Fungal-type carbohydrate binding modules from the coccolithophore Emiliania huxleyi show binding affinity to cellulose and chitin.

PubMed

Rooijakkers, Bart J M; Ikonen, Martina S; Linder, Markus B

2018-01-01

Six fungal-type cellulose binding domains were found in the genome of the coccolithophore Emiliania huxleyi and cloned and expressed in Escherichia coli. Sequence comparison indicate high similarity to fungal cellulose binding domains, raising the question of why these domains exist in coccolithophores. The proteins were tested for binding with cellulose and chitin as ligands, which resulted in the identification of two functional carbohydrate binding modules: EHUX2 and EHUX4. Compared to benchmark fungal cellulose binding domain Cel7A-CBM1 from Trichoderma reesei, these proteins showed slightly lower binding to birch and bacterial cellulose, but were more efficient chitin binders. Finally, a set of cellulose binding domains was created based on the shuffling of one well-functioning and one non-functional domain. These were characterized in order to get more information of the binding domain's sequence-function relationship, indicating characteristic differences between the molecular basis of cellulose versus chitin recognition. As previous reports have showed the presence of cellulose in coccoliths and here we find functional cellulose binding modules, a possible connection is discussed.

Development of enzymatically-active bacterial cellulose membranes through stable immobilization of an engineered β-galactosidase.

PubMed

Estevinho, Berta N; Samaniego, Nuria; Talens-Perales, David; Fabra, Maria José; López-Rubio, Amparo; Polaina, Julio; Marín-Navarro, Julia

2018-08-01

Enzymatically-active bacterial cellulose (BC) was prepared by non-covalent immobilization of a hybrid enzyme composed by a β-galactosidase from Thermotoga maritima (TmLac) and a carbohydrate binding module (CBM2) from Pyrococcus furiosus. TmLac-CBM2 protein was bound to BC, with higher affinity at pH 6.5 than at pH 8.5 and with high specificity compared to the non-engineered enzyme. Both hydrated (HBC) and freeze-dried (DBC) bacterial cellulose showed equivalent enzyme binding efficiencies. Initial reaction rate of HBC-bound enzyme was higher than DBC-bound and both of them were lower than the free enzyme. However, enzyme performance was similar in all three cases for the hydrolysis of 5% lactose to a high extent. Reuse of the immobilized enzyme was limited by the stability of the β-galactosidase module, whereas the CBM2 module provided stable attachment of the hybrid enzyme to the BC support, after long incubation periods (3 h) at 75 °C. Copyright © 2018 Elsevier B.V. All rights reserved.

Inter-domain Synergism Is Required for Efficient Feeding of Cellulose Chain into Active Site of Cellobiohydrolase Cel7A.

PubMed

Kont, Riin; Kari, Jeppe; Borch, Kim; Westh, Peter; Väljamäe, Priit

2016-12-09

Structural polysaccharides like cellulose and chitin are abundant and their enzymatic degradation to soluble sugars is an important route in green chemistry. Processive glycoside hydrolases (GHs), like cellobiohydrolase Cel7A of Trichoderma reesei (TrCel7A) are key components of efficient enzyme systems. TrCel7A consists of a catalytic domain (CD) and a smaller carbohydrate-binding module (CBM) connected through the glycosylated linker peptide. A tunnel-shaped active site rests in the CD and contains 10 glucose unit binding sites. The active site of TrCel7A is lined with four Trp residues with two of them, Trp-40 and Trp-38, in the substrate binding sites near the tunnel entrance. Although addressed in numerous studies the elucidation of the role of CBM and active site aromatics has been obscured by a complex multistep mechanism of processive GHs. Here we studied the role of the CBM-linker and Trp-38 of TrCel7A with respect to binding affinity, on- and off-rates, processivity, and synergism with endoglucanase. The CBM-linker increased the on-rate and substrate affinity of the enzyme. The Trp-38 to Ala substitution resulted in increased off-rates and decreased processivity. The effect of the Trp-38 to Ala substitution on on-rates was strongly dependent on the presence of the CBM-linker. This compensation between CBM-linker and Trp-38 indicates synergism between CBM-linker and CD in feeding the cellulose chain into the active site. The inter-domain synergism was pre-requisite for the efficient degradation of cellulose in the presence of endoglucanase. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Inter-domain Synergism Is Required for Efficient Feeding of Cellulose Chain into Active Site of Cellobiohydrolase Cel7A*

PubMed Central

Kont, Riin; Kari, Jeppe; Borch, Kim; Westh, Peter; Väljamäe, Priit

2016-01-01

Structural polysaccharides like cellulose and chitin are abundant and their enzymatic degradation to soluble sugars is an important route in green chemistry. Processive glycoside hydrