqueous ammonia pretreatment and two-phase simultaneouos saccharification and fermentation (TPSSF)

    USDA-ARS?s Scientific Manuscript database

    An integrated bioconversion process was developed to convert corn-stover derived pentose and hexose to ethanol effectively. In this study, corn stover was pretreated by soaking in aqueous ammonia (SAA), which resulted in high retention of glucan (~100%) and xylan (>80%) in the solids. The pretreated...

  20. Broad diversity and newly cultured bacterial isolates from enrichment of pig feces on complex polysaccharides

    USDA-ARS?s Scientific Manuscript database

    Microbial fermentation of plant cell wall components to short chain fatty acids in the large intestine provides energy to both humans and pigs. To better understand plant cell wall fermentation in the pig and human intestine, we isolated cellulose, xylan, and pectin fermenting bacteria from pig and ...

  1. Cloning and characterization of alpha-glucuronidase enzymes from mixed cultures

    USDA-ARS?s Scientific Manuscript database

    Hemicellulose is second to cellulose as the most common carbohydrate source on the planet. Efficient utilization of this resource is essential to the economic viability of biomass refineries. Xylan, a primary component of hemicellulose, is a polymer of beta-1,4-linked xylose sugars. This polymer ...

  2. Newly cultured bacteria with broad diversity isolated from 8 week continuous culture enrichments of cow feces on complex polysaccharides

    USDA-ARS?s Scientific Manuscript database

    One of the fascinating functions of the mammalian intestinal microbiota is the fermentation of plant cell wall components. Eight week continuous culture enrichments of cow feces with cellulose and xylan/pectin were used to isolate bacteria from this community. A total of 459 bacterial isolates were ...

  3. Bacterial isolates from polysaccharide enrichments cluster by host origin for Firmicutes but not Bacteroidetes.

    USDA-ARS?s Scientific Manuscript database

    The intestinal microbiota allows mammals to recover energy stored in plant biomass through fermentation of plant cell walls, primarily cellulose and hemicellulose. Bacteria were isolated from 8 week continuous culture enrichments with cellulose and xylan/pectin from cow (C, n=4), goat (G, n=4), huma...

  4. Cellulases and hemicellulases from endophytic Acremonium species and its application on sugarcane bagasse hydrolysis

    USDA-ARS?s Scientific Manuscript database

    The aim of this work was to have cellulase activity and hemicellulase activity screenings of endophyte Acremonium species (Acremonium zeae EA0802 and Acremonium sp. EA0810). Both fungi were cultivated in submerged culture (SC) containing L-arabinose, D-xylose, oat spelt xylan, sugarcane bagasse, or...

  5. Using a combined hydrolysis factor to optimize high titer ethanol production from sulfite-pretreated poplar without detoxification

    Treesearch

    Jingzhi Zhang; Feng Gu; J.Y. Zhu; Ronald S. Zalesny Jr.

    2015-01-01

    Sulfite pretreatment to overcome the recalcitrance of lignocelluloses (SPORL) was applied to poplar NE222 chips in a range of chemical loadings, temperatures, and times. The combined hydrolysis factor (CHF) as a pretreatment severity accurately predicted xylan dissolution by SPORL. Good correlations between CHF and pretreated...

  6. Quantification of crystalline cellulose in lignocellulosic biomass using sum frequency generation (SFG) vibration spectroscopy and comparison with other analytical methods.

    PubMed

    Barnette, Anna L; Lee, Christopher; Bradley, Laura C; Schreiner, Edward P; Park, Yong Bum; Shin, Heenae; Cosgrove, Daniel J; Park, Sunkyu; Kim, Seong H

    2012-07-01

    The non-centrosymmetry requirement of sum frequency generation (SFG) vibration spectroscopy allows the detection and quantification of crystalline cellulose in lignocellulose biomass without spectral interferences from hemicelluloses and lignin. This paper shows a correlation between the amount of crystalline cellulose in biomass and the SFG signal intensity. Model biomass samples were prepared by mixing commercially available cellulose, xylan, and lignin to defined concentrations. The SFG signal intensity was found sensitive to a wide range of crystallinity, but varied non-linearly with the mass fraction of cellulose in the samples. This might be due to the matrix effects such as light scattering and absorption by xylan and lignin, as well as the non-linear density dependence of the SFG process itself. Comparison with other techniques such as XRD, FT-Raman, FT-IR and NMR demonstrate that SFG can be a complementary and sensitive tool to assess crystalline cellulose in biomass. Copyright © 2012 Elsevier Ltd. All rights reserved.

  7. Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Thompson, David N.; Apel, William A.; Thompson, Vicki S.

    A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extractsmore » are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.« less

  8. Thermophilic acetylxylan esterase genes and enzymes from alicyclobacillus acidocaldarius and related organisms and methods

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Thompson, Vicki S.; Thompson, David N.; Reed, David W.

    A genetically modified organism comprising at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharide, lignocellulose, hemicellulose, lignin, chitin, heteroxylan, and/or xylan-decorating group; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extracts are utilized to convert biomass into a product. Further provided are methodsmore » of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.« less

  9. Effect of hot-water extraction (HWE) severity on bleached pulp based biorefinery performance of eucalyptus during the HWE-Kraft-ECF bleaching process.

    PubMed

    Liu, Jing; Li, Meng; Luo, Xiaolin; Chen, Lihui; Huang, Liulian

    2015-04-01

    The effectiveness of a biorefinery based on an HWE-Kraft-ECF bleaching process and the end use of pulp was systematically evaluated. Using a P-factor of 198, nearly 30% of xylan-based sugars were recovered. The resulting pulp and paper properties were found to be comparable with the control. A maximum xylan-based sugar recovery of nearly 50% was achieved at a P-factor of 738. Although the strength of this P-factor induced handsheet was lower than that of the control by about 20%, the corresponding pulp was sufficient for dissolving pulp application. However, once the P-factor rose above 1189, hemicellulose sugars were significantly degraded into furans; pulp and paper properties were also deteriorated due to cellulose degradation, lignin deposition and condensation. Thus, considering the different end use of pulps, the performance of an HWE-based biorefinery could be balanced by its HWE severity. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Biorefining of wheat straw using an acetic and formic acid based organosolv fractionation process.

    PubMed

    Snelders, Jeroen; Dornez, Emmie; Benjelloun-Mlayah, Bouchra; Huijgen, Wouter J J; de Wild, Paul J; Gosselink, Richard J A; Gerritsma, Jort; Courtin, Christophe M

    2014-03-01

    To assess the potential of acetic and formic acid organosolv fractionation of wheat straw as basis of an integral biorefinery concept, detailed knowledge on yield, composition and purity of the obtained streams is needed. Therefore, the process was performed, all fractions extensively characterized and the mass balance studied. Cellulose pulp yield was 48% of straw dry matter, while it was 21% and 27% for the lignin and hemicellulose-rich fractions. Composition analysis showed that 67% of wheat straw xylan and 96% of lignin were solubilized during the process, resulting in cellulose pulp of 63% purity, containing 93% of wheat straw cellulose. The isolated lignin fraction contained 84% of initial lignin and had a purity of 78%. A good part of wheat straw xylan (58%) ended up in the hemicellulose-rich fraction, half of it as monomeric xylose, together with proteins (44%), minerals (69%) and noticeable amounts of acids used during processing. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Effect of additives on adsorption and desorption behavior of xylanase on acid-insoluble lignin from corn stover and wheat straw.

    PubMed

    Li, Yanfei; Ge, Xiaoyan; Sun, Zongping; Zhang, Junhua

    2015-06-01

    The competitive adsorption between cellulases and additives on lignin in the hydrolysis of lignocelluloses has been confirmed, whereas the effect of additives on the interaction between xylanase and lignin is not clear. In this work, the effects of additives, poly(ethylene glycol) 2000, poly(ethylene glycol) 6000, Tween 20, and Tween 80, on the xylanase adsorption/desorption onto/from acid-insoluble lignin from corn stover (CS-lignin) and wheat straw (WS-lignin) were investigated. The results indicated that the additives could adsorb onto isolated lignin and reduce the xylanase adsorption onto lignin. Compared to CS-lignin, more additives could adsorb onto WS-lignin, making less xylanase adsorbed onto WS-lignin. In addition, the additives could enhance desorption of xylanase from lignin, which might be due to the competitive adsorption between xylanase and additives on lignin. The released xylanase from lignin still exhibited hydrolytic capacity in the hydrolysis of isolated xylan and xylan in corn stover. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Direct production of feruloyl oligosaccharides and hemicellulase inducement and distribution in a newly isolated Aureobasidium pullulans strain.

    PubMed

    Yu, Xiao-hong; Gu, Zhen-xin

    2014-02-01

    Studies were carried out to screen and identify strains that are able to directly produce ferulic oligosaccharides (FOs) from wheat bran (WB). The inducement and distribution of hemicellulases from strain 2012, which was identified as a non-melanin secreting strain of Aureobasidium pullulans (A. pullulans), were also determined. In a 60 g/L WB solution, A. pullulans could produce 545 nmol/L FOs, 64.12 IU/mL xylanase and 0.14 IU/mL ferulic acid esterase (FAE). A. pullulans was cultivated in media with WB, glucose, xylose, sucrose, lactose or xylan as the carbon source, and hemicellulases were mainly induced by xylan and WB and inhibited by glucose and sucrose. Xylanase and FAE were mainly present in the culture filtrate, xylosidase in the hyphal filaments and arabinofuranosidase was a membrane-bound enzyme. The yield of FOs was positively correlated to the hemicellulases activity, and significantly positively (P < 0.05) correlated to the xylanase activity (r = 0.992).

  13. Comparison of alkaline- and fungi-assisted wet-storage of corn stover.

    PubMed

    Cui, Zhifang; Shi, Jian; Wan, Caixia; Li, Yebo

    2012-04-01

    Storage of lignocellulosic biomass is critical for a year-round supply of feedstock for a biorefinery. Compared with dry storage, wet storage is a promising alternative technology, providing several advantages including reduced dry matter loss and fire risk and improved feedstock digestibility after storage. This study investigated the concurrent pretreatment and wet-storage of corn stover with the assistance of NaOH or a lignin-degrading fungus, Ceriporiopsis subvermispora, during a 90-d period. Compared with ensilage, adding NaOH or inoculation with C. subvermispora significantly enhanced the enzymatic degradability of corn stover by 2-3-fold after 90-d wet storage. Lignin and xylan removal during NaOH pretreatment and wet-storage were influenced by NaOH loading and moisture. NaOH pretreatment retarded the production of organic acids during storage and the acetate release correlated with lignin and xylan removal. Further study is needed to reduce cellulose degradation during the late stage of fungal treatment. Copyright © 2012 Elsevier Ltd. All rights reserved.

  14. Furfural production using ionic liquids: A review.

    PubMed

    Peleteiro, Susana; Rivas, Sandra; Alonso, José Luis; Santos, Valentín; Parajó, Juan Carlos

    2016-02-01

    Furfural, a platform chemical with a bright future, is commercially obtained by acidic processing of xylan-containing biomass in aqueous media. Ionic liquids (ILs) can be employed in processed for furfural manufacture as additives, as catalysts and/or as reaction media. Depending on the IL utilized, externally added catalysts (usually, Lewis acids, Brönsted acids and/or solid acid catalysts) can be necessary to achieve high reaction yields. Oppositely, acidic ionic liquids (AILs) can perform as both solvents and catalysts, enabling the direct conversion of suitable substrates (pentoses, pentosans or xylan-containing biomass) into furfural. Operating in IL-containing media, the furfural yields can be improved when the product is continuously removed along the reaction (for example, by stripping or extraction), to avoid unwanted side-reactions leading to furfural consumption. These topics are reviewed, as well as the major challenges involved in the large scale utilization of ILs for furfural production. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Fibrillar assembly of bacterial cellulose in the presence of wood-based hemicelluloses.

    PubMed

    Penttilä, Paavo A; Imai, Tomoya; Sugiyama, Junji

    2017-09-01

    Composite materials mimicking the plant cell wall structure were made by culturing cellulose-producing bacteria together with secondary-wall hemicelluloses from wood. The effects of spruce galactoglucomannan (GGM) and beech xylan on the nanoscale morphology of bacterial cellulose were studied in the original, hydrated state with small-angle X-ray scattering (SAXS). The SAXS intensities were fitted with a model covering multiple levels of the hierarchical structure. Additional information on the structure of dried samples was obtained using scanning and transmission electron microscopy and infra-red spectroscopy. Both hemicelluloses induced a partial conversion of the cellulose crystal structure from I α to I β and a reduction of the cross-sectional dimensions of the cellulose microfibrils, thereby affecting also their packing into bundles. The differences were more pronounced in samples with xylan instead of GGM, and they became more significant with higher hemicellulose concentrations. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Enzymatic hydrolysis of biomimetic bacterial cellulose-hemicellulose composites.

    PubMed

    Penttilä, Paavo A; Imai, Tomoya; Hemming, Jarl; Willför, Stefan; Sugiyama, Junji

    2018-06-15

    The production of biofuels and other chemicals from lignocellulosic biomass is limited by the inefficiency of enzymatic hydrolysis. Here a biomimetic composite material consisting of bacterial cellulose and wood-based hemicelluloses was used to study the effects of hemicelluloses on the enzymatic hydrolysis with a commercial cellulase mixture. Bacterial cellulose synthesized in the presence of hemicelluloses, especially xylan, was found to be more susceptible to enzymatic hydrolysis than hemicellulose-free bacterial cellulose. The reason for the easier hydrolysis could be related to the nanoscale structure of the substrate, particularly the packing of cellulose microfibrils into ribbons or bundles. In addition, small-angle X-ray scattering was used to show that the average nanoscale morphology of bacterial cellulose remained unchanged during the enzymatic hydrolysis. The reported easier enzymatic hydrolysis of bacterial cellulose produced in the presence of wood-based xylan offers new insights to overcome biomass recalcitrance through genetic engineering. Copyright © 2018 Elsevier Ltd. All rights reserved.

  17. The type of carbohydrates specifically selects microbial community structures and fermentation patterns.

    PubMed

    Chatellard, Lucile; Trably, Eric; Carrère, Hélène

    2016-12-01

    The impact on dark fermentation of seven carbohydrates as model substrates of lignocellulosic fractions (glucose, cellobiose, microcrystalline cellulose, arabinose, xylose, xylan and wheat straw) was investigated. Metabolic patterns and bacterial communities were characterized at the end of batch tests inoculated with manure digestate. It was found that hydrogen production was linked to the sugar type (pentose or hexose) and the degree of polymerisation. Hexoses produced less hydrogen, with a specific selection of lactate-producing bacterial community structures. Maximal hydrogen production was five times higher on pentose-based substrates, with specific bacterial community structures producing acetate and butyrate as main metabolites. Low hydrogen amounts accumulated from complex sugars (cellulose, xylan and wheat straw). A relatively high proportion of the reads was affiliated to Ruminococcaceae suggesting an efficient hydrolytic activity. Knowing that the bacterial community structure is very specific to a particular substrate offers new possibilities to design more efficient H 2 -producing biological systems. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes

    DOEpatents

    Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Ward, Thomas E.

    2016-03-22

    A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extracts are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.

  19. Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes

    DOEpatents

    Thompson, David N; Apel, William A; Thompson, Vicki S; Ward, Thomas E

    2013-07-23

    A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extracts are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.

  20. Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes

    DOEpatents

    Thompson, David N; Apel, William A; Thompson, Vicki S; Ward, Thomas E

    2014-04-08

    A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extracts are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.

  1. Comparison of autohydrolysis and ionic liquid 1-butyl-3-methylimidazolium acetate pretreatment to enhance enzymatic hydrolysis of sugarcane bagasse

    DOE PAGES

    Hashim, Muzna; Univ. of Tennessee, Knoxville, TN; Sun, Qining; ...

    2016-11-02

    The aim of this work was to evaluate the efficiency of an ionic liquid (IL) 1-butyl-3-methylimidazolium acetate ([C4mim][OAc]) pretreatment (110 C for 30 min) in comparison to high severity autohydrolysis pretreatment in terms of delignification, cellulose crystallinity and enzymatic digestibility. The increase in severity of autohydrolysis pretreatment had positive effect on glucan digestibility, but was limited by the crystallinity of cellulose. [C4mim][OAc] pretreated sugarcane bagasse exhibited a substantial decrease in lignin content, reduced cellulose crystallinity, and enhanced glucan and xylan digestibility. Glucan and xylan digestibility was determined as 97.4% and 98.6% from [C4mim][OAc] pretreated bagasse, and 62.1% and 57.5% frommore » the bagasse autohydrolyzed at 205 C for 6 min, respectively. The results indicated the improved digestibility and hydrolysis rates after [C4mim][OAc] pretreatment when compared against a comparable autohydrolyzed biomass.« less

  2. Generating bifunctional fusion enzymes composed of heat-active endoglucanase (Cel5A) and endoxylanase (XylT).

    PubMed

    Rizk, Mazen; Elleuche, Skander; Antranikian, Garabed

    2015-01-01

    Bifunctional enzyme constructs were generated comprising two genes encoding heat-active endoglucanase (cel5A) and endoxylanase (xylT). The fused proteins Cel5A-XylT and XylT-Cel5A were active on both β-glucan and beechwood xylan. An improvement in endoglucanase and endoxylanase catalytic activities was observed. The specific activity of the fusion towards xylan was significantly raised when compared to XylT. The fusion constructs were active from 40 to 100 °C for endoglucanase and from 40 to 90 °C for endoxylanase, but the temperature optima were lowered from 90 to 80 °C for the endoglucanase and from 80 to 70 °C for the endoxylanase. XylT in the construct XylT-Cel5A was less stable at higher temperatures compared to Cel5A-XylT. Due to the enzymatic performance, these fusion enzymes are attractive candidates for applications in biorefineries based on plant waste.

  3. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hashim, Muzna; Univ. of Tennessee, Knoxville, TN; Sun, Qining

    The aim of this work was to evaluate the efficiency of an ionic liquid (IL) 1-butyl-3-methylimidazolium acetate ([C4mim][OAc]) pretreatment (110 C for 30 min) in comparison to high severity autohydrolysis pretreatment in terms of delignification, cellulose crystallinity and enzymatic digestibility. The increase in severity of autohydrolysis pretreatment had positive effect on glucan digestibility, but was limited by the crystallinity of cellulose. [C4mim][OAc] pretreated sugarcane bagasse exhibited a substantial decrease in lignin content, reduced cellulose crystallinity, and enhanced glucan and xylan digestibility. Glucan and xylan digestibility was determined as 97.4% and 98.6% from [C4mim][OAc] pretreated bagasse, and 62.1% and 57.5% frommore » the bagasse autohydrolyzed at 205 C for 6 min, respectively. The results indicated the improved digestibility and hydrolysis rates after [C4mim][OAc] pretreatment when compared against a comparable autohydrolyzed biomass.« less

  4. Nanoreinforced xylan–cellulose composite foams by freeze-casting

    Treesearch

    Tobias Köhnke; Angela Lin; Thomas Elder; Hans Theliander; Arthur J. Ragauskas

    2012-01-01

    Structured biofoams have been prepared from the readily available renewable biopolymer xylan by employing an ice-templating technique, where the pore morphology of the material can be controlled by the solidification conditions and the molecular structure of the polysaccharide. Furthermore, reinforcement of these biodegradable foams using cellulose nanocrystals shows...

  5. Quantitative predictions of bioconversion of aspen by dilute acid and SPORL pretreatments using a unified combined hydrolysis factor (CHF)

    Treesearch

    W. Zhu; Carl J. Houtman; J.Y. Zhu; Roland Gleisner; K.F. Chen

    2012-01-01

    A combined hydrolysis factor (CHF) was developed to predict xylan hydrolysis during pretreatments of native aspen (Populus tremuloides) wood chips. A natural extension of previously developed kinetic models allowed us to account for the effect of catalysts by dilute acid and two sulfite pretreatments at different pH values....

  6. Correlating Detergent Fiber Analysis and Dietary Fiber Analysis Data for Corn Stover

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wolfrum, E. J.; Lorenz, A. J.; deLeon, N.

    There exist large amounts of detergent fiber analysis data [neutral detergent fiber (NDF), acid detergent fiber (ADF), acid detergent lignin (ADL)] for many different potential cellulosic ethanol feedstocks, since these techniques are widely used for the analysis of forages. Researchers working in the area of cellulosic ethanol are interested in the structural carbohydrates in a feedstock (principally glucan and xylan), which are typically determined by acid hydrolysis of the structural fraction after multiple extractions of the biomass. These so-called dietary fiber analysis methods are significantly more involved than detergent fiber analysis methods. The purpose of this study was to determinemore » whether it is feasible to correlate detergent fiber analysis values to glucan and xylan content determined by dietary fiber analysis methods for corn stover. In the detergent fiber analysis literature cellulose is often estimated as the difference between ADF and ADL, while hemicellulose is often estimated as the difference between NDF and ADF. Examination of a corn stover dataset containing both detergent fiber analysis data and dietary fiber analysis data predicted using near infrared spectroscopy shows that correlations between structural glucan measured using dietary fiber techniques and cellulose estimated using detergent techniques, and between structural xylan measured using dietary fiber techniques and hemicellulose estimated using detergent techniques are high, but are driven largely by the underlying correlation between total extractives measured by fiber analysis and NDF/ADF. That is, detergent analysis data is correlated to dietary fiber analysis data for structural carbohydrates, but only indirectly; the main correlation is between detergent analysis data and solvent extraction data produced during the dietary fiber analysis procedure.« less

  7. Efficient Degradation of Lignocellulosic Plant Biomass, without Pretreatment, by the Thermophilic Anaerobe “Anaerocellum thermophilum” DSM 6725▿

    PubMed Central

    Yang, Sung-Jae; Kataeva, Irina; Hamilton-Brehm, Scott D.; Engle, Nancy L.; Tschaplinski, Timothy J.; Doeppke, Crissa; Davis, Mark; Westpheling, Janet; Adams, Michael W. W.

    2009-01-01

    Very few cultivated microorganisms can degrade lignocellulosic biomass without chemical pretreatment. We show here that “Anaerocellum thermophilum” DSM 6725, an anaerobic bacterium that grows optimally at 75°C, efficiently utilizes various types of untreated plant biomass, as well as crystalline cellulose and xylan. These include hardwoods such as poplar, low-lignin grasses such as napier and Bermuda grasses, and high-lignin grasses such as switchgrass. The organism did not utilize only the soluble fraction of the untreated biomass, since insoluble plant biomass (as well as cellulose and xylan) obtained after washing at 75°C for 18 h also served as a growth substrate. The predominant end products from all growth substrates were hydrogen, acetate, and lactate. Glucose and cellobiose (on crystalline cellulose) and xylose and xylobiose (on xylan) also accumulated in the growth media during growth on the defined substrates but not during growth on the plant biomass. A. thermophilum DSM 6725 grew well on first- and second-spent biomass derived from poplar and switchgrass, where spent biomass is defined as the insoluble growth substrate recovered after the organism has reached late stationary phase. No evidence was found for the direct attachment of A. thermophilum DSM 6725 to the plant biomass. This organism differs from the closely related strain A. thermophilum Z-1320 in its ability to grow on xylose and pectin. Caldicellulosiruptor saccharolyticus DSM 8903 (optimum growth temperature, 70°C), a close relative of A. thermophilum DSM 6725, grew well on switchgrass but not on poplar, indicating a significant difference in the biomass-degrading abilities of these two otherwise very similar organisms. PMID:19465524

  8. Function of the family-9 and family-22 carbohydrate-binding modules in a modular beta-1,3-1,4-glucanase/xylanase derived from Clostridium stercorarium Xyn10B.

    PubMed

    Zhao, Guangshan; Ali, Ehsan; Araki, Rie; Sakka, Makiko; Kimura, Tetsuya; Sakka, Kazuo

    2005-08-01

    Clostridium stercorarium Xyn10B having hydrolytic activities on xylan and beta-1,3-1,4-glucan is a modular enzyme composed of two family-22 carbohydrate-binding modules (CBMs), a family-10 catalytic module of the glycoside hydrolases, a family-9 CBM, and two S-layer homologous modules, consecutively from the N-terminus. We investigated the function of family-9 and family-22 CBMs in a modular enzyme by comparing the enzymatic properties of a truncated enzyme composed of two family-22 CBMs and the catalytic module (rCBM22-CM), an enzyme composed of the catalytic module and family-9 CBM (rCM-CBM9), an enzyme composed of two family-22 CBMs, the catalytic module, and family-9 CBM (rCBM22-CM-CBM9), and the catalytic module polypeptide (rCM). Although the addition of family-9 CBM to rCM and rCBM22-CM did not significantly change catalytic activity toward xylan and beta-1,3-1,4-glucan, the addition of family-22 CBM to rCM and rCM-CBM9 drastically enhanced catalytic activity toward xylan and especially beta-1,3-1,4-glucan. Furthermore, the addition of family-22 CBM to rCM and rCM-CBM9 shifted the optimum temperature from 65 degrees C to 75 degrees C, but that of family-9 CBM to rCM and rCBM22-CM did not affect the optimum temperature. These facts suggest that the enzyme properties of Xyn10B were mainly dependent on the presence of the family-22 CBMs but not family-9 CBM.

  9. Investigation of Pleurotus ostreatus pretreatment on switchgrass for ethanol production

    NASA Astrophysics Data System (ADS)

    Slavens, Shelyn Gehle

    Fungal pretreatment using the white-rot fungus Pleurotus ostreatus on switchgrass for ethanol production was studied. In a small-scale storage study, small switchgrass bales were inoculated with fungal spawn and automatically watered to maintain moisture. Sampled at 25, 53, and 81 d, the switchgrass composition was determined and liquid hot water (LHW) pretreatment was conducted. Fungal pretreatment significantly decreased the xylan and lignin content; glucan was not significantly affected by fungal loading. The glucan, xylan, and lignin contents significantly decreased with increased fungal pretreatment time. The effects of the fungal pretreatment were not highly evident after the LHW pretreatment, showing only changes based on sampling time. Although other biological activity within the bales increased cellulose degradation, the fungal pretreatment successfully reduced the switchgrass lignin and hemicellulose contents. In a laboratory-scale nutrient supplementation study, copper, manganese, glucose, or water was added to switchgrass to induce production of ligninolytic enzymes by P. ostreatus. After 40 d, ligninolytic enzyme activities and biomass composition were determined and simultaneous saccharification and fermentation (SSF) was conducted to determine ethanol yield. Laccase activity was similar for all supplements and manganese peroxidase (MnP) activity was significantly less in copper-treated samples than in the other fungal-inoculated samples. The fungal pretreatment reduced glucan, xylan, and lignin content, while increasing extractable sugars content. The lowest lignin contents occurred in the water-fungal treated samples and produced the greatest ethanol yields. The greatest lignin contents occurred in the copper-fungal treated samples and produced the lowest ethanol yields. Manganese-fungal and glucose-fungal treated samples had similar, intermediate lignin contents and produced similar, intermediate ethanol yields. Ethanol yields from switchgrass

  10. Separation and characterization of acetyl and non-acetyl hemicelluloses of Arundo donax by ammonium sulfate precipitation.

    PubMed

    Peng, Feng; Bian, Jing; Peng, Pai; Xiao, Huan; Ren, Jun-Li; Xu, Feng; Sun, Run-Cang

    2012-04-25

    Delignified Arundo donax was sequentially extracted with DMSO, saturated barium hydroxide, and 1.0 M aqueous NaOH solution. The yields of the soluble fractions were 10.2, 6.7, and 10.0% (w/w), respectively, of the dry Arundo donax materials. The DMSO-, Ba(OH)(2)- and NaOH-soluble hemicellulosic fractions were further fractionated into two subfractions by gradient 50% and 80% saturation ammonium sulfate precipitation, respectively. Monosaccharide, molecular weight, FT-IR, and 1D ((1)H and (13)C) and 2D (HSQC) NMR analysis revealed the differences in structural characteristics and physicochemical properties among the subfractions. The subfractions precipitated with 50% saturation ammonium sulfate had lower arabinose/xylose and glucuronic acid/xylose ratios but had higher molecular weight than those of the subfractions precipitated by 80% saturation ammonium sulfate. FT-IR and NMR analysis revealed that the highly acetylated DMSO-soluble hemicellulosic subfraction (H(D50)) could be precipitated with a relatively lower concentration of 50% saturated ammonium sulfate, and thus the gradient ammonium sulfate precipitation technique could discriminate acetyl and non-acetyl hemicelluloses. It was found that the DMSO-soluble subfraction H(D50) precipitated by 50% saturated ammonium sulfate mainly consisted of poorly substituted O-acetyl arabino-4-O-methylglucurono xylan with terminal units of arabinose linked on position 3 of xylose, 4-O-methylglucuronic acid residues linked on position 2 of the xylan bone, and the acetyl groups (degree of acetylation, 37%) linked on position 2 or 3. The DMSO-soluble subfraction H(D80) precipitated by 80% saturated ammonium sulfate was mainly composed of highly substituted arabino-4-O-methylglucurono xylan and β-d-glucan.

  11. Efficient degradation of lignocellulosic plant biomass without pretreatment by the 9 thermophilic anaerobe, Anaerocellum thermophilum DSM 6725

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yang, Sung-Jae; Kataeva, Irina; Hamilton-Brehm, Scott

    2009-01-01

    Very few cultivated microorganisms can degrade lignocellulosic biomass without chemical pretreatment. We show here that 'Anaerocellum thermophilum' DSM 6725, an anaerobic bacterium that grows optimally at 75 C, efficiently utilizes various types of untreated plant biomass, as well as crystalline cellulose and xylan. These include hardwoods such as poplar, low-lignin grasses such as napier and Bermuda grasses, and high-lignin grasses such as switchgrass. The organism did not utilize only the soluble fraction of the untreated biomass, since insoluble plant biomass (as well as cellulose and xylan) obtained after washing at 75 C for 18 h also served as a growthmore » substrate. The predominant end products from all growth substrates were hydrogen, acetate, and lactate. Glucose and cellobiose (on crystalline cellulose) and xylose and xylobiose (on xylan) also accumulated in the growth media during growth on the defined substrates but not during growth on the plant biomass. A. thermophilum DSM 6725 grew well on first- and second-spent biomass derived from poplar and switchgrass, where spent biomass is defined as the insoluble growth substrate recovered after the organism has reached late stationary phase. No evidence was found for the direct attachment of A. thermophilum DSM 6725 to the plant biomass. This organism differs from the closely related strain A. thermophilum Z-1320 in its ability to grow on xylose and pectin. Caldicellulosiruptor saccharolyticus DSM 8903 (optimum growth temperature, 70 C), a close relative of A. thermophilum DSM 6725, grew well on switchgrass but not on poplar, indicating a significant difference in the biomass-degrading abilities of these two otherwise very similar organisms.« less

  12. Coupling alkaline pre-extraction with alkaline-oxidative post-treatment of corn stover to enhance enzymatic hydrolysis and fermentability.

    PubMed

    Liu, Tongjun; Williams, Daniel L; Pattathil, Sivakumar; Li, Muyang; Hahn, Michael G; Hodge, David B

    2014-04-03

    A two-stage chemical pretreatment of corn stover is investigated comprising an NaOH pre-extraction followed by an alkaline hydrogen peroxide (AHP) post-treatment. We propose that conventional one-stage AHP pretreatment can be improved using alkaline pre-extraction, which requires significantly less H2O2 and NaOH. To better understand the potential of this approach, this study investigates several components of this process including alkaline pre-extraction, alkaline and alkaline-oxidative post-treatment, fermentation, and the composition of alkali extracts. Mild NaOH pre-extraction of corn stover uses less than 0.1 g NaOH per g corn stover at 80°C. The resulting substrates were highly digestible by cellulolytic enzymes at relatively low enzyme loadings and had a strong susceptibility to drying-induced hydrolysis yield losses. Alkaline pre-extraction was highly selective for lignin removal over xylan removal; xylan removal was relatively minimal (~20%). During alkaline pre-extraction, up to 0.10 g of alkali was consumed per g of corn stover. AHP post-treatment at low oxidant loading (25 mg H2O2 per g pre-extracted biomass) increased glucose hydrolysis yields by 5%, which approached near-theoretical yields. ELISA screening of alkali pre-extraction liquors and the AHP post-treatment liquors demonstrated that xyloglucan and β-glucans likely remained tightly bound in the biomass whereas the majority of the soluble polymeric xylans were glucurono (arabino) xylans and potentially homoxylans. Pectic polysaccharides were depleted in the AHP post-treatment liquor relative to the alkaline pre-extraction liquor. Because the already-low inhibitor content was further decreased in the alkaline pre-extraction, the hydrolysates generated by this two-stage pretreatment were highly fermentable by Saccharomyces cerevisiae strains that were metabolically engineered and evolved for xylose fermentation. This work demonstrates that this two-stage pretreatment process is well suited for

  13. Mapping N-linked glycosylation of carbohydrate-active enzymes in the secretome of Aspergillus nidulans grown on lignocellulose.

    PubMed

    Rubio, Marcelo Ventura; Zubieta, Mariane Paludetti; Franco Cairo, João Paulo Lourenço; Calzado, Felipe; Paes Leme, Adriana Franco; Squina, Fabio Marcio; Prade, Rolf Alexander; de Lima Damásio, André Ricardo

    2016-01-01

    The genus Aspergillus includes microorganisms that naturally degrade lignocellulosic biomass, secreting large amounts of carbohydrate-active enzymes (CAZymes) that characterize their saprophyte lifestyle. Aspergillus has the capacity to perform post-translational modifications (PTM), which provides an additional advantage for the use of these organisms as a host for the production of heterologous proteins. In this study, the N-linked glycosylation of CAZymes identified in the secretome of Aspergillus nidulans grown on lignocellulose was mapped. Aspergillus nidulans was grown in glucose, xylan and pretreated sugarcane bagasse (SCB) for 96 h, after which glycoproteomics and glycomics were carried out on the extracellular proteins (secretome). A total of 265 proteins were identified, with 153, 210 and 182 proteins in the glucose, xylan and SCB substrates, respectively. CAZymes corresponded to more than 50 % of the total secretome in xylan and SCB. A total of 182 N-glycosylation sites were identified, of which 121 were detected in 67 CAZymes. A prevalence of the N-glyc sequon N-X-T (72.2 %) was observed in N-glyc sites compared with N-X-S (27.8 %). The amino acids flanking the validated N-glyc sites were mainly composed of hydrophobic and polar uncharged amino acids. Selected proteins were evaluated for conservation of the N-glyc sites in Aspergilli homologous proteins, but a pattern of conservation was not observed. A global analysis of N-glycans released from the proteins secreted by A. nidulans was also performed. While the proportion of N-glycans with Hex5 to Hex9 was similar in the xylan condition, a prevalence of Hex5 was observed in the SCB and glucose conditions. The most common and frequent N-glycosylated motifs, an overview of the N-glycosylation of the CAZymes and the number of mannoses found in N-glycans were analyzed. There are many bottlenecks in protein production by filamentous fungi, such as folding, transport by vesicles and secretion, but N

  14. Coupling alkaline pre-extraction with alkaline-oxidative post-treatment of corn stover to enhance enzymatic hydrolysis and fermentability

    PubMed Central

    2014-01-01

    Background A two-stage chemical pretreatment of corn stover is investigated comprising an NaOH pre-extraction followed by an alkaline hydrogen peroxide (AHP) post-treatment. We propose that conventional one-stage AHP pretreatment can be improved using alkaline pre-extraction, which requires significantly less H2O2 and NaOH. To better understand the potential of this approach, this study investigates several components of this process including alkaline pre-extraction, alkaline and alkaline-oxidative post-treatment, fermentation, and the composition of alkali extracts. Results Mild NaOH pre-extraction of corn stover uses less than 0.1 g NaOH per g corn stover at 80°C. The resulting substrates were highly digestible by cellulolytic enzymes at relatively low enzyme loadings and had a strong susceptibility to drying-induced hydrolysis yield losses. Alkaline pre-extraction was highly selective for lignin removal over xylan removal; xylan removal was relatively minimal (~20%). During alkaline pre-extraction, up to 0.10 g of alkali was consumed per g of corn stover. AHP post-treatment at low oxidant loading (25 mg H2O2 per g pre-extracted biomass) increased glucose hydrolysis yields by 5%, which approached near-theoretical yields. ELISA screening of alkali pre-extraction liquors and the AHP post-treatment liquors demonstrated that xyloglucan and β-glucans likely remained tightly bound in the biomass whereas the majority of the soluble polymeric xylans were glucurono (arabino) xylans and potentially homoxylans. Pectic polysaccharides were depleted in the AHP post-treatment liquor relative to the alkaline pre-extraction liquor. Because the already-low inhibitor content was further decreased in the alkaline pre-extraction, the hydrolysates generated by this two-stage pretreatment were highly fermentable by Saccharomyces cerevisiae strains that were metabolically engineered and evolved for xylose fermentation. Conclusions This work demonstrates that this two

  15. Purification and characterization of xylooligosaccharides (XOS) from Miscanthus x giganteus

    USDA-ARS?s Scientific Manuscript database

    Our previous investigation showed that xylooligosaccharides (XOS) could be produced effectively from Miscanthus x giganteus (MxG). Using autohydrolysis, an XOS yield of to 13.5% (w/w) of initial biomass and xylan yield of 69.2% (w/w) was observed. In this study, we investigated the purification of X...

  16. Ligand bound structures of a glycosyl hydrolase family 30 glucuronoxylan xylanohydrolase

    Treesearch

    Franz St. Johns; Jason C. Hurlbert; John D. Rice; James F. Preston; Edwin Pozharski

    2011-01-01

    Xylanases of glycosyl hydrolase family 30 (GH30) have been shown to cleave β-1,4 linkages of 4-O-methylglucuronoxylan (MeGXn) as directed by the position along the xylan chain of an α-1,2-linked 4-O-methylglucuronate (MeGA) moiety. Complete hydrolysis of MeGXn by...

  17. Thermal and acid tolerant beta xylosidases, arabinofuranosidases, genes encoding, related organisms, and methods

    DOEpatents

    Thompson, David N; Thompson, Vicki S; Schaller, Kastli D; Apel, William A; Reed, David W; Lacey, Jeffrey A

    2013-04-30

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius and variations thereof are provided. Further provided are methods of at least partially degrading xylotriose, xylobiose, and/or arabinofuranose-substituted xylan using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius and variations thereof.

  18. Ethanol production from non-detoxified whole slurry of sulfite-pretreated empty fruit bunches at a low cellulase loading

    Treesearch

    Jinlan Cheng; Shao-Yuan Leu; J.Y. Zhu; Thomas W. Jeffries

    2014-01-01

    Sulfite pretreatment to overcome the recalcitrance of lignocelluloses (SPORL) was applied to an empty fruit bunches (EFB) for ethanol production. SPORL facilitated delignification through lignin sulfonation and dissolution of xylan to result in a highly digestible substrate. The pretreated whole slurry was enzymatically saccharified at a solids loading of 18% using a...

  19. Enzymatic hydrolysis of steam-pretreated lignocellulosic materials with Trichoderma atroviride enzymes produced in-house

    PubMed Central

    Kovacs, Krisztina; Macrelli, Stefano; Szakacs, George; Zacchi, Guido

    2009-01-01

    Background Improvement of the process of cellulase production and development of more efficient lignocellulose-degrading enzymes are necessary in order to reduce the cost of enzymes required in the biomass-to-bioethanol process. Results Lignocellulolytic enzyme complexes were produced by the mutant Trichoderma atroviride TUB F-1663 on three different steam-pretreated lignocellulosic substrates, namely spruce, wheat straw and sugarcane bagasse. Filter paper activities of the enzymes produced on the three materials were very similar, while β-glucosidase and hemicellulase activities were more dependent on the nature of the substrate. Hydrolysis of the enzyme preparations investigated produced similar glucose yields. However, the enzymes produced in-house proved to degrade the xylan and the xylose oligomers less efficiently than a commercial mixture of cellulase and β-glucosidase. Furthermore, accumulation of xylose oligomers was observed when the TUB F-1663 supernatants were applied to xylan-containing substrates, probably due to the low β-xylosidase activity of the enzymes. The efficiency of the enzymes produced in-house was enhanced by supplementation with extra commercial β-glucosidase and β-xylosidase. When the hydrolytic capacities of various mixtures of a commercial cellulase and a T. atroviride supernatant produced in the lab were investigated at the same enzyme loading, the glucose yield appeared to be correlated with the β-glucosidase activity, while the xylose yield seemed to be correlated with the β-xylosidase level in the mixtures. Conclusion Enzyme supernatants produced by the mutant T. atroviride TUB F-1663 on various pretreated lignocellulosic substrates have good filter paper activity values combined with high levels of β-glucosidase activities, leading to cellulose conversion in the enzymatic hydrolysis that is as efficient as with a commercial cellulase mixture. On the other hand, in order to achieve good xylan conversion, the supernatants

  20. Anti-inflammatory effect of microbial consortia during the utilization of dietary polysaccharides.

    PubMed

    Thomson, Pamela; Medina, Daniel A; Ortúzar, Verónica; Gotteland, Martín; Garrido, Daniel

    2018-07-01

    The gut microbiome has a significant impact on host health, especially at the metabolic level. Dietary compounds arriving at the colon have a large influence on the composition of the gut microbiome. High fiber diets have been associated to health benefits that are mediated in great part by short chain fatty acids (SCFA). Gut microbial interactions are relevant for the utilization of complex carbohydrates in the gut microbiome. In this work we characterized the utilization of two dietary polysaccharides by combinations of representative adult gut microbes, and the impact of their activities on a cellular inflammation model. Paired combinations of Bifidobacterium adolescentis, Bacteroides dorei, Lactobacillus plantarum, Escherichia coli and Clostridium symbiosum were grown in inulin or xylan as carbon source. Their relative abundance, substrate consumption and major SCFAs produced were determined. Higher cell growth was observed during inulin consumption, and B. adolescentis and L. plantarum were dominant in co-cultures. The co-culture of B. dorei and C. symbiosum was dominant in xylan. In several cases the combined bacterial growth was lower in co-cultures than monocultures, with a few exceptions of synergistic growth between microorganisms. Inulin fermentation resulted in larger acetate and lactate concentrations, and several combinations grown in xylan containing C. symbiosum were characterized by high amounts of butyrate. These microbial consortia were scaled to batch bioreactor fermentations reaching high cell densities and similar profiles to co-culture experiments. Interestingly, a microbial combination producing high amounts of butyrate was able to reduce IL-8 expression in HT-29 cells co-incubated with TNFα. In summary, this work shows that microbial interactions during the utilization of dietary polysaccharides are complex and substrate dependent. Moreover, certain combinations deploy potent anti-inflammatory effects, which are independent of individual

  1. Differential Expression of Three α-Galactosidase Genes and a Single β-Galactosidase Gene from Aspergillus niger

    PubMed Central

    de Vries, Ronald P.; van den Broeck, Hetty C.; Dekkers, Ester; Manzanares, Paloma; de Graaff, Leo H.; Visser, Jaap

    1999-01-01

    A gene encoding a third α-galactosidase (AglB) from Aspergillus niger has been cloned and sequenced. The gene consists of an open reading frame of 1,750 bp containing six introns. The gene encodes a protein of 443 amino acids which contains a eukaryotic signal sequence of 16 amino acids and seven putative N-glycosylation sites. The mature protein has a calculated molecular mass of 48,835 Da and a predicted pI of 4.6. An alignment of the AglB amino acid sequence with those of other α-galactosidases revealed that it belongs to a subfamily of α-galactosidases that also includes A. niger AglA. A. niger AglC belongs to a different subfamily that consists mainly of prokaryotic α-galactosidases. The expression of aglA, aglB, aglC, and lacA, the latter of which encodes an A. niger β-galactosidase, has been studied by using a number of monomeric, oligomeric, and polymeric compounds as growth substrates. Expression of aglA is only detected on galactose and galactose-containing oligomers and polymers. The aglB gene is expressed on all of the carbon sources tested, including glucose. Elevated expression was observed on xylan, which could be assigned to regulation via XlnR, the xylanolytic transcriptional activator. Expression of aglC was only observed on glucose, fructose, and combinations of glucose with xylose and galactose. High expression of lacA was detected on arabinose, xylose, xylan, and pectin. Similar to aglB, the expression on xylose and xylan can be assigned to regulation via XlnR. All four genes have distinct expression patterns which seem to mirror the natural substrates of the encoded proteins. PMID:10347026

  2. A plasmid borne, functionally novel glycoside hydrolase family 30 subfamily 8 endoxylanase from solventogenic Clostridium

    PubMed Central

    Dietrich, Diane; Crooks, Casey; Balogun, Peter; deSerrano, Vesna; Pozharski, Edwin; Smith, James Kennon; Bales, Elizabeth; Hurlbert, Jason

    2018-01-01

    Glycoside hydrolase family 30 subfamily 8 (GH30-8) β-1,4-endoxylanases are known for their appendage-dependent function requiring recognition of an α-1,2-linked glucuronic acid (GlcA) common to glucuronoxylans for hydrolysis. Structural studies have indicated that the GlcA moiety of glucuronoxylans is coordinated through six hydrogen bonds and a salt bridge. These GlcA-dependent endoxylanases do not have significant activity on xylans that do not bear GlcA substitutions such as unsubstituted linear xylooligosaccharides or cereal bran arabinoxylans. In the present study, we present the structural and biochemical characteristics of xylanase 30A from Clostridium acetobutylicum (CaXyn30A) which was originally selected for study due to predicted structural differences within the GlcA coordination loops. Amino acid sequence comparisons indicated that this Gram-positive-derived GH30-8 more closely resembles Gram-negative derived forms of these endoxylanases: a hypothesis borne out in the developed crystallographic structure model of the CaXyn30A catalytic domain (CaXyn30A-CD). CaXyn30A-CD hydrolyzes xylans to linear and substituted oligoxylosides showing the greatest rate with the highly arabinofuranose (Araf)-substituted cereal arabinoxylans. CaXyn30A-CD hydrolyzes xylooligosaccharides larger than xylotriose and shows an increased relative rate of hydrolysis for xylooligosaccharides containing α-1,2-linked arabinofuranose substitutions. Biochemical analysis confirms that CaXyn30A benefits from five xylose-binding subsites which extend from the −3 subsite to the +2 subsite of the binding cleft. These studies indicate that CaXyn30A is a GlcA-independent endoxylanase that may have evolved for the preferential recognition of α-1,2-Araf substitutions on xylan chains. PMID:29626157

  3. Polypeptide having acetyl xylan esterase activity and uses thereof

    DOEpatents

    Schoonneveld-Bergmans, Margot Elisabeth Francoise; Heijne, Wilbert Herman Marie; Los, Alrik Pieter

    2015-10-20

    The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 82% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 82% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

  4. Co-production of bio-ethanol, xylonic acid and slow-release nitrogen fertilizer from low-cost straw pulping solid residue.

    PubMed

    Huang, Chen; Ragauskas, Arthur J; Wu, Xinxing; Huang, Yang; Zhou, Xuelian; He, Juan; Huang, Caoxing; Lai, Chenhuan; Li, Xin; Yong, Qiang

    2018-02-01

    A novel bio-refinery sequence yielding varieties of co-products was developed using straw pulping solid residue. This process utilizes neutral sulfite pretreatment which under optimal conditions (160 °C and 3% (w/v) sulfite charge) provides 64.3% delignification while retaining 90% of cellulose and 67.3% of xylan. The pretreated solids exhibited excellent enzymatic digestibility, with saccharification yields of 86.9% and 81.1% for cellulose and xylan, respectively. After pretreatment, the process of semi-simultaneous saccharification and fermentation (S-SSF) and bio-catalysis was investigated. The results revealed that decreased ethanol yields were achieved when solid loading increased from 5% to 30%. An acceptable ethanol yield of 76.8% was obtained at 20% solid loading. After fermentation, bio-catalysis of xylose remaining in fermentation broth resulted in near 100% xylonic acid (XA) yield at varied solid loadings. To complete the co-product portfolio, oxidation ammoniation of the dissolved lignin successfully transformed it into biodegradable slow-release nitrogen fertilizer with excellent agricultural properties. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. MALDI-TOF MS analysis of cellodextrins and xylo-oligosaccharides produced by hindgut homogenates of Reticulitermes santonensis.

    PubMed

    Brasseur, Catherine; Bauwens, Julien; Tarayre, Cédric; Mattéotti, Christel; Thonart, Philippe; Destain, Jacqueline; Francis, Frédéric; Haubruge, Eric; Portetelle, Daniel; Vandenbol, Micheline; Focant, Jean-François; De Pauw, Edwin

    2014-04-11

    Hindgut homogenates of the termite Reticulitermes santonensis were incubated with carboxymethyl cellulose (CMC), crystalline celluloses or xylan substrates. Hydrolysates were analyzed with matrix-assisted laser desorption/ionization coupled to time-of-flight mass spectrometry (MALDI-TOF MS). The method was first set up using acid hydrolysis analysis to characterize non-enzymatic profiles. Commercial enzymes of Trichoderma reesei or T. longibrachiatum were also tested to validate the enzymatic hydrolysis analysis. For CMC hydrolysis, data processing and visual display were optimized to obtain comprehensive profiles and allow rapid comparison and evaluation of enzymatic selectivity, according to the number of substituents of each hydrolysis product. Oligosaccharides with degrees of polymerization (DPs) ranging from three to 12 were measured from CMC and the enzymatic selectivity was demonstrated. Neutral and acidic xylo-oligosaccharides with DPs ranging from three to 11 were measured from xylan substrate. These results are of interest for lignocellulose biomass valorization and demonstrated the potential of termites and their symbiotic microbiota as a source of interesting enzymes for oligosaccharides production.

  6. Enhancement of enzymatic hydrolysis and Klason lignin removal of corn stover using photocatalyst-assisted ammonia pretreatment.

    PubMed

    Yoo, Chang Geun; Wang, Chao; Yu, Chenxu; Kim, Tae Hyun

    2013-03-01

    Photocatalyst-assisted ammonia pretreatment was explored to improve lignin removal of the lignocellulosic biomass for effective sugar conversion. Corn stover was treated with 5.0-12.5 wt.% ammonium hydroxide, two different photocatalysts (TiO(2) and ZnO) in the presence of molecular oxygen in a batch reactor at 60 °C. Various solid-to-liquid ratios (1:20-1:50) were also tested. Ammonia pretreatment assisted by TiO(2)-catalyzed photo-degradation removed 70 % of Klason lignin under the optimum condition (12.5 % ammonium hydroxide, 60 °C, 24 h, solid/liquid=1:20, photocatalyst/biomass=1:10 with oxygen atmosphere). The enzymatic digestibilities of pretreated corn stover were 85 % for glucan and 75 % for xylan with NH(3)-TiO(2)-treated solid and 82 % for glucan and 77 % for xylan with NH(3)-ZnO-treated solid with 15 filter paper units/g-glucan of cellulase and 30 cellobiase units/g-glucan of β-glucosidase, a 2-13 % improvement over ammonia pretreatment alone.

  7. Wood mimetic hydrogel beads for enzyme immobilization.

    PubMed

    Park, Saerom; Kim, Sung Hee; Won, Keehoon; Choi, Joon Weon; Kim, Yong Hwan; Kim, Hyung Joo; Yang, Yung-Hun; Lee, Sang Hyun

    2015-01-22

    Wood component-based composite hydrogels have potential applications in biomedical fields owing to their low cost, biodegradability, and biocompatibility. The controllable properties of wood mimetic composites containing three major wood components are useful for enzyme immobilization. Here, lipase from Candida rugosa was entrapped in wood mimetic beads containing cellulose, xylan, and lignin by dissolving wood components with lipase in [Emim][Ac], followed by reconstitution. Lipase entrapped in cellulose/xylan/lignin beads in a 5:3:2 ratio showed the highest activity; this ratio is very similar to that in natural wood. The lipase entrapped in various wood mimetic beads showed increased thermal and pH stability. The half-life times of lipase entrapped in cellulose/alkali lignin hydrogel were 31- and 82-times higher than those of free lipase during incubation under denaturing conditions of high temperature and low pH, respectively. Owing to their biocompatibility, biodegradability, and controllable properties, wood mimetic hydrogel beads can be used to immobilize various enzymes for applications in the biomedical, bioelectronic, and biocatalytic fields. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Effects of enzyme loading and β-glucosidase supplementation on enzymatic hydrolysis of switchgrass processed by leading pretreatment technologies.

    PubMed

    Pallapolu, Venkata Ramesh; Lee, Y Y; Garlock, Rebecca J; Balan, Venkatesh; Dale, Bruce E; Kim, Youngmi; Mosier, Nathan S; Ladisch, Michael R; Falls, Matthew; Holtzapple, Mark T; Sierra-Ramirez, Rocio; Shi, Jian; Ebrik, Mirvat A; Redmond, Tim; Yang, Bin; Wyman, Charles E; Donohoe, Bryon S; Vinzant, Todd B; Elander, Richard T; Hames, Bonnie; Thomas, Steve; Warner, Ryan E

    2011-12-01

    The objective of this work is to investigate the effects of cellulase loading and β-glucosidase supplementation on enzymatic hydrolysis of pretreated Dacotah switchgrass. To assess the difference among various pretreatment methods, the profiles of sugars and intermediates were determined for differently treated substrates. For all pretreatments, 72 h glucan/xylan digestibilities increased sharply with enzyme loading up to 25mg protein/g-glucan, after which the response varied depending on the pretreatment method. For a fixed level of enzyme loading, dilute sulfuric acid (DA), SO(2), and Lime pretreatments exhibited higher digestibility than the soaking in aqueous ammonia (SAA) and ammonia fiber expansion (AFEX). Supplementation of Novozyme-188 to Spezyme-CP improved the 72 h glucan digestibility only for the SAA treated samples. The effect of β-glucosidase supplementation was discernible only at the early phase of hydrolysis where accumulation of cellobiose and oligomers is significant. Addition of β-glucosidase increased the xylan digestibility of alkaline treated samples due to the β-xylosidase activity present in Novozyme-188. Copyright © 2011 Elsevier Ltd. All rights reserved.

  9. Biobutanol production by Clostridium acetobutylicum using xylose recovered from birch Kraft black liquor.

    PubMed

    Kudahettige-Nilsson, Rasika L; Helmerius, Jonas; Nilsson, Robert T; Sjöblom, Magnus; Hodge, David B; Rova, Ulrika

    2015-01-01

    Acetone-butanol-ethanol (ABE) fermentation was studied using acid-hydrolyzed xylan recovered from hardwood Kraft black liquor by CO2 acidification as the only carbon source. Detoxification of hydrolyzate using activated carbon was conducted to evaluate the impact of inhibitor removal and fermentation. Xylose hydrolysis yields as high as 18.4% were demonstrated at the highest severity hydrolysis condition. Detoxification using active carbon was effective for removal of both phenolics (76-81%) and HMF (38-52%). Batch fermentation of the hydrolyzate and semi-defined P2 media resulted in a total solvent yield of 0.12-0.13g/g and 0.34g/g, corresponding to a butanol concentration of 1.8-2.1g/L and 7.3g/L respectively. This work is the first study of a process for the production of a biologically-derived biofuel from hemicelluloses solubilized during Kraft pulping and demonstrates the feasibility of utilizing xylan recovered directly from industrial Kraft pulping liquors as a feedstock for biological production of biofuels such as butanol. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  10. Direct ethanol production from cellulosic materials by consolidated biological processing using the wood rot fungus Schizophyllum commune.

    PubMed

    Horisawa, Sakae; Ando, Hiromasa; Ariga, Osamu; Sakuma, Yoh

    2015-12-01

    In the present study, ethanol production from polysaccharides or wood chips was conducted in a single reactor under anaerobic conditions using the white rot fungus Schizophyllum commune NBRC 4928, which produces enzymes that degrade lignin, cellulose and hemicellulose. The ethanol yields produced from glucose and xylose were 80.5%, and 52.5%, respectively. The absolute yields of ethanol per microcrystalline cellulose (MCC), xylan and arabinogalactan were 0.26g/g-MCC, 0.0419g/g-xylan and 0.0508g/g-arabinogalactan, respectively. By comparing the actual ethanol yields from polysaccharides with monosaccharide fermentation, it was shown that the rate of saccharification was slower than that in fermentation. S. commune NBRC 4928 is concluded to be suitable for CBP because it can produce ethanol from various types of sugar. From the autoclaved cedar chip, only little ethanol was produced by S. commune NBRC 4928 alone but ethanol production was enhanced by combined use of ethanol fermenting and lignin degrading fungi. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Potential of a gypsum-free composting process of wheat straw for mushroom production.

    PubMed

    Mouthier, Thibaut M B; Kilic, Baris; Vervoort, Pieter; Gruppen, Harry; Kabel, Mirjam A

    2017-01-01

    Wheat straw based composting generates a selective substrate for mushroom production. The first phase of this process requires 5 days, and a reduction in time is wished. Here, we aim at understanding the effect of gypsum on the duration of the first phase and the mechanism behind it. Hereto, the regular process with gypsum addition and the same process without gypsum were studied during a 5-day period. The compost quality was evaluated based on compost lignin composition analysed by py-GC/MS and its degradability by a commercial (hemi-)cellulolytic enzyme cocktail. The composting phase lead to the decrease of the pyrolysis products 4-vinylphenol and 4-vinylguaiacol that can be associated with p-coumarates and ferulates linking xylan and lignin. In the regular compost, the enzymatic conversion reached 32 and 39% for cellulose, and 23 and 32% for xylan after 3 and 5 days, respectively. In absence of gypsum similar values were reached after 2 and 4 days, respectively. Thus, our data show that in absence of gypsum the desired compost quality was reached 20% earlier compared to the control process.

  12. Quantitative investigations of xylose and arabinose substituents in hydroxypropylated and hydroxyvinylethylated arabinoxylans.

    PubMed

    Lorenz, Dominic; Knöpfle, Anna; Akil, Youssef; Saake, Bodo

    2017-11-01

    The chemical structures obtained by the modification of arabinoxylans with the cyclic carbonates propylene carbonate (PC) and 4-vinyl-1,3-dioxolan-2-one (VEC) with varying degrees of substitution were investigated. Therefore, a new analytical method was developed that is based on a microwave-assisted hydrolysis of the polysaccharides with trifluoroacetic acid and the reductive amination with 2-aminobenzoic acid. The peak assignment was achieved by HPLC-MS and the carbohydrate derivatives were quantified by HPLC-fluorescence. The obtained maximum molar substitution of PC-derivatized xylan (X HP ) was 1.8; the molar substitution of VEC-derivatized xylan (X HVE ) was 2.3. Investigations of xylose and arabinose based mono- and disubstituted derivatives revealed a preferred reaction of the cyclic carbonates with arabinose. Conversion rates were up to 2.4 times higher for monosubstitution and up to 3.0 times for disubstitution compared to xylose. Furthermore, the reaction with VEC was preferred due to higher reactivity of the newly introduced side chains. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Improving hydrocarbon yield from catalytic fast co-pyrolysis of hemicellulose and plastic in the dual-catalyst bed of CaO and HZSM-5.

    PubMed

    Ding, Kuan; Zhong, Zhaoping; Wang, Jia; Zhang, Bo; Fan, Liangliang; Liu, Shiyu; Wang, Yunpu; Liu, Yuhuan; Zhong, Daoxu; Chen, Paul; Ruan, Roger

    2018-08-01

    The high concentration of oxygenated compounds in pyrolytic products prohibits the conversion of hemicellulose to important biofuels and chemicals via fast pyrolysis. Herein a dual-catalyst bed of CaO and HZSM-5 was developed to convert acids in the pyrolytic products of xylan to valuable hydrocarbons. Meanwhile, LLDPE was co-pyrolyzed with xylan to supplement hydrogen during the catalysis of HZSM-5. The results showed that CaO could effectively transform acids into ketones. A minimum yield of acids (2.74%) and a maximum yield of ketones (42.93%) were obtained at a catalyst to feedstock ratio of 2:1. The dual-catalyst bed dramatically increased the yield of aromatics. Moreover, hydrogen-rich fragments derived from LLDPE promoted the Diels-Alder reactions of furans and participated in the hydrocarbon pool reactions of non-furanic compounds. As a result, a higher yield of hydrocarbons was achieved. This study provides a fundamental for recovering energy and chemicals from pyrolysis of hemicellulose. Copyright © 2018 Elsevier Ltd. All rights reserved.

  14. A comparative study of mucilage and pulp polysaccharides from tamarillo fruit (Solanum betaceum Cav.).

    PubMed

    do Nascimento, Georgia Erdmann; Iacomini, Marcello; Cordeiro, Lucimara M C

    2016-07-01

    A comparative study of mucilage (locular tissue) and pulp polysaccharides from ripe tamarillo fruits (Solanum betaceum Cav.) was carried out. After aqueous and alkaline extractions and various purification steps (freeze-thaw and α-amylase - EC 3.2.1.1 treatments, Fehling precipitation and ultrafiltration through 50 kDa cut-off membrane), the obtained fractions from mucilage were analyzed by sugar composition, HPSEC, and NMR spectroscopy analyses. The results showed that the mucilage of tamarillo contains a highly methoxylated homogalacturonans mixed with type I arabinogalactans, a linear (1 → 5)-linked α-L-arabinan, and a linear (1 → 4)-β-D-xylan. A comparison with polysaccharides extracted from the pulp revealed that differences were observed in the yield and in the ratio of extracted polysaccharides. Moreover, structural differences between pulp and mucilage polysaccharides were also observed, such as in the length of side chains of the pectins, and in the degree of branching of the xylans. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  15. Large Scale Green Synthesis of 1,2,4-Butanetriol

    DTIC Science & Technology

    2007-03-31

    processing Corn fiber was pretreated by AFEX and the resultant glucan was enzymatically converted to monosaccharide . Saccharification of the cellulose...After removing the residual solids from the hydrolyzate solution, solubilized hemicellulose and monosaccharides were measured in solution, where the...resulting hemicellulose was 62% polysachharide by mass. The component polysaccharide content of the hemicellulose was 35% xylan, 18% arabinan, 6

  16. Bacterial extracellular lignin peroxidase

    DOEpatents

    Crawford, Donald L.; Ramachandra, Muralidhara

    1993-01-01

    A newly discovered lignin peroxidase enzyme is provided. The enzyme is obtained from a bacterial source and is capable of degrading the lignin portion of lignocellulose in the presence of hydrogen peroxide. The enzyme is extracellular, oxidative, inducible by lignin, larch wood xylan, or related substrates and capable of attacking certain lignin substructure chemical bonds that are not degradable by fungal lignin peroxidases.

  17. Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Thompson, David N.; Apel, William A.; Thompson, Vicki S.

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

  18. Mechanisms for kappa reduction and color removal by xylanases

    Treesearch

    Thomas W. Jeffries; Mark Davis; Brian Rosin; Larry L. Landucci

    1998-01-01

    Xylanases reduce kappa and release UV- and visibly absorptive materials from kraft pulps. The extents of these actions depend on the origin and processing of the pulp, access of enzymes to the substrate, and the natures of the enzymes. Hexeneuronic acid (HexA) is a component of kraft pulp xylans that accounts for a fraction of the kappa content. It absorbs strongly in...

  19. Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

    DOEpatents

    Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Reed, David W.; Lacey, Jeffrey A.; Henriksen, Emily D.

    2015-06-02

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

  20. Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

    DOEpatents

    Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Reed, David W.; Lacey, Jeffrey A.

    2013-10-15

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

  1. Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from alicyclobacillus acidocaldarius and related organisms, methods

    DOEpatents

    Thompson, David N [Idaho Falls, ID; Apel, William A [Jackson, WY; Thompson, Vicki S [Idaho Falls, ID; Reed, David W [Idaho Falls, ID; Lacey, Jeffrey A [Idaho Falls, ID; Henriksen, Emily D [Idaho Falls, ID

    2012-06-19

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

  2. Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

    DOEpatents

    Thompson, David N; Apel, William A; Thompson, Vicki S; Reed, David W; Lacey, Jeffrey A; Henriksen, Emily D

    2013-04-23

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

  3. Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from alicyclobacillus acidocaldarius and related organisms, methods

    DOEpatents

    Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Reed, David W.; Lacey, Jeffrey A.; Henriksen, Emily D.

    2010-12-28

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

  4. Thermophilic and thermoacidophilic biopolymer-degrading genes and enzymes from alicyclobacillus acidocaldarius and related organisms, methods

    DOEpatents

    Thompson, David N; Apel, William A; Thompson, Vicki S; Reed, David W; Lacey, Jeffrey A; Henriksen, Emily D

    2013-07-30

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

  5. Thermophilic and thermoacidophilic biopolymer degrading genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Thompson, David N; Apel, William A; Thompson, Vicki S

    Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of at least partially degrading, cleaving, or removing polysaccharides, lignocellulose, cellulose, hemicellulose, lignin, starch, chitin, polyhydroxybutyrate, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups using isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius.

  6. Rapid 2,2'-bicinchoninic-based xylanase assay compatible with high throughput screening

    Treesearch

    William R. Kenealy; Thomas W. Jeffries

    2003-01-01

    High-throughput screening requires simple assays that give reliable quantitative results. A microplate assay was developed for reducing sugar analysis that uses a 2,2'-bicinchoninic-based protein reagent. Endo-1,4-â-D-xylanase activity against oat spelt xylan was detected at activities of 0.002 to 0.011 IU ml−1. The assay is linear for sugar...

  7. Influence of carbon source on cell surface topology of Thermomonospora curvata.

    PubMed Central

    Hostalka, F; Moultrie, A; Stutzenberger, F

    1992-01-01

    The appearance of cell surface protuberances in Thermomonospora curvata correlated with cell-bound exoenzymes which could be removed by brief sonication. Mycelia grown on cellulose or xylan had numerous protuberances and retained 20 to 25% of endoglucanase and endoxylanase at cell surfaces, while those grown on pectin or starch had few protuberances and negligible bound pectinase or amylase. Images PMID:1400256

  8. Evaluation of ethanol production from corncob using Scheffersomyces (Pichia) stipitis CBS 6054 by volumetric scale-up

    Treesearch

    Jae-Won Lee; J.Y. Zhu; Danilo Scordia; Thomas W. Jeffries

    2011-01-01

    In scale-up, the potential of ethanol production by dilute sulfuric acid pretreatment using corncob was investigated. Pretreatments were performed at 170 °C with various acid concentrations ranging from 0% to 1.656% based on oven dry weight. Following pretreatment, pretreated biomass yield ranged from 59% to 67%. More than 90% of xylan was removed at 0.828% of sulfuric...

  9. Production of beta-xylanase by a Thermomyces lanuginosus MC 134 mutant on corn cobs and its application in biobleaching of bagasse pulp.

    PubMed

    Kumar, Kuttanpillai Santhosh; Manimaran, Ayyachamy; Permaul, Kugen; Singh, Suren

    2009-05-01

    The production of hemicellulases by Thermomyces lanuginosus SK using oatspelts xylan was examined during submerged cultivation. A high level of extracellular xylanase (346+/-10 U ml(-1)) production was observed on the fifth day; however, accessory enzyme levels were low. T. lanuginosus SK was further subjected to UV and N-methyl-N-nitro-N-nitrosoguanidine mutagenesis. The T. lanuginosus MC 134 mutant showed a 1.5 fold increase in xylanase production on oatspelts xylan, compared to the wild type strain. Xylanase production was further enhanced to 3299+/-95 U ml(-1) by using corn cobs under optimized growth conditions. A reduction in xylanase production was observed in a 5 L fermenter. Also, the biobleaching efficiency of crude xylanase was evaluated on bagasse pulp, and a brightness of 46.07+/-0.05% was observed with the use of 50 U of crude xylanase per gram of pulp. This brightness was 3.6 points higher than that of the untreated samples. Reducing sugars (25.78+/-0.14 mg g(-1)) and UV-absorbing lignin-derived compound values were considerably higher in xylanase-treated samples. T. lanuginosus MC 134 has a potential application in the pulp and paper industries.

  10. Comparative study of alkaline hydrogen peroxide and organosolv pretreatments of sugarcane bagasse to improve the overall sugar yield.

    PubMed

    Yu, Hailong; You, Yanzhi; Lei, Fuhou; Liu, Zuguang; Zhang, Weiming; Jiang, Jianxin

    2015-01-01

    Green liquor (GL) combined with H2O2 (GL-H2O2) and green liquor (GL) combined with ethanol (GL-ethanol) were chosen for treating sugarcane bagasse. Results showed that the glucose yield (calculated from the glucose content as a percentage of the theoretical glucose available in the substrates)of sugarcane bagasse from GL-ethanol pretreatment (97.7%) was higher than that from GL-H2O2 pretreatment (41.7%) after 72h hydrolysis with 18 filter paper unit (FPU)/g-cellulose for cellulase, 27,175 cellobiase units (CBU)/g-cellulose for β-glucosidase. Furthermore, about 94.1% of xylan was converted to xylose after GL-ethanol pretreatment without additional xylanase, while the xylose yield was only 29.2% after GL-H2O2 pretreatment. Scanning electron microscopy showed that GL-ethanol pretreatment could break up the fiber severely. Moreover, GL-ethanol pretreated substrate was more accessible to cellulase and more hydrophilic than that of GL-H2O2 pretreated. Therefore, GL-ethanol pretreatment is a promising method for improving the overall sugar (glucose and xylan) yield of sugarcane bagasse. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Complete genome sequence of Geobacillus thermoglucosidasius C56-YS93, a novel biomass degrader isolated from obsidian hot spring in Yellowstone National Park.

    PubMed

    Brumm, Phillip J; Land, Miriam L; Mead, David A

    2015-01-01

    Geobacillus thermoglucosidasius C56-YS93 was one of several thermophilic organisms isolated from Obsidian Hot Spring, Yellowstone National Park, Montana, USA under permit from the National Park Service. Comparison of 16 S rRNA sequences confirmed the classification of the strain as a G. thermoglucosidasius species. The genome was sequenced, assembled, and annotated by the DOE Joint Genome Institute and deposited at the NCBI in December 2011 (CP002835). The genome of G. thermoglucosidasius C56-YS93 consists of one circular chromosome of 3,893,306 bp and two circular plasmids of 80,849 and 19,638 bp and an average G + C content of 43.93 %. G. thermoglucosidasius C56-YS93 possesses a xylan degradation cluster not found in the other G. thermoglucosidasius sequenced strains. This cluster appears to be related to the xylan degradation cluster found in G. stearothermophilus. G. thermoglucosidasius C56-YS93 possesses two plasmids not found in the other two strains. One plasmid contains a novel gene cluster coding for proteins involved in proline degradation and metabolism, the other contains a collection of mostly hypothetical proteins.

  12. Potential of a gypsum-free composting process of wheat straw for mushroom production

    PubMed Central

    Mouthier, Thibaut M. B.; Kilic, Baris; Vervoort, Pieter; Gruppen, Harry

    2017-01-01

    Wheat straw based composting generates a selective substrate for mushroom production. The first phase of this process requires 5 days, and a reduction in time is wished. Here, we aim at understanding the effect of gypsum on the duration of the first phase and the mechanism behind it. Hereto, the regular process with gypsum addition and the same process without gypsum were studied during a 5-day period. The compost quality was evaluated based on compost lignin composition analysed by py-GC/MS and its degradability by a commercial (hemi-)cellulolytic enzyme cocktail. The composting phase lead to the decrease of the pyrolysis products 4-vinylphenol and 4-vinylguaiacol that can be associated with p-coumarates and ferulates linking xylan and lignin. In the regular compost, the enzymatic conversion reached 32 and 39% for cellulose, and 23 and 32% for xylan after 3 and 5 days, respectively. In absence of gypsum similar values were reached after 2 and 4 days, respectively. Thus, our data show that in absence of gypsum the desired compost quality was reached 20% earlier compared to the control process. PMID:28982119

  13. Regulation of the Feruloyl Esterase (faeA) Gene from Aspergillus niger

    PubMed Central

    de Vries, Ronald P.; Visser, Jaap

    1999-01-01

    Feruloyl esterases can remove aromatic residues (e.g., ferulic acid) from plant cell wall polysaccharides (xylan, pectin) and are essential for complete degradation of these polysaccharides. Expression of the feruloyl esterase-encoding gene (faeA) from Aspergillus niger depends on d-xylose (expression is mediated by XlnR, the xylanolytic transcriptional activator) and on a second system that responds to aromatic compounds with a defined ring structure, such as ferulic acid and vanillic acid. Several compounds were tested, and all of the inducing compounds contained a benzene ring which had a methoxy group at C-3 and a hydroxy group at C-4 but was not substituted at C-5. Various aliphatic groups occurred at C-1. faeA expression in the presence of xylose or ferulic acid was repressed by glucose. faeA expression in the presence of ferulic acid and xylose was greater than faeA expression in the presence of either compound alone. The various inducing systems allow A. niger to produce feruloyl esterase not only during growth on xylan but also during growth on other ferulic acid-containing cell wall polysaccharides, such as pectin. PMID:10584009

  14. Simultaneous saccharification and co-fermentation of paper sludge to ethanol by Saccharomyces cerevisiae RWB222--Part I: kinetic modeling and parameters.

    PubMed

    Zhang, Jiayi; Shao, Xiongjun; Townsend, Oliver V; Lynd, Lee R

    2009-12-01

    A kinetic model was developed to predict batch simultaneous saccharification and co-fermentation (SSCF) of paper sludge by the xylose-utilizing yeast Saccharomyces cerevisiae RWB222 and the commercial cellulase preparation Spezyme CP. The model accounts for cellulose and xylan enzymatic hydrolysis and competitive uptake of glucose and xylose. Experimental results show that glucan and xylan enzymatic hydrolysis are highly correlated, and that the low concentrations of xylose encountered during SSCF do not have a significant inhibitory effect on enzymatic hydrolysis. Ethanol is found to not only inhibit the specific growth rate, but also to accelerate cell death. Glucose and xylose uptake rates were found to be competitively inhibitory, but this did not have a large impact during SSCF because the sugar concentrations are low. The model was used to evaluate which constants had the greatest impact on ethanol titer for a fixed substrate loading, enzyme loading, and fermentation time. The cellulose adsorption capacity and cellulose hydrolysis rate constants were found to have the greatest impact among enzymatic hydrolysis related constants, and ethanol yield and maximum ethanol tolerance had the greatest impact among fermentation related constants.

  15. Separation of Glucose and Pentose Sugars by Selective Enzyme Hydrolysis of AFEX-Treated Corn Fiber

    NASA Astrophysics Data System (ADS)

    Hanchar, Robert J.; Teymouri, Farzaneh; Nielson, Chandra D.; McCalla, Darold; Stowers, Mark D.

    A process was developed to fractionate corn fiber into glucose- and pentose-rich fractions. Corn fiber was ammonia fiber explosion treated at 90°C, using 1 g anhydrous ammonia per gram of dry biomass, 60% moisture, and 30-min residence time. Twenty four hour hydrolysis of ammonia fiber explosion-treated corn fiber with cellulase converted 83% of available glucanto-glucose. In this hydrolysis the hemicellulose was partially broken down with 81% of the xylan and 68% of the arabinan being contained in the hydrolysate after filtration to remove lignin and other insoluble material. Addition of ethanol was used to precipitate and recover the solubilized hemicellulose from the hydrolysate, followed by hydrolysis with 2% (v/v) sulfuric acid to convert the recovered xylan and arabinan to monomeric sugars. Using this method, 57% of xylose and 54% of arabinose available in corn fiber were recovered in a pentose-rich stream. The carbohydrate composition of the pentose-enriched stream was 5% glucose, 57% xylose, 27% arabinose, and 11% galactose. The carbohydrate composition of the glucose-enriched stream was 87% glucose, 5% xylose, 6% arabinose, and 1% galactose, and contained 83% of glucose available from the corn fiber.

  16. Complete genome sequence of Geobacillus thermoglucosidasius C56-YS93, a novel biomass degrader isolated from obsidian hot spring in Yellowstone National Park

    DOE PAGES

    Brumm, Phillip J.; Land, Miriam L.; Mead, David A.

    2015-10-05

    Geobacillus thermoglucosidasius C56-YS93 was one of several thermophilic organisms isolated from Obsidian Hot Spring, Yellowstone National Park, Montana, USA under permit from the National Park Service. Comparison of 16 S rRNA sequences confirmed the classification of the strain as a G. thermoglucosidasius species. We sequenced the genome, assembled, and annotated by the DOE Joint Genome Institute and deposited at the NCBI in December 2011 (CP002835). Moreover, the genome of G. thermoglucosidasius C56-YS93 consists of one circular chromosome of 3,893,306 bp and two circular plasmids of 80,849 and 19,638 bp and an average G + C content of 43.93 %. G.more » thermoglucosidasius C56-YS93 possesses a xylan degradation cluster not found in the other G. thermoglucosidasius sequenced strains. Furthermore this cluster appears to be related to the xylan degradation cluster found in G. stearothermophilus. G. thermoglucosidasius C56-YS93 possesses two plasmids not found in the other two strains. One plasmid contains a novel gene cluster coding for proteins involved in proline degradation and metabolism, the other contains a collection of mostly hypothetical proteins.« less

  17. Complete genome sequence of Geobacillus thermoglucosidasius C56-YS93, a novel biomass degrader isolated from obsidian hot spring in Yellowstone National Park

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Brumm, Phillip J.; Land, Miriam L.; Mead, David A.

    Geobacillus thermoglucosidasius C56-YS93 was one of several thermophilic organisms isolated from Obsidian Hot Spring, Yellowstone National Park, Montana, USA under permit from the National Park Service. Comparison of 16 S rRNA sequences confirmed the classification of the strain as a G. thermoglucosidasius species. We sequenced the genome, assembled, and annotated by the DOE Joint Genome Institute and deposited at the NCBI in December 2011 (CP002835). Moreover, the genome of G. thermoglucosidasius C56-YS93 consists of one circular chromosome of 3,893,306 bp and two circular plasmids of 80,849 and 19,638 bp and an average G + C content of 43.93 %. G.more » thermoglucosidasius C56-YS93 possesses a xylan degradation cluster not found in the other G. thermoglucosidasius sequenced strains. Furthermore this cluster appears to be related to the xylan degradation cluster found in G. stearothermophilus. G. thermoglucosidasius C56-YS93 possesses two plasmids not found in the other two strains. One plasmid contains a novel gene cluster coding for proteins involved in proline degradation and metabolism, the other contains a collection of mostly hypothetical proteins.« less

  18. Degradable Bone Graft Substitute for Effective Delivery of Multiple Growth Factors in the Treatment of Nonunion Fractures

    DTIC Science & Technology

    2011-10-01

    During this reporting period, a more general set of hydrogel synthesis steps were defined which enables the incorporation of chitosan from multiple...sources and suppliers and still produce a consistent material. Functional behavior of the hydrogel was confirmed with a new source of chitosan ...inducing tissue ingrowth into a subcutaneously injected scaffold loaded with the composite xylan/ chitosan hydrogel. Delivery of new hydrogel treatment for

  19. A Multi-omics Approach to Understand the Microbial Transformation of Lignocellulosic Materials in the Digestive System of the Wood-Feeding Beetle Odontotaenius disjunctus

    NASA Astrophysics Data System (ADS)

    Ceja Navarro, J. A.; Karaoz, U.; White, R. A., III; Lipton, M. S.; Adkins, J.; Mayali, X.; Blackwell, M.; Pett-Ridge, J.; Brodie, E.; Hao, Z.

    2015-12-01

    Odontotaenius disjuctus is a wood feeding beetle that processes large amounts of hardwoods and plays an important role in forest carbon cycling. In its gut, plant material is transformed into simple molecules by sequential processing during passage through the insect's digestive system. In this study, we used multiple 'omics approaches to analyze the distribution of microbial communities and their specific functions in lignocellulose deconstruction within the insect's gut. Fosmid clones were selected and sequenced from a pool of clones based on their expression of plant polymer degrading enzymes, allowing the identification of a wide range of carbohydrate degrading enzymes. Comparison of metagenomes of all gut regions demonstrated the distribution of genes across the beetle gut. Cellulose, starch, and xylan degradation genes were particularly abundant in the midgut and posterior hindgut. Genes involved in hydrogenotrophic production of methane and nitrogenases were more abundant in the anterior hindgut. Assembled contigs were binned into 127 putative genomes representing Bacteria, Archaea, Fungi and Nematodes. Eleven complete genomes were reconstructed allowing to identify linked functions/traits, including organisms with cellulosomes, and a combined potential for cellulose, xylan and starch hydrolysis and nitrogen fixation. A metaproteomic study was conducted to test the expression of the pathways identified in the metagenomic study. Preliminary analyses suggest enrichment of pathways related to hemicellulosic degradation. A complete xylan degradation pathway was reconstructed and GC-MS/MS based metabolomics identified xylobiose and xylose as major metabolite pools. To relate microbial identify to function in the beetle gut, Chip-SIP isotope tracing was conducted with RNA extracted from beetles fed 13C-cellulose. Multiple 13C enriched bacterial groups were detected, mainly in the midgut. Our multi-omics approach has allowed us to characterize the contribution of

  20. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Warnecke, Falk; Warnecke, Falk; Luginbuhl, Peter

    From the standpoints of both basic research and biotechnology, there is considerable interest in reaching a clearer understanding of the diversity of biological mechanisms employed during lignocellulose degradation. Globally, termites are an extremely successful group of wood-degrading organisms and are therefore important both for their roles in carbon turnover in the environment and as potential sources of biochemical catalysts for efforts aimed at converting wood into biofuels. Only recently have data supported any direct role for the symbiotic bacteria in the gut of the termite in cellulose and xylan hydrolysis. Here we use a metagenomic analysis of the bacterial communitymore » resident in the hindgut paunch of a wood-feeding Nasutitermes species to show the presence of a large, diverse set of bacterial genes for cellulose and xylan hydrolysis. Many of these genes were expressed in vivo or had cellulase activity in vitro, and further analyses implicate spirochete and fibrobacter species in gut lignocellulose degradation. New insights into other important symbiotic functions including H{sub 2} metabolism, CO{sub 2}-reductive acetogenesis and N{sub 2} fixation are also provided by this first system-wide gene analysis of a microbial community specialized towards plant lignocellulose degradation. Our results underscore how complex even a 1-{micro}l environment can be.« less

  1. Attack on Lignified Grass Cell Walls by a Facultatively Anaerobic Bacterium

    PubMed Central

    Akin, Danny E.

    1980-01-01

    A filamentous, facultatively anaerobic microorganism that attacked lignified tissue in forage grasses was isolated from rumen fluid with a Bermuda grass-containing anaerobic medium in roll tubes. The microbe, designated 7-1, demonstrated various colony and cellular morphologies under different growth conditions. Scanning electron microscopy revealed that 7-1 attacked lignified cell walls in aerobic and anaerobic culture. 7-1 predominately degraded tissues reacting positively for lignin with the chlorine-sulfite stain (i.e., sclerenchyma in leaf blades and parenchyma in stems) rather than the more resistant acid phloroglucinol-positive tissues (i.e., lignified vascular tissue and sclerenchyma ring in stems), although the latter tissues were occasionally attacked. Turbidimetric tests showed that 7-1 in anaerobic culture grew optimally at 39°C at a pH of 7.4 to 8.0. Tests for growth on plant cell wall carbohydrates showed that 7-1 grew on xylan and pectin slowly in aerobic cultures but not with pectin and only slightly with xylan in anaerobic culture. 7-1 was noncellulolytic as shown by filter paper tests. The microbe used the phenolic acids sinapic, ferulic, and p-coumaric acids as substrates for growth; the more highly methoxylated acids were used more effectively. Images PMID:16345651

  2. Structural differences between the lignin-carbohydrate complexes present in wood and in chemical pulps.

    PubMed

    Lawoko, Martin; Henriksson, Gunnar; Gellerstedt, Göran

    2005-01-01

    Lignin-carbohydrate complexes (LCCs) were prepared in quantitative yield from spruce wood and from the corresponding kraft and oxygen-delignified pulps and were separated into different fractions on the basis of their carbohydrate composition. To obtain an understanding of the differences in lignin structure and reactivity within the various LCC fractions, thioacidolysis in combination with gas chromatography was used to quantify the content of beta-O-4 structures in the lignin. Periodate oxidation followed by determination of methanol was used to quantify the phenolic hydroxyl groups. Furthermore, size exclusion chromatography (SEC) of the thioacidolysis fractions was used to monitor any differences between the original molecular size distribution and that after the delignification processes. Characteristic differences between the various LCC fractions were observed, clearly indicating that two different forms of lignin are present in the wood fiber wall. These forms are linked to glucomannan and xylan, respectively. On pulping, the different LCCs have different reactivities. The xylan-linked lignin is to a large extent degraded, whereas the glucomannan-linked lignin undergoes a partial condensation to form more high molecular mass material. The latter seems to be rather unchanged during a subsequent oxygen-delignification stage. On the basis of these findings, a modified arrangement of the fiber wall polymers is suggested.

  3. Multifunctional cellulase and hemicellulase

    DOEpatents

    Fox, Brian G.; Takasuka, Taichi; Bianchetti, Christopher M.

    2015-09-29

    A multifunctional polypeptide capable of hydrolyzing cellulosic materials, xylan, and mannan is disclosed. The polypeptide includes the catalytic core (cc) of Clostridium thermocellum Cthe_0797 (CelE), the cellulose-specific carbohydrate-binding module CBM3 of the cellulosome anchoring protein cohesion region (CipA) of Clostridium thermocellum (CBM3a), and a linker region interposed between the catalytic core and the cellulose-specific carbohydrate binding module. Methods of using the multifunctional polypeptide are also disclosed.

  4. Screening and production study of microbial xylanase producers from Brazilian Cerrado.

    PubMed

    Alves-Prado, Heloiza Ferreira; Pavezzi, Fabiana Carina; Leite, Rodrigo Simões Ribeiro; de Oliveira, Valéria Maia; Sette, Lara Durães; Dasilva, Roberto

    2010-05-01

    Hemicelluloses are polysaccharides of low molecular weight containing 100 to 200 glycosidic residues. In plants, the xylans or the hemicelluloses are situated between the lignin and the collection of cellulose fibers underneath. The xylan is the most common hemicellulosic polysaccharide in cell walls of land plants, comprising a backbone of xylose residues linked by beta-1,4-glycosidic bonds. So, xylanolytic enzymes from microorganism have attracted a great deal of attention in the last decade, particularly because of their biotechnological characteristics in various industrial processes, related to food, feed, ethanol, pulp, and paper industries. A microbial screening of xylanase producer was carried out in Brazilian Cerrado area in Selviria city, Mato Grosso do Sul State, Brazil. About 50 bacterial strains and 15 fungal strains were isolated from soil sample at 35 degrees C. Between these isolated microorganisms, a bacterium Lysinibacillus sp. and a fungus Neosartorya spinosa as good xylanase producers were identified. Based on identification processes, Lysinibacillus sp. is a new species and the xylanase production by this bacterial genus was not reported yet. Similarly, it has not reported about xylanase production from N. spinosa. The bacterial strain P5B1 identified as Lysinibacillus sp. was cultivated on submerged fermentation using as substrate xylan, wheat bran, corn straw, corncob, and sugar cane bagasse. Corn straw and wheat bran show a good xylanase activity after 72 h of fermentation. A fungus identified as N. spinosa (strain P2D16) was cultivated on solid-state fermentation using as substrate source wheat bran, wheat bran plus sawdust, corn straw, corncob, cassava bran, and sugar cane bagasse. Wheat bran and corncobs show the better xylanase production after 72 h of fermentation. Both crude xylanases were characterized and a bacterial xylanase shows optimum pH for enzyme activity at 6.0, whereas a fungal xylanase has optimum pH at 5.0-5.5. They were

  5. Developing a xylanase XYNZG from Plectosphaerella cucumerina for baking by heterologously expressed in Kluyveromyces lactis.

    PubMed

    Zhan, Fei Xiang; Wang, Qin Hong; Jiang, Si Jing; Zhou, Yu Ling; Zhang, Gui Min; Ma, Yan He

    2014-12-16

    Xylanase can replace chemical additives to improve the volume and sensory properties of bread in the baking. Suitable baking xylanase with improved yield will promote the application of xylanase in baking industry. The xylanase XYNZG from the Plectosphaerella cucumerina has been previously characterized by heterologous expression in Pichia pastoris. However, P. pastoris is not a suitable host for xylanase to be used in the baking process since P. pastoris does not have GRAS (Generally Regarded As Safe) status and requires large methanol supplement during the fermentation in most conditions, which is not allowed to be used in the food industry. Kluyveromyces lactis, as another yeast expression host, has a GRAS status, which has been successfully used in food and feed applications. No previous work has been reported concerning the heterologous expression of xylanase gene xynZG in K. lactis with an aim for application in baking. The xylanase gene xynZG from the P. cucumerina was heterologously expressed in K. lactis. The recombinant protein XYNZG in K. lactis presented an approximately 19 kDa band on SDS-PAGE and zymograms analysis. Transformant with the highest halo on the plate containing the RBB-xylan (Remazol Brilliant Blue-xylan) was selected for the flask fermentation in different media. The results indicated that the highest activity of 115 U/ml at 72 h was obtained with the YLPU medium. The mass spectrometry analysis suggested that the hydrolytic products of xylan by XYNZG were mainly xylobiose and xylotriose. The results of baking trials indicated that the addition of XYNZG could reduce the kneading time of dough, increase the volume of bread, improve the texture, and have more positive effects on the sensory properties of bread. Xylanase XYNZG is successfully expressed in K. lactis, which exhibits the highest activity among the published reports of the xylanase expression in K. lactis. The recombinant XYNZG can be used to improve the volume and sensory

  6. Characterization of hemicellulase and cellulase from the extremely thermophilic bacterium Caldicellulosiruptor owensensis and their potential application for bioconversion of lignocellulosic biomass without pretreatment.

    PubMed

    Peng, Xiaowei; Qiao, Weibo; Mi, Shuofu; Jia, Xiaojing; Su, Hong; Han, Yejun

    2015-01-01

    Pretreatment is currently the common approach for improving the efficiency of enzymatic hydrolysis on lignocellulose. However, the pretreatment process is expensive and will produce inhibitors such as furan derivatives and phenol derivatives. If the lignocellulosic biomass can efficiently be saccharified by enzymolysis without pretreatment, the bioconversion process would be simplified. The genus Caldicellulosiruptor, an obligatory anaerobic and extreme thermophile can produce a diverse set of glycoside hydrolases (GHs) for deconstruction of lignocellulosic biomass. It gives potential opportunities for improving the efficiency of converting native lignocellulosic biomass to fermentable sugars. Both of the extracellular (extra-) and intracellular (intra-) enzymes of C. owensensis cultivated on corncob xylan or xylose had cellulase (including endoglucanase, cellobiohydrolase and β-glucosidase) and hemicellulase (including xylanase, xylosidase, arabinofuranosidase and acetyl xylan esterase) activities. The enzymes of C. owensensis had high ability for degrading hemicellulose of native corn stover and corncob with the conversion rates of xylan 16.7 % and araban 60.0 %. Moreover, they had remarkable synergetic function with the commercial enzyme cocktail Cellic CTec2 (Novoyzmes). When the native corn stover and corncob were respectively, sequentially hydrolyzed by the extra-enzymes of C. owensensis and CTec2, the glucan conversion rates were 31.2 and 37.9 %,which were 1.7- and 1.9-fold of each control (hydrolyzed by CTec2 alone), whereas the glucan conversion rates of the steam-exploded corn stover and corncob hydrolyzed by CTec2 alone on the same loading rate were 38.2 and 39.6 %, respectively. These results show that hydrolysis by the extra-enzyme of C. owensensis made almost the same contribution as steam-exploded pretreatment on degradation of native lignocellulosic biomass. A new process for saccharification of lignocellulosic biomass by sequential hydrolysis

  7. The Dynamics of Plant Cell-Wall Polysaccharide Decomposition in Leaf-Cutting Ant Fungus Gardens

    PubMed Central

    Harholt, Jesper; Willats, William G. T.; Boomsma, Jacobus J.

    2011-01-01

    The degradation of live plant biomass in fungus gardens of leaf-cutting ants is poorly characterised but fundamental for understanding the mutual advantages and efficiency of this obligate nutritional symbiosis. Controversies about the extent to which the garden-symbiont Leucocoprinus gongylophorus degrades cellulose have hampered our understanding of the selection forces that induced large scale herbivory and of the ensuing ecological footprint of these ants. Here we use a recently established technique, based on polysaccharide microarrays probed with antibodies and carbohydrate binding modules, to map the occurrence of cell wall polymers in consecutive sections of the fungus garden of the leaf-cutting ant Acromyrmex echinatior. We show that pectin, xyloglucan and some xylan epitopes are degraded, whereas more highly substituted xylan and cellulose epitopes remain as residuals in the waste material that the ants remove from their fungus garden. These results demonstrate that biomass entering leaf-cutting ant fungus gardens is only partially utilized and explain why disproportionally large amounts of plant material are needed to sustain colony growth. They also explain why substantial communities of microbial and invertebrate symbionts have evolved associations with the dump material from leaf-cutting ant nests, to exploit decomposition niches that the ant garden-fungus does not utilize. Our approach thus provides detailed insight into the nutritional benefits and shortcomings associated with fungus-farming in ants. PMID:21423735

  8. Profile of Secreted Hydrolases, Associated Proteins, and SlpA in Thermoanaerobacterium saccharolyticum during the Degradation of Hemicellulose

    PubMed Central

    Currie, D. H.; Guss, A. M.; Herring, C. D.; Giannone, R. J.; Johnson, C. M.; Lankford, P. K.; Brown, S. D.; Hettich, R. L.

    2014-01-01

    Thermoanaerobacterium saccharolyticum, a Gram-positive thermophilic anaerobic bacterium, grows robustly on insoluble hemicellulose, which requires a specialized suite of secreted and transmembrane proteins. We report here the characterization of proteins secreted by this organism. Cultures were grown on hemicellulose, glucose, xylose, starch, and xylan in pH-controlled bioreactors, and samples were analyzed via spotted microarrays and liquid chromatography-mass spectrometry. Key hydrolases and transporters employed by T. saccharolyticum for growth on hemicellulose were, for the most part, hitherto uncharacterized and existed in two clusters (Tsac_1445 through Tsac_1464 for xylan/xylose and Tsac_1344 through Tsac_1349 for starch). A phosphotransferase system subunit, Tsac_0032, also appeared to be exclusive to growth on glucose. Previously identified hydrolases that showed strong conditional expression changes included XynA (Tsac_1459), XynC (Tsac_0897), and a pullulanase, Apu (Tsac_1342). An omnipresent transcript and protein making up a large percentage of the overall secretome, Tsac_0361, was tentatively identified as the primary S-layer component in T. saccharolyticum, and deletion of the Tsac_0361 gene resulted in gross morphological changes to the cells. The view of hemicellulose degradation revealed here will be enabling for metabolic engineering efforts in biofuel-producing organisms that degrade cellulose well but lack the ability to catabolize C5 sugars. PMID:24907337

  9. Profile of secreted hydrolases, associated proteins, and SlpA in Thermoanaerobacterium saccharolyticum during the degradation of hemicellulose.

    PubMed

    Currie, D H; Guss, A M; Herring, C D; Giannone, R J; Johnson, C M; Lankford, P K; Brown, S D; Hettich, R L; Lynd, L R

    2014-08-01

    Thermoanaerobacterium saccharolyticum, a Gram-positive thermophilic anaerobic bacterium, grows robustly on insoluble hemicellulose, which requires a specialized suite of secreted and transmembrane proteins. We report here the characterization of proteins secreted by this organism. Cultures were grown on hemicellulose, glucose, xylose, starch, and xylan in pH-controlled bioreactors, and samples were analyzed via spotted microarrays and liquid chromatography-mass spectrometry. Key hydrolases and transporters employed by T. saccharolyticum for growth on hemicellulose were, for the most part, hitherto uncharacterized and existed in two clusters (Tsac_1445 through Tsac_1464 for xylan/xylose and Tsac_1344 through Tsac_1349 for starch). A phosphotransferase system subunit, Tsac_0032, also appeared to be exclusive to growth on glucose. Previously identified hydrolases that showed strong conditional expression changes included XynA (Tsac_1459), XynC (Tsac_0897), and a pullulanase, Apu (Tsac_1342). An omnipresent transcript and protein making up a large percentage of the overall secretome, Tsac_0361, was tentatively identified as the primary S-layer component in T. saccharolyticum, and deletion of the Tsac_0361 gene resulted in gross morphological changes to the cells. The view of hemicellulose degradation revealed here will be enabling for metabolic engineering efforts in biofuel-producing organisms that degrade cellulose well but lack the ability to catabolize C5 sugars. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  10. Structural changes of corn stover lignin during acid pretreatment.

    PubMed

    Moxley, Geoffrey; Gaspar, Armindo Ribeiro; Higgins, Don; Xu, Hui

    2012-09-01

    In this study, raw corn stover was subjected to dilute acid pretreatments over a range of severities under conditions similar to those identified by the National Renewable Energy Laboratory (NREL) in their techno-economic analysis of biochemical conversion of corn stover to ethanol. The pretreated corn stover then underwent enzymatic hydrolysis with yields above 70 % at moderate enzyme loading conditions. The enzyme exhausted lignin residues were characterized by ³¹P NMR spectroscopy and functional moieties quantified and correlated to enzymatic hydrolysis yields. Results from this study indicated that both xylan solubilization and lignin degradation are important for improving the enzyme accessibility and digestibility of dilute acid pretreated corn stover. At lower pretreatment temperatures, there is a good correlation between xylan solubilization and cellulose accessibility. At higher pretreatment temperatures, lignin degradation correlated better with cellulose accessibility, represented by the increase in phenolic groups. During acid pretreatment, the ratio of syringyl/guaiacyl functional groups also gradually changed from less than 1 to greater than 1 with the increase in pretreatment temperature. This implies that more syringyl units are released from lignin depolymerization of aryl ether linkages than guaiacyl units. The condensed phenolic units are also correlated with the increase in pretreatment temperature up to 180 °C, beyond which point condensation reactions may overtake the hydrolysis of aryl ether linkages as the dominant reactions of lignin, thus leading to decreased cellulose accessibility.

  11. Profile of Secreted Hydrolases, Associated Proteins, and SlpA in Thermoanaerobacterium saccharolyticum during the Degradation of Hemicellulose

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Currie, Devin; Guss, Adam M; Herring, Christopher

    2014-01-01

    Thermoanaerobacterium saccharolyticum, a Gram-positive thermophilic anaerobic bacterium, grows robustly on insoluble hemicellulose, which requires a specialized suite of secreted and transmembrane proteins. We report here the characterization of proteins secreted by this organism. Cultures were grown on hemicellulose, glucose, xylose, starch, and xylan in pH-controlled bioreactors, and samples were analyzed via spotted microarrays and liquid chromatography-mass spectrometry. Key hydrolases and transporters employed by T. saccharolyticum for growth on hemicellulose were, for the most part, hitherto uncharacterized and existed in two clusters (Tsac_1445 through Tsac_1464 for xylan/xylose and Tsac_1344 through Tsac_1349 for starch). A phosphotransferase system subunit, Tsac_0032, also appeared tomore » be exclusive to growth on glucose. Previously identified hydrolases that showed strong conditional expression changes included XynA (Tsac_1459), XynC (Tsac_0897), and a pullulanase, Apu (Tsac_1342). An omnipresent transcript and protein making up a large percentage of the overall secretome, Tsac_0361, was tentatively identified as the primary S-layer component in T. saccharolyticum, and deletion of the Tsac_0361 gene resulted in gross morphological changes to the cells. The view of hemicellulose degradation revealed here will be enabling for metabolic engineering efforts in biofuel-producing organisms that degrade cellulose well but lack the ability to catabolize C5 sugars« less

  12. Pore-scale dynamics of enzyme adsorption, swelling and reactive dissolution determine sugar yield in hemicellulose hydrolysis for biofuel production

    PubMed Central

    Dutta, Sajal Kanti; Chakraborty, Saikat

    2016-01-01

    Hemicelluloses are the earth’s second most abundant structural polymers, found in lignocellulosic biomass. Efficient enzymatic depolymerization of xylans by cleaving their β-(1 → 4)-glycosidic bonds to produce soluble sugars is instrumental to the cost-effective production of liquid biofuels. Here we show that the multi-scale two-phase process of enzymatic hydrolysis of amorphous hemicelluloses is dominated by its smallest scale–the pores. In the crucial first five hours, two to fourfold swelling of the xylan particles allow the enzymes to enter the pores and undergo rapid non-equilibrium adsorption on the pore surface before they hydrolyze the solid polymers, albeit non-competitively inhibited by the products xylose and xylobiose. Rapid pore-scale reactive dissolution increases the solid carbohydrate’s porosity to 80–90%. This tightly coupled experimental and theoretical study quantifies the complex temporal dynamics of the transport and reaction processes coupled across scales and phases to show that this unique pore-scale phenomenon can be exploited to accelerate the depolymerization of hemicelluloses to monomeric sugars in the first 5–6 h. We find that an ‘optimal substrate loading’ of 5 mg/ml (above which substrate inhibition sets in) accelerates non-equilibrium enzyme adsorption and solid hemicellulose depolymerization at the pore-scale, which contributes three-quarters of the soluble sugars produced for bio-alcohol fermentation. PMID:27905534

  13. Two-stage statistical medium optimization for augmented cellulase production via solid-state fermentation by newly isolated Aspergillus niger HN-1 and application of crude cellulase consortium in hydrolysis of rice straw.

    PubMed

    Sandhu, Simranjeet Kaur; Oberoi, Harinder Singh; Babbar, Neha; Miglani, Kanupriya; Chadha, Bhupinder Singh; Nanda, Dhiraj Kumar

    2013-12-26

    Cellulolytic enzyme production by newly isolated Aspergillus niger HN-1 was statistically optimized using Plackett-Burman and central composite design (CCD). Optimum concentrations of 2, 0.40, 0.01, and 0.60 g L (-1) for KH2PO4, urea, trace elements solution, and CaCl2·2H2O, respectively, were suggested by Design-Expert software. The two-stage optimization process led to a 3- and 2-fold increases in the filter paper cellulase (FP) and β-glucosidase activities, respectively. FP, β-glucosidase, endoglucanase, exopolygalaturonase, cellobiohydrolase, xylanase, α-l-arabinofuranosidase, β-xylosidase, and xylan esterase activities of 36.7 ± 1.54 FPU gds(-1), 252.3 ± 7.4 IU gds(-1), 416.3 ± 22.8 IU gds(-1), 111.2 ± 5.4 IU gds(-1), 8.9 ± 0.50 IU gds(-1), 2593.5 ± 78.9 IU gds(-1), 79.4 ± 4.3 IU gds(-1), 180.8 ± 9.3 IU gds(-1), and 288.7 ± 11.8 IU gds(-1), respectively, were obtained through solid-state fermentation during the validation studies. Hydrolysis of alkali-treated rice straw with crude cellulases resulted in about 84% glucan to glucose, 89% xylan to xylose, and 91% arabinan to arabinose conversions, indicating potential for biomass hydrolysis by the crude cellulase consortium obtained in this study.

  14. A xylanase with broad pH and temperature adaptability from Streptomyces megasporus DSM 41476, and its potential application in brewing industry.

    PubMed

    Qiu, Zhenhua; Shi, Pengjun; Luo, Huiying; Bai, Yingguo; Yuan, Tiezheng; Yang, Peilong; Liu, Suchun; Yao, Bin

    2010-05-05

    A xylanase gene, xynAM6, was isolated from the genomic DNA library of Streptomyces megasporus DSM 41476 using colony PCR screening method. The 1440-bp full-length gene encodes a 479-amino acid peptide consisting of a putative signal peptide of 36 residues, a family 10 glycoside hydrolase domain and a family 2 carbohydrate-binding module. The mature peptide of xynAM6 was successfully expressed in Pichia pastoris GS115. The optimal pH and temperature were pH 5.5 and 70°C, respectively. The enzyme showed broad temperature adaptability (>60% of the maximum activity at 50-80°C), had good thermostability at 60°C and 70°C, remained stable at pH 4.0-11.0, and was resistant to most proteases. The Km and Vmax values for oat spelt xylan were 1.68mgml(-1) and 436.76μmolmin(-1)mg(-1), respectively, and 2.33mgml(-1) and 406.93μmolmin(-1)mg(-1) for birchwood xylan, respectively. The hydrolysis products of XYNAM6 were mainly xylose and xylobiose. Addition of XYNAM6 (80U) to the brewery mash significantly reduced the filtration rate and viscosity by 36.33% and 35.51%, respectively. These favorable properties probably make XYNAM6 a good candidate for application in brewing industry. Copyright © 2010 Elsevier Inc. All rights reserved.

  15. The Clarinet Reed: AN Introduction to its Biology, Chemistry, and Physics

    NASA Astrophysics Data System (ADS)

    Casadonte, Donald Jay

    Although clarinet reeds have been used for over two-hundred years, there has been little scientific study of the reed, either from a material science or engineering perspective. This document is intended to be the first large-scale study of the clarinet reed covering its biology, chemistry and physics. The reed is made, most often, from cane--Arundo donax. We present a complete atlas of the anatomy of Arundo donax, and examine the role of each of the cellular components in the clarinet reed performance. We examine the three principal chemical components of the processed clarinet reed: cellulose, xylan, and lignin through the use of instrumental analysis. We examine the breakdown pathways of the clarinet reed, and isolate five: (1) decrystallization of the cellulose microstructure, (2) removal of xylan by saliva, (3) plasticization of the reed material due to alkalai attack in saliva, (4) the culturing of a bacterium, Staph Epidermitis, in the cell wall matrix, (5) density changes due to salival coating of the reed. The physics of the reed is examined, and a finite element model of the modal shapes is presented. We present a theoretical treatment of the two modes of excitation of the reed, a low frequency mode (normal playing mode) due to vortex shedding, and a high frequency mode which is associated with reed squeak.

  16. Heterologous Expression of Aspergillus niger β-d-Xylosidase (XlnD): Characterization on Lignocellulosic Substrates

    NASA Astrophysics Data System (ADS)

    Selig, Michael J.; Knoshaug, Eric P.; Decker, Stephen R.; Baker, John O.; Himmel, Michael E.; Adney, William S.

    The gene encoding a glycosyl hydrolase family 3 xylan 1,4-beta-xylosidase, xlnD, was successfully cloned from Aspergillus niger strain ATCC 10864. The recombinant product was expressed in Aspergillus awamori, purified by column chromatography, and verified by matrix-assisted laser desorption ionization, tandem time of flight (MALDI-TOF/ TOF) mass spectroscopy of tryptic digests. The T max was determined using differential scanning microcalorimetry (DSC) to be 78.2 °C; the K m and k cat were found to be 255 μM and 13.7 s-1, respectively, using pNP-β-d-xylopyranoside as substrate. End-product inhibition by d-xylose was also verified and shown to be competitive; the K i for this inhibition was estimated to be 3.3 mM. XlnD was shown to efficiently hydrolyze small xylo-oligomers to monomeric xylose, making it a critical hydrolytic activity in cases where xylose is to be recovered from biomass conversion processes. In addition, the presence of the XlnD was shown to synergistically enhance the ability of an endoxylanase, XynA from Thermomyces lanuginosus, to convert xylan present in selected pretreated lignocellulosic substrates. Furthermore, the addition of the XynA/XlnD complex was effective in enhancing the ability of a simplified cellulase complex to convert glucan present in the substrates.

  17. Compositional evaluation of selected agro-industrial wastes as valuable sources for the recovery of complex carbohydrates.

    PubMed

    Vojvodić, Aleksandra; Komes, Draženka; Vovk, Irena; Belščak-Cvitanović, Ana; Bušić, Arijana

    2016-11-01

    Re-utilization of various agro-industrial wastes is of growing importance from many aspects. Considering the variety and complexity of such materials, compositional data and compliant methodology is still undergoing many updates and improvements. Present study evaluated sugar beet pulp (SBP), walnut shell (WS), cocoa bean husk (CBH), onion peel (OP) and pea pods (PP) as potentially valuable materials for carbohydrate recovery. Macrocomponent analyses revealed carbohydrate fraction as the most abundant, dominating in dietary fibres. Upon complete acid hydrolysis of sample alcohol insoluble residues, developed procedures of high performance thin-layer chromatography (HPTLC) and high performance liquid chromatography (HPLC) coupled with 3-methyl-1-phenyl-2-pyrazolin-5-one pre-column derivatization (PMP-derivatization) were used for carbohydrate monomeric composition determination. HPTLC exhibited good qualitative features useful for multi-sample rapid analysis, while HPLC superior separation and quantification characteristics. Distinctive monomeric patterns were obtained among samples. OP, SBP and CBH, due to the high galacturonic acid content (20.81%, 13.96% and 6.90% dry matter basis, respectively), may be regarded as pectin sources, while WS and PP as materials abundant in xylan-rich hemicellulose (total xylan content 15.53%, 9.63% dry matter basis, respectively). Present study provides new and valuable compositional data for different plant residual materials and a reference for the application of established methodology. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Pore-scale dynamics of enzyme adsorption, swelling and reactive dissolution determine sugar yield in hemicellulose hydrolysis for biofuel production

    NASA Astrophysics Data System (ADS)

    Dutta, Sajal Kanti; Chakraborty, Saikat

    2016-12-01

    Hemicelluloses are the earth’s second most abundant structural polymers, found in lignocellulosic biomass. Efficient enzymatic depolymerization of xylans by cleaving their β-(1 → 4)-glycosidic bonds to produce soluble sugars is instrumental to the cost-effective production of liquid biofuels. Here we show that the multi-scale two-phase process of enzymatic hydrolysis of amorphous hemicelluloses is dominated by its smallest scale-the pores. In the crucial first five hours, two to fourfold swelling of the xylan particles allow the enzymes to enter the pores and undergo rapid non-equilibrium adsorption on the pore surface before they hydrolyze the solid polymers, albeit non-competitively inhibited by the products xylose and xylobiose. Rapid pore-scale reactive dissolution increases the solid carbohydrate’s porosity to 80-90%. This tightly coupled experimental and theoretical study quantifies the complex temporal dynamics of the transport and reaction processes coupled across scales and phases to show that this unique pore-scale phenomenon can be exploited to accelerate the depolymerization of hemicelluloses to monomeric sugars in the first 5-6 h. We find that an ‘optimal substrate loading’ of 5 mg/ml (above which substrate inhibition sets in) accelerates non-equilibrium enzyme adsorption and solid hemicellulose depolymerization at the pore-scale, which contributes three-quarters of the soluble sugars produced for bio-alcohol fermentation.

  19. Heterologous expression of Aspergillus niger beta-D-xylosidase (XlnD): characterization on lignocellulosic substrates.

    PubMed

    Selig, Michael J; Knoshaug, Eric P; Decker, Stephen R; Baker, John O; Himmel, Michael E; Adney, William S

    2008-03-01

    The gene encoding a glycosyl hydrolase family 3 xylan 1,4-beta-xylosidase, xlnD, was successfully cloned from Aspergillus niger strain ATCC 10864. The recombinant product was expressed in Aspergillus awamori, purified by column chromatography, and verified by matrix-assisted laser desorption ionization, tandem time of flight (MALDI-TOF/TOF) mass spectroscopy of tryptic digests. The T (max) was determined using differential scanning microcalorimetry (DSC) to be 78.2 degrees C; the K (m) and k (cat) were found to be 255 microM and 13.7 s(-1), respectively, using pNP-beta-D-xylopyranoside as substrate. End-product inhibition by D-xylose was also verified and shown to be competitive; the K (i) for this inhibition was estimated to be 3.3 mM. XlnD was shown to efficiently hydrolyze small xylo-oligomers to monomeric xylose, making it a critical hydrolytic activity in cases where xylose is to be recovered from biomass conversion processes. In addition, the presence of the XlnD was shown to synergistically enhance the ability of an endoxylanase, XynA from Thermomyces lanuginosus, to convert xylan present in selected pretreated lignocellulosic substrates. Furthermore, the addition of the XynA/XlnD complex was effective in enhancing the ability of a simplified cellulase complex to convert glucan present in the substrates.

  20. Heterologous Expression of Aspergillus Niger --beta--D-Xylosidase (XInD): Characterization on Lignocellulosic Substrates

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Selig, M. J.; Knoshaug, E. P.; Decker, S. R.

    2008-01-01

    The gene encoding a glycosyl hydrolase family 3 xylan 1,4-beta-xylosidase, xlnD, was successfully cloned from Aspergillus niger strain ATCC 10864. The recombinant product was expressed in Aspergillus awamori, purified by column chromatography, and verified by matrix-assisted laser desorption ionization, tandem time of flight (MALDI-TOF/TOF) mass spectroscopy of tryptic digests. The T{sub max} was determined using differential scanning microcalorimetry (DSC) to be 78.2 C; the K{sub m} and k{sub cat} were found to be 255 {micro}M and 13.7 s{sup -1}, respectively, using {rho}NP-{Beta}-d-xylopyranoside as substrate. End-product inhibition by d-xylose was also verified and shown to be competitive; the K{sub i} formore » this inhibition was estimated to be 3.3 mM. XlnD was shown to efficiently hydrolyze small xylo-oligomers to monomeric xylose, making it a critical hydrolytic activity in cases where xylose is to be recovered from biomass conversion processes. In addition, the presence of the XlnD was shown to synergistically enhance the ability of an endoxylanase, XynA from Thermomyces lanuginosus, to convert xylan present in selected pretreated lignocellulosic substrates. Furthermore, the addition of the XynA/XlnD complex was effective in enhancing the ability of a simplified cellulase complex to convert glucan present in the substrates.« less

  1. Pretreatment of corn stover and hybrid poplar by sodium hydroxide and hydrogen peroxide.

    PubMed

    Gupta, Rajesh; Lee, Y Y

    2010-01-01

    Sodium hydroxide and its derivatives are used as pulping reagents, wherein the spent NaOH is recovered in salt form and reused. In this study, use of low concentration NaOH (1-5%) in pretreatment of corn stover and hybrid poplar was investigated. It was done with the understanding that NaOH can be recovered. One of the main objectives in this study is to explore the potential of H(2)O(2) with NaOH for pretreatment of high lignin substrate such as hybrid poplar. Pretreatment time has not been optimized in this study but held constant at 24 h. Corn stover, after treatment with NaOH under moderate conditions, attains near quantitative glucan digestibility. On the other hand, hybrid poplar requires treatment at higher temperature and NaOH concentration to attain acceptable level of digestibility. Supplementation of hydrogen peroxide in the pretreatment significantly raises delignification and digestibility of hybrid poplar. It was also helpful in retaining the carbohydrates in the treated solids. Retention of hemicellulose after pretreatment provides a significant economic benefit as it eliminates the need for detoxifying hemicellulose sugars. As the residual xylan remaining after pretreatment is an impediment to enzymatic digestion of glucan, supplementation of xylanase has significantly increased the digestibility of glucan as well as xylan of the treated hybrid poplar. (c) 2010 American Institute of Chemical Engineers

  2. Transcription factors for modification of lignin content in plants

    DOEpatents

    Wang, Huanzhong; Chen, Fang; Dixon, Richard A.

    2015-06-02

    The invention provides methods for modifying lignin, cellulose, xylan, and hemicellulose content in plants, and for achieving ectopic lignification and, for instance, secondary cell wall synthesis in pith cells, by altered regulation of a WRKY transcription factor. Nucleic acid constructs for altered WRKY-TF expression are described. Transgenic plants are provided that comprise modified pith cell walls, and lignin, cellulose, and hemicellulose content. Plants described herein may be used, for example, as improved biofuel feedstock and as highly digestible forage crops.

  3. The effect of liquid hot water pretreatment on the chemical-structural alteration and the reduced recalcitrance in poplar.

    PubMed

    Li, Mi; Cao, Shilin; Meng, Xianzhi; Studer, Michael; Wyman, Charles E; Ragauskas, Arthur J; Pu, Yunqiao

    2017-01-01

    Hydrothermal pretreatment using liquid hot water (LHW) is capable of substantially reducing the cell wall recalcitrance of lignocellulosic biomass. It enhances the saccharification of polysaccharides, particularly cellulose, into glucose with relatively low capital required. Due to the close association with biomass recalcitrance, the structural change of the components of lignocellulosic materials during the pretreatment is crucial to understand pretreatment chemistry and advance the bio-economy. Although the LHW pretreatment has been extensively applied and studied, the molecular structural alteration during pretreatment and its significance to reduced recalcitrance have not been well understood. We investigated the effects of LHW pretreatment with different severity factors (log R 0 ) on the structural changes of fast-grown poplar ( Populus trichocarpa ). With the severity factor ranging from 3.6 to 4.2, LHW pretreatment resulted in a substantial xylan solubilization by 50-77% ( w/w , dry matter). The molecular weights of the remained hemicellulose in pretreated solids also have been significantly reduced by 63-75% corresponding to LHW severity factor from 3.6 to 4.2. In addition, LHW had a considerable impact on the cellulose structure. The cellulose crystallinity increased 6-9%, whereas its degree of polymerization decreased 35-65% after pretreatment. We found that the pretreatment severity had an empirical linear correlation with the xylan solubilization ( R 2  = 0.98, r  = + 0.99), hemicellulose molecular weight reduction ( R 2  = 0.97, r  = - 0.96 and R 2  = 0.93, r  = - 0.98 for number-average and weight-average degree of polymerization, respectively), and cellulose crystallinity index increase ( R 2  = 0.98, r  = + 0.99). The LHW pretreatment also resulted in small changes in lignin structure such as decrease of β- O -4' ether linkages and removal of cinnamyl alcohol end group and acetyl group, while the S/G ratio of lignin in LHW

  4. Structure-Specificity Relationships of an Intracellular Xylanase from Geobacillus stearothermophilus

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Solomon,V.; Teplitsky, A.; Shulami, S.

    2007-01-01

    Geobacillus stearothermophilus T-6 is a thermophilic Gram-positive bacterium that produces two selective family 10 xylanases which both take part in the complete degradation and utilization of the xylan polymer. The two xylanases exhibit significantly different substrate specificities. While the extracellular xylanase (XT6; MW 43.8 kDa) hydrolyzes the long and branched native xylan polymer, the intracellular xylanase (IXT6; MW 38.6 kDa) preferentially hydrolyzes only short xylo-oligosaccharides. In this study, the detailed three-dimensional structure of IXT6 is reported, as determined by X-ray crystallography. It was initially solved by molecular replacement and then refined at 1.45 {angstrom} resolution to a final R factormore » of 15.0% and an R{sub free} of 19.0%. As expected, the structure forms the classical ({alpha}/{beta}){sub 8} fold, in which the two catalytic residues (Glu134 and Glu241) are located on the inner surface of the central cavity. The structure of IXT6 was compared with the highly homologous extracellular xylanase XT6, revealing a number of structural differences between the active sites of the two enzymes. In particular, structural differences derived from the unique subdomain in the carboxy-terminal region of XT6, which is completely absent in IXT6. These structural modifications may account for the significant differences in the substrate specificities of these otherwise very similar enzymes.« less

  5. Optimization of alkaline sulfite pretreatment and comparative study with sodium hydroxide pretreatment for improving enzymatic digestibility of corn stover.

    PubMed

    Liu, Huan; Pang, Bo; Wang, Haisong; Li, Haiming; Lu, Jie; Niu, Meihong

    2015-04-01

    In this study, alkaline sulfite pretreatment of corn stover was optimized. The influences of pretreatments on solid yield, delignification, and carbohydrate recovery under different pretreatment conditions and subsequent enzymatic hydrolysis were investigated. The effect of pretreatment was evaluated by enzymatic hydrolysis efficiency and the total sugar yield. The optimum pretreatment conditions were obtained, as follows: the total titratable alkali (TTA) of 12%, liquid/solid ratio of 6:1, temperature of 140 °C, and holding time of 20 min. Under those conditions, the solid yield was 55.24%, and the removal of lignin was 82.68%. Enzymatic hydrolysis rates of glucan and xylan for pretreated corn stover were 85.38% and 70.36%, and the total sugar yield was 74.73% at cellulase loading of 20 FPU/g and β-glucosidase loading of 10 IU/g for 48 h. Compared with sodium hydroxide pretreatment with the same amount of total titratable alkali, the total sugar yield was raised by about 10.43%. Additionally, the corn stover pretreated under the optimum pretreatment conditions was beaten by PFI at 1500 revolutions. After beating, enzymatic hydrolysis rates of glucan and xylan were 89.74% and 74.06%, and the total sugar yield was 78.58% at the same enzymatic hydrolysis conditions. Compared with 1500 rpm of PFI beating after sodium pretreatment with the same amount of total titratable alkali, the total sugar yield was raised by about 14.05%.

  6. Direct comparison of gluco-oligosaccharide oxidase variants and glucose oxidase: substrate range and H2O2 stability.

    PubMed

    Vuong, Thu V; Foumani, Maryam; MacCormick, Benjamin; Kwan, Rachel; Master, Emma R

    2016-11-21

    Glucose oxidase (GO) activity is generally restricted to glucose and is susceptible to inactivation by H 2 O 2 . By comparison, the Y300A variant of gluco-oligosaccharide oxidase (GOOX) from Sarocladium strictum showed broader substrate range and higher H 2 O 2 stability. Specifically, Y300A exhibited up to 40 times higher activity on all tested sugars except glucose, compared to GO. Moreover, fusion of the Y300A variant to a family 22 carbohydrate binding module from Clostridium thermocellum (CtCBM22A) nearly doubled its catalytic efficiency on glucose, while retaining significant activity on oligosaccharides. In the presence of 200 mM of H 2 O 2 , the recombinant CtCBM22A_Y300A retained 80% of activity on glucose and 100% of activity on cellobiose, the preferred substrate for this enzyme. By contrast, a commercial glucose oxidase reported to contain ≤0.1 units catalase/ mg protein, retained 60% activity on glucose under the same conditions. GOOX variants appear to undergo a different mechanism of inactivation, as a loss of histidine instead of methionine was observed after H 2 O 2 incubation. The addition of CtCBM22A also promoted functional binding of the fusion enzyme to xylan, facilitating its simultaneous purification and immobilization using edible oat spelt xylan, which might benefit the usage of this enzyme preparation in food and baking applications.

  7. Bioprospecting and evolving alternative xylose and arabinose pathway enzymes for use in Saccharomyces cerevisiae.

    PubMed

    Lee, Sun-Mi; Jellison, Taylor; Alper, Hal S

    2016-03-01

    Bioprospecting is an effective way to find novel enzymes from strains with desirable phenotypes. Such bioprospecting has enabled organisms such as Saccharomyces cerevisiae to utilize nonnative pentose sugars. Yet, the efficiency of this pentose catabolism (especially for the case of arabinose) remains suboptimal. Thus, further pathway optimization or identification of novel, optimal pathways is needed. Previously, we identified a novel set of xylan catabolic pathway enzymes from a superior pentose-utilizing strain of Ustilago bevomyces. These enzymes were used to successfully engineer a xylan-utilizing S. cerevisiae through a blended approach of bioprospecting and evolutionary engineering. Here, we expanded this approach to xylose and arabinose catabolic pathway engineering and demonstrated that bioprospected xylose and arabinose catabolic pathways from U. bevomyces offer alternative choices for enabling efficient pentose catabolism in S. cerevisiae. By introducing a novel set of xylose catabolic genes from U. bevomyces, growth rates were improved up to 85 % over a set of traditional Scheffersomyces stipitis pathway genes. In addition, we suggested an alternative arabinose catabolic pathway which, after directed evolution and pathway engineering, enabled S. cerevisiae to grow on arabinose as a sole carbon source in minimal medium with growth rates upwards of 0.05 h(-1). This pathway represents the most efficient growth of yeast on pure arabinose minimal medium. These pathways provide great starting points for further strain development and demonstrate the utility of bioprospecting from U. bevomyces.

  8. Description of a new anaerobic thermophilic bacterium, Thermoanaerobacterium butyriciformans sp. nov.

    PubMed

    López, G; Cañas-Duarte, S J; Pinzón-Velasco, A M; Vega-Vela, N E; Rodríguez, M; Restrepo, S; Baena, S

    2017-03-01

    Strain USBA-019 T , an anaerobic and thermophilic strain, was identified as a new member of the genus Thermoanaerobacterium. USBA-019 T cells are gram-positive, strictly anaerobic, thermophilic, chemoorganotrophic, moderately acidophilic, non-motile, endospore-forming, slightly curved, and rod-shaped. Cells measure 0.4×3.0-7.0μm. Optimal growth occurs at 50-55°C (35-65°C). Optimum pH is 5.0-5.5 (4.0-8.5). Thiosulfate, elemental sulfur and nitrate were utilized as electron acceptors. Fermentation of glucose, lactose, cellobiose, galactose, arabinose, xylose, starch and xylan primarily produced acetate and butyrate. Xylan, starch and cellobiose produced ethanol and starch, cellobiose, galactose, arabinose and mannose produced lactic acid. Phylogenetic analyses based on 16S rRNA gene sequence comparison and genomic relatedness indices show the close relation of USBA-019 T to Thermoanaerobacterium thermostercoris and Thermoanaerobacterium aotearoense (similarity value: 99%). Hybridization of USBA-019 T , Th. thermostercoris DSM22141 T and Th. aotearoense DMS10170 T found DNA-DNA relatedness of 33.2% and 18.2%, respectively. Based on phenotypic, chemotaxonomic and phylogenetic evidence, along with low identity at whole genome level, USBA-019 T is a novel species of the genus Thermoanaerobacterium which we propose to name Thermoanaerobacterium butyriciformans sp. nov. The type strain is USBA-019 T (=CMPUJ U-019 T =DSM 101588 T ). Copyright © 2016 Elsevier GmbH. All rights reserved.

  9. Isolation of Paenibacillus sp. and Variovorax sp. strains from decaying woods and characterization of their potential for cellulose deconstruction.

    PubMed

    Ghio, Silvina; Lorenzo, Gonzalo Sabarís Di; Lia, Verónica; Talia, Paola; Cataldi, Angel; Grasso, Daniel; Campos, Eleonora

    2012-01-01

    Prospection of cellulose-degrading bacteria in natural environments allows the identification of novel cellulases and hemicellulases that could be useful in second-generation bioethanol production. In this work, cellulolytic bacteria were isolated from decaying native forest soils by enrichment on cellulose as sole carbon source. There was a predominance of Gram positive isolates that belonged to the phyla Proteobacteria and Firmicutes. Many primary isolates with cellulolytic activity were not pure cultures. From these consortia, isolation of pure constituents was attempted in order to test the hypothesis whether microbial consortia are needed for full degradation of complex substrates. Two isolates, CB1-2-A-5 and VG-4-A-2, were obtained as the pure constituents of CB1-2 and VG-4 consortia, respectively. Based on 16S RNA sequence, they could be classified as Variovorax paradoxus and Paenibacillus alvei. Noteworthy, only VG-4 consortium showed measurable xylan degrading capacity and signs of filter paper degradation. However, no xylan or filter paper degrading capacities were observed for the pure cultures isolated from it, suggesting that other members of this consortium were necessary for these hydrolyzing activities. Our results indicated that Paenibacillus sp. and Variovorax sp. as well as VG-4 consortium, might be a useful source of hydrolytic enzymes. Moreover, although Variovorax sp. had been previously identified in metagenomic studies of cellulolytic communities, this is the first report on the isolation and characterization of this microorganism as a cellulolytic genus.

  10. Isolation of Paenibacillus sp. and Variovorax sp. strains from decaying woods and characterization of their potential for cellulose deconstruction

    PubMed Central

    Ghio, Silvina; Lorenzo, Gonzalo Sabarís Di; Lia, Verónica; Talia, Paola; Cataldi, Angel; Grasso, Daniel; Campos, Eleonora

    2012-01-01

    Prospection of cellulose-degrading bacteria in natural environments allows the identification of novel cellulases and hemicellulases that could be useful in second-generation bioethanol production. In this work, cellulolytic bacteria were isolated from decaying native forest soils by enrichment on cellulose as sole carbon source. There was a predominance of Gram positive isolates that belonged to the phyla Proteobacteria and Firmicutes. Many primary isolates with cellulolytic activity were not pure cultures. From these consortia, isolation of pure constituents was attempted in order to test the hypothesis whether microbial consortia are needed for full degradation of complex substrates. Two isolates, CB1-2-A-5 and VG-4-A-2, were obtained as the pure constituents of CB1-2 and VG-4 consortia, respectively. Based on 16S RNA sequence, they could be classified as Variovorax paradoxus and Paenibacillus alvei. Noteworthy, only VG-4 consortium showed measurable xylan degrading capacity and signs of filter paper degradation. However, no xylan or filter paper degrading capacities were observed for the pure cultures isolated from it, suggesting that other members of this consortium were necessary for these hydrolyzing activities. Our results indicated that Paenibacillus sp. and Variovorax sp. as well as VG-4 consortium, might be a useful source of hydrolytic enzymes. Moreover, although Variovorax sp. had been previously identified in metagenomic studies of cellulolytic communities, this is the first report on the isolation and characterization of this microorganism as a cellulolytic genus. PMID:23301200

  11. A novel member of glycoside hydrolase family 30 subfamily 8 with altered substrate specificity

    PubMed Central

    St John, Franz J.; Dietrich, Diane; Crooks, Casey; Pozharski, Edwin; González, Javier M.; Bales, Elizabeth; Smith, Kennon; Hurlbert, Jason C.

    2014-01-01

    Endoxylanases classified into glycoside hydrolase family 30 subfamily 8 (GH30-8) are known to hydrolyze the hemicellulosic polysaccharide glucuronoxylan (GX) but not arabinoxylan or neutral xylooligosaccharides. This is owing to the specificity of these enzymes for the α-1,2-linked glucuronate (GA) appendage of GX. Limit hydrolysis of this substrate produces a series of aldouronates each containing a single GA substituted on the xylose penultimate to the reducing terminus. In this work, the structural and biochemical characterization of xylanase 30A from Clostridium papyro­solvens (CpXyn30A) is presented. This xylanase possesses a high degree of amino-acid identity to the canonical GH30-8 enzymes, but lacks the hallmark β8–α8 loop region which in part defines the function of this GH30 subfamily and its role in GA recognition. CpXyn30A is shown to have a similarly low activity on all xylan substrates, while hydrolysis of xylohexaose revealed a competing transglycosylation reaction. These findings are directly compared with the model GH30-8 enzyme from Bacillus subtilis, XynC. Despite its high sequence identity to the GH30-8 enzymes, CpXyn30A does not have any apparent specificity for the GA appendage. These findings confirm that the typically conserved β8–α8 loop region of these enzymes influences xylan substrate specificity but not necessarily β-1,4-xylanase function. PMID:25372685

  12. Reaction Kinetic Model of Dilute Acid-Catalyzed Hemicellulose Hydrolysis of Corn Stover under High-Solid Conditions

    DOE PAGES

    Shi, Suan; Guan, Wenjian; Kang, Li; ...

    2017-09-13

    High solid conditions are desirable in pretreatment of lignocellulosic biomass. An advanced dilute-acid pretreatment reactor has been developed at National Renewable Energy Laboratory (NREL). It is a continuous auger-driven reactor that can be operated with high-solid charge at high temperature and with short residence time resulting high productivity and high sugar concentration. Here, we investigated the kinetics of the reactions associated with dilute-acid pretreatment of corn stover, covering the reaction conditions of the NREL reactor operation: 155-185 C, 1-2 wt% sulfuric acid concentration, and 1:2 solid to liquid ratio. The experimental data were fitted to a first-order biphasic model whichmore » assumes that xylan is comprised of two different fragments: fast and slow reacting fractions. Due to the high solid loading condition, significant amount of xylose oligomers was observed during the pretreatment. We also included the oligomers as an intermediate entity in the kinetic model. The effect of acid concentration was incorporated into the pre-exponential factor of Arrhenius equation. The kinetic model with bestfit kinetic parameters has shown good agreement with experimental data. The kinetic parameter values of the proposed model were noticeably different from those previously reported. The activation energies of xylan hydrolysis are lower and the acid exponents are higher than the average of literature values. The proposed model can serve as a useful tool for design and operation of pretreatment system pertaining to corn stover.« less

  13. Reaction Kinetic Model of Dilute Acid-Catalyzed Hemicellulose Hydrolysis of Corn Stover under High-Solid Conditions

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Shi, Suan; Guan, Wenjian; Kang, Li

    High solid conditions are desirable in pretreatment of lignocellulosic biomass. An advanced dilute-acid pretreatment reactor has been developed at National Renewable Energy Laboratory (NREL). It is a continuous auger-driven reactor that can be operated with high-solid charge at high temperature and with short residence time resulting high productivity and high sugar concentration. Here, we investigated the kinetics of the reactions associated with dilute-acid pretreatment of corn stover, covering the reaction conditions of the NREL reactor operation: 155-185 C, 1-2 wt% sulfuric acid concentration, and 1:2 solid to liquid ratio. The experimental data were fitted to a first-order biphasic model whichmore » assumes that xylan is comprised of two different fragments: fast and slow reacting fractions. Due to the high solid loading condition, significant amount of xylose oligomers was observed during the pretreatment. We also included the oligomers as an intermediate entity in the kinetic model. The effect of acid concentration was incorporated into the pre-exponential factor of Arrhenius equation. The kinetic model with bestfit kinetic parameters has shown good agreement with experimental data. The kinetic parameter values of the proposed model were noticeably different from those previously reported. The activation energies of xylan hydrolysis are lower and the acid exponents are higher than the average of literature values. The proposed model can serve as a useful tool for design and operation of pretreatment system pertaining to corn stover.« less

  14. Characterization of a novel xylanase gene from rumen content of Hu sheep.

    PubMed

    Wang, Qian; Luo, Yang; He, Bo; Jiang, Lin-Shu; Liu, Jian-Xin; Wang, Jia-Kun

    2015-12-01

    A novel xylanase gene, xyn-lxy, was cloned from a metagenomic fosmid library, which was previously constructed from the rumen contents of Hu sheep and was functionally characterized in Escherichia coli. The open reading frame was composed of 1923 bp and encoded for 640 amino acids, including a catalytic domain of glycosyl hydrolase family 10 and carbohydrate-binding module 9. The gene showed 97 % identity with uncultured bacterium Contig1552 but low similarity with xylanases from known cellulolytic-degrading microorganisms in the rumen. The recombinant XYN-LXY showed a specific activity of 664.7 U mg(-1). The optimal temperature and pH of the enzyme were 50 °C and 6.0, respectively. Specifically, XYN-LXY was exclusively activated by Mn(2+) among all of the cations and reducing agents tested in this study. An enzymatic hydrolysis assay revealed that XYN-LXY degraded birchwood xylan into xylooligosaccharide with a low degree of polymerization. After incubation for 4 h, the concentration of the dominant product, xylobiose, was 2.297 ± 0.175 mg ml(-1) (74.07 % of total product) followed by xylose with a concentration of 0.656 ± 0.010 mg ml(-1) (21.14 % of total product). The XYN-LXY exhibited deep degradation effects on the xylan substrate, which were rarely observed with endo-xylanase, making it a promising candidate for industrial application, especially in biofuel production.

  15. Chemicals and ruminant feed from lignocelluloses by the steaming-extraction process

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Puls, J.; Ayla, C.; Dietrichs, H.H.

    1983-01-01

    Steaming is applicable to lignocelluloses with lower lignin content such as hardwoods and most agricultural residues. The cellulose of steamed fiber materials becomes accessible for enzymatic degradation in spite of the presence of lignin. The hemicelluloses become water soluble. The lignin can be extracted with alkaline or organic solvents. Without further treatment, the steamed material can be used as highly digestible ruminant feed. Steam treatment, however, is most effective after separation of the hemicelluloses. Depending on the starting material, 10-25% hemicelluloses with xylose contents up to 80% can be recovered by aqueous extraction of the fiber material. The xylans andmore » xylan fragments can be used as substrates for chemical, biochemical, or microbial processes. The residual fiber material consists only of cellulose and lignin in highly accessible form for rumen bacteria and fungal cellulases. They are digested by ruminants up to 70-80% and degraded by cellulases without further treatment to 50-60%. In a second extraction step, the lignin can be removed from the fiber material. After controlled steaming at 190/sup 0/C with an optimum yield of hemicellulose, two-thirds of the original lignin present in the starting material can be extracted with dilute alkali. A relatively mild steaming with additional alkaline extraction of lignin is recommended when total utilization of the components including the hemicelluloses is desired. The extracted lignin can be used as a chemical feedstock. 16 references, 6 figures, 3 tables.« less

  16. Structural analysis of cell wall polysaccharides using PACE

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mortimer, Jennifer C.

    The plant cell wall is composed of many complex polysaccharides. The composition and structure of the polysaccharides affect various cell properties including cell shape, cell function and cell adhesion. Many techniques to characterize polysaccharide structure are complicated, requiring expensive equipment and specialized operators e.g. NMR, MALDI-MS. PACE (Polysaccharide Analysis using Carbohydrate gel Electrophoresis) uses a simple, rapid technique to analyze polysaccharide quantity and structure (Goubet et al. 2002). Whilst the method here describes xylan analysis, it can be applied (by use of the appropriate glycosyl hydrolase) to any cell wall polysaccharide.

  17. Use of inedible wheat residues from the KSC-CELSS breadboard facility for production of fungal cellulase

    NASA Technical Reports Server (NTRS)

    Strayer, R. F.; Brannon, M. A.; Garland, J. L.

    1990-01-01

    Cellulose and xylan (a hemicellulose) comprise 50 percent of inedible wheat residue (which is 60 percent of total wheat biomass) produced in the Kennedy Space Center Closed Ecological Life Support System (CELSS) Breadboard Biomass Production Chamber (BPC). These polysaccharides can be converted by enzymatic hydrolysis into useful monosaccharides, thus maximizing the use of BPC volume and energy, and minimizing waste material to be treated. The evaluation of CELSS-derived wheat residues for production for cellulase enzyme complex by Trichoderma reesei and supplemental beta-glucosidase by Aspergillus phoenicis is in progress. Results to date are given.

  18. Maize pollen coat xylanase facilitates pollen tube penetration into silk during sexual reproduction.

    PubMed

    Suen, Der Fen; Huang, Anthony H C

    2007-01-05

    Cell wall hydrolases are well documented to be present on pollen, but their roles on the stigma during sexual reproduction have not been previously demonstrated. We explored the function of the tapetum-synthesized xylanase, ZmXYN1, on maize (Zea mays L.) pollen. Transgenic lines (xyl-less) containing little or no xylanase in the pollen coat were generated with use of an antisense construct of the xylanase gene-coding region driven by the XYN1 gene promoter. Xyl-less and wild-type plants had similar vegetative growth. Electron microscopy revealed no appreciable morphological difference in anther cells and pollen between xyl-less lines and the wild type, whereas immunofluorescence microscopy and biochemical analyses indicated an absence of xylanase on xyl-less pollen. Xyl-less pollen germinated as efficiently as wild-type pollen in vitro in a liquid medium but less so on gel media of increasing solidity or on silk, which is indicative of partial impaired water uptake. Once germinated in vitro or on silk, the xyl-less and wild-type pollen tubes elongated at comparable rates. Tubes of germinated xyl-less pollen on silk did not penetrate into the silk as efficiently as tubes of wild-type pollen, and this lower efficiency could be overcome by the addition of xylanase to the silk. For wild-type pollen, coat xylanase activity on oat spelled xylan in vitro and tube penetration into silk were inhibited by xylose but not glucose. The overall findings indicate that maize pollen coat xylanase facilitates pollen tube penetration into silk via enzymatic xylan hydrolysis.

  19. Genome Sequence of Microbulbifer mangrovi DD-13T Reveals Its Versatility to Degrade Multiple Polysaccharides.

    PubMed

    Imran, Md; Pant, Poonam; Shanbhag, Yogini P; Sawant, Samir V; Ghadi, Sanjeev C

    2017-02-01

    Microbulbifer mangrovi strain DD-13 T is a novel-type species isolated from the mangroves of Goa, India. The draft genome sequence of strain DD-13 comprised 4,528,106 bp with G+C content of 57.15%. Out of 3479 open reading frames, functions for 3488 protein coding sequences were predicted on the basis of similarity with the cluster of orthologous groups. In addition to protein coding sequences, 34 tRNA genes and 3 rRNA genes were detected. Analysis of nucleotide sequence of predicted gene using a Carbohydrate-Active Enzymes (CAZymes) Analysis Toolkit indicates that strain DD-13 encodes a large set of CAZymes including 255 glycoside hydrolases, 76 carbohydrate esterases, 17 polysaccharide lyases, and 113 carbohydrate-binding modules (CBMs). Many genes from strain DD-13 were annotated as carbohydrases specific for degradation of agar, alginate, carrageenan, chitin, xylan, pullulan, cellulose, starch, β-glucan, pectin, etc. Some of polysaccharide-degrading genes were highly modular and were appended at least with one CBM indicating the versatility of strain DD-13 to degrade complex polysaccharides. The cell growth of strain DD-13 was validated using pure polysaccharides such as agarose or alginate as carbon source as well as by using red and brown seaweed powder as substrate. The homologous carbohydrase produced by strain DD-13 during growth degraded the polysaccharide, ensuring the production of metabolizable reducing sugars. Additionally, several other polysaccharides such as carrageenan, xylan, pullulan, pectin, starch, and carboxymethyl cellulose were also corroborated as growth substrate for strain DD-13 and were associated with concomitant production of homologous carbohydrase.

  20. Emended descriptions of Prevotella denticola, Prevotella loescheii, Prevotella veroralis, and Prevotella melaninogenica.

    PubMed

    Wu, C C; Johnson, J L; Moore, W E; Moore, L V

    1992-10-01

    During studies of human periodontal disease, a number of bacterial strains were encountered that, on the basis of results of standard biochemical tests, appeared to be Prevotella buccalis, Prevotella denticola, Prevotella melaninogenica, or Prevotella loescheii. However, use of the standard biochemical tests, cellular fatty acid analyses, and the polyacrylamide gel electrophoresis patterns of soluble proteins resulted in conflicting identifications of these strains. The results of tests for cellobiose fermentation, inulin fermentation, and pigment production were responsible for most of the discordant results. Cellular fatty acid analyses in which the Microbial Identification System was used did not differentiate these strains from validly described species, even though separate library entries were created for them. DNA reassociation determinations in which the S1 nuclease procedure was used showed that cellobiose fermentation and pigment production are variable among strains of P. melaninogenica and P. denticola and that fermentation of xylan is not a reliable characteristic for differentiating P. buccalis from Prevotella veroralis. In contrast to previous indications, most strains of P. veroralis do not ferment xylan. These species can be differentiated by DNA-DNA reassociation and by cellular fatty acid analysis, using the Microbial Identification System, but differentiation by currently described phenotypic characteristics is not reliable. Similarly, P. loescheii and the genetically distinct (but closely related) D1C-20 group cannot be distinguished reliably from each other or from P. veroralis, P. denticola, and P. melaninogenica on the basis of currently described phenotypic tests other than cellular fatty acid composition or, for some species, electrophoretic patterns of soluble whole-cell proteins.

  1. Understanding how the complex molecular architecture of mannan-degrading hydrolases contributes to plant cell wall degradation.

    PubMed

    Zhang, Xiaoyang; Rogowski, Artur; Zhao, Lei; Hahn, Michael G; Avci, Utku; Knox, J Paul; Gilbert, Harry J

    2014-01-24

    Microbial degradation of plant cell walls is a central component of the carbon cycle and is of increasing importance in environmentally significant industries. Plant cell wall-degrading enzymes have a complex molecular architecture consisting of catalytic modules and, frequently, multiple non-catalytic carbohydrate binding modules (CBMs). It is currently unclear whether the specificities of the CBMs or the topology of the catalytic modules are the primary drivers for the specificity of these enzymes against plant cell walls. Here, we have evaluated the relationship between CBM specificity and their capacity to enhance the activity of GH5 and GH26 mannanases and CE2 esterases against intact plant cell walls. The data show that cellulose and mannan binding CBMs have the greatest impact on the removal of mannan from tobacco and Physcomitrella cell walls, respectively. Although the action of the GH5 mannanase was independent of the context of mannan in tobacco cell walls, a significant proportion of the polysaccharide was inaccessible to the GH26 enzyme. The recalcitrant mannan, however, was fully accessible to the GH26 mannanase appended to a cellulose binding CBM. Although CE2 esterases display similar specificities against acetylated substrates in vitro, only CjCE2C was active against acetylated mannan in Physcomitrella. Appending a mannan binding CBM27 to CjCE2C potentiated its activity against Physcomitrella walls, whereas a xylan binding CBM reduced the capacity of esterases to deacetylate xylan in tobacco walls. This work provides insight into the biological significance for the complex array of hydrolytic enzymes expressed by plant cell wall-degrading microorganisms.

  2. Benefits from Tween during enzymic hydrolysis of corn stover

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kaar, W.E.; Holtzapple, M.T.

    1998-08-20

    Corn stover is a potential substrate for fermentation processes. Previous work with corn stover demonstrated that lime pretreatment rendered it digestible by cellulase; however, high sugar yields required very high enzyme loadings. Because cellulase is a significant cost in biomass conversion processes, the present study focused on improving the enzyme efficiency using Tween 20 and Tween 80; Tween 20 is slightly more effective than Tween 80. The recommended pretreatment conditions for the biomass remained unchanged regardless of whether Tween was added during the hydrolysis. The recommended Tween loading was 0.15 g Tween/g dry biomass. The critical relationship was the Tweenmore » loading on the biomass, not the Tween concentration in solution. The 72-h enzymic conversion of pretreated corn stover using 5 FPU cellulase/g dry biomass at 50 C with Tween 20 as part of the medium was 0.85 g/g for cellulose, 0.66 g/g for xylan, and 0.75 for total polysaccharide; addition of Tween improved the cellulose, xylan, and total polysaccharide conversions by 42, 40, and 42%, respectively. Kinetic analyses showed that Tween improved the enzymic absorption constants, which increased the effective hydrolysis rate compared to hydrolysis without Tween. Furthermore, Tween prevented thermal deactivation of the enzymes, which allows for the kinetic advantage of higher temperature hydrolysis. Ultimate digestion studies showed higher conversions for samples containing Tween, indicating a substrate effect. It appears that Tween improves corn stover hydrolysis through three effects: enzyme stabilizer, lignocellulose disrupter, and enzyme effector.« less

  3. Secondary cell walls: biosynthesis, patterned deposition and transcriptional regulation.

    PubMed

    Zhong, Ruiqin; Ye, Zheng-Hua

    2015-02-01

    Secondary walls are mainly composed of cellulose, hemicelluloses (xylan and glucomannan) and lignin, and are deposited in some specialized cells, such as tracheary elements, fibers and other sclerenchymatous cells. Secondary walls provide strength to these cells, which lend mechanical support and protection to the plant body and, in the case of tracheary elements, enable them to function as conduits for transporting water. Formation of secondary walls is a complex process that requires the co-ordinated expression of secondary wall biosynthetic genes, biosynthesis and targeted secretion of secondary wall components, and patterned deposition and assembly of secondary walls. Here, we provide a comprehensive review of genes involved in secondary wall biosynthesis and deposition. Most of the genes involved in the biosynthesis of secondary wall components, including cellulose, xylan, glucomannan and lignin, have been identified and their co-ordinated activation has been shown to be mediated by a transcriptional network encompassing the secondary wall NAC and MYB master switches and their downstream transcription factors. It has been demonstrated that cortical microtubules and microtubule-associated proteins play important roles in the targeted secretion of cellulose synthase complexes, the oriented deposition of cellulose microfibrils and the patterned deposition of secondary walls. Further investigation of many secondary wall-associated genes with unknown functions will provide new insights into the mechanisms controlling the formation of secondary walls that constitute the bulk of plant biomass. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  4. Genomics Review of Holocellulose Deconstruction by Aspergilli

    PubMed Central

    Segato, Fernando; Damásio, André R. L.; de Lucas, Rosymar C.; Squina, Fabio M.

    2014-01-01

    SUMMARY Biomass is constructed of dense recalcitrant polymeric materials: proteins, lignin, and holocellulose, a fraction constituting fibrous cellulose wrapped in hemicellulose-pectin. Bacteria and fungi are abundant in soil and forest floors, actively recycling biomass mainly by extracting sugars from holocellulose degradation. Here we review the genome-wide contents of seven Aspergillus species and unravel hundreds of gene models encoding holocellulose-degrading enzymes. Numerous apparent gene duplications followed functional evolution, grouping similar genes into smaller coherent functional families according to specialized structural features, domain organization, biochemical activity, and genus genome distribution. Aspergilli contain about 37 cellulase gene models, clustered in two mechanistic categories: 27 hydrolyze and 10 oxidize glycosidic bonds. Within the oxidative enzymes, we found two cellobiose dehydrogenases that produce oxygen radicals utilized by eight lytic polysaccharide monooxygenases that oxidize glycosidic linkages, breaking crystalline cellulose chains and making them accessible to hydrolytic enzymes. Among the hydrolases, six cellobiohydrolases with a tunnel-like structural fold embrace single crystalline cellulose chains and cooperate at nonreducing or reducing end termini, splitting off cellobiose. Five endoglucanases group into four structural families and interact randomly and internally with cellulose through an open cleft catalytic domain, and finally, seven extracellular β-glucosidases cleave cellobiose and related oligomers into glucose. Aspergilli contain, on average, 30 hemicellulase and 7 accessory gene models, distributed among 9 distinct functional categories: the backbone-attacking enzymes xylanase, mannosidase, arabinase, and xyloglucanase, the short-side-chain-removing enzymes xylan α-1,2-glucuronidase, arabinofuranosidase, and xylosidase, and the accessory enzymes acetyl xylan and feruloyl esterases. PMID:25428936

  5. Enhancing anaerobic digestibility and phosphorus recovery of dairy manure through microwave-based thermochemical pretreatment.

    PubMed

    Jin, Ying; Hu, Zhenhu; Wen, Zhiyou

    2009-08-01

    Anaerobic digestion and struvite precipitation are two effective ways of treating dairy manure for recovering biogas and phosphorus. Anaerobic digestion of dairy manure is commonly limited by slow fiber degradation, while struvite precipitation is limited by the availability of orthophosphate. The aim of this work is to study the possibility of using microwave-based thermochemical pretreatment to simultaneously enhance manure anaerobic digestibility (through fiber degradation) and struvite precipitation (through phosphorus solubilization). Microwave heating combined with different chemicals (NaOH, CaO, H(2)SO(4), or HCl) enhanced solubilization of manure and degradation of glucan/xylan in dairy manure. However, sulfuric acid-based pretreatment resulted in a low anaerobic digestibility, probably due to the sulfur inhibition and Maillard side reaction. The pretreatments released 20-40% soluble phosphorus and 9-14% ammonium. However, CaO-based pretreatment resulted in lower orthophosphate releases and struvite precipitation efficiency as calcium interferes with phosphate to form calcium phosphate. Collectively, microwave heating combined with NaOH or HCl led to a high anaerobic digestibility and phosphorus recovery. Using these two chemicals, the performance of microwave- and conventional-heating in thermochemical pretreatment was further compared. The microwave heating resulted in a better performance in terms of COD solubilization, glucan/xylan reduction, phosphorus solubilization and anaerobic digestibility. Lastly, temperature and heating time used in microwave treatment were optimized. The optimal values of temperature and heating time were 147 degrees C and 25.3 min for methane production, and 135 degrees C and 26 min for orthophosphate release, respectively.

  6. Tubulin perturbation leads to unexpected cell wall modifications and affects stomatal behaviour in Populus

    DOE PAGES

    Swamy, Prashant S.; Hu, Hao; Pattathil, Sivakumar; ...

    2015-08-05

    Cortical microtubules are integral to plant morphogenesis, cell wall synthesis, and stomatal behaviour, presumably by governing cellulose microfibril orientation. Genetic manipulation of tubulins often leads to abnormal plant development, making it difficult to probe additional roles of cortical microtubules in cell wall biogenesis. Here, it is shown that expressing post-translational C-terminal modification mimics of α-tubulin altered cell wall characteristics and guard cell dynamics in transgenic Populus tremula x alba that otherwise appear normal. 35S promoter-driven transgene expression was high in leaves but unusually low in xylem, suggesting high levels of tubulin transgene expression were not tolerated in wood-forming tissues duringmore » regeneration of transformants. Cellulose, hemicellulose, and lignin contents were unaffected in transgenic wood, but expression of cell wall-modifying enzymes, and extractability of lignin-bound pectin and xylan polysaccharides were increased in developing xylem. The results suggest that pectin and xylan polysaccharides deposited early during cell wall biogenesis are more sensitive to subtle tubulin perturbation than cellulose and matrix polysaccharides deposited later. Tubulin perturbation also affected guard cell behaviour, delaying drought-induced stomatal closure as well as light-induced stomatal opening in leaves. Pectins have been shown to confer cell wall flexibility critical for reversible stomatal movement, and results presented here are consistent with microtubule involvement in this process. In conclusion, taken together, the data show the value of growth-compatible tubulin perturbations for discerning microtubule functions, and add to the growing body of evidence for microtubule involvement in non-cellulosic polysaccharide assembly during cell wall biogenesis.« less

  7. Cell-wall properties contributing to improved deconstruction by alkaline pre-treatment and enzymatic hydrolysis in diverse maize (Zea mays L.) lines

    PubMed Central

    Li, Muyang; Heckwolf, Marlies; Crowe, Jacob D.; Williams, Daniel L.; Magee, Timothy D.; Kaeppler, Shawn M.; de Leon, Natalia; Hodge, David B.

    2015-01-01

    A maize (Zea mays L. subsp. mays) diversity panel consisting of 26 maize lines exhibiting a wide range of cell-wall properties and responses to hydrolysis by cellulolytic enzymes was employed to investigate the relationship between cell-wall properties, cell-wall responses to mild NaOH pre-treatment, and enzymatic hydrolysis yields. Enzymatic hydrolysis of the cellulose in the untreated maize was found to be positively correlated with the water retention value, which is a measure of cell-wall susceptibility to swelling. It was also positively correlated with the lignin syringyl/guaiacyl ratio and negatively correlated with the initial cell-wall lignin, xylan, acetate, and p-coumaric acid (pCA) content, as well as pCA released from the cell wall by pre-treatment. The hydrolysis yield following pre-treatment exhibited statistically significant negative correlations to the lignin content after pre-treatment and positive correlations to the solubilized ferulic acid and pCA. Several unanticipated results were observed, including a positive correlation between initial lignin and acetate content, lack of correlation between acetate content and initial xylan content, and negative correlation between each of these three variables to the hydrolysis yields for untreated maize. Another surprising result was that pCA release was negatively correlated with hydrolysis yields for untreated maize and, along with ferulic acid release, was positively correlated with the pre-treated maize hydrolysis yields. This indicates that these properties that may negatively contribute to the recalcitrance in untreated cell walls may positively contribute to their deconstruction by alkaline pre-treatment. PMID:25871649

  8. Cell-wall properties contributing to improved deconstruction by alkaline pre-treatment and enzymatic hydrolysis in diverse maize ( Zea mays L.) lines

    DOE PAGES

    Li, Muyang; Heckwolf, Marlies; Crowe, Jacob D.; ...

    2015-02-20

    A maize (Zea mays L. subsp. mays) diversity panel consisting of 26 maize lines exhibiting a wide range of cell-wall properties and responses to hydrolysis by cellulolytic enzymes was employed to investigate the relationship between cell-wall properties, cell-wall responses to mild NaOH pre-treatment, and enzymatic hydrolysis yields. Enzymatic hydrolysis of the cellulose in the untreated maize was found to be positively correlated with the water retention value, which is a measure of cell-wall susceptibility to swelling. It was also positively correlated with the lignin syringyl/guaiacyl ratio and negatively correlated with the initial cell-wall lignin, xylan, acetate, and p-coumaric acid (pCA)more » content, as well as pCA released from the cell wall by pre-treatment. The hydrolysis yield following pre-treatment exhibited statistically significant negative correlations to the lignin content after pre-treatment and positive correlations to the solubilized ferulic acid and pCA. Several unanticipated results were observed, including a positive correlation between initial lignin and acetate content, lack of correlation between acetate content and initial xylan content, and negative correlation between each of these three variables to the hydrolysis yields for untreated maize. Also, another surprising result was that pCA release was negatively correlated with hydrolysis yields for untreated maize and, along with ferulic acid release, was positively correlated with the pre-treated maize hydrolysis yields. In conclusion, this indicates that these properties that may negatively contribute to the recalcitrance in untreated cell walls may positively contribute to their deconstruction by alkaline pre-treatment« less

  9. Development and biotechnological application of a novel endoxylanase family GH10 identified from sugarcane soil metagenome.

    PubMed

    Alvarez, Thabata M; Goldbeck, Rosana; dos Santos, Camila Ramos; Paixão, Douglas A A; Gonçalves, Thiago A; Franco Cairo, João Paulo L; Almeida, Rodrigo Ferreira; de Oliveira Pereira, Isabela; Jackson, George; Cota, Junio; Büchli, Fernanda; Citadini, Ana Paula; Ruller, Roberto; Polo, Carla Cristina; de Oliveira Neto, Mario; Murakami, Mário T; Squina, Fabio M

    2013-01-01

    Metagenomics has been widely employed for discovery of new enzymes and pathways to conversion of lignocellulosic biomass to fuels and chemicals. In this context, the present study reports the isolation, recombinant expression, biochemical and structural characterization of a novel endoxylanase family GH10 (SCXyl) identified from sugarcane soil metagenome. The recombinant SCXyl was highly active against xylan from beechwood and showed optimal enzyme activity at pH 6,0 and 45°C. The crystal structure was solved at 2.75 Å resolution, revealing the classical (β/α)8-barrel fold with a conserved active-site pocket and an inherent flexibility of the Trp281-Arg291 loop that can adopt distinct conformational states depending on substrate binding. The capillary electrophoresis analysis of degradation products evidenced that the enzyme displays unusual capacity to degrade small xylooligosaccharides, such as xylotriose, which is consistent to the hydrophobic contacts at the +1 subsite and low-binding energies of subsites that are distant from the site of hydrolysis. The main reaction products from xylan polymers and phosphoric acid-pretreated sugarcane bagasse (PASB) were xylooligosaccharides, but, after a longer incubation time, xylobiose and xylose were also formed. Moreover, the use of SCXyl as pre-treatment step of PASB, prior to the addition of commercial cellulolytic cocktail, significantly enhanced the saccharification process. All these characteristics demonstrate the advantageous application of this enzyme in several biotechnological processes in food and feed industry and also in the enzymatic pretreatment of biomass for feedstock and ethanol production.

  10. Cell-wall properties contributing to improved deconstruction by alkaline pre-treatment and enzymatic hydrolysis in diverse maize ( Zea mays L.) lines

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Li, Muyang; Heckwolf, Marlies; Crowe, Jacob D.

    A maize (Zea mays L. subsp. mays) diversity panel consisting of 26 maize lines exhibiting a wide range of cell-wall properties and responses to hydrolysis by cellulolytic enzymes was employed to investigate the relationship between cell-wall properties, cell-wall responses to mild NaOH pre-treatment, and enzymatic hydrolysis yields. Enzymatic hydrolysis of the cellulose in the untreated maize was found to be positively correlated with the water retention value, which is a measure of cell-wall susceptibility to swelling. It was also positively correlated with the lignin syringyl/guaiacyl ratio and negatively correlated with the initial cell-wall lignin, xylan, acetate, and p-coumaric acid (pCA)more » content, as well as pCA released from the cell wall by pre-treatment. The hydrolysis yield following pre-treatment exhibited statistically significant negative correlations to the lignin content after pre-treatment and positive correlations to the solubilized ferulic acid and pCA. Several unanticipated results were observed, including a positive correlation between initial lignin and acetate content, lack of correlation between acetate content and initial xylan content, and negative correlation between each of these three variables to the hydrolysis yields for untreated maize. Also, another surprising result was that pCA release was negatively correlated with hydrolysis yields for untreated maize and, along with ferulic acid release, was positively correlated with the pre-treated maize hydrolysis yields. In conclusion, this indicates that these properties that may negatively contribute to the recalcitrance in untreated cell walls may positively contribute to their deconstruction by alkaline pre-treatment« less

  11. Global Microarray Analysis of Carbohydrate Use in Alkaliphilic Hemicellulolytic Bacterium Bacillus sp. N16-5

    PubMed Central

    Song, Yajian; Xue, Yanfen; Ma, Yanhe

    2013-01-01

    The alkaliphilic hemicellulolytic bacterium Bacillus sp. N16-5 has a broad substrate spectrum and exhibits the capacity to utilize complex carbohydrates such as galactomannan, xylan, and pectin. In the monosaccharide mixture, sequential utilization by Bacillus sp. N16-5 was observed. Glucose appeared to be its preferential monosaccharide, followed by fructose, mannose, arabinose, xylose, and galactose. Global transcription profiles of the strain were determined separately for growth on six monosaccharides (glucose, fructose, mannose, galactose, arabinose, and xylose) and four polysaccharides (galactomannan, xylan, pectin, and sodium carboxymethylcellulose) using one-color microarrays. Numerous genes potentially related to polysaccharide degradation, sugar transport, and monosaccharide metabolism were found to respond to a specific substrate. Putative gene clusters for different carbohydrates were identified according to transcriptional patterns and genome annotation. Identification and analysis of these gene clusters contributed to pathway reconstruction for carbohydrate utilization in Bacillus sp. N16-5. Several genes encoding putative sugar transporters were highly expressed during growth on specific sugars, suggesting their functional roles. Two phosphoenolpyruvate-dependent phosphotransferase systems were identified as candidate transporters for mannose and fructose, and a major facilitator superfamily transporter was identified as a candidate transporter for arabinose and xylose. Five carbohydrate uptake transporter 1 family ATP-binding cassette transporters were predicted to participate in the uptake of hemicellulose and pectin degradation products. Collectively, microarray data improved the pathway reconstruction involved in carbohydrate utilization of Bacillus sp. N16-5 and revealed that the organism precisely regulates gene transcription in response to fluctuations in energy resources. PMID:23326578

  12. Conduction properties of thin films from a water soluble carbon nanotube/hemicellulose complex

    NASA Astrophysics Data System (ADS)

    Shao, Dongkai; Yotprayoonsak, Peerapong; Saunajoki, Ville; Ahlskog, Markus; Virtanen, Jorma; Kangas, Veijo; Volodin, Alexander; Van Haesendonck, Chris; Burdanova, Maria; Mosley, Connor D. W.; Lloyd-Hughes, James

    2018-04-01

    We have examined the conductive properties of carbon nanotube based thin films, which were prepared via dispersion in water by non-covalent functionalization of the nanotubes with xylan, a type of hemicellulose. Measurements of low temperature conductivity, Kelvin probe force microscopy, and high frequency (THz) conductivity elucidated the intra-tube and inter-tube charge transport processes in this material. The measurements show excellent conductive properties of the as prepared thin films, with bulk conductivity up to 2000 S cm-1. The transport results demonstrate that the hemicellulose does not seriously interfere with the inter-tube conductance.

  13. Fungi of virgin and cultivated soil of Salhiah Desert, Egypt.

    PubMed

    el-Gindy, A A; Saad, R R

    1990-01-01

    27 species and 13 genera of fungi were identified from virgin and cultivated soil of Salhiah. The most abundant species of phosphate solubilizing fungi were Aspergillus nidulans, A. niger, A flavus, Penicillium lilacinum, P. frequentans and Fusarium moniliforme. On cellulose agar the most prevalent species were Chaetomium bostrychodes, C. olivaceum, Humicola fuscoatra, Aspergillus flavus, A. nidulans, A. niger, A. ochraceus, Fusarium solani and F. oxysporum. On xylan agar Aspergillus fumigatus, A. ochraceus, A. niger, A. versicolor and Penicillium frequentans were the most frequent species. On lignin agar only two species were isolated. These are Aspergillus niger and Humicola fuscoatra.

  14. Lignocellulose-Adapted Endo-Cellulase Producing Streptomyces Strains for Bioconversion of Cellulose-Based Materials.

    PubMed

    Ventorino, Valeria; Ionata, Elena; Birolo, Leila; Montella, Salvatore; Marcolongo, Loredana; de Chiaro, Addolorata; Espresso, Francesco; Faraco, Vincenza; Pepe, Olimpia

    2016-01-01

    Twenty-four Actinobacteria strains, isolated from Arundo donax, Eucalyptus camaldulensis and Populus nigra biomass during natural biodegradation and with potential enzymatic activities specific for the degradation of lignocellulosic materials, were identified by a polyphasic approach. All strains belonged to the genus Streptomyces ( S .) and in particular, the most highly represented species was Streptomyces argenteolus representing 50% of strains, while 8 strains were identified as Streptomyces flavogriseus (synonym S. flavovirens ) and Streptomyces fimicarius (synonyms Streptomyces acrimycini, Streptomyces baarnensis, Streptomyces caviscabies , and Streptomyces flavofuscus ), and the other four strains belonged to the species Streptomyces drozdowiczii, Streptomyces rubrogriseus, Streptomyces albolongus , and Streptomyces ambofaciens . Moreover, all Streptomyces strains, tested for endo and exo-cellulase, cellobiase, xylanase, pectinase, ligninase, peroxidase, and laccase activities using qualitative and semi-quantitative methods on solid growth medium, exhibited multiple enzymatic activities (from three to six). The 24 strains were further screened for endo-cellulase activity in liquid growth medium and the four best endo-cellulase producers ( S. argenteolus AE58P, S. argenteolus AE710A, S. argenteolus AE82P, and S. argenteolus AP51A) were subjected to partial characterization and their enzymatic crude extracts adopted to perform saccharification experiments on A. donax pretreated biomass. The degree of cellulose and xylan hydrolysis was evaluated by determining the kinetics of glucose and xylose release during 72 h incubation at 50°C from the pretreated biomass in the presence of cellulose degrading enzymes (cellulase and β-glucosidase) and xylan related activities (xylanase and β-xylosidase). The experiments were carried out utilizing the endo-cellulase activities from the selected S. argenteolus strains supplemented with commercial β-gucosidase and

  15. Switchgrass and Giant Miscanthus Biomass and Theoretical Ethanol Production from Reclaimed Mine Lands

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wolfrum, Edward J; Scagline-Mellor, Steffany; Griggs, Thomas

    Switchgrass (Panicum virgatum L.) and giant miscanthus (Miscanthus x giganteus Greef & Deuter ex Hodkinson & Renvoize) are productive on marginal lands in the eastern USA, but their productivity and composition have not been compared on mine lands. Our objectives were to compare biomass production, composition, and theoretical ethanol yield (TEY) and production (TEP) of these grasses on a reclaimed mined site. Following 25 years of herbaceous cover, vegetation was killed and plots of switchgrass cultivars Kanlow and BoMaster and miscanthus lines Illinois and MBX-002 were planted in five replications. Annual switchgrass and miscanthus yields averaged 5.8 and 8.9 Mgmore » dry matter ha-1, respectively, during 2011 to 2015. Cell wall carbohydrate composition was analyzed via near-infrared reflectance spectroscopy with models based on switchgrass or mixed herbaceous samples including switchgrass and miscanthus. Concentrations were higher for glucan and lower for xylan in miscanthus than in switchgrass but TEY did not differ (453 and 450 L Mg-1, respectively). In response to biomass production, total ethanol production was greater for miscanthus than for switchgrass (5594 vs 3699 L ha-1), did not differ between Kanlow and BoMaster switchgrass (3880 and 3517 L ha-1, respectively), and was higher for MBX-002 than for Illinois miscanthus (6496 vs 4692 L ha-1). Relative to the mixed feedstocks model, the switchgrass model slightly underpredicted glucan and slightly overpredicted xylan concentrations. Estimated TEY was slightly lower from the switchgrass model but both models distinguished genotype, year, and interaction effects similarly. Biomass productivity and TEP were similar to those from agricultural sites with marginal soils.« less

  16. Cell-wall properties contributing to improved deconstruction by alkaline pre-treatment and enzymatic hydrolysis in diverse maize (Zea mays L.) lines.

    PubMed

    Li, Muyang; Heckwolf, Marlies; Crowe, Jacob D; Williams, Daniel L; Magee, Timothy D; Kaeppler, Shawn M; de Leon, Natalia; Hodge, David B

    2015-07-01

    A maize (Zea mays L. subsp. mays) diversity panel consisting of 26 maize lines exhibiting a wide range of cell-wall properties and responses to hydrolysis by cellulolytic enzymes was employed to investigate the relationship between cell-wall properties, cell-wall responses to mild NaOH pre-treatment, and enzymatic hydrolysis yields. Enzymatic hydrolysis of the cellulose in the untreated maize was found to be positively correlated with the water retention value, which is a measure of cell-wall susceptibility to swelling. It was also positively correlated with the lignin syringyl/guaiacyl ratio and negatively correlated with the initial cell-wall lignin, xylan, acetate, and p-coumaric acid (pCA) content, as well as pCA released from the cell wall by pre-treatment. The hydrolysis yield following pre-treatment exhibited statistically significant negative correlations to the lignin content after pre-treatment and positive correlations to the solubilized ferulic acid and pCA. Several unanticipated results were observed, including a positive correlation between initial lignin and acetate content, lack of correlation between acetate content and initial xylan content, and negative correlation between each of these three variables to the hydrolysis yields for untreated maize. Another surprising result was that pCA release was negatively correlated with hydrolysis yields for untreated maize and, along with ferulic acid release, was positively correlated with the pre-treated maize hydrolysis yields. This indicates that these properties that may negatively contribute to the recalcitrance in untreated cell walls may positively contribute to their deconstruction by alkaline pre-treatment. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  17. Analysis of the Arabidopsis IRX9/IRX9-L and IRX14/IRX14-L pairs of glycosyltransferase genes reveals critical contributions to biosynthesis of the hemicellulose glucuronoxylan.

    PubMed

    Wu, Ai-Min; Hörnblad, Emma; Voxeur, Aline; Gerber, Lorenz; Rihouey, Christophe; Lerouge, Patrice; Marchant, Alan

    2010-06-01

    The hemicellulose glucuronoxylan (GX) is a major component of plant secondary cell walls. However, our understanding of GX synthesis remains limited. Here, we identify and analyze two new genes from Arabidopsis (Arabidopsis thaliana), IRREGULAR XYLEM9-LIKE (IRX9-L) and IRX14-LIKE (IRX14-L) that encode glycosyltransferase family 43 members proposed to function during xylan backbone elongation. We place IRX9-L and IRX14-L in a genetic framework with six previously described glycosyltransferase genes (IRX9, IRX10, IRX10-L, IRX14, FRAGILE FIBER8 [FRA8], and FRA8 HOMOLOG [F8H]) and investigate their function in GX synthesis. Double-mutant analysis identifies IRX9-L and IRX14-L as functional homologs of IRX9 and IRX14, respectively. Characterization of irx9 irx10 irx14 fra8 and irx9-L irx10-L irx14-L f8h quadruple mutants allows definition of a set of genes comprising IRX9, IRX10, IRX14, and FRA8 that perform the main role in GX synthesis during vegetative development. The IRX9-L, IRX10-L, IRX14-L, and F8H genes are able to partially substitute for their respective homologs and normally perform a minor function. The irx14 irx14-L double mutant virtually lacks xylan, whereas irx9 irx9-L and fra8 f8h double mutants form lowered amounts of GX displaying a greatly reduced degree of backbone polymerization. Our findings reveal two distinct sets of four genes each differentially contributing to GX biosynthesis.

  18. The impacts of deacetylation prior to dilute acid pretreatment on the bioethanol process

    PubMed Central

    2012-01-01

    Background Dilute acid pretreatment is a promising pretreatment technology for the biochemical production of ethanol from lignocellulosic biomass. During dilute acid pretreatment, xylan depolymerizes to form soluble xylose monomers and oligomers. Because the xylan found in nature is highly acetylated, the formation of xylose monomers requires two steps: 1) cleavage of the xylosidic bonds, and 2) cleavage of covalently bonded acetyl ester groups. Results In this study, we show that the latter may be the rate limiting step for xylose monomer formation. Furthermore, acetyl groups are also found to be a cause of biomass recalcitrance and hydrolyzate toxicity. While the removal of acetyl groups from native corn stover by alkaline de-esterification prior to pretreatment improves overall process yields, the exact impact is highly dependent on the corn stover variety in use. Xylose monomer yields in pretreatment generally increases by greater than 10%. Compared to pretreated corn stover controls, the deacetylated corn stover feedstock is approximately 20% more digestible after pretreatment. Finally, by lowering hydrolyzate toxicity, xylose utilization and ethanol yields are further improved during fermentation by roughly 10% and 7%, respectively. In this study, several varieties of corn stover lots were investigated to test the robustness of the deacetylation-pretreatment-saccharification-fermentation process. Conclusions Deacetylation shows significant improvement on glucose and xylose yields during pretreatment and enzymatic hydrolysis, but it also reduces hydrolyzate toxicity during fermentation, thereby improving ethanol yields and titer. The magnitude of effect is dependent on the selected corn stover variety, with several varieties achieving improvements of greater than 10% xylose yield in pretreatment, 20% glucose yield in low solids enzymatic hydrolysis and 7% overall ethanol yield. PMID:22369467

  19. The Complex Cell Wall Composition of Syncytia Induced by Plant Parasitic Cyst Nematodes Reflects Both Function and Host Plant.

    PubMed

    Zhang, Li; Lilley, Catherine J; Imren, Mustafa; Knox, J Paul; Urwin, Peter E

    2017-01-01

    Plant-parasitic cyst nematodes induce the formation of specialized feeding structures, syncytia, within their host roots. These unique plant organs serve as the sole nutrient resource for development and reproduction throughout the biotrophic interaction. The multinucleate syncytium, which arises through local dissolution of cell walls and protoplast fusion of multiple adjacent cells, has dense cytoplasm containing numerous organelles, surrounded by thickened outer cell walls that must withstand high turgor pressure. However, little is known about how the constituents of the syncytial cell wall and their conformation support its role during nematode parasitism. We used a set of monoclonal antibodies, targeted to a range of plant cell wall components, to reveal the microstructures of syncytial cell walls induced by four of the most economically important cyst nematode species, Globodera pallida , Heterodera glycines , Heterodera avenae and Heterodera filipjevi , in their respective potato, soybean, and spring wheat host roots. In situ fluorescence analysis revealed highly similar cell wall composition of syncytia induced by G. pallida and H. glycines . Both consisted of abundant xyloglucan, methyl-esterified homogalacturonan and pectic arabinan. In contrast, the walls of syncytia induced in wheat roots by H. avenae and H. filipjevi contain little xyloglucan but are rich in feruloylated xylan and arabinan residues, with variable levels of mixed-linkage glucan. The overall chemical composition of syncytial cell walls reflected the general features of root cell walls of the different host plants. We relate specific components of syncytial cell walls, such as abundant arabinan, methyl-esterification status of pectic homogalacturonan and feruloylation of xylan, to their potential roles in forming a network to support both the strength and flexibility required for syncytium function.

  20. Analysis of the Arabidopsis IRX9/IRX9-L and IRX14/IRX14-L Pairs of Glycosyltransferase Genes Reveals Critical Contributions to Biosynthesis of the Hemicellulose Glucuronoxylan1[C][W

    PubMed Central

    Wu, Ai-Min; Hörnblad, Emma; Voxeur, Aline; Gerber, Lorenz; Rihouey, Christophe; Lerouge, Patrice; Marchant, Alan

    2010-01-01

    The hemicellulose glucuronoxylan (GX) is a major component of plant secondary cell walls. However, our understanding of GX synthesis remains limited. Here, we identify and analyze two new genes from Arabidopsis (Arabidopsis thaliana), IRREGULAR XYLEM9-LIKE (IRX9-L) and IRX14-LIKE (IRX14-L) that encode glycosyltransferase family 43 members proposed to function during xylan backbone elongation. We place IRX9-L and IRX14-L in a genetic framework with six previously described glycosyltransferase genes (IRX9, IRX10, IRX10-L, IRX14, FRAGILE FIBER8 [FRA8], and FRA8 HOMOLOG [F8H]) and investigate their function in GX synthesis. Double-mutant analysis identifies IRX9-L and IRX14-L as functional homologs of IRX9 and IRX14, respectively. Characterization of irx9 irx10 irx14 fra8 and irx9-L irx10-L irx14-L f8h quadruple mutants allows definition of a set of genes comprising IRX9, IRX10, IRX14, and FRA8 that perform the main role in GX synthesis during vegetative development. The IRX9-L, IRX10-L, IRX14-L, and F8H genes are able to partially substitute for their respective homologs and normally perform a minor function. The irx14 irx14-L double mutant virtually lacks xylan, whereas irx9 irx9-L and fra8 f8h double mutants form lowered amounts of GX displaying a greatly reduced degree of backbone polymerization. Our findings reveal two distinct sets of four genes each differentially contributing to GX biosynthesis. PMID:20424005

  1. Screening of Thermophilic Bacteria Produce Xylanase from Sapan Sungai Aro Hot Spring South Solok

    NASA Astrophysics Data System (ADS)

    Irdawati, I.; Syamsuardi, S.; Agustien, A.; Rilda, Y.

    2018-04-01

    xylanase is one of the enzymes with great prospects as hemicellulose hydrolyzing enzyme. Global annual market demand for this enzyme reach US 200 million. This enzyme catalyzes the xylan (hemicellulose) reactions breaking into xilooligosakarida and xylose. Xylanase can be applied to various industrial sectors such as bread, sugar xylose, biofuels, especially in bleaching paper (bleaching) pulp. Xylanase Isable to replace conventional chemical bleaching using chlorine that is not friendly for the environment. Currently xylanase production is extracted from the thermophilic bacteria for enzyme stability at high temperatures that are suitable for industrial applications. Thermophilic bacteria can be isolated from a hot spring, one of the which is a source of Sapan Sungai Aro Hot Spring, located in the district South Solok. The aim of this study was to select and identification of thermophilic bacteria can produce xylanase.This roomates is a descriptive study, which was Carried out in the Laboratory of Microbiology, Mathematic and Science Faculty of Padang State University, and Laboratory of Bacteriology, BasoVeterinary Research Center. The research procedure consisted of the preparation and sterilization of materials and tools, medium manufacturing, regeneration, selection and identification. Selection is performed by using a semiquantitative screening plate that contains xylan substrate. Identification is based on microscopic and biochemical characteristics until the genus level.Selection results Showed 12 out of 16 isolates had xilanolitik activity, with the highest activity is SSA2 with xilanolitik index of 0.74. The top five index producehigestxilanolitik isolates that are SSA2, SSA3 and SSA4 identified as Bacillus sp. 1., and SSAS6 and SSA7 is Bacillus sp. 2.

  2. Effects of helix and fingertip mutations on the thermostability of xyn11A investigated by molecular dynamics simulations and enzyme activity assays.

    PubMed

    Sutthibutpong, Thana; Rattanarojpong, Triwit; Khunrae, Pongsak

    2017-12-04

    Local conformational changes and global unfolding pathways of wildtype xyn11A recombinant and its mutated structures were studied through a series of atomistic molecular dynamics (MD) simulations, along with enzyme activity assays at three incubation temperatures to investigate the effects of mutations at three different sites to the thermostability. The first mutation was to replace an unstable negatively charged residue at a surface beta turn near the active site (D32G) by a hydrophobic residue. The second mutation was to create a disulphide bond (S100C/N147C) establishing a strong connection between an alpha helix and a distal beta hairpin associated with the thermally sensitive Thumb loop, and the third mutation add an extra hydrogen bond (A155S) to the same alpha helix. From the MD simulations performed, MM/PBSA energy calculations of the unfolding energy were in a good agreement with the enzyme activities measured from the experiment, as all mutated structures demonstrated the improved thermostability, especially the S100C/N147C proved to be the most stable mutant both by the simulations and the experiment. Local conformational analysis at the catalytic sites and the xylan access region also suggested that mutated xyn11A structures could accommodate xylan binding. However, the analysis of global unfolding pathways showed that structural disruptions at the beta sheet regions near the N-terminal were still imminent. These findings could provide the insight on the molecular mechanisms underlying the enhanced thermostability due to mutagenesis and changes in the protein unfolding pathways for further protein engineering of the GH11 family xylanase enzymes.

  3. Partial Optimization of Endo-1, 4-Β-Xylanase Production by Aureobasidium pullulans Using Agro-Industrial Residues

    PubMed Central

    Nasr, Shaghayegh; Soudi, Mohammad Reza; Hatef Salmanian, Ali; Ghadam, Parinaz

    2013-01-01

    Objective(s) : Although bacteria and molds are the pioneering microorganisms for production of many enzymes, yet yeasts provide safe and reliable sources of enzymes with applications in food and feed. Materials and Methods: Single xylanase producer yeast was isolated from plant residues based on formation of transparent halo zones on xylan agar plates. The isolate showed much greater endo-1, 4-β-xylanase activity of 2.73 IU/ml after optimization of the initial extrinsic conditions. It was shown that the strain was also able to produce β-xylosidase (0.179 IU/ml) and α-arabinofuranosidase (0.063 IU/ml). Identification of the isolate was carried out and the endo-1, 4-β-xylanaseproduction by feeding the yeast cells on agro-industrial residues was optimized using one factor at a time approach. Results: The enzyme producer strain was identified as Aureobasidiumpullulans. Based on the optimization approach, an incubation time of 48 hr at 27°C, inoculum size of 2% (v/v), initial pH value of 4 and agitation rate of 90 rpm were found to be the optimal conditions for achieving maximum yield of the enzyme. Xylan, containing agricultural residues, was evaluated as low-cost alternative carbon source for production of xylanolytic enzymes. The production of xylanase enzyme in media containing wheat bran as the sole carbon source was very similar to that of the medium containing pure beechwoodxylan. Conclusion:This finding indicates the feasibility of growing of A. pullulans strain SN090 on wheat bran as an alternate economical substrate in order for reducing the costs of enzyme production and using this fortified agro-industrial byproduct in formulation of animal feed. PMID:24570830

  4. The Complex Cell Wall Composition of Syncytia Induced by Plant Parasitic Cyst Nematodes Reflects Both Function and Host Plant

    PubMed Central

    Zhang, Li; Lilley, Catherine J.; Imren, Mustafa; Knox, J. Paul; Urwin, Peter E.

    2017-01-01

    Plant–parasitic cyst nematodes induce the formation of specialized feeding structures, syncytia, within their host roots. These unique plant organs serve as the sole nutrient resource for development and reproduction throughout the biotrophic interaction. The multinucleate syncytium, which arises through local dissolution of cell walls and protoplast fusion of multiple adjacent cells, has dense cytoplasm containing numerous organelles, surrounded by thickened outer cell walls that must withstand high turgor pressure. However, little is known about how the constituents of the syncytial cell wall and their conformation support its role during nematode parasitism. We used a set of monoclonal antibodies, targeted to a range of plant cell wall components, to reveal the microstructures of syncytial cell walls induced by four of the most economically important cyst nematode species, Globodera pallida, Heterodera glycines, Heterodera avenae and Heterodera filipjevi, in their respective potato, soybean, and spring wheat host roots. In situ fluorescence analysis revealed highly similar cell wall composition of syncytia induced by G. pallida and H. glycines. Both consisted of abundant xyloglucan, methyl-esterified homogalacturonan and pectic arabinan. In contrast, the walls of syncytia induced in wheat roots by H. avenae and H. filipjevi contain little xyloglucan but are rich in feruloylated xylan and arabinan residues, with variable levels of mixed-linkage glucan. The overall chemical composition of syncytial cell walls reflected the general features of root cell walls of the different host plants. We relate specific components of syncytial cell walls, such as abundant arabinan, methyl-esterification status of pectic homogalacturonan and feruloylation of xylan, to their potential roles in forming a network to support both the strength and flexibility required for syncytium function. PMID:28680436

  5. Homologue expression of a β-xylosidase from native Aspergillus niger.

    PubMed

    Amaro-Reyes, A; García-Almendárez, B E; Vázquez-Mandujano, D G; Amaya-Llano, S; Castaño-Tostado, E; Guevara-González, R G; Loera, O; Regalado, C

    2011-09-01

    Xylan constitutes the second most abundant source of renewable organic carbon on earth and is located in the cell walls of hardwood and softwood plants in the form of hemicellulose. Based on its availability, there is a growing interest in production of xylanolytic enzymes for industrial applications. β-1,4-xylan xylosidase (EC 3.2.1.37) hydrolyses from the nonreducing end of xylooligosaccharides arising from endo-1,4-β-xylanase activity. This work reports the partial characterization of a purified β-xylosidase from the native strain Aspergillus niger GS1 expressed by means of a fungal system. A gene encoding β-xylosidase, xlnD, was successfully cloned from a native A. niger GS1 strain. The recombinant enzyme was expressed in A. niger AB4.1 under control of A. nidulans gpdA promoter and trpC terminator. β-xylosidase was purified by affinity chromatography, with an apparent molecular weight of 90 kDa, and showed a maximum activity of 4,280 U mg protein(-1) at 70°C, pH 3.6. Half-life was 74 min at 70°C, activation energy was 58.9 kJ mol(-1), and at 50°C optimum stability was shown at pH 4.0-5.0. β-xylosidase kept residual activity >83% in the presence of dithiothreitol (DTT), β-mercaptoethanol, sodium dodecyl sulfate (SDS), ethylenediaminetetraacetate (EDTA), and Zn(2+). Production of a hemicellulolytic free xylosidase showed some advantages in applications, such as animal feed, enzymatic synthesis, and the fruit-juice industry where the presence of certain compounds, high temperatures, and acid media is unavoidable in the juice-making process.

  6. Enzymatic production of ferulic acid from defatted rice bran by using a combination of bacterial enzymes.

    PubMed

    Uraji, Misugi; Kimura, Masayo; Inoue, Yosikazu; Kawakami, Kayoko; Kumagai, Yuya; Harazono, Koichi; Hatanaka, Tadashi

    2013-11-01

    Ferulic acid (FA), which is present in the cell walls of some plants, is best known for its antioxidant property. By combining a commercial enzyme that shows FA esterase activity with several Streptomyces carbohydrate-hydrolyzing enzymes, we succeeded in enhancing the enzymatic production of FA from defatted rice bran. In particular, the combination of three xylanases, an α-L-arabinofuranosidase, and an acetyl xylan esterase from Streptomyces spp. produced the highest increase in the amount of released FAs among all the enzymes in the Streptomyces enzymes library. This enzyme combination also had an effect on FA production from other biomasses, such as raw rice bran, wheat bran, and corncob.

  7. Automated synthesis of arabinoxylan-oligosaccharides enables characterization of antibodies that recognize plant cell wall glycans.

    PubMed

    Schmidt, Deborah; Schuhmacher, Frank; Geissner, Andreas; Seeberger, Peter H; Pfrengle, Fabian

    2015-04-07

    Monoclonal antibodies that recognize plant cell wall glycans are used for high-resolution imaging, providing important information about the structure and function of cell wall polysaccharides. To characterize the binding epitopes of these powerful molecular probes a library of eleven plant arabinoxylan oligosaccharides was produced by automated solid-phase synthesis. Modular assembly of oligoarabinoxylans from few building blocks was enabled by adding (2-naphthyl)methyl (Nap) to the toolbox of orthogonal protecting groups for solid-phase synthesis. Conjugation-ready oligosaccharides were obtained and the binding specificities of xylan-directed antibodies were determined on microarrays. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Immobilization of Aspergillus niger xylanase on magnetic latex beads.

    PubMed

    Tyagi, R; Gupta, M N

    1995-04-01

    Xylanase from Pectinex 3XL was purified 70-fold by precipitation with an enteric polymer, Eudragit S-100. The purified xylanase was immobilized on magnetic latex beads via carbodi-imide coupling. The immobilized preparation showed 80% of the total activity bound to the beads. The pH optimum remained unchanged at 6.0 and the Km increased from 0.25 g/100 ml (free enzyme) to 0.39 g/100 ml on immobilization. Immobilization resulted in significant thermal stability at 60 degrees C. The time course of hydrolysis of xylan at 60 degrees C by free enzyme as well as immobilized enzyme was also studied.

  9. Partial structural characterization and antioxidant activity of a phenolic-xylan from Castanea sativa hardwood.

    PubMed

    Renault, Emmanuel; Barbat-Rogeon, Aline; Chaleix, Vincent; Calliste, Claude-Alain; Colas, Cyril; Gloaguen, Vincent

    2014-09-01

    4-O-Methylglucuronoxylans (MGX) were isolated from chestnut wood sawdust using two different procedures: chlorite delignification followed by the classical alkaline extraction step, and an unusual green chemistry process of delignification using phthalocyanine/H2O2 followed by a simple extraction with hot water. Antioxidant properties of both MGX were evaluated against the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) by electronic spin resonance (ESR). IC50 of water-extracted MGX was found to be less than 225 μg mL(-1), in contrast with alkali-extracted MGX for which no radical scavenging was observed. Characterization of extracts by colorimetric assay, GC, LC-MS and NMR spectroscopy provided some clues to understanding structure-function relationships of MGX in connection with their antioxidant activity. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Helicoidal pattern in secondary cell walls and possible role of xylans in their construction.

    PubMed

    Reis, Danièle; Vian, Brigitte

    2004-01-01

    The helicoidal organization of secondary cell walls is overviewed from several examples. Both the plywood texture and the occurrence of characteristic defects strongly suggest that the wall ordering is relevant of a cholesteric liquid-crystal assembly that is rapidly and strongly consolidated by lignification. A preferential localization of glucuronoxylans, major matrix components, and in vitro re-association experiments emphasize their preeminent role: (1) during the construction of the composite as directing the cellulose microfibrils in a helicoidal array; (2) during the lignification of the composite as a host structure for lignin precursors.

  11. Virus-induced gene silencing (VIGS)-mediated functional characterization of two genes involved in lignocellulosic secondary cell wall formation.

    PubMed

    Pandey, Shashank K; Nookaraju, Akula; Fujino, Takeshi; Pattathil, Sivakumar; Joshi, Chandrashekhar P

    2016-11-01

    Functional characterization of two tobacco genes, one involved in xylan synthesis and the other, a positive regulator of secondary cell wall formation, is reported. Lignocellulosic secondary cell walls (SCW) provide essential plant materials for the production of second-generation bioethanol. Therefore, thorough understanding of the process of SCW formation in plants is beneficial for efficient bioethanol production. Recently, we provided the first proof-of-concept for using virus-induced gene silencing (VIGS) approach for rapid functional characterization of nine genes involved in cellulose, hemicellulose and lignin synthesis during SCW formation. Here, we report VIGS-mediated functional characterization of two tobacco genes involved in SCW formation. Stems of VIGS plants silenced for both selected genes showed increased amount of xylem formation but thinner cell walls than controls. These results were further confirmed by production of stable transgenic tobacco plants manipulated in expression of these genes. Stems of stable transgenic tobacco plants silenced for these two genes showed increased xylem proliferation with thinner walls, whereas transgenic tobacco plants overexpressing these two genes showed increased fiber cell wall thickness but no change in xylem proliferation. These two selected genes were later identified as possible members of DUF579 family involved in xylan synthesis and KNAT7 transcription factor family involved in positive regulation of SCW formation, respectively. Glycome analyses of cell walls showed increased polysaccharide extractability in 1 M KOH extracts of both VIGS-NbDUF579 and VIGS-NbKNAT7 lines suggestive of cell wall loosening. Also, VIGS-NbDUF579 and VIGS-NbKNAT7 lines showed increased saccharification rates (74.5 and 40 % higher than controls, respectively). All these properties are highly desirable for producing higher quantities of bioethanol from lignocellulosic materials of bioenergy plants.

  12. Cellulose-Hemicellulose Interactions at Elevated Temperatures Increase Cellulose Recalcitrance to Biological Conversion

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mittal, Ashutosh; Himmel, Michael E; Kumar, Rajeev

    It has been previously shown that cellulose-lignin droplets' strong interactions, resulting from lignin coalescence and redisposition on cellulose surface during thermochemical pretreatments, increase cellulose recalcitrance to biological conversion, especially at commercially viable low enzyme loadings. However, information on the impact of cellulose-hemicellulose interactions on cellulose recalcitrance following relevant pretreatment conditions are scarce. Here, to investigate the effects of plausible hemicellulose precipitation and re-association with cellulose on cellulose conversion, different pretreatments were applied to pure Avicel(R) PH101 cellulose alone and Avicel mixed with model hemicellulose compounds followed by enzymatic hydrolysis of resulting solids at both low and high enzyme loadings. Solidsmore » produced by pretreatment of Avicel mixed with hemicelluloses (AMH) were found to contain about 2 to 14.6% of exogenous, precipitated hemicelluloses and showed a remarkably much lower digestibility (up to 60%) than their respective controls. However, the exogenous hemicellulosic residues that associated with Avicel following high temperature pretreatments resulted in greater losses in cellulose conversion than those formed at low temperatures, suggesting that temperature plays a strong role in the strength of cellulose-hemicellulose association. Molecular dynamics simulations of hemicellulosic xylan and cellulose were found to further support this temperature effect as the xylan-cellulose interactions were found to substantially increase at elevated temperatures. Furthermore, exogenous, precipitated hemicelluloses in pretreated AMH solids resulted in a larger drop in cellulose conversion than the delignified lignocellulosic biomass containing comparably much higher natural hemicellulose amounts. Increased cellulase loadings or supplementation of cellulase with xylanases enhanced cellulose conversion for most pretreated AMH solids; however, this

  13. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Walker, Johnnie A.; Takasuka, Taichi E.; Deng, Kai

    Carbohydrate binding modules (CBMs) bind polysaccharides and help target glycoside hydrolases catalytic domains to their appropriate carbohydrate substrates. To better understand how CBMs can improve cellulolytic enzyme reactivity, representatives from each of the 18 families of CBM found in Ruminoclostridium thermocellum were fused to the multifunctional GH5 catalytic domain of CelE (Cthe_0797, CelEcc), which can hydrolyze numerous types of polysaccharides including cellulose, mannan, and xylan. Since CelE is a cellulosomal enzyme, none of these fusions to a CBM previously existed. CelEcc_CBM fusions were assayed for their ability to hydrolyze cellulose, lichenan, xylan, and mannan. Several CelEcc_CBM fusions showed enhanced hydrolyticmore » activity with different substrates relative to the fusion to CBM3a from the cellulosome scaffoldin, which has high affinity for binding to crystalline cellulose. Additional binding studies and quantitative catalysis studies using nanostructure-initiator mass spectrometry (NIMS) were carried out with the CBM3a, CBM6, CBM30, and CBM44 fusion enzymes. In general, and consistent with observations of others, enhanced enzyme reactivity was correlated with moderate binding affinity of the CBM. Numerical analysis of reaction time courses showed that CelEcc_CBM44, a combination of a multifunctional enzyme domain with a CBM having broad binding specificity, gave the fastest rates for hydrolysis of both the hexose and pentose fractions of ionic-liquid pretreated switchgrass. In conclusion, we have shown that fusions of different CBMs to a single multifunctional GH5 catalytic domain can increase its rate of reaction with different pure polysaccharides and with pretreated biomass. This fusion approach, incorporating domains with broad specificity for binding and catalysis, provides a new avenue to improve reactivity of simple combinations of enzymes within the complexity of plant biomass.« less

  14. Identification of microbial populations driving biopolymer degradation in acidic peatlands by metatranscriptomic analysis.

    PubMed

    Ivanova, Anastasia A; Wegner, Carl-Eric; Kim, Yongkyu; Liesack, Werner; Dedysh, Svetlana N

    2016-10-01

    Northern peatlands play a crucial role in the global carbon balance, serving as a persistent sink for atmospheric CO2 and a global carbon store. Their most extensive type, Sphagnum-dominated acidic peatlands, is inhabited by microorganisms with poorly understood degradation capabilities. Here, we applied a combination of barcoded pyrosequencing of SSU rRNA genes and Illumina RNA-Seq of total RNA (metatranscriptomics) to identify microbial populations and enzymes involved in degrading the major components of Sphagnum-derived litter and exoskeletons of peat-inhabiting arthropods: cellulose, xylan, pectin and chitin. Biopolymer addition to peat induced a threefold to fivefold increase in bacterial cell numbers. Functional community profiles of assembled mRNA differed between experimental treatments. In particular, pectin and xylan triggered increased transcript abundance of genes involved in energy metabolism and central carbon metabolism, such as glycolysis and TCA cycle. Concurrently, the substrate-induced activity of bacteria on these two biopolymers stimulated grazing of peat-inhabiting protozoa. Alveolata (ciliates) was the most responsive protozoa group as confirmed by analysis of both SSU rRNA genes and SSU rRNA. A stimulation of alphaproteobacterial methanotrophs on pectin was consistently shown by rRNA and mRNA data. Most likely, their significant enrichment was due to the utilization of methanol released during the degradation of pectin. Analysis of SSU rRNA and total mRNA revealed a specific response of Acidobacteria and Actinobacteria to chitin and pectin, respectively. Relatives of Telmatobacter bradus were most responsive among the Acidobacteria, while the actinobacterial response was primarily affiliated with Frankiales and Propionibacteriales. The expression of a wide repertoire of carbohydrate-active enzymes (CAZymes) corresponded well to the detection of a highly diverse peat-inhabiting microbial community, which is dominated by yet uncultivated

  15. Evaluation of Relationships between Growth Rate, Tree Size, Lignocellulose Composition, and Enzymatic Saccharification in Interspecific Corymbia Hybrids and Parental Taxa.

    PubMed

    Healey, Adam L; Lee, David J; Lupoi, Jason S; Papa, Gabriella; Guenther, Joel M; Corno, Luca; Adani, Fabrizio; Singh, Seema; Simmons, Blake A; Henry, Robert J

    2016-01-01

    In order for a lignocellulosic bioenergy feedstock to be considered sustainable, it must possess a high rate of growth to supply biomass for conversion. Despite the desirability of a fast growth rate for industrial application, it is unclear what effect growth rate has on biomass composition or saccharification. We characterized Klason lignin, glucan, and xylan content with response to growth in Corymbia interspecific F1 hybrid families (HF) and parental species Corymbia torelliana and C. citriodora subspecies variegata and measured the effects on enzymatic hydrolysis from hydrothermally pretreated biomass. Analysis of biomass composition within Corymbia populations found similar amounts of Klason lignin content (19.7-21.3%) among parental and hybrid populations, whereas glucan content was clearly distinguished within C. citriodora subspecies variegata (52%) and HF148 (60%) as compared to other populations (28-38%). Multiple linear regression indicates that biomass composition is significantly impacted by tree size measured at the same age, with Klason lignin content increasing with diameter breast height (DBH) (+0.12% per cm DBH increase), and glucan and xylan typically decreasing per DBH cm increase (-0.7 and -0.3%, respectively). Polysaccharide content within C. citriodora subspecies variegata and HF-148 were not significantly affected by tree size. High-throughput enzymatic saccharification of hydrothermally pretreated biomass found significant differences among Corymbia populations for total glucose production from biomass, with parental Corymbia torelliana and hybrids HF-148 and HF-51 generating the highest amounts of glucose (~180 mg/g biomass, respectively), with HF-51 undergoing the most efficient glucan-to-glucose conversion (74%). Based on growth rate, biomass composition, and further optimization of enzymatic saccharification yield, high production Corymbia hybrid trees are potentially suitable for fast-rotation bioenergy or biomaterial production.

  16. Evaluation of Relationships between Growth Rate, Tree Size, Lignocellulose Composition, and Enzymatic Saccharification in Interspecific Corymbia Hybrids and Parental Taxa

    PubMed Central

    Healey, Adam L.; Lee, David J.; Lupoi, Jason S.; Papa, Gabriella; Guenther, Joel M.; Corno, Luca; Adani, Fabrizio; Singh, Seema; Simmons, Blake A.; Henry, Robert J.

    2016-01-01

    In order for a lignocellulosic bioenergy feedstock to be considered sustainable, it must possess a high rate of growth to supply biomass for conversion. Despite the desirability of a fast growth rate for industrial application, it is unclear what effect growth rate has on biomass composition or saccharification. We characterized Klason lignin, glucan, and xylan content with response to growth in Corymbia interspecific F1 hybrid families (HF) and parental species Corymbia torelliana and C. citriodora subspecies variegata and measured the effects on enzymatic hydrolysis from hydrothermally pretreated biomass. Analysis of biomass composition within Corymbia populations found similar amounts of Klason lignin content (19.7–21.3%) among parental and hybrid populations, whereas glucan content was clearly distinguished within C. citriodora subspecies variegata (52%) and HF148 (60%) as compared to other populations (28–38%). Multiple linear regression indicates that biomass composition is significantly impacted by tree size measured at the same age, with Klason lignin content increasing with diameter breast height (DBH) (+0.12% per cm DBH increase), and glucan and xylan typically decreasing per DBH cm increase (-0.7 and -0.3%, respectively). Polysaccharide content within C. citriodora subspecies variegata and HF-148 were not significantly affected by tree size. High-throughput enzymatic saccharification of hydrothermally pretreated biomass found significant differences among Corymbia populations for total glucose production from biomass, with parental Corymbia torelliana and hybrids HF-148 and HF-51 generating the highest amounts of glucose (~180 mg/g biomass, respectively), with HF-51 undergoing the most efficient glucan-to-glucose conversion (74%). Based on growth rate, biomass composition, and further optimization of enzymatic saccharification yield, high production Corymbia hybrid trees are potentially suitable for fast-rotation bioenergy or biomaterial production

  17. Evaluation of Relationships between Growth Rate, Tree Size, Lignocellulose Composition, and Enzymatic Saccharification in Interspecific Corymbia Hybrids and Parental Taxa

    DOE PAGES

    Healey, Adam L.; Lee, David J.; Lupoi, Jason S.; ...

    2016-11-18

    In order for a lignocellulosic bioenergy feedstock to be considered sustainable,it must possess a high rate of growth to supply biomass for conversion. Despite the desirability of a fast growth rate for industrial application,it is unclear what effect growth rate has on biomass composition or saccharification. We characterized Klason lignin,glucan,and xylan content with response to growth in Corymbia interspecific F1 hybrid families (HF) and parental species Corymbia torelliana and C. citriodora subspecies variegata and measured the effects on enzymatic hydrolysis from hydrothermally pretreated biomass. Analysis of biomass composition within Corymbia populations found similar amounts of Klason lignin content (19.7–21.3%) amongmore » parental and hybrid populations,whereas glucan content was clearly distinguished within C. citriodora subspecies variegata (52%) and HF148 (60%) as compared to other populations (28–38%). Multiple linear regression indicates that biomass composition is significantly impacted by tree size measured at the same age,with Klason lignin content increasing with diameter breast height (DBH) (+0.12% per cm DBH increase),and glucan and xylan typically decreasing per DBH cm increase (-0.7 and -0.3%,respectively). Polysaccharide content within C. citriodora subspecies variegata and HF-148 were not significantly affected by tree size. High-throughput enzymatic saccharification of hydrothermally pretreated biomass found significant differences among Corymbia populations for total glucose production from biomass,with parental Corymbia torelliana and hybrids HF-148 and HF-51 generating the highest amounts of glucose (~180 mg/g biomass,respectively),with HF-51 undergoing the most efficient glucan-to-glucose conversion (74%). Based on growth rate,biomass composition,and further optimization of enzymatic saccharification yield,high production Corymbia hybrid trees are potentially suitable for fast-rotation bioenergy or biomaterial production.« less

  18. EcXyl43 β-xylosidase: molecular modeling, activity on natural and artificial substrates, and synergism with endoxylanases for lignocellulose deconstruction.

    PubMed

    Ontañon, Ornella M; Ghio, Silvina; Marrero Díaz de Villegas, Rubén; Piccinni, Florencia E; Talia, Paola M; Cerutti, María L; Campos, Eleonora

    2018-06-06

    Biomass hydrolysis constitutes a bottleneck for the biotransformation of lignocellulosic residues into bioethanol and high-value products. The efficient deconstruction of polysaccharides to fermentable sugars requires multiple enzymes acting concertedly. GH43 β-xylosidases are among the most interesting enzymes involved in hemicellulose deconstruction into xylose. In this work, the structural and functional properties of β-xylosidase EcXyl43 from Enterobacter sp. were thoroughly characterized. Molecular modeling suggested a 3D structure formed by a conserved N-terminal catalytic domain linked to an ancillary C-terminal domain. Both domains resulted essential for enzymatic activity, and the role of critical residues, from the catalytic and the ancillary modules, was confirmed by mutagenesis. EcXyl43 presented β-xylosidase activity towards natural and artificial substrates while arabinofuranosidase activity was only detected on nitrophenyl α-L-arabinofuranoside (pNPA). It hydrolyzed xylobiose and purified xylooligosaccharides (XOS), up to degree of polymerization 6, with higher activity towards longer XOS. Low levels of activity on commercial xylan were also observed, mainly on the soluble fraction. The addition of EcXyl43 to GH10 and GH11 endoxylanases increased the release of xylose from xylan and pre-treated wheat straw. Additionally, EcXyl43 exhibited high efficiency and thermal stability under its optimal conditions (40 °C, pH 6.5), with a half-life of 58 h. Therefore, this enzyme could be a suitable additive for hemicellulases in long-term hydrolysis reactions. Because of its moderate inhibition by monomeric sugars but its high inhibition by ethanol, EcXyl43 could be particularly more useful in separate hydrolysis and fermentation (SHF) than in simultaneous saccharification and co-fermentation (SSCF) or consolidated bioprocessing (CBP).

  19. The hemicellulolytic enzyme arsenal of Thermobacillus xylanilyticus depends on the composition of biomass used for growth

    PubMed Central

    2012-01-01

    Background Thermobacillus xylanilyticus is a thermophilic and highly xylanolytic bacterium. It produces robust and stable enzymes, including glycoside hydrolases and esterases, which are of special interest for the development of integrated biorefineries. To investigate the strategies used by T. xylanilyticus to fractionate plant cell walls, two agricultural by-products, wheat bran and straw (which differ in their chemical composition and tissue organization), were used in this study and compared with glucose and xylans. The ability of T. xylanilyticus to grow on these substrates was studied. When the bacteria used lignocellulosic biomass, the production of enzymes was evaluated and correlated with the initial composition of the biomass, as well as with the evolution of any residues during growth. Results Our results showed that T. xylanilyticus is not only able to use glucose and xylans as primary carbon sources but can also use wheat bran and straw. The chemical compositions of both lignocellulosic substrates were modified by T. xylanilyticus after growth. The bacteria were able to consume 49% and 20% of the total carbohydrates in bran and straw, respectively, after 24 h of growth. The phenolic and acetyl ester contents of these substrates were also altered. Bacterial growth on both lignocellulosic biomasses induced hemicellulolytic enzyme production, and xylanase was the primary enzyme secreted. Debranching activities were differentially produced, as esterase activities were more important to bacterial cultures grown on wheat straw; arabinofuranosidase production was significantly higher in bacterial cultures grown on wheat bran. Conclusion This study provides insight into the ability of T. xylanilyticus to grow on abundant agricultural by-products, which are inexpensive carbon sources for enzyme production. The composition of the biomass upon which the bacteria grew influenced their growth, and differences in the biomass provided resulted in dissimilar enzyme

  20. Evaluation of Relationships between Growth Rate, Tree Size, Lignocellulose Composition, and Enzymatic Saccharification in Interspecific Corymbia Hybrids and Parental Taxa

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Healey, Adam L.; Lee, David J.; Lupoi, Jason S.

    In order for a lignocellulosic bioenergy feedstock to be considered sustainable,it must possess a high rate of growth to supply biomass for conversion. Despite the desirability of a fast growth rate for industrial application,it is unclear what effect growth rate has on biomass composition or saccharification. We characterized Klason lignin,glucan,and xylan content with response to growth in Corymbia interspecific F1 hybrid families (HF) and parental species Corymbia torelliana and C. citriodora subspecies variegata and measured the effects on enzymatic hydrolysis from hydrothermally pretreated biomass. Analysis of biomass composition within Corymbia populations found similar amounts of Klason lignin content (19.7–21.3%) amongmore » parental and hybrid populations,whereas glucan content was clearly distinguished within C. citriodora subspecies variegata (52%) and HF148 (60%) as compared to other populations (28–38%). Multiple linear regression indicates that biomass composition is significantly impacted by tree size measured at the same age,with Klason lignin content increasing with diameter breast height (DBH) (+0.12% per cm DBH increase),and glucan and xylan typically decreasing per DBH cm increase (-0.7 and -0.3%,respectively). Polysaccharide content within C. citriodora subspecies variegata and HF-148 were not significantly affected by tree size. High-throughput enzymatic saccharification of hydrothermally pretreated biomass found significant differences among Corymbia populations for total glucose production from biomass,with parental Corymbia torelliana and hybrids HF-148 and HF-51 generating the highest amounts of glucose (~180 mg/g biomass,respectively),with HF-51 undergoing the most efficient glucan-to-glucose conversion (74%). Based on growth rate,biomass composition,and further optimization of enzymatic saccharification yield,high production Corymbia hybrid trees are potentially suitable for fast-rotation bioenergy or biomaterial production.« less

  1. The Effects of Noncellulosic Compounds on the Nanoscale Interaction Forces Measured between Carbohydrate-Binding Module and Lignocellulosic Biomass.

    PubMed

    Arslan, Baran; Colpan, Mert; Ju, Xiaohui; Zhang, Xiao; Kostyukova, Alla; Abu-Lail, Nehal I

    2016-05-09

    The lack of fundamental understanding of the types of forces that govern how cellulose-degrading enzymes interact with cellulosic and noncellulosic components of lignocellulosic surfaces limits the design of new strategies for efficient conversion of biomass to bioethanol. In a step to improve our fundamental understanding of such interactions, nanoscale forces acting between a model cellulase-a carbohydrate-binding module (CBM) of cellobiohydrolase I (CBH I)-and a set of lignocellulosic substrates with controlled composition were measured using atomic force microscopy (AFM). The three model substrates investigated were kraft (KP), sulfite (SP), and organosolv (OPP) pulped substrates. These substrates varied in their surface lignin coverage, lignin type, and xylan and acetone extractives' content. Our results indicated that the overall adhesion forces of biomass to CBM increased linearly with surface lignin coverage with kraft lignin showing the highest forces among lignin types investigated. When the overall adhesion forces were decoupled into specific and nonspecific component forces via the Poisson statistical model, hydrophobic and Lifshitz-van der Waals (LW) forces dominated the binding forces of CBM to kraft lignin, whereas permanent dipole-dipole interactions and electrostatic forces facilitated the interactions of lignosulfonates to CBM. Xylan and acetone extractives' content increased the attractive forces between CBM and lignin-free substrates, most likely through hydrogen bonding forces. When the substrates treated differently were compared, it was found that both the differences in specific and nonspecific forces between lignin-containing and lignin-free substrates were the least for OPP. Therefore, cellulase enzymes represented by CBM would weakly bind to organosolv lignin. This will facilitate an easy enzyme recovery compared to other substrates treated with kraft or sulfite pulping. Our results also suggest that altering the surface hydrophobicity

  2. Novel thermophilic hemicellulases for the conversion of lignocellulose for second generation biorefineries.

    PubMed

    Cobucci-Ponzano, Beatrice; Strazzulli, Andrea; Iacono, Roberta; Masturzo, Giuseppe; Giglio, Rosa; Rossi, Mosè; Moracci, Marco

    2015-10-01

    The biotransformation of lignocellulose biomasses into fermentable sugars is a very complex procedure including, as one of the most critical steps, the (hemi) cellulose hydrolysis by specific enzymatic cocktails. We explored here, the potential of stable glycoside hydrolases from thermophilic organisms, so far not used in commercial enzymatic preparations, for the conversion of glucuronoxylan, the major hemicellulose of several energy crops. Searches in the genomes of thermophilic bacteria led to the identification, efficient production, and detailed characterization of novel xylanase and α-glucuronidase from Alicyclobacillus acidocaldarius (GH10-XA and GH67-GA, respectively) and a α-glucuronidase from Caldicellulosiruptor saccharolyticus (GH67-GC). Remarkably, GH10-XA, if compared to other thermophilic xylanases from this family, coupled good specificity on beechwood xylan and the best stability at 65 °C (3.5 days). In addition, GH67-GC was the most stable α-glucuronidases from this family and the first able to hydrolyse both aldouronic acid and aryl-α-glucuronic acid substrates. These enzymes, led to the very efficient hydrolysis of beechwood xylan by using 7- to 9-fold less protein (concentrations <0.3 μM) and in much less reaction time (2h vs 12h) if compared to other known biotransformations catalyzed by thermophilic enzymes. In addition, remarkably, together with a thermophilic β-xylosidase, they catalyzed the production of xylose from the smart cooking pre-treated biomass of one of the most promising energy crops for second generation biorefineries. We demonstrated that search by the CAZy Data Bank of currently available genomes and detailed enzymatic characterization of recombinant enzymes allow the identification of glycoside hydrolases with novel and interesting properties and applications. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Multifunctional cellulase catalysis targeted by fusion to different carbohydrate-binding modules

    DOE PAGES

    Walker, Johnnie A.; Takasuka, Taichi E.; Deng, Kai; ...

    2015-12-21

    Carbohydrate binding modules (CBMs) bind polysaccharides and help target glycoside hydrolases catalytic domains to their appropriate carbohydrate substrates. To better understand how CBMs can improve cellulolytic enzyme reactivity, representatives from each of the 18 families of CBM found in Ruminoclostridium thermocellum were fused to the multifunctional GH5 catalytic domain of CelE (Cthe_0797, CelEcc), which can hydrolyze numerous types of polysaccharides including cellulose, mannan, and xylan. Since CelE is a cellulosomal enzyme, none of these fusions to a CBM previously existed. CelEcc_CBM fusions were assayed for their ability to hydrolyze cellulose, lichenan, xylan, and mannan. Several CelEcc_CBM fusions showed enhanced hydrolyticmore » activity with different substrates relative to the fusion to CBM3a from the cellulosome scaffoldin, which has high affinity for binding to crystalline cellulose. Additional binding studies and quantitative catalysis studies using nanostructure-initiator mass spectrometry (NIMS) were carried out with the CBM3a, CBM6, CBM30, and CBM44 fusion enzymes. In general, and consistent with observations of others, enhanced enzyme reactivity was correlated with moderate binding affinity of the CBM. Numerical analysis of reaction time courses showed that CelEcc_CBM44, a combination of a multifunctional enzyme domain with a CBM having broad binding specificity, gave the fastest rates for hydrolysis of both the hexose and pentose fractions of ionic-liquid pretreated switchgrass. In conclusion, we have shown that fusions of different CBMs to a single multifunctional GH5 catalytic domain can increase its rate of reaction with different pure polysaccharides and with pretreated biomass. This fusion approach, incorporating domains with broad specificity for binding and catalysis, provides a new avenue to improve reactivity of simple combinations of enzymes within the complexity of plant biomass.« less

  4. GH51 Arabinofuranosidase and Its Role in the Methylglucuronoarabinoxylan Utilization System in Paenibacillus sp. Strain JDR-2

    PubMed Central

    Sawhney, Neha

    2014-01-01

    Methylglucuronoarabinoxylan (MeGAXn) from agricultural residues and energy crops is a significant yet underutilized biomass resource for production of biofuels and chemicals. Mild thermochemical pretreatment of bagasse yields MeGAXn requiring saccharifying enzymes for conversion to fermentable sugars. A xylanolytic bacterium, Paenibacillus sp. strain JDR-2, produces an extracellular cell-associated GH10 endoxylanse (XynA1) which efficiently depolymerizes methylglucuronoxylan (MeGXn) from hardwoods coupled with assimilation of oligosaccharides for further processing by intracellular GH67 α-glucuronidase, GH10 endoxylanase, and GH43 β-xylosidase. This process has been ascribed to genes that comprise a xylan utilization regulon that encodes XynA1 and includes a gene cluster encoding transcriptional regulators, ABC transporters, and intracellular enzymes that convert assimilated oligosaccharides to fermentable sugars. Here we show that Paenibacillus sp. JDR-2 utilized MeGAXn without accumulation of oligosaccharides in the medium. The Paenibacillus sp. JDR-2 growth rate on MeGAXn was 3.1-fold greater than that on oligosaccharides generated from MeGAXn by XynA1. Candidate genes encoding GH51 arabinofuranosidases with potential roles were identified. Following growth on MeGAXn, quantitative reverse transcription-PCR identified a cluster of genes encoding a GH51 arabinofuranosidase (AbfB) and transcriptional regulators which were coordinately expressed along with the genes comprising the xylan utilization regulon. The action of XynA1 on MeGAXn generated arabinoxylobiose, arabinoxylotriose, xylobiose, xylotriose, and methylglucuronoxylotriose. Recombinant AbfB processed arabinoxylooligosaccharides to xylooligosaccharides and arabinose. MeGAXn processing by Paenibacillus sp. JDR-2 may be achieved by extracellular depolymerization by XynA1 coupled to assimilation of oligosaccharides and further processing by intracellular enzymes, including AbfB. Paenibacillus sp. JDR-2

  5. Enzymatic Hydrolysis of Pretreated Fibre Pressed Oil Palm Frond by using Sacchariseb C6

    NASA Astrophysics Data System (ADS)

    Hashim, F. S.; Yussof, H. W.; Zahari, M. A. K. M.; Rahman, R. A.; Illias, R. M.

    2017-06-01

    Enzymatic hydrolysis becomes a prominent technology for conversion of cellulosic biomass to its glucose monomers that requires an action of cellulolytic enzymes in a sequential and synergistic manner. In this study, the effect of agitation speed, glucan loading, enzyme loading, temperature and reaction time on the production of glucose from fibre pressed oil palm frond (FPOPF) during enzymatic hydrolysis was screened by a half factorial design 25-1 using Response Surface Methodology (RSM). The FPOPF sample was first delignified by alkaline pretreatment at 4.42 (w/v) sodium hydroxide for an hour prior to enzymatic hydrolysis using commercial cellulase enzyme, Sacchariseb C6. The effect of enzymatic hydrolysis on the structural of FPOPF has been evaluated by Scanning Electron Microscopy (SEM) analysis. Characterization of raw FPOPF comprised of 4.5 extractives, 40.7 glucan, 26.1 xylan, 26.2 lignin and 1.8 ash, whereas for pretreated FPOPF gave 0.3 extractives, 61.4 glucan, 20.4 xylan, 13.3 lignin and 1.3 ash. From this study, it was found that the best enzymatic hydrolysis condition yielded 33.01 ± 0.73 g/L of glucose when performed at 200 rpm of agitation speed, 60 FPU/mL of enzyme loading, 4 (w/w) of glucan loading, temperature at 55 □ and 72 hours of reaction time. The model obtained was significant with p-value <0.0001 as verified by the analysis of variance (ANOVA). The coefficient of determination (R2) from ANOVA study was 0.9959. Overall, it can be concluded that addition of Sacchariseb C6 during enzymatic hydrolysis from pretreated FPOPF produce high amount of glucose that enhances it potential for industrial application. This glucose can be further used to produce high-value products.

  6. Simultaneous and Sequential Integration by Cre/loxP Site-Specific Recombination in Saccharomyces cerevisiae.

    PubMed

    Choi, Ho-Jung; Kim, Yeon-Hee

    2018-05-28

    A Cre/ loxP -δ-integration system was developed to allow sequential and simultaneous integration of a multiple gene expression cassette in Saccharomyces cerevisiae . To allow repeated integrations, the reusable Candida glabrata MARKER ( CgMARKER ) carrying loxP sequences was used, and the integrated CgMARKER was efficiently removed by inducing Cre recombinase. The XYLP and XYLB genes encoding endoxylanase and β-xylosidase, respectively, were used as model genes for xylan metabolism in this system, and the copy number of these genes was increased to 15.8 and 16.9 copies/cell, respectively, by repeated integration. This integration system is a promising approach for the easy construction of yeast strains with enhanced metabolic pathways through multicopy gene expression.

  7. Xylanases, nucleic acids encoding them and methods for making and using them

    DOEpatents

    Gray, Kevin A; Dirmeier, Reinhard

    2013-07-16

    The invention relates to enzymes having xylanase, mannanase and/or glucanase activity, e.g., catalyzing hydrolysis of internal .beta.-1,4-xylosidic linkages or endo-.beta.-1,4-glucanase linkages; and/or degrading a linear polysaccharide beta-1,4-xylan into xylose. Thus, the invention provides methods and processes for breaking down hemicellulose, which is a major component of the cell wall of plants, including methods and processes for hydrolyzing hemicelluloses in any plant or wood or wood product, wood waste, paper pulp, paper product or paper waste or byproduct. In addition, methods of designing new xylanases, mannanases and/or glucanases and methods of use thereof are also provided. The xylanases, mannanases and/or glucanases have increased activity and stability at increased pH and temperature.

  8. Carbohydrase Systems of Saccharophagus degradans Degrading Marine Complex Polysaccharides

    PubMed Central

    Hutcheson, Steven W.; Zhang, Haitao; Suvorov, Maxim

    2011-01-01

    Saccharophagus degradans 2–40 is a γ-subgroup proteobacterium capable of using many of the complex polysaccharides found in the marine environment for growth. To utilize these complex polysaccharides, this bacterium produces a plethora of carbohydrases dedicated to the processing of a carbohydrate class. Aiding in the identification of the contributing genes and enzymes is the known genome sequence for this bacterium. This review catalogs the genes and enzymes of the S. degradans genome that are likely to function in the systems for the utilization of agar, alginate, α- and β-glucans, chitin, mannans, pectins, and xylans and discusses the cell biology and genetics of each system as it functions to transfer carbon back to the bacterium. PMID:21731555

  9. Applicability of thermo-alkali-stable and cellulase-free xylanase from a novel thermo-halo-alkaliphilic Bacillus halodurans in producing xylooligosaccharides.

    PubMed

    Kumar, Vikash; Satyanarayana, T

    2011-11-01

    An alkaliphilic, moderately thermophilic and halophilic bacterial isolate capable of producing a high titer of extracellular thermo-alkali-stable, cellulase-free endoxylanase was isolated from the paper mill effluents. It was identified as Bacillus halodurans. The purified xylanase was active from pH 7 to 12 and 30 to 100°C with optimal activity at pH 9.0 and 80°C. It had T(1/2) values of 40 and 15 min at 70 and 80°C, respectively. Activity was stimulated by dithiothreitol but strongly inhibited by N-bromosuccinimide. Its action on birchwood xylan and agro-residues liberated xylooligosaccharides of 2-7 degree of polymerization, and thus, the mode of action is similar to endoxylanases of the family 10 glucoside hydrolases.

  10. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Odelson, D.A.; Breznak, J.A.

    Crude extracts of the anaerobic, cellulolytic protozoan Trichomitopsis termopsidis possessed endo-..beta..-1,4-glucanase and cellobiase activities, as evidenced by hydrolytic action on carboxymethyl cellulose and cellobiose, respectively. Cell extracts also hydrolyzed microcrystalline cellulose. Hydrolysis of microcrystalline cellulose displayed optima at pH 5 and at 30 degrees C, and glucose was the sole product liberated. Cellulolytic activities of T. termopsidis appeared to be entirely cell associated. Hydrolytic activity was also detected against Douglas fir wood powder, xylan, starch, and protein, but not chitin. The importance of these enyzmes in the nutrition of T. termopsidis is discussed in terms of the natural habitat ofmore » this protozoan (the hindgut of wood-eating termites). 31 references.« less

  11. Molecular Analysis of Primary Vapor and Char Products during Stepwise Pyrolysis of Poplar Biomass

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jones, Roger W.; Reinot, Tonu; McClelland, John F.

    2010-08-03

    Pyrolysis of biomass produces both pyrolysis oil and solid char. In this study, poplar has been pyrolyzed in a stepwise fashion over a series of temperatures from 200 to 500°C, and both the primary products contributing to pyrolysis oil and the changes in the pyrolyzing poplar surface leading toward char have been characterized at each step. The primary products were identified by direct analysis in real time (DART) mass spectrometry, and the changes in the poplar surface were monitored using Fourier transform infrared (FTIR) photoacoustic spectroscopy, with a sampling depth of a few micrometers. The primary products from pyrolyzing cellulose,more » xylan, and lignin under similar conditions were also characterized to identify the sources of the poplar products.« less

  12. Molecular Analysis of Primary Vapor and Char Products during Stepwise Pyrolysis of Poplar Biomass

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jones, Roger W.; Reinot, Tonu; McClelland, John F.

    2010-08-30

    Pyrolysis of biomass produces both pyrolysis oil and solid char. In this study, poplar has been pyrolyzed in a stepwise fashion over a series of temperatures from 200 to 500 C, and both the primary products contributing to pyrolysis oil and the changes in the pyrolyzing poplar surface leading toward char have been characterized at each step. The primary products were identified by direct analysis in real time (DART) mass spectrometry, and the changes in the poplar surface were monitored using Fourier transform infrared (FTIR) photoacoustic spectroscopy, with a sampling depth of a few micrometers. The primary products from pyrolyzingmore » cellulose, xylan, and lignin under similar conditions were also characterized to identify the sources of the poplar products.« less

  13. Ensiling and hydrothermal pretreatment of grass: consequences for enzymatic biomass conversion and total monosaccharide yields

    PubMed Central

    2014-01-01

    Background Ensiling may act as a pretreatment of fresh grass biomass and increase the enzymatic conversion of structural carbohydrates to fermentable sugars. However, ensiling does not provide sufficient severity to be a standalone pretreatment method. Here, ensiling of grass is combined with hydrothermal treatment (HTT) with the aim of improving the enzymatic biomass convertibility and decrease the required temperature of the HTT. Results Grass silage (Festulolium Hykor) was hydrothermally treated at temperatures of 170, 180, and 190°C for 10 minutes. Relative to HTT treated dry grass, ensiling increased the solubilization of dry matter (DM) during HTT and gave increased glucan content, but lower lignin in the insoluble fiber fraction. Ensiling improved glucose yields in the enzymatic hydrolysis of the washed solid fiber fraction at the lower HTT temperatures. At 170°C glucose yield improved from 17 to 24 (w/w)% (45 to 57% cellulose convertibility), and at 180°C glucose yield improved from 22 to 29 (w/w)% (54 to 69% cellulose convertibility). Direct HTT of grass at 190°C gave the same high glucose yield as for grass silage (35 (w/w)% (77% cellulose convertibility)) and improved xylan yields (27% xylan convertibility). The effect of ensiling of grass prior to HTT improved the enzymatic conversion of cellulose for HTT at 170 and 180°C, but the increased glucose release did not make up for the loss of water soluble carbohydrates (WSC) during ensiling. Overall, sugar yields (C6 + C5) were similar for HTT of grass and grass silage at both 170 and 180°C, but at 190°C the overall sugar yield was better for HTT of dry grass. Conclusions This study unequivocally establishes that ensiling of grass as a biomass pretreatment method comes with a loss of WSC. The loss of WSC by ensiling is not necessarily compensated for by providing a lower temperature requirement for HTT for high enzymatic monosaccharide release. However, ensiling can be an advantageous storage

  14. Effects of Diet on Resource Utilization by a Model Human Gut Microbiota Containing Bacteroides cellulosilyticus WH2, a Symbiont with an Extensive Glycobiome

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    McNulty, Nathan; Wu, Meng; Erickson, Alison L

    The human gut microbiota is an important metabolic organ, yet little is known about how its individual species interact, establish dominant positions, and respond to changes in environmental factors such as diet. In this study, gnotobiotic mice were colonized with an artificial microbiota comprising 12 sequenced human gut bacterial species and fed oscillating diets of disparate composition. Rapid, reproducible, and reversible changes in the structure of this assemblage were observed. Time-series microbial RNA-Seq analyses revealed staggered functional responses to diet shifts throughout the assemblage that were heavily focused on carbohydrate and amino acid metabolism. High-resolution shotgun metaproteomics confirmed many ofmore » these responses at a protein level. One member, Bacteroides cellulosilyticus WH2, proved exceptionally fit regardless of diet. Its genome encoded more carbohydrate active enzymes than any previously sequenced member of the Bacteroidetes. Transcriptional profiling indicated that B. cellulosilyticus WH2 is an adaptive forager that tailors its versatile carbohydrate utilization strategy to available dietary polysaccharides, with a strong emphasis on plant-derived xylans abundant in dietary staples like cereal grains. Two highly expressed, diet-specific polysaccharide utilization loci (PULs) in B. cellulosilyticus WH2 were identified, one with characteristics of xylan utilization systems. Introduction of a B. cellulosilyticus WH2 library comprising .90,000 isogenic transposon mutants into gnotobiotic mice, along with the other artificial community members, confirmed that these loci represent critical diet-specific fitness determinants. Carbohydrates that trigger dramatic increases in expression of these two loci and many of the organism s 111 other predicted PULs were identified by RNA-Seq during in vitro growth on 31 distinct carbohydrate substrates, allowing us to better interpret in vivo RNA-Seq and proteomics data. These results offer

  15. Effects of Diet on Resource Utilization by a Model Human Gut Microbiota Containing Bacteroides cellulosilyticus WH2, a Symbiont with an Extensive Glycobiome

    PubMed Central

    McNulty, Nathan P.; Wu, Meng; Erickson, Alison R.; Pan, Chongle; Erickson, Brian K.; Martens, Eric C.; Pudlo, Nicholas A.; Muegge, Brian D.; Henrissat, Bernard; Hettich, Robert L.; Gordon, Jeffrey I.

    2013-01-01

    The human gut microbiota is an important metabolic organ, yet little is known about how its individual species interact, establish dominant positions, and respond to changes in environmental factors such as diet. In this study, gnotobiotic mice were colonized with an artificial microbiota comprising 12 sequenced human gut bacterial species and fed oscillating diets of disparate composition. Rapid, reproducible, and reversible changes in the structure of this assemblage were observed. Time-series microbial RNA-Seq analyses revealed staggered functional responses to diet shifts throughout the assemblage that were heavily focused on carbohydrate and amino acid metabolism. High-resolution shotgun metaproteomics confirmed many of these responses at a protein level. One member, Bacteroides cellulosilyticus WH2, proved exceptionally fit regardless of diet. Its genome encoded more carbohydrate active enzymes than any previously sequenced member of the Bacteroidetes. Transcriptional profiling indicated that B. cellulosilyticus WH2 is an adaptive forager that tailors its versatile carbohydrate utilization strategy to available dietary polysaccharides, with a strong emphasis on plant-derived xylans abundant in dietary staples like cereal grains. Two highly expressed, diet-specific polysaccharide utilization loci (PULs) in B. cellulosilyticus WH2 were identified, one with characteristics of xylan utilization systems. Introduction of a B. cellulosilyticus WH2 library comprising >90,000 isogenic transposon mutants into gnotobiotic mice, along with the other artificial community members, confirmed that these loci represent critical diet-specific fitness determinants. Carbohydrates that trigger dramatic increases in expression of these two loci and many of the organism's 111 other predicted PULs were identified by RNA-Seq during in vitro growth on 31 distinct carbohydrate substrates, allowing us to better interpret in vivo RNA-Seq and proteomics data. These results offer insight

  16. Identification of a novel fungus, Leptosphaerulina chartarum SJTU59 and characterization of its xylanolytic enzymes.

    PubMed

    Wu, Qiong; Li, Yaqian; Li, Yingying; Gao, Shigang; Wang, Meng; Zhang, Tailong; Chen, Jie

    2013-01-01

    Xylanolytic enzymes are widely used in processing industries, e.g., pulp and paper, food, livestock feeds, and textile. Furthermore, certain xylanotic enzymes have demonstrated the capability to improve the resistance and immunity of plants. Screening of high-yield microbial xylanolytic enzyme producers is significant for improving large-scale cost-effective xylanolytic enzyme production. This study provided new evidence of high-level xylanolytic enzyme production by a novel fungus, designated Leptosphaerulina chartarum SJTU59. Under laboratory conditions, L. chartarum SJTU59 produced xylanolytic enzymes of up to 17.566 U/mL (i.e., 878.307 U/g substrate). The enzyme solution was relatively stable over a wide range of pH (pH 3.0 to pH 9.0) and temperature (40°C to 65°C) while showing high resistance to the majority of metal ions tested. Composition analysis of the hydrolytic products of xylan showed sufficient degradation by xylanolytic enzymes from L. chartarum SJTU59, mainly the monosaccharide xylose, and a small amount of xylobiose were enzymatically produced; whereas in the presence of sufficient xylan substrates, mainly xylooligosaccharides, an emerging prebiotic used in food industry, were produced. In addition, the xylanolytic enzyme preparation from L. chartarum SJTU59 could initiate tissue necrosis and oxidative burst in tobacco leaves, which may be related to enhanced plant defense to adversity and disease. L. chartarum SJTU59 possessed a complex xylanolytic enzyme system, from which two novel endo-β-1,4-xylanases of the glycoside hydrolase (GH) family 10, one novel endo-β-1,4-xylanase of the GH family 11, and one novel β-xylosidase of the GH family 43 were obtained via rapid amplification of complementary DNA ends. Given the high yield and stable properties of xylanolytic enzymes produced by L. chartarum SJTU59, future studies will be conducted to characterize the properties of individual xylanolytic enzymes from L. chartarum SJTU59. xylanolytic

  17. Long-Term Warming Alters Carbohydrate Degradation Potential in Temperate Forest Soils.

    PubMed

    Pold, Grace; Billings, Andrew F; Blanchard, Jeff L; Burkhardt, Daniel B; Frey, Serita D; Melillo, Jerry M; Schnabel, Julia; van Diepen, Linda T A; DeAngelis, Kristen M

    2016-11-15

    As Earth's climate warms, soil carbon pools and the microbial communities that process them may change, altering the way in which carbon is recycled in soil. In this study, we used a combination of metagenomics and bacterial cultivation to evaluate the hypothesis that experimentally raising soil temperatures by 5°C for 5, 8, or 20 years increased the potential for temperate forest soil microbial communities to degrade carbohydrates. Warming decreased the proportion of carbohydrate-degrading genes in the organic horizon derived from eukaryotes and increased the fraction of genes in the mineral soil associated with Actinobacteria in all studies. Genes associated with carbohydrate degradation increased in the organic horizon after 5 years of warming but had decreased in the organic horizon after warming the soil continuously for 20 years. However, a greater proportion of the 295 bacteria from 6 phyla (10 classes, 14 orders, and 34 families) isolated from heated plots in the 20-year experiment were able to depolymerize cellulose and xylan than bacterial isolates from control soils. Together, these findings indicate that the enrichment of bacteria capable of degrading carbohydrates could be important for accelerated carbon cycling in a warmer world. The massive carbon stocks currently held in soils have been built up over millennia, and while numerous lines of evidence indicate that climate change will accelerate the processing of this carbon, it is unclear whether the genetic repertoire of the microbes responsible for this elevated activity will also change. In this study, we showed that bacteria isolated from plots subject to 20 years of 5°C of warming were more likely to depolymerize the plant polymers xylan and cellulose, but that carbohydrate degradation capacity is not uniformly enriched by warming treatment in the metagenomes of soil microbial communities. This study illustrates the utility of combining culture-dependent and culture-independent surveys of

  18. Multi-scale processes of beech wood disintegration and pretreatment with 1-ethyl-3-methylimidazolium acetate/water mixtures.

    PubMed

    Viell, Jörn; Inouye, Hideyo; Szekely, Noemi K; Frielinghaus, Henrich; Marks, Caroline; Wang, Yumei; Anders, Nico; Spiess, Antje C; Makowski, Lee

    2016-01-01

    The valorization of biomass for chemicals and fuels requires efficient pretreatment. One effective strategy involves the pretreatment with ionic liquids which enables enzymatic saccharification of wood within a few hours under mild conditions. This pretreatment strategy is, however, limited by water and the ionic liquids are rather expensive. The scarce understanding of the involved effects, however, challenges the design of alternative pretreatment concepts. This work investigates the multi length-scale effects of pretreatment of wood in 1-ethyl-3-methylimidazolium acetate (EMIMAc) in mixtures with water using spectroscopy, X-ray and neutron scattering. The structure of beech wood is disintegrated in EMIMAc/water mixtures with a water content up to 8.6 wt%. Above 10.7 wt%, the pretreated wood is not disintegrated, but still much better digested enzymatically compared to native wood. In both regimes, component analysis of the solid after pretreatment shows an extraction of few percent of lignin and hemicellulose. In concentrated EMIMAc, xylan is extracted more efficiently and lignin is defunctionalized. Corresponding to the disintegration at macroscopic scale, SANS and XRD show isotropy and a loss of crystallinity in the pretreated wood, but without distinct reflections of type II cellulose. Hence, the microfibril assembly is decrystallized into rather amorphous cellulose within the cell wall. The molecular and structural changes elucidate the processes of wood pretreatment in EMIMAc/water mixtures. In the aqueous regime with >10.7 wt% water in EMIMAc, xyloglucan and lignin moieties are extracted, which leads to coalescence of fibrillary cellulose structures. Dilute EMIMAc/water mixtures thus resemble established aqueous pretreatment concepts. In concentrated EMIMAc, the swelling due to decrystallinization of cellulose, dissolution of cross-linking xylan, and defunctionalization of lignin releases the mechanical stress to result in macroscopic disintegration of

  19. Characterization of a recombinant α-glucuronidase from Aspergillus fumigatus.

    PubMed

    Rosa, Lorena; Ravanal, María Cristina; Mardones, Wladimir; Eyzaguirre, Jaime

    2013-05-01

    The degradation of xylan requires the action of glycanases and esterases which hydrolyse, in a synergistic fashion, the main chain and the different substituents which decorate its structure. Among the xylanolytic enzymes acting on side-chains are the α-glucuronidases (AguA) (E.C. 3.2.1.139) which release methyl glucuronic acid residues. These are the least studies among the xylanolytic enzymes. In this work, the gene and cDNA of an α-glucuronidase from a newly isolated strain of Aspergillus fumigatus have been sequenced, and the gene has been expressed in Pichia pastoris. The gene is 2523 bp long, has no introns and codes for a protein of 840 amino acid residues including a putative signal peptide of 19 residues. The mature protein has a calculated molecular weight of 91,725 and shows 99 % identity with a putative α-glucuronidase from A. fumigatus A1163. The recombinant enzyme was expressed with a histidine tag and was purified to near homogeneity with a nickel nitriloacetic acid (Ni-NTA) column. The purified enzyme has a molecular weight near 100,000. It is inactive using birchwood glucuronoxylan as substrate. Activity is observed in the presence of xylooligosaccharides generated from this substrate by a family 10 endoxylanase and when a mixture of aldouronic acids are used as substrates. If, instead, family 11 endoxylanase is used to generate oligosaccharides, no activity is detected, indicating a different specificity in the cleavage of xylan by family 10 and 11 endoxylanases. Enzyme activity is optimal at 37 °C and pH 4.5-5. The enzyme binds cellulose, thus it likely possesses a carbohydrate binding module. Based on its properties and sequence similarities the catalytic module of the newly described α-glucuronidase can be classified in family 67 of the glycosyl hydrolases. The recombinant enzyme may be useful for biotechnological applications of α-glucuronidases. Copyright © 2013 The British Mycological Society. Published by Elsevier Ltd. All rights

  20. Long-Term Warming Alters Carbohydrate Degradation Potential in Temperate Forest Soils

    PubMed Central

    Billings, Andrew F.; Blanchard, Jeff L.; Burkhardt, Daniel B.; Frey, Serita D.; Melillo, Jerry M.; Schnabel, Julia; van Diepen, Linda T. A.

    2016-01-01

    ABSTRACT As Earth's climate warms, soil carbon pools and the microbial communities that process them may change, altering the way in which carbon is recycled in soil. In this study, we used a combination of metagenomics and bacterial cultivation to evaluate the hypothesis that experimentally raising soil temperatures by 5°C for 5, 8, or 20 years increased the potential for temperate forest soil microbial communities to degrade carbohydrates. Warming decreased the proportion of carbohydrate-degrading genes in the organic horizon derived from eukaryotes and increased the fraction of genes in the mineral soil associated with Actinobacteria in all studies. Genes associated with carbohydrate degradation increased in the organic horizon after 5 years of warming but had decreased in the organic horizon after warming the soil continuously for 20 years. However, a greater proportion of the 295 bacteria from 6 phyla (10 classes, 14 orders, and 34 families) isolated from heated plots in the 20-year experiment were able to depolymerize cellulose and xylan than bacterial isolates from control soils. Together, these findings indicate that the enrichment of bacteria capable of degrading carbohydrates could be important for accelerated carbon cycling in a warmer world. IMPORTANCE The massive carbon stocks currently held in soils have been built up over millennia, and while numerous lines of evidence indicate that climate change will accelerate the processing of this carbon, it is unclear whether the genetic repertoire of the microbes responsible for this elevated activity will also change. In this study, we showed that bacteria isolated from plots subject to 20 years of 5°C of warming were more likely to depolymerize the plant polymers xylan and cellulose, but that carbohydrate degradation capacity is not uniformly enriched by warming treatment in the metagenomes of soil microbial communities. This study illustrates the utility of combining culture-dependent and culture

  1. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Cui, Z. F.; Wan, C. X.; Shi, J.

    Corn stover fractions (leaves, cobs, and stalks) were studied for enzymatic digestibility after pretreatment with a white rot fungus, Ceriporiopsis subvermispora. Among the three fractions, leaves had the least recalcitrance to fungal pretreatment and the lignin degradation reached 45% after 30 days of pretreatment. The lignin degradation of stalks and cobs was similar but was significantly lower than that of leaves (p < 0.05). For all fractions, xylan and glucan degradation followed a pattern similar to lignin degradation, with leaves having a significantly higher percentage of degradation (p < 0.05). Hydrolytic enzyme activity also revealed that the fungus was moremore » active in the degradation of carbohydrates in leaves. As a result of fungal pretreatment, the highest sugar yield, however, was obtained with corn cobs.« less

  2. Improving enzymatic hydrolysis of industrial hemp ( Cannabis sativa L.) by electron beam irradiation

    NASA Astrophysics Data System (ADS)

    Shin, Soo-Jeong; Sung, Yong Joo

    2008-09-01

    The electron beam irradiation was applied as a pretreatment of the enzymatic hydrolysis of hemp biomass with doses of 150, 300 and 450 kGy. The higher irradiation dose resulted in the more extraction with hot-water extraction or 1% sodium hydroxide solution extraction. The higher solubility of the treated sample was originated from the chains scission during irradiation, which was indirectly demonstrated by the increase of carbonyl groups as shown in diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) spectra. The changes in the micro-structure of hemp resulted in the better response to enzymatic hydrolysis with commercial cellulases (Celluclast 1.5L and Novozym 342). The improvement in enzymatic hydrolysis by the irradiation was more evident in the hydrolysis of the xylan than in that of the cellulose.

  3. Composition and ethanol production potential of cotton gin residues.

    PubMed

    Agblevor, Foster A; Batz, Sandra; Trumbo, Jessica

    2003-01-01

    Cotton gin residue (CGR) collected from five cotton gins was fractionated and characterized for summative composition. The major fractions of the CGR varied widely between cotton gins and consisted of clean lint (5-12%),hulls (16-48%), seeds (6-24%), motes (16-24%), and leaves (14-30%). The summative composition varied within and between cotton gins and consisted of ash (7.9-14.6%), acid-insoluble material (18-26%), xylan (4-15%),and cellulose (20-38%). Overlimed steam-exploded cotton gin waste was readily fermented to ethanol by Escherichia coli KO11. Ethanol yields were feedstock and severity dependent and ranged from 58 to 92.5% of the theoretical yields. The highest ethanol yield was 191 L (50 gal)/t, and the lowest was 120 L (32 gal)/t.

  4. Xylanases, nucleic acids encoding them and methods for making and using them

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gray, Kevin A.; Dirmeier, Richard

    The invention relates to enzymes having xylanase, mannanase and/or glucanase activity, e.g., catalyzing hydrolysis of internal .beta.-1,4-xylosidic linkages or endo-.beta.-1,4-glucanase linkages; and/or degrading a linear polysaccharide beta-1,4-xylan into xylose. Thus, the invention provides methods and processes for breaking down hemicellulose, which is a major component of the cell wall of plants, including methods and processes for hydrolyzing hemicelluloses in any plant or wood or wood product, wood waste, paper pulp, paper product or paper waste or byproduct. In addition, methods of designing new xylanases, mannanases and/or glucanases and methods of use thereof are also provided. The xylanases, mannanases and/or glucanasesmore » have increased activity and stability at increased pH and temperature.« less

  5. Production of Enzymes from Marine Actinobacteria.

    PubMed

    Zhao, X Q; Xu, X N; Chen, L Y

    Marine actinobacteria are well recognized for their capabilities to produce valuable natural products, which have great potential for applications in medical, agricultural, and fine chemical industries. In addition to producing unique enzymes responsible for biosynthesis of natural products, many marine actinobacteria also produce hydrolytic enzymes which are able to degrade various biopolymers, such as cellulose, xylan, and chitin. These enzymes are important to produce biofuels and biochemicals of interest from renewable biomass. In this chapter, the recent reports of novel enzymes produced by marine actinobacteria are reviewed, and advanced technologies that can be applied to search for novel marine enzymes as well as for improved enzyme production by marine actinobacteria are summarized, which include ribosome engineering, genome mining, as well as synthetic biology studies. © 2016 Elsevier Inc. All rights reserved.

  6. Sodium hydroxide pretreatment and enzymatic hydrolysis of coastal Bermuda grass.

    PubMed

    Wang, Ziyu; Keshwani, Deepak R; Redding, Arthur P; Cheng, Jay J

    2010-05-01

    Coastal Bermuda grass was pretreated with NaOH at concentrations from 0.5% to 3% (w/v) for a residence time from 15 to 90min at 121 degrees C. The pretreatments were evaluated based on total lignin removal and production of total reducing sugars, glucose and xylose from enzymatic hydrolysis of the pretreated biomass. Up to 86% lignin removal was observed. The optimal NaOH pretreatment conditions at 121 degrees C for total reducing sugars production as well as glucose and xylose yields are 15min and 0.75% NaOH. Under these optimal pretreatment conditions, total reducing sugars yield was about 71% of the theoretical maximum, and the overall conversion efficiencies for glucan and xylan were 90.43% and 65.11%, respectively. Copyright 2009 Elsevier Ltd. All rights reserved.

  7. Evaluation of Brazilian Sugarcane Bagasse Characterization: An Interlaboratory Comparison Study

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sluiter, Justin B.; Chum, Helena; Gomes, Absai C.

    2016-05-01

    This paper describes a study of the variability of measured composition for a single bulk sugarcane bagasse conducted across eight laboratories using similar analytical methods, with the purpose of determining the expected variation for compositional analysis performed by different laboratories. The results show good agreement of measured composition within a single laboratory, but greater variability when results are compared among laboratories. These interlaboratory variabilities do not seem to be associated with a specific method or technique or any single piece of instrumentation. The summary censored statistics provide mean values and pooled standard deviations as follows: total extractives 6.7% (0.6%), wholemore » ash 1.5% (0.2%), glucan 42.3% (1.2%), xylan 22.3% (0.5%), total lignin 21.3% (0.4%), and total mass closure 99.4% (2.9%).« less

  8. Accounting for all sugars produced during integrated production of ethanol from lignocellulosic biomass

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Schell, Daniel J.; Dowe, Nancy; Chapeaux, Alexandre

    This study explored integrated conversion of corn stover to ethanol and highlights techniques for accurate yield calculations. Acid pretreated corn stover (PCS) produced in a pilot-scale reactor was enzymatically hydrolyzed and the resulting sugars were fermented to ethanol by the glucose–xylose fermenting bacteria, Zymomonas mobilis 8b. The calculations account for high solids operation and oligomeric sugars produced during pretreatment, enzymatic hydrolysis, and fermentation, which, if not accounted for, leads to overestimating ethanol yields. The calculations are illustrated for enzymatic hydrolysis and fermentation of PCS at 17.5% and 20.0% total solids achieving 80.1% and 77.9% conversion of cellulose and xylan tomore » ethanol and ethanol titers of 63 g/L and 69 g/L, respectively. In the future, these techniques, including the TEA results, will be applied to fully integrated pilot-scale runs.« less

  9. Enzymatic hydrolysis of lignocellulosic biomass by Kitasatospora sp. to produce xylo-oligosaccharides (XOS)

    NASA Astrophysics Data System (ADS)

    Rahmani, Nanik; Jannah, Alifah Mafatikhul; Lisdiyanti, Puspita; Prasetya, Bambang; Yopi

    2017-11-01

    The optimizations of enzymatic hydrolysis to produce of xylo-oligosaccharides (XOs) from three different lignocellulosic biomasses were investigated. Sugarcane bagasse, oil palm empty fruit bunch, and rice straw contain rich hemicelluloses especially hetero-xylan which can be hydrolyzes by endo-xylanase enzyme. Enzymatic hydrolysis of sugarcane bagasse by endo-xylanase from Kitasatospora sp. was optimum at temperature hydrolysis 30 °C using 16 U of enzyme concentrations and 4 % substrate concentrations, while oil palm empty fruit bunchwas optimum at temperature hydrolysis 30 °C using 16 U of enzyme concentrations and 5 % substrate concentrations, and rice straw was optimum at 40 °C temperature hydrolysis using 16 U of enzyme concentrations and 4 % substrate concentrations. The hydrolysis products were analyzed by TLC and HPLC. The main product hydrolysis for sugarcane bagasse, oil palm empty fruit bunch and rice straw are xylobiose.

  10. Accounting for all sugars produced during integrated production of ethanol from lignocellulosic biomass

    DOE PAGES

    Schell, Daniel J.; Dowe, Nancy; Chapeaux, Alexandre; ...

    2016-01-19

    This study explored integrated conversion of corn stover to ethanol and highlights techniques for accurate yield calculations. Acid pretreated corn stover (PCS) produced in a pilot-scale reactor was enzymatically hydrolyzed and the resulting sugars were fermented to ethanol by the glucose–xylose fermenting bacteria, Zymomonas mobilis 8b. The calculations account for high solids operation and oligomeric sugars produced during pretreatment, enzymatic hydrolysis, and fermentation, which, if not accounted for, leads to overestimating ethanol yields. The calculations are illustrated for enzymatic hydrolysis and fermentation of PCS at 17.5% and 20.0% total solids achieving 80.1% and 77.9% conversion of cellulose and xylan tomore » ethanol and ethanol titers of 63 g/L and 69 g/L, respectively. In the future, these techniques, including the TEA results, will be applied to fully integrated pilot-scale runs.« less

  11. Influences of Media Compositions on Characteristics of Isolated Bacteria Exhibiting Lignocellulolytic Activities from Various Environmental Sites.

    PubMed

    Gong, Gyeongtaek; Lee, Sun-Mi; Woo, Han Min; Park, Tai Hyun; Um, Youngsoon

    2017-11-01

    Efficient isolation of lignocellulolytic bacteria is essential for the utilization of lignocellulosic biomass. In this study, bacteria with cellulolytic, xylanolytic, and lignolytic activities were isolated from environmental sites such as mountain, wetland, and mudflat using isolation media containing the combination of lignocellulose components (cellulose, xylan, and lignin). Eighty-nine isolates from the isolation media were characterized by analyzing taxonomic ranks and cellulolytic, xylanolytic, and lignolytic activities. Most of the cellulolytic bacteria showed multienzymatic activities including xylanolytic activity. The isolation media without lignin were efficient in isolating bacteria exhibiting multienzymatic activities even including lignolytic activity, whereas a lignin-containing medium was effective to isolate bacteria exhibiting lignolytic activity only. Multienzymatic activities were mainly observed in Bacillus and Streptomyces, while Burkholderia was the most abundant genus with lignolytic activity only. This study provides insight into isolation medium for efficient isolation of lignocellulose-degrading microorganisms.

  12. β-Glucuronidase-coupled assays of glucuronoyl esterases.

    PubMed

    Fraňová, Lucia; Puchart, Vladimír; Biely, Peter

    2016-10-01

    Glucuronoyl esterases (GEs) are microbial enzymes with potential to cleave the ester bonds between lignin alcohols and xylan-bound 4-O-methyl-d-glucuronic acid in plant cell walls. This activity renders GEs attractive research targets for biotechnological applications. One of the factors impeding the progress in GE research is the lack of suitable substrates. In this work, we report a facile preparation of methyl esters of chromogenic 4-nitrophenyl and 5-bromo-4-chloro-3-indolyl β-D-glucuronides for qualitative and quantitative GE assay coupled with β-glucuronidase as the auxiliary enzyme. The indolyl derivative affording a blue indigo-type product is suitable for rapid and sensitive assay of GE in commercial preparations as well as for high throughput screening of microorganisms and genomic and metagenomic libraries. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Dual effect of soluble materials in pretreated lignocellulose on simultaneous saccharification and co-fermentation process for the bioethanol production.

    PubMed

    Qin, Lei; Li, Xia; Liu, Li; Zhu, Jia-Qing; Guan, Qi-Man; Zhang, Man-Tong; Li, Wen-Chao; Li, Bing-Zhi; Yuan, Ying-Jin

    2017-01-01

    In this study, wash liquors isolated from ethylenediamine and dry dilute acid pretreated corn stover were used to evaluate the effect of soluble materials in pretreated biomass on simultaneous saccharification and co-fermentation (SSCF) for ethanol production, respectively. Both of the wash liquors had different impacts on enzymatic hydrolysis and fermentation. Enzymatic conversions of glucan and xylan monotonically decreased as wash liquor concentration increased. Whereas, with low wash liquor concentrations, xylose consumption rate, cell viability and ethanol yield were maximally stimulated in fermentation without nutrient supplementary. Soluble lignins were found as the key composition which promoted sugars utilization and cell viability without nutrient supplementary. The dual effects of soluble materials on enzymatic hydrolysis and fermentation resulted in the reduction of ethanol yield as soluble materials increased in SSCF. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Early-branching Gut Fungi Possess A Large, And Comprehensive Array Of Biomass-Degrading Enzymes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Solomon, Kevin V.; Haitjema, Charles; Henske, John K.

    The fungal kingdom is the source of almost all industrial enzymes in use for lignocellulose bioprocessing. Its more primitive members, however, remain relatively unexploited. We developed a systems-level approach that integrates RNA-Seq, proteomics, phenotype and biochemical studies of relatively unexplored early-branching free-living fungi. Anaerobic gut fungi isolated from herbivores produce a large array of biomass-degrading enzymes that synergistically degrade crude, unpretreated plant biomass, and are competitive with optimized commercial preparations from Aspergillus and Trichoderma. Compared to these model platforms, gut fungal enzymes are unbiased in substrate preference due to a wealth of xylan-degrading enzymes. These enzymes are universally catabolite repressed,more » and are further regulated by a rich landscape of noncoding regulatory RNAs. Furthermore, we identified several promising sequence divergent enzyme candidates for lignocellulosic bioprocessing.« less

  15. Improving enzymatic hydrolysis efficiency of wheat straw through sequential autohydrolysis and alkaline post-extraction.

    PubMed

    Wu, Xinxing; Huang, Chen; Zhai, Shengcheng; Liang, Chen; Huang, Caoxing; Lai, Chenhuan; Yong, Qiang

    2018-03-01

    In this work, a two-step pretreatment process of wheat straw was established by combining autohydrolysis pretreatment and alkaline post-extraction. The results showed that employing alkaline post-extraction to autohydrolyzed wheat straw could significantly improve its enzymatic hydrolysis efficiency from 36.0% to 83.7%. Alkaline post-extraction lead to the changes of the structure characteristics of autohydrolyzed wheat straw. Associations between enzymatic hydrolysis efficiency and structure characteristics were also studied. The results showed that the factors of structure characteristics such as delignification, xylan removal yield, crystallinity, accessibility and hydrophobicity are positively related to enzymatic hydrolysis efficiency within a certain range for alkaline post-extracted wheat straw. The results demonstrated that autohydrolysis coupled with alkaline post-extraction is an effective and promising method to gain fermentable sugars from biomass. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Role in pathogenesis of two endo-beta-1,4-xylanase genes from the vascular wilt fungus Fusarium oxysporum.

    PubMed

    Gómez-Gómez, E; Ruíz-Roldán, M C; Di Pietro, A; Roncero, M I G; Hera, C

    2002-04-01

    A gene, xyl4, whose predicted amino acid sequence shows significant homology with family 11 xylanases, was identified from the tomato vascular wilt fungus Fusarium oxysporum f. sp. lycopersici. Expression of xyl4 is induced on oat spelt xylan as the carbon source, subject to carbon catabolite repression and preferentially expressed at alkaline ambient pH. Transcript levels of xyl4 on an inducing carbon source are differentially regulated by the nature and concentration of the nitrogen source. As shown by RT-PCR, xyl4 is expressed by F. oxysporum during the entire cycle of infection on tomato plants. Targeted inactivation of xyl4 and of xyl3, a previously identified gene of F. oxysporum f. sp. lycopersici encoding a family 10 xylanase, had no detectable effect on virulence on tomato plants, demonstrating that both genes are not essential for pathogenicity.

  17. Pretreatment of corn stover using low-moisture anhydrous ammonia (LMAA) process.

    PubMed

    Yoo, Chang Geun; Nghiem, Nhuan P; Hicks, Kevin B; Kim, Tae Hyun

    2011-11-01

    A simple pretreatment method using anhydrous ammonia was developed to minimize water and ammonia inputs for cellulosic ethanol production, termed the low moisture anhydrous ammonia (LMAA) pretreatment. In this method, corn stover with 30-70% moisture was contacted with anhydrous ammonia in a reactor under nearly ambient conditions. After the ammoniation step, biomass was subjected to a simple pretreatment step at moderate temperatures (40-120°C) for 48-144 h. Pretreated biomass was saccharified and fermented without an additional washing step. With 3% glucan loading of LMAA-treated corn stover under best treatment conditions (0.1g-ammonia+1.0 g-water per g biomass, 80°C, and 84 h), simultaneous saccharification and cofermentation test resulted in 24.9 g/l (89% of theoretical ethanol yield based on glucan+xylan in corn stover). Copyright © 2011 Elsevier Ltd. All rights reserved.

  18. Development and Characterization of a High-Solids Deacetylation Process

    DOE PAGES

    Shekiro, III, Joseph; Chen, Xiaowen; Smith, Holly; ...

    2016-05-20

    Dilute-acid pretreatment has proven to be a robust means of converting herbaceous feedstock to fermentable sugars. However, it also releases acetic acid, a known fermentation inhibitor, from acetyl groups present in the biomass. A mild, dilute alkaline extraction stage was implemented prior to acid pretreatment to separate acetic acid from the hydrolysate sugar stream. This step, termed deacetylation, improved the glucose and xylose yields from enzymatic hydrolysis and ethanol yields from fermentation of the sugars relative to the control experiments using dilute-acid pretreatment of native corn stover without deacetylation. While promising, deacetylation as it was historically practiced is conducted atmore » low solids loadings, and at fixed conditions. Thus, many questions have been left unanswered, including the relationship between sodium hydroxide and solids loading, and acetate and xylan solubilization, as well as the impact of temperature and residence time on the process efficacy. A central composite experiment was designed to evaluate the impact of solids loading, sodium hydroxide loading, reaction time and temperature during deacetylation on the acetate and xylan solubilization of corn stover. Using the ANOVA test, it became apparent that neither of the responses was significantly impacted by the solids loading, while the reaction time was a minor factor - the responses were largely driven by reaction temperature and the sodium hydroxide loading. Based on the results, we successfully demonstrated the ability to transition the low-solids (10 % w/w) deacetylation process to a higher-solids process (30 % w/w) with minimal impact on the ability to extract acetate from biomass. Conditions were selected to minimize xylose loss during deacetylation, while also removing 70 % of acetyl groups. The impact of selected conditions on the enzymatic hydrolysis and fermentation was further investigated. In conclusion, evaluation of the whole-process impact demonstrated

  19. A novel cost-effective technology to convert sucrose and homocelluloses in sweet sorghum stalks into ethanol.

    PubMed

    Li, Jihong; Li, Shizhong; Han, Bing; Yu, Menghui; Li, Guangming; Jiang, Yan

    2013-11-29

    Sweet sorghum is regarded as a very promising energy crop for ethanol production because it not only supplies grain and sugar, but also offers lignocellulosic resource. Cost-competitive ethanol production requires bioconversion of all carbohydrates in stalks including of both sucrose and lignocellulose hydrolyzed into fermentable sugars. However, it is still a main challenge to reduce ethanol production cost and improve feasibility of industrial application. An integration of the different operations within the whole process is a potential solution. An integrated process combined advanced solid-state fermentation technology (ASSF) and alkaline pretreatment was presented in this work. Soluble sugars in sweet sorghum stalks were firstly converted into ethanol by ASSF using crushed stalks directly. Then, the operation combining ethanol distillation and alkaline pretreatment was performed in one distillation-reactor simultaneously. The corresponding investigation indicated that the addition of alkali did not affect the ethanol recovery. The effect of three alkalis, NaOH, KOH and Ca(OH)2 on pretreatment were investigated. The results indicated the delignification of lignocellulose by NaOH and KOH was more significant than that by Ca(OH)2, and the highest removal of xylan was caused by NaOH. Moreover, an optimized alkali loading of 10% (w/w DM) NaOH was determined. Under this favorable pretreatment condition, enzymatic hydrolysis of sweet sorghum bagasse following pretreatment was investigated. 92.0% of glucan and 53.3% of xylan conversion were obtained at enzyme loading of 10 FPU/g glucan. The fermentation of hydrolyzed slurry was performed using an engineered stain, Zymomonas mobilis TSH-01. A mass balance of the overall process was calculated, and 91.9 kg was achieved from one tonne of fresh sweet sorghum stalk. A low energy-consumption integrated technology for ethanol production from sweet sorghum stalks was presented in this work. Energy consumption for raw

  20. Mining for hemicellulases in the fungus-growing termite Pseudacanthotermes militaris using functional metagenomics.

    PubMed

    Bastien, Géraldine; Arnal, Grégory; Bozonnet, Sophie; Laguerre, Sandrine; Ferreira, Fernando; Fauré, Régis; Henrissat, Bernard; Lefèvre, Fabrice; Robe, Patrick; Bouchez, Olivier; Noirot, Céline; Dumon, Claire; O'Donohue, Michael

    2013-05-14

    The metagenomic analysis of gut microbiomes has emerged as a powerful strategy for the identification of biomass-degrading enzymes, which will be no doubt useful for the development of advanced biorefining processes. In the present study, we have performed a functional metagenomic analysis on comb and gut microbiomes associated with the fungus-growing termite, Pseudacanthotermes militaris. Using whole termite abdomens and fungal-comb material respectively, two fosmid-based metagenomic libraries were created and screened for the presence of xylan-degrading enzymes. This revealed 101 positive clones, corresponding to an extremely high global hit rate of 0.49%. Many clones displayed either β-d-xylosidase (EC 3.2.1.37) or α-l-arabinofuranosidase (EC 3.2.1.55) activity, while others displayed the ability to degrade AZCL-xylan or AZCL-β-(1,3)-β-(1,4)-glucan. Using secondary screening it was possible to pinpoint clones of interest that were used to prepare fosmid DNA. Sequencing of fosmid DNA generated 1.46 Mbp of sequence data, and bioinformatics analysis revealed 63 sequences encoding putative carbohydrate-active enzymes, with many of these forming parts of sequence clusters, probably having carbohydrate degradation and metabolic functions. Taxonomic assignment of the different sequences revealed that Firmicutes and Bacteroidetes were predominant phyla in the gut sample, while microbial diversity in the comb sample resembled that of typical soil samples. Cloning and expression in E. coli of six enzyme candidates identified in the libraries provided access to individual enzyme activities, which all proved to be coherent with the primary and secondary functional screens. This study shows that the gut microbiome of P. militaris possesses the potential to degrade biomass components, such as arabinoxylans and arabinans. Moreover, the data presented suggests that prokaryotic microorganisms present in the comb could also play a part in the degradation of biomass within the

  1. Sexual crossing of thermophilic fungus Myceliophthora heterothallica improved enzymatic degradation of sugar beet pulp.

    PubMed

    Aguilar-Pontes, Maria Victoria; Zhou, Miaomiao; van der Horst, Sjors; Theelen, Bart; de Vries, Ronald P; van den Brink, Joost

    2016-01-01

    Enzymatic degradation of plant biomass requires a complex mixture of many different enzymes. Like most fungi, thermophilic Myceliophthora species therefore have a large set of enzymes targeting different linkages in plant polysaccharides. The majority of these enzymes have not been functionally characterized, and their role in plant biomass degradation is unknown. The biotechnological challenge is to select the right set of enzymes to efficiently degrade a particular biomass. This study describes a strategy using sexual crossing and screening with the thermophilic fungus Myceliophthora heterothallica to identify specific enzymes associated with improved sugar beet pulp saccharification. Two genetically diverse M. heterothallica strains CBS 203.75 and CBS 663.74 were used to generate progenies with improved growth on sugar beet pulp. One progeny, named SBP.F1.2.11, had a different genetic pattern from the parental strains and had improved saccharification activity after the growth on 3 % sugar beet pulp. The improved SBP saccharification was not explained by altered activities of the major (hemi-)cellulases. Exo-proteome analysis of progeny and parental strains after 7-day growth on sugar beet pulp showed that only 17 of the 133 secreted CAZy enzymes were more abundant in progeny SBP.F1.2.11. Particularly one enzyme belonging to the carbohydrate esterase family 5 (CE5) was more abundant in SBP.F1.2.11. This CE5-CBM1 enzyme, named as Axe1, was phylogenetically related to acetyl xylan esterases. Biochemical characterization of Axe1 confirmed de-acetylation activity with optimal activities at 75-85 °C and pH 5.5-6.0. Supplementing Axe1 to CBS 203.75 enzyme set improved release of xylose and glucose from sugar beet pulp. This study identified beneficial enzymes for sugar beet pulp saccharification by selecting progeny with improved growth on this particular substrate. Saccharification of sugar beet pulp was improved by supplementing enzyme mixtures with a previously

  2. Structure determination of the extracellular xylanase from Geobacillus stearothermophilus by selenomethionyl MAD phasing.

    PubMed

    Teplitsky, A; Mechaly, A; Stojanoff, V; Sainz, G; Golan, G; Feinberg, H; Gilboa, R; Reiland, V; Zolotnitsky, G; Shallom, D; Thompson, A; Shoham, Y; Shoham, G

    2004-05-01

    Xylanases are hemicellulases that hydrolyze the internal beta-1,4-glycoside bonds of xylan. The extracellular thermostable endo-1,4-beta-xylanase (EC 3.2.1.8; XT6) produced by the thermophilic bacterium Geobacillus stearothermophilus T-6 was shown to bleach pulp optimally at pH 9 and 338 K and was successfully used in a large-scale biobleaching mill trial. The xylanase gene was cloned and sequenced. The mature enzyme consists of 379 amino acids, with a calculated molecular weight of 43 808 Da and a pI of 9.0. Crystallographic studies of XT6 were performed in order to study the mechanism of catalysis and to provide a structural basis for the rational introduction of enhanced thermostability by site-specific mutagenesis. XT6 was crystallized in the primitive trigonal space group P3(2)21, with unit-cell parameters a = b = 112.9, c = 122.7 A. A full diffraction data set for wild-type XT6 has been measured to 2.4 A resolution on flash-frozen crystals using synchrotron radiation. A fully exchanged selenomethionyl XT6 derivative (containing eight Se atoms per XT6 molecule) was also prepared and crystallized in an isomorphous crystal form, providing full selenium MAD data at three wavelengths and enabling phase solution and structure determination. The structure of wild-type XT6 was refined at 2.4 A resolution to a final R factor of 15.6% and an R(free) of 18.6%. The structure demonstrates that XT6 is made up of an eightfold TIM-barrel containing a deep active-site groove, consistent with its 'endo' mode of action. The two essential catalytic carboxylic residues (Glu159 and Glu265) are located at the active site within 5.5 A of each other, as expected for 'retaining' glycoside hydrolases. A unique subdomain was identified in the carboxy-terminal part of the enzyme and was suggested to have a role in xylan binding. The three-dimensional structure of XT6 is of great interest since it provides a favourable starting point for the rational improvement of its already high

  3. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Shekiro, III, Joseph; Chen, Xiaowen; Smith, Holly

    Dilute-acid pretreatment has proven to be a robust means of converting herbaceous feedstock to fermentable sugars. However, it also releases acetic acid, a known fermentation inhibitor, from acetyl groups present in the biomass. A mild, dilute alkaline extraction stage was implemented prior to acid pretreatment to separate acetic acid from the hydrolysate sugar stream. This step, termed deacetylation, improved the glucose and xylose yields from enzymatic hydrolysis and ethanol yields from fermentation of the sugars relative to the control experiments using dilute-acid pretreatment of native corn stover without deacetylation. While promising, deacetylation as it was historically practiced is conducted atmore » low solids loadings, and at fixed conditions. Thus, many questions have been left unanswered, including the relationship between sodium hydroxide and solids loading, and acetate and xylan solubilization, as well as the impact of temperature and residence time on the process efficacy. A central composite experiment was designed to evaluate the impact of solids loading, sodium hydroxide loading, reaction time and temperature during deacetylation on the acetate and xylan solubilization of corn stover. Using the ANOVA test, it became apparent that neither of the responses was significantly impacted by the solids loading, while the reaction time was a minor factor - the responses were largely driven by reaction temperature and the sodium hydroxide loading. Based on the results, we successfully demonstrated the ability to transition the low-solids (10 % w/w) deacetylation process to a higher-solids process (30 % w/w) with minimal impact on the ability to extract acetate from biomass. Conditions were selected to minimize xylose loss during deacetylation, while also removing 70 % of acetyl groups. The impact of selected conditions on the enzymatic hydrolysis and fermentation was further investigated. In conclusion, evaluation of the whole-process impact demonstrated

  4. Kinetic compensation effect in logistic distributed activation energy model for lignocellulosic biomass pyrolysis.

    PubMed

    Xu, Di; Chai, Meiyun; Dong, Zhujun; Rahman, Md Maksudur; Yu, Xi; Cai, Junmeng

    2018-06-04

    The kinetic compensation effect in the logistic distributed activation energy model (DAEM) for lignocellulosic biomass pyrolysis was investigated. The sum of square error (SSE) surface tool was used to analyze two theoretically simulated logistic DAEM processes for cellulose and xylan pyrolysis. The logistic DAEM coupled with the pattern search method for parameter estimation was used to analyze the experimental data of cellulose pyrolysis. The results showed that many parameter sets of the logistic DAEM could fit the data at different heating rates very well for both simulated and experimental processes, and a perfect linear relationship between the logarithm of the frequency factor and the mean value of the activation energy distribution was found. The parameters of the logistic DAEM can be estimated by coupling the optimization method and isoconversional kinetic methods. The results would be helpful for chemical kinetic analysis using DAEM. Copyright © 2018 Elsevier Ltd. All rights reserved.

  5. WRKY13 acts in stem development in Arabidopsis thaliana.

    PubMed

    Li, Wei; Tian, Zhaoxia; Yu, Diqiu

    2015-07-01

    Stems are important for plants to grow erectly. In stems, sclerenchyma cells must develop secondary cell walls to provide plants with physical support. The secondary cell walls are mainly composed of lignin, xylan and cellulose. Deficiency of overall stem development could cause weakened stems. Here we prove that WRKY13 acts in stem development. The wrky13 mutants take on a weaker stem phenotype. The number of sclerenchyma cells, stem diameter and the number of vascular bundles were reduced in wrky13 mutants. Lignin-synthesis-related genes were repressed in wrky13 mutants. Chromatin immunoprecipitation assays proved that WRKY13 could directly bind to the promoter of NST2. Taken together, we proposed that WRKY13 affected the overall development of stem. Identification of the role of WRKY13 may help to resolve agricultural problems caused by weaker stems. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  6. Differential reinforcement of enzymatic hydrolysis by adding chemicals and accessory proteins to high solid loading substrates with different pretreatments.

    PubMed

    Du, Jian; Song, Wenxia; Zhang, Xiu; Zhao, Jian; Liu, Guodong; Qu, Yinbo

    2018-04-23

    High dosage of enzyme is required to achieve effective lignocellulose hydrolysis, especially at high-solid loadings, which is a significant barrier to large-scale bioconversion of lignocellulose. Here, we screened four chemical additives and three accessory proteins for their effects on the enzymatic hydrolysis of various lignocellulosic materials. The effects were found to be highly dependent on the composition and solid loadings of substrates. For xylan-extracted lignin-rich corncob residue, the enhancing effect of PEG 6000 was most pronounced and negligibly affected by solid content, which reduced more than half of enzyme demand at 20% dry matter (DM). Lytic polysaccharide monooxygenase enhanced the hydrolysis of ammonium sulfite wheat straw pulp, and its addition reduced about half of protein demand at the solid loading of 20% DM. Supplementation of the additives in the hydrolysis of pure cellulose and complex lignocellulosic materials revealed that their effects are tightly linked to pretreatment strategies.

  7. Impact of lignins isolated from pretreated lignocelluloses on enzymatic cellulose saccharification.

    PubMed

    Barsberg, Søren; Selig, Michael Joseph; Felby, Claus

    2013-02-01

    Lignins were enzymatically isolated from corn stover and wheat straw samples and subjected to hydrothermal or wet oxidation pretreatments for enzyme adsorption experimentations. Lignin contents of the isolates ranged from 26 to 71 % (w/w); cellulose ranged from 3 to 22 % (w/w); xylan from 0.7 to 6 % (w/w) and ash was from 5.8 to 30 % (w/w). ATR-IR analyses indicated significant and similar levels of calcium in all lignin isolates. Commercial cellulase adsorption studies showed that the presence of these lignins had no significant impact on the total amount of adsorbed enzyme in cellulose and cellulose-lignin systems. Consequently, the presence of the lignins had minimal effect, if any, on enzymatic cellulose conversion. Furthermore, this result, coupled with significant calcium levels in the isolated lignins, supports previous work suggesting lignin-calcium complexes reduce enzyme-lignin interactions.

  8. Prewashing enhances the liquid hot water pretreatment efficiency of waste wheat straw with high free ash content.

    PubMed

    Huang, Chen; Wu, Xinxing; Huang, Yang; Lai, Chenhuan; Li, Xin; Yong, Qiang

    2016-11-01

    The effect of prewashing process prior to the liquid hot water (LHW) pretreatment of high free ash content waste wheat straw (WWS) was investigated. It was found that prewashing process decreased the ash content of WWS greatly, from 29.48% to 9.82%. This contributed to the lower pH value of prehydrolyzate and higher xylan removal in the following LHW pretreatment. More importantly, the prewashing process effectively increased the cellulose enzymatic hydrolysis efficiency of pretreated WWS, from 53.04% to 84.15%. The acid buffering capacity (ABC) and cation exchange capacity (CEC) of raw and prewashed WWS were examined. The majority of free ash removal from WWS by prewashing resulted in the decrease of the ABC of the WWS from 211.74 to 61.81mmol/pH-kg, and potentially enhancing the efficiency of the follow-up LHW pretreatment. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Occurrence and function of enzymes for lignocellulose degradation in commercial Agaricus bisporus cultivation.

    PubMed

    Kabel, Mirjam A; Jurak, Edita; Mäkelä, Miia R; de Vries, Ronald P

    2017-06-01

    The white button mushroom Agaricus bisporus is economically the most important commercially produced edible fungus. It is grown on carbon- and nitrogen-rich substrates, such as composted cereal straw and animal manure. The commercial mushroom production process is usually performed in buildings or tunnels under highly controlled environmental conditions. In nature, the basidiomycete A. bisporus has a significant impact on the carbon cycle in terrestrial ecosystems as a saprotrophic decayer of leaf litter. In this mini-review, the fate of the compost plant cell wall structures, xylan, cellulose and lignin, is discussed. A comparison is made from the structural changes observed to the occurrence and function of enzymes for lignocellulose degradation present, with a special focus on the extracellular enzymes produced by A. bisporus. In addition, recent advancements in whole genome level molecular studies in various growth stages of A. bisporus in compost are reviewed.

  10. The composition of accessory enzymes of Penicillium chrysogenum P33 revealed by secretome and synergistic effects with commercial cellulase on lignocellulose hydrolysis.

    PubMed

    Yang, Yi; Yang, Jinshui; Liu, Jiawen; Wang, Ruonan; Liu, Liang; Wang, Fengqin; Yuan, Hongli

    2018-06-01

    Herein, we report the secretome of Penicillium chrysogenum P33 under induction of lignocellulose for the first time. A total of 356 proteins were identified, including complete cellulases and numerous hemicellulases. Supplementing a commercial cellulase with increasing dosage of P33 enzyme cocktail from 1 to 5 mg/g substrate increased the release of reducing sugars from delignified corn stover by 21.4% to 106.8%. When 50% cellulase was replaced by P33 enzyme cocktail, release of reducing sugars was 78.6% higher than with cellulase alone. Meanwhile, glucan and xylan conversion was increased by 37% and 106%, respectively. P33 enzyme cocktail also enhanced commercial cellulase hydrolysis against four different delignified lignocellulosic biomass. These findings demonstrate that mixing appropriate amount of P33 cocktail with cellulase improves polysaccharide hydrolysis, suggesting P33 enzymes have great potential for industrial applications. Copyright © 2018 Elsevier Ltd. All rights reserved.

  11. Combining hydrothermal pretreatment with enzymes de-pectinates and exposes the innermost xyloglucan-rich hemicellulose layers of wine grape pomace.

    PubMed

    Zietsman, Anscha J J; Moore, John P; Fangel, Jonatan U; Willats, William G T; Vivier, Melané A

    2017-10-01

    Chardonnay grape pomace was treated with pressurized heat followed by enzymatic hydrolysis, with commercial or pure enzymes, in buffered conditions. The pomace was unfermented as commonly found for white winemaking wastes and treatments aimed to simulate biovalorization processing. Cell wall profiling techniques showed that the pretreatment led to depectination of the outer layers thereby exposing xylan polymers and increasing the extractability of arabinans, galactans, arabinogalactan proteins and mannans. This higher extractability is believed to be linked with partial degradation and opening-up of cell wall networks. Pectinase-rich enzyme preparations were presumably able to access the inner rhamnogalacturonan I dominant coating layers due to the hydrothermal pretreatment. Patterns of epitope abundance and the sequential release of cell wall polymers with specific combinations of enzymes led to a working model of the hitherto, poorly understood innermost xyloglucan-rich hemicellulose layers of unfermented grape pomace. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Surfactant-assisted pretreatment and enzymatic hydrolysis of spent mushroom compost for the production of sugars.

    PubMed

    Kapu, N U S; Manning, M; Hurley, T B; Voigt, J; Cosgrove, D J; Romaine, C P

    2012-06-01

    Spent mushroom compost (SMC), a byproduct of commercial mushroom cultivation, poses serious environmental problems that have hampered the growth of this important agro-industry. In an effort to develop new applications for SMC, we explored its use as a feedstock for bioethanol production. SMC constitutes approximately 30%w/w polysaccharides, 66% of which is glucan. Following dilute-acid pretreatment and enzymatic hydrolysis, both in the presence of PEG 6000, 97% of glucan and 44% of xylan in SMC were converted into the corresponding monosaccharides. Incorporation of PEG 6000 reduced the cellulase requirement by 77%. Zwittergent 3-12 and 3-14 also significantly increased the efficacy of acid pretreatment and enzymatic hydrolysis. The use of SMC in bioethanol production represents a potential mitigation solution for the critical environmental issues associated with the stockpiling of the major byproduct of the mushroom industry. Copyright © 2012 Elsevier Ltd. All rights reserved.

  13. Isolation, purification and characterisation of low molecular weight xylanase from Bacillus pumilus SSP-34.

    PubMed

    Subramaniyan, S

    2012-04-01

    Low molecular weight endo-xylanase from Bacillus pumilus SSP-34 was purified to homogeneity using ion exchange and size exclusion chromatographies. Xylanases were isolated by novel purification protocol which includes the use of anion exchange matrix such as DEAE Sepharose CL 6B with less affinity towards enzyme protein. The purified B. pumilus SSP-34 have a molecular weight of 20 kDa, with optimum pH and temperature at 6.0 and 50 °C, respectively. The enzyme was stable at 50 °C for 30 min. It showed remarkable stability at pH values ranging from 4.5 to 9 when the reaction was carried out at 50 °C. K (m) and V (max) values, determined with oats spelts xylan were 6.5 mg ml⁻¹ and 1,233 μmol min⁻¹ mg⁻¹ protein, respectively, and the specific activity was 1,723 U mg⁻¹.

  14. The roles of xylan and lignin in oxalic acid pretreated corncob during separate enzymatic hydrolysis and ethanol fermentation

    Treesearch

    Jae-Won Lee; Rita C.L.B. Rodrigues; Hyun Joo Kim; In-Gyu Choi; Thomas W. Jeffries

    2010-01-01

    High yields of hemicellulosic and cellulosic sugars are critical in obtaining economical conversion of agricultural residues to ethanol. To optimize pretreatment conditions, we evaluated oxalic acid loading rates, treatment temperatures and times in a 23 full factorial design. Response-surface analysis revealed an optimal oxalic acid pretreatment...

  15. Combination of Xylanase and Debranching Enzymes Specific to Wheat Arabinoxylan Improve the Growth Performance and Gut Health of Broilers.

    PubMed

    Lei, Zhao; Shao, Yuxin; Yin, Xiaonan; Yin, Dafei; Guo, Yuming; Yuan, Jianmin

    2016-06-22

    Arabinoxylan (AX) is the major antinutritional factor of wheat. This study evaluated the synergistic effects of xylanase and debranching enzymes (arabinofuranosidase [ABF] and feruloyl esterase [FAE]) on AX. During in vitro tests, the addition of ABF or FAE accelerated the hydrolysis of water-soluble AX (WE-AX) and water-insoluble AX (WU-AX) and produced more xylan oligosaccharides (XOS) than xylanase alone. XOS obtained from WE-AX stimulated greater proliferation of Lactobacillus brevis and Bacillus subtilis than did fructo-oligosaccharides (FOS) and glucose. During in vivo trials, xylanase increased the average daily growth (ADG), decreased the feed-conversion ratio (FCR), and reduced the digesta viscosity of jejunum and intestinal lesions of broilers fed a wheat-based diet on day 36. ABF or FAE additions further improved these effects. Broilers fed a combination of xylanase, ABF, and FAE exhibited the best growth. In conclusion, the synergistic effects among xylanase, ABF, and FAE increased AX degradation, which improve the growth performance and gut health of broilers.

  16. Insights into cell wall structure of Sida hermaphrodita and its influence on recalcitrance.

    PubMed

    Damm, Tatjana; Pattathil, Sivakumar; Günl, Markus; Jablonowski, Nicolai David; O'Neill, Malcolm; Grün, Katharina Susanne; Grande, Philipp Michael; Leitner, Walter; Schurr, Ulrich; Usadel, Björn; Klose, Holger

    2017-07-15

    The perennial plant Sida hermaphrodita (Sida) is attracting attention as potential energy crop. Here, the first detailed view on non-cellulosic Sida cell wall polysaccharide composition, structure and architecture is given. Cell walls were prepared from Sida stems and sequentially extracted with aqueous buffers and alkali. The structures of the quantitatively predominant polysaccharides present in each fraction were determined by biochemical characterization, glycome profiling and mass spectrometry. The amounts of glucose released by Accellerase-1500 ® treatment of the cell wall and the cell wall residue remaining after each extraction were used to assess the roles of pectin and hemicellulose in the recalcitrance of Sida biomass. 4-O-Methyl glucuronoxylan with a low proportion of side substitutions was identified as the major non-cellulosic glycan component of Sida stem cell walls. Pectic polysaccharides and xylans were found to be associated with lignin, suggesting that these polysaccharides have roles in Sida cell wall recalcitrance to enzymatic hydrolysis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Phosphoric acid pretreatment of Achyranthes aspera and Sida acuta weed biomass to improve enzymatic hydrolysis.

    PubMed

    Siripong, Premjet; Duangporn, Premjet; Takata, Eri; Tsutsumi, Yuji

    2016-03-01

    Achyranthes aspera and Sida acuta, two types of weed biomass are abundant and waste in Thailand. We focus on them as novel feedstock for bio-ethanol production because they contain high-cellulose content (45.9% and 46.9%, respectively) and unutilized material. Phosphoric acid (70%, 75%, and 80%) was employed for the pretreatment to improve by enzymatic hydrolysis. The pretreatment process removed most of the xylan and a part of the lignin from the weeds, while most of the glucan remained. The cellulose conversion to glucose was greater for pretreated A. aspera (86.2 ± 0.3%) than that of the pretreated S. acuta (82.2 ± 1.1%). Thus, the removal of hemicellulose significantly affected the efficiency of the enzymatic hydrolysis. The scanning electron microscopy images showed the exposed fibrous cellulose on the cell wall surface, and this substantial change of the surface structure contributed to improving the enzyme accessibility. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Utilization of deoiled Jatropha curcas seed cake for production of xylanase from thermophilic Scytalidium thermophilum.

    PubMed

    Joshi, Chetna; Khare, S K

    2011-01-01

    Jatropha curcas is a major biodiesel crop. Large amount of deoiled cake is generated as by-product during biodiesel production from its seeds. Deoiled J. curcas seed cake was assessed as substrate for the production of xylanase from thermophilic fungus Scytalidium thermophilum by solid-state fermentation. The seed cake was efficiently utilized by S. thermophilum for its growth during which it produced good amount of heat stable extracellular xylanase. The solid-state fermentation conditions were optimized for maximum xylanase production. Under the optimized conditions viz. deoiled seed cake supplemented with 1% oat-spelt xylan, adjusted to pH 9.0, moisture content 1:3 w/v, inoculated with 1×10(6) spores per 5 g cake and incubated at 45 °C, 1455 U xylanase/g deoiled seed cake was obtained. The xylanase was useful in biobleaching of paper pulp. Solid-state fermentation of deoiled cake appears a potentially viable approach for its effective utilization. Copyright © 2010 Elsevier Ltd. All rights reserved.

  19. Comparative genomic analysis of the thermophilic biomass-degrading fungi Myceliophthora thermophila and Thielavia terrestris

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Berka, Randy M.; Grigoriev, Igor V.; Otillar, Robert

    2011-10-02

    Thermostable enzymes and thermophilic cell factories may afford economic advantages in the production of many chemicals and biomass-based fuels. Here we describe and compare the genomes of two thermophilic fungi, Myceliophthora thermophila and Thielavia terrestris. To our knowledge, these genomes are the first described for thermophilic eukaryotes and the first complete telomere-to-telomere genomes for filamentous fungi. Genome analyses and experimental data suggest that both thermophiles are capable of hydrolyzing all major polysaccharides found in biomass. Examination of transcriptome data and secreted proteins suggests that the two fungi use shared approaches in the hydrolysis of cellulose and xylan but distinct mechanismsmore » in pectin degradation. Characterization of the biomass-hydrolyzing activity of recombinant enzymes suggests that these organisms are highly efficient in biomass decomposition at both moderate and high temperatures. Furthermore, we present evidence suggesting that aside from representing a potential reservoir of thermostable enzymes, thermophilic fungi are amenable to manipulation using classical and molecular genetics.« less

  20. Hydrothermal Pretreatment of Date Palm (Phoenix dactylifera L.) Leaflets and Rachis to Enhance Enzymatic Digestibility and Bioethanol Potential

    PubMed Central

    Fang, Chuanji; Cybulska, Iwona; Brudecki, Grzegorz P.; Frankær, Christian Grundahl; Thomsen, Mette Hedegaard

    2015-01-01

    Date palm residues are one of the most promising lignocellulosic biomass for bioethanol production in the Middle East. In this study, leaflets and rachis were subjected to hydrothermal pretreatment to overcome the recalcitrance of the biomass for enzymatic conversion. Evident morphological, structural, and chemical changes were observed by scanning electron microscopy, X-ray diffraction, and infrared spectroscopy after pretreatment. High glucan (>90% for both leaflets and rachis) and xylan (>75% for leaflets and >79% for rachis) recovery were achieved. Under the optimal condition of hydrothermal pretreatment (210°C/10 min) highly digestible (glucan convertibility, 100% to leaflets, 78% to rachis) and fermentable (ethanol yield, 96% to leaflets, 80% to rachis) solid fractions were obtained. Fermentability test of the liquid fractions proved that no considerable inhibitors to Saccharomyces cerevisiae were produced in hydrothermal pretreatment. Given the high sugar recovery, enzymatic digestibility, and ethanol yield, production of bioethanol by hydrothermal pretreatment could be a promising way of valorization of date palm residues in this region. PMID:26347878

  1. Development of rapid bioconversion with integrated recycle technology for ethanol production from extractive ammonia pretreated corn stover.

    PubMed

    Jin, Mingjie; Liu, Yanping; da Costa Sousa, Leonardo; Dale, Bruce E; Balan, Venkatesh

    2017-08-01

    High enzyme loading and low productivity are two major issues impeding low cost ethanol production from lignocellulosic biomass. This work applied rapid bioconversion with integrated recycle technology (RaBIT) and extractive ammonia (EA) pretreatment for conversion of corn stover (CS) to ethanol at high solids loading. Enzymes were recycled via recycling unhydrolyzed solids. Enzymatic hydrolysis with recycled enzymes and fermentation with recycled yeast cells were studied. Both enzymatic hydrolysis time and fermentation time were shortened to 24 h. Ethanol productivity was enhanced by two times and enzyme loading was reduced by 30%. Glucan and xylan conversions reached as high as 98% with an enzyme loading of as low as 8.4 mg protein per g glucan. The overall ethanol yield was 227 g ethanol/kg EA-CS (191 g ethanol/kg untreated CS). Biotechnol. Bioeng. 2017;114: 1713-1720. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  2. Effects of Kraft lignin on hydrolysis/dehydration of sugars, cellulosic and lignocellulosic biomass under hot compressed water.

    PubMed

    Daorattanachai, Pornlada; Viriya-empikul, Nawin; Laosiripojana, Navadol; Faungnawakij, Kajornsak

    2013-09-01

    The effect of Kraft lignin presenting on the hydrolysis and dehydration of C5 and C6 sugars, cellulose, hemicelluloses and biomass under hot compressed water (HCW) in the presence of H3PO4 catalyst was intensively studied. The lignin strongly inhibited the acid hydrolysis of cellulose and hemicellulose to glucose and xylose, respectively. Interestingly, the admixed lignin markedly promoted the isomerization of glucose to fructose, and dehydration of fructose (except at the low catalyst loading), resulting in high 5-hydroxymethylfurfural yields. Nonetheless, lignin inhibited the hydrolysis of xylan to xylose and dehydration of xylose to furfural. Moreover, the acidity of the system significantly affects the hydrolysis/dehydration of biomass. It was revealed that the presence of lignin strongly interfered the yields of sugars and furans produced from raw corncob, while the delignified corncob provided significant improvement of product yields, confirming the observed role of lignin in the biomass conversion system via sugar platforms. Copyright © 2013 Elsevier Ltd. All rights reserved.

  3. Nitrogen and Sulfur Requirements for Clostridium thermocellum and Caldicellulosiruptor bescii on Cellulosic Substrates in Minimal Nutrient Media

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kridelbaugh, Donna M; Nelson, Josh C; Engle, Nancy L

    2013-01-01

    Growth media for cellulolytic Clostridium thermocellum and Caldicellulosiruptor bescii bacteria usually contain excess nutrients that would increase costs for consolidated bioprocessing for biofuel production and create a waste stream with nitrogen, sulfur and phosphate. C. thermocellum was grown on crystalline cellulose with varying concentrations of nitrogen and sulfur compounds, and growth rate and alcohol production response curves were determined. Both bacteria assimilated sulfate in the presence of ascorbate reductant, increasing the ratio of oxidized to reduced fermentation products. From these results, a low ionic strength, defined minimal nutrient medium with decreased nitrogen, sulfur, phosphate and vitamin supplements was developed formore » the fermentation of cellobiose, cellulose and acid-pretreated Populus. Carbon and electron balance calculations indicate the unidentified residual fermentation products must include highly reduced molecules. Both bacterial populations were maintained in co-cultures with substrates containing xylan or hemicellulose in defined medium with sulfate and basal vitamin supplements.« less

  4. Structural and Functional Analyses of a Glycoside Hydrolase Family 5 Enzyme with an Unexpected [beta]-Fucosidase Activity

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yoshida, Shosuke; Park, David S.; Bae, Brian

    2012-02-15

    We present characterization of PbFucA, a family 5 glycoside hydrolase (GH5) from Prevotella bryantii B{sub 1}4. While GH5 members typically are xylanases, PbFucA shows no activity toward xylan polysaccharides. A screen against a panel of p-nitrophenol coupled sugars identifies PbFucA as a {beta}-D-fucosidase. We also present the 2.2 {angstrom} resolution structure of PbFucA and use structure-based mutational analysis to confirm the role of catalytically essential residues. A comparison of the active sites of PbFucA with those of family 5 and 51 glycosidases reveals that while the essential catalytic framework is identical between these enzymes, the steric contours of the respectivemore » active site clefts are distinct and likely account for substrate discrimination. Our results show that members of this cluster of orthologous group (COG) 5520 have {beta}-D-fucosidase activities, despite showing an overall sequence and structural similarity to GH-5 xylanases.« less

  5. Simultaneous pretreatment and enzymatic hydrolysis of forage biomass

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Henk, L.; Linden, J.C.

    1993-12-31

    Sweet sorghum is an attractive fermentation feedstock because as much as 40% of the dry weight consists of readily femented sugars such as sucrose, glucose and frutose. Cellulose and hemicellulose comprise another 50%. However, if this material is to be used a year-round feedstock for ethanol production, a stable method of storage must be developed to maintain the sugar content. A modified version of the traditional ensiling process is made effective by the addition of cellulolytic/hemicellulolytic enzymes and lactic acid bacteria to freshly chopped sweet sorghum prior to the production of silage. In situ hydrolysis of cellulose and hemicellulose occursmore » concurrently with the acidic ensiling fementation. By hydolyzing the acetyl groups using acetyl xylan esterase and 3-0-methyl glucuronyl side chains using pectinase from hemicellulose, cellulose becomes accessible to hydrolysis by cellulase, both during in situ ensiling with enzymes and in the simultaneous saccharification and fermentation (SSF) to ethanol.« less

  6. Comparative genomic analysis of the thermophilic biomass-degrading fungi Myceliophthora thermophila and Thielavia terrestris

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Berka, Randy M.; Grigoriev, Igor V.; Otillar, Robert

    2011-05-16

    Thermostable enzymes and thermophilic cell factories may afford economic advantages in the production of many chemicals and biomass-based fuels. Here we describe and compare the genomes of two thermophilic fungi, Myceliophthora thermophila and Thielavia terrestris. To our knowledge, these genomes are the first described for thermophilic eukaryotes and the first complete telomere-to-telomere genomes for filamentous fungi. Genome analyses and experimental data suggest that both thermophiles are capable of hydrolyzing all major polysaccharides found in biomass. Examination of transcriptome data and secreted proteins suggests that the two fungi use shared approaches in the hydrolysis of cellulose and xylan but distinct mechanismsmore » in pectin degradation. Characterization of the biomass-hydrolyzing activity of recombinant enzymes suggests that these organisms are highly efficient in biomass decomposition at both moderate and high temperatures. Furthermore, we present evidence suggesting that aside from representing a potential reservoir of thermostable enzymes, thermophilic fungi are amenable to manipulation using classical and molecular genetics.« less

  7. Hot-compressed water extraction of polysaccharides from soy hulls.

    PubMed

    Liu, Hua-Min; Wang, Fei-Yun; Liu, Yu-Lan

    2016-07-01

    The polysaccharides of soy hulls were extracted by hot-compressed water at temperatures of 110 from 180°C and various treatment times (10-150min) in a batch system. It was determined that a moderate temperature and short time are suitable for the preparation of polysaccharides. The structure of xylan and the inter- and intra-chain hydrogen bonding of cellulose fibrils in the soy hulls were not significantly broken down. The polysaccharides obtained were primarily composed of α-L-arabinofuranosyl units, 4-O-methyl-glucuronic acid units and α-D-galactose units attached with substituted units. A sugar analysis indicated that arabinose was the major component, constituting 35.6-46.9% of the polysaccharide products extracted at 130°C, 140°C, and 150°C. This investigation contributes to the knowledge of the polysaccharides of soy by-products, which can reduce the environmental impact of waste from the food industries. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Solid-State Treatment of Castor Cake Employing the Enzymatic Cocktail Produced from Pleurotus djamor Fungi.

    PubMed

    Sánchez-Cantú, Manuel; Ortiz-Moreno, Liliana; Ramos-Cassellis, María E; Marín-Castro, Marco; De la Cerna-Hernández, C

    2018-06-01

    In this work, the enzymatic cocktail produced by Pleurotus djamor fungi extracted at pH of 4.8 and 5.3 was employed for castor cake solid-state treatment. Proximal, X-ray powder diffraction and scanning electron microscopy analysis of the pristine castor cake were carried out. First, Pleurotus djamor stain was inoculated in castor cake for the enzymatic production and the enzymatic activity was determined. The maximum enzymatic activity was identified at days 14 (65.9 UI/gss) and 11 (140.3 UI/gss) for the enzymatic cocktail obtained at pH 5.3 and 4.8, respectively. Then, the enzymatic cocktail obtained at the highest enzymatic activity days was employed directly over castor cake. Lignin was degraded throughout incubation time achieving a 47 and 45% decrease for the cocktail produced at pH 4.8 and 5.3, correspondingly. These results were corroborated by the SEM and XRD analysis where a higher porosity and xylan degradation were perceived throughout the enzymatic treatment.

  9. Improvement in the productivity of xylooligosaccharides from waste medium after mushroom cultivation by hydrothermal treatment with suitable pretreatment.

    PubMed

    Sato, Nobuaki; Shinji, Kazunori; Mizuno, Masahiro; Nozaki, Kouichi; Suzuki, Masayuki; Makishima, Satoshi; Shiroishi, Masahiro; Onoda, Takeru; Takahashi, Fumihiro; Kanda, Takahisa; Amano, Yoshihiko

    2010-08-01

    The effective xylooligosaccharides (XOs) production from the waste medium after mushroom cultivation (WM) was investigated. The WM contains rich nutrients (protein, etc.) which induce Maillard reaction with reducing sugars under hydrothermal conditions. To improve the productivity of XOs, the suitable pretreatment combined with washing and grinding was investigated, and subsequently hydrothermal treatment was demonstrated with batch type and continuous flow type reactor. The washing pretreatment with hot water of 60 degrees C was effective to remove nutrients from the WM, and it led to prevent brownish discoloration on the hydrothermal treatment. On the basis of experimental data, industrial XOs production processes consisting of the pretreatment, hydrothermal treatment and purification step was designed. During the designed process, 2.3 kg-dry of the purified XOs was produced from 30 kg-wet of the WM (15% yield as dry basis weight). Theoretical yield of XOs attained to 48% as xylan weight in the WM. (c) 2010 Elsevier Ltd. All rights reserved.

  10. The mechanisms of hydrothermal deconstruction of lignocellulose: New insights from thermal–analytical and complementary studies

    PubMed Central

    Ibbett, Roger; Gaddipati, Sanyasi; Davies, Scott; Hill, Sandra; Tucker, Greg

    2011-01-01

    Differential Scanning Calorimetry, Dynamic Mechanical Thermal Analysis, gravimetric and chemical techniques have been used to study hydrothermal reactions of straw biomass. Exothermic degradation initiates above 195 °C, due to breakdown of the xylose ring from hemicellulose, which may be similar to reactions occurring during the early stage pyrolysis of dry biomass, though activated at lower temperature through water mediation. The temperature and magnitude of the exotherm reduce with increasing acid concentration, suggesting a reduction in activation energy and a change in the balance of reaction pathways. The presence of xylan oligomers in auto-catalytic hydrolysates is believed to be due to a low rate constant rather than a specific reaction mechanism. The loss of the lignin glass transition indicates that the lignin phase is reorganised under high temperature auto-catalytic conditions, but remains partially intact under lower temperature acid-catalytic conditions. This shows that lignin degradation reactions are activated thermally but are not effectively catalysed by aqueous acid. PMID:21763128

  11. Benefits from additives and xylanase during enzymatic hydrolysis of bamboo shoot and mature bamboo.

    PubMed

    Li, Kena; Wang, Xiao; Wang, Jingfeng; Zhang, Junhua

    2015-09-01

    Effects of additives (BSA, PEG 6000, and Tween 80) on enzymatic hydrolysis of bamboo shoot and mature bamboo fractions (bamboo green, bamboo timber, bamboo yellow, bamboo node, and bamboo branches) by cellulases and/or xylanase were evaluated. The addition of additives was comparable to the increase of cellulase loadings in the conversion of cellulose and xylan in bamboo fractions. Supplementation of xylanase (1 mg/g DM) with cellulases (10 FPU/g DM) in the hydrolysis of bamboo fractions was more efficient than addition of additives in the production of glucose and xylose. Moreover, addition of additives could further increase the glucose release from different bamboo fractions by cellulases and xylanase. Bamboo green exhibited the lowest hydrolyzability. Almost all of the polysaccharides in pretreated bamboo shoot fractions were hydrolyzed by cellulases with the addition of additives or xylanase. Additives and xylanase showed great potential for reducing cellulase requirement in the hydrolysis of bamboo. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Investigation of the pellets produced from sugarcane bagasse during liquid hot water pretreatment and their impact on the enzymatic hydrolysis.

    PubMed

    Wang, Wen; Zhuang, Xinshu; Yuan, Zhenhong; Yu, Qiang; Qi, Wei

    2015-08-01

    In the process of liquid hot water (LHW) pretreatment, there are numbers of pellets formed on the lignocellulosic surface. The characteristics and effect of pellets on the enzymatic hydrolysis of LHW-treated sugarcane bagasse (SCB) were investigated. After SCB was treated with LHW at 180°C, the pellets deposited on the surface of solid residues were extracted gently with 1% sodium hydroxide (NaOH) solution. They were composed of 81.0% lignin, 7.0% glucan, and 3.2% xylan. The LHW pretreatment solution (PS) was sprayed to the filter paper, and the pellets were observed on its surface. Fourier transform infrared spectroscopy (FTIR) data showed that lignin was also the main component of the PS pellets. The effect of the pellets on enzymatic hydrolysis was chiefly attributed to the steric hindrance, not the cellulase adsorption. The structural characteristics of LHW-treated SCB might play a more important role in influencing the enzymatic hydrolysis than the pellets. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Simultaneous saccharification and cofermentation of peracetic acid-pretreated biomass.

    PubMed

    Teixeira, L C; Linden, J C; Schroeder, H A

    2000-01-01

    Previous work in our laboratories has demonstrated the effectiveness of peracetic acid for improving enzymatic digestibility of lignocellulosic materials. The use of dilute alkali solutions as a pre-pretreatment prior to peracetic acid lignin oxidation increased carbohydrate hydrolysis yields in a synergistic as opposed to additive manner. Deacetylation of xylan is easily achieved using dilute alkali solutions under mild conditions. In this article, we evaluate the effectiveness of peracetic acid combined with an alkaline pre-pretreatment through simultaneous saccharification and cofermentation (SSCF) of pretreated hybrid poplar wood and sugar cane bagasse. Respective ethanol yields of 92.8 and 91.9% of theoretical are achieved using 6% NaOH/15% peracetic acid-pretreated substrates and recombinant Zymomonas mobilis CP4/pZB5. Reduction of acetyl groups of the lignocellulosic materials is demonstrated following alkaline pre-pretreatments. Such processing may be helpful in reducing peracetic acid requirements. The influence of deacetylation is more significant in combined pretreatments using lower peracetic acid loadings.

  14. Evaluation of agave bagasse recalcitrance using AFEX™, autohydrolysis, and ionic liquid pretreatments.

    PubMed

    Perez-Pimienta, Jose A; Flores-Gómez, Carlos A; Ruiz, Héctor A; Sathitsuksanoh, Noppadon; Balan, Venkatesh; da Costa Sousa, Leonardo; Dale, Bruce E; Singh, Seema; Simmons, Blake A

    2016-07-01

    A comparative analysis of the response of agave bagasse (AGB) to pretreatment by ammonia fiber expansion (AFEX™), autohydrolysis (AH) and ionic liquid (IL) was performed using 2D nuclear magnetic resonance (NMR) spectroscopy, wet chemistry, enzymatic saccharification and mass balances. It has been found that AFEX pretreatment preserved all carbohydrates in the biomass, whereas AH removed 62.4% of xylan and IL extracted 25% of lignin into wash streams. Syringyl and guaiacyl lignin ratio of untreated AGB was 4.3, whereas for the pretreated biomass the ratios were 4.2, 5.0 and 4.7 for AFEX, AH and IL, respectively. Using NMR spectra, the intensity of β-aryl ether units in aliphatic, anomeric, and aromatic regions decreased in all three pretreated samples when compared to untreated biomass. Yields of glucose plus xylose in the major hydrolysate stream were 42.5, 39.7 and 26.9kg per 100kg of untreated AGB for AFEX, IL and AH, respectively. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Key Microbiota Identification Using Functional Gene Analysis during Pepper (Piper nigrum L.) Peeling.

    PubMed

    Zhang, Jiachao; Hu, Qisong; Xu, Chuanbiao; Liu, Sixin; Li, Congfa

    2016-01-01

    Pepper pericarp microbiota plays an important role in the pepper peeling process for the production of white pepper. We collected pepper samples at different peeling time points from Hainan Province, China, and used a metagenomic approach to identify changes in the pericarp microbiota based on functional gene analysis. UniFrac distance-based principal coordinates analysis revealed significant changes in the pericarp microbiota structure during peeling, which were attributed to increases in bacteria from the genera Selenomonas and Prevotella. We identified 28 core operational taxonomic units at each time point, mainly belonging to Selenomonas, Prevotella, Megasphaera, Anaerovibrio, and Clostridium genera. The results were confirmed by quantitative polymerase chain reaction. At the functional level, we observed significant increases in microbial features related to acetyl xylan esterase and pectinesterase for pericarp degradation during peeling. These findings offer a new insight into biodegradation for pepper peeling and will promote the development of the white pepper industry.

  16. Key Microbiota Identification Using Functional Gene Analysis during Pepper (Piper nigrum L.) Peeling

    PubMed Central

    Xu, Chuanbiao; Liu, Sixin; Li, Congfa

    2016-01-01

    Pepper pericarp microbiota plays an important role in the pepper peeling process for the production of white pepper. We collected pepper samples at different peeling time points from Hainan Province, China, and used a metagenomic approach to identify changes in the pericarp microbiota based on functional gene analysis. UniFrac distance-based principal coordinates analysis revealed significant changes in the pericarp microbiota structure during peeling, which were attributed to increases in bacteria from the genera Selenomonas and Prevotella. We identified 28 core operational taxonomic units at each time point, mainly belonging to Selenomonas, Prevotella, Megasphaera, Anaerovibrio, and Clostridium genera. The results were confirmed by quantitative polymerase chain reaction. At the functional level, we observed significant increases in microbial features related to acetyl xylan esterase and pectinesterase for pericarp degradation during peeling. These findings offer a new insight into biodegradation for pepper peeling and will promote the development of the white pepper industry. PMID:27768750

  17. Complete genome sequence of Paenibacillus sp. strain JDR-2

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chow, Virginia; Nong, Guang; St. John, Franz J.

    2012-01-01

    Paenibacillus sp. strain JDR-2, an aggressively xylanolytic bacterium isolated from sweetgum (Liquidambar styraciflua) wood, is able to efficiently depolymerize, assimilate and metabolize 4-O-methylglucuronoxylan, the predominant structural component of hardwood hemicelluloses. A basis for this capability was first supported by the identification of genes and characterization of encoded enzymes and has been further defined by the sequencing and annotation of the complete genome, which we describe. In addition to genes implicated in the utilization of -1,4-xylan, genes have also been identified for the utilization of other hemicellulosic polysaccharides. The genome of Paenibacillus sp. JDR-2 contains 7,184,930 bp in a single repliconmore » with 6,288 protein-coding and 122 RNA genes. Uniquely prominent are 874 genes encoding proteins involved in carbohydrate transport and metabolism. The prevalence and organization of these genes support a metabolic potential for bioprocessing of hemicellulose fractions derived from lignocellulosic resources.« less

  18. Maleic acid treatment of biologically detoxified corn stover liquor.

    PubMed

    Kim, Daehwan; Ximenes, Eduardo A; Nichols, Nancy N; Cao, Guangli; Frazer, Sarah E; Ladisch, Michael R

    2016-09-01

    Elimination of microbial and enzyme inhibitors from pretreated lignocellulose is critical for effective cellulose conversion and yeast fermentation of liquid hot water (LHW) pretreated corn stover. In this study, xylan oligomers were hydrolyzed using either maleic acid or hemicellulases, and other soluble inhibitors were eliminated by biological detoxification. Corn stover at 20% (w/v) solids was LHW pretreated LHW (severity factor: 4.3). The 20% solids (w/v) pretreated corn stover derived liquor was recovered and biologically detoxified using the fungus Coniochaeta ligniaria NRRL30616. After maleic acid treatment, and using 5 filter paper units of cellulase/g glucan (8.3mg protein/g glucan), 73% higher cellulose conversion from corn stover was obtained for biodetoxified samples compared to undetoxified samples. This corresponded to 87% cellulose to glucose conversion. Ethanol production by yeast of pretreated corn stover solids hydrolysate was 1.4 times higher than undetoxified samples, with a reduction of 3h in the fermentation lag phase. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Characterization of structural cell wall polysaccharides in cattail (Typha latifolia): Evaluation as potential biofuel feedstock.

    PubMed

    Rebaque, Diego; Martínez-Rubio, Romina; Fornalé, Silvia; García-Angulo, Penélope; Alonso-Simón, Ana; Álvarez, Jesús M; Caparros-Ruiz, David; Acebes, José L; Encina, Antonio

    2017-11-01

    Second generation bioethanol produced from lignocellulosic biomass is attracting attention as an alternative energy source. In this study, a detailed knowledge of the composition and structure of common cattail (Typha latifolia L.) cell wall polysaccharides, obtained from stem or leaves, has been conducted using a wide set of techniques to evaluate this species as a potential bioethanol feedstock. Our results showed that common cattail cellulose content was high for plants in the order Poales and was accompanied by a small amount of cross-linked polysaccharides. A high degree of arabinose-substitution in xylans, a high syringyl/guaiacyl ratio in lignin and a low level of cell wall crystallinity could yield a good performance for lignocellulose saccharification. These results identify common cattail as a promising plant for use as potential bioethanol feedstock. To the best of our knowledge, this is the first in-depth analysis to be conducted of lignocellulosic material from common cattail. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Isolation of mesophilic solvent-producing clostridia from Colombian sources: physiological characterization, solvent production and polysaccharide hydrolysis.

    PubMed

    Montoya, D; Spitia, S; Silva, E; Schwarz, W H

    2000-04-28

    One hundred and seventy-eight new butanol-acetone producing bacteria related to saccharolytic clostridia were isolated from agricultural sources in Colombia and their fermentation potential was evaluated. Thirteen isolates produced more total solvents from glucose than Clostridium acetobutylicum ATCC 824. The isolates with the highest single solvent production were IBUN 125C and IBUN 18A with 0.46 mol butanol and 0.96 mol ethanol formed from 1 mol glucose, yielding 25. 2 and 29.1 g l(-1) total solvents, respectively, which is close to the maximum values described to date. Most of the new isolates produced exoenzymes for the hydrolysis of starch, carboxymethyl cellulose, xylan, polygalacturonic acid, inulin and chitosan. Together with the high efficiency of solvent production, these hydrolytic isolates may be useful for the direct fermentation of biomass. According to their physiological profile, the most solvent-productive isolates could be classified as strains of C. acetobutylicum, Clostridium beijerinckii, and Clostridium NCP262.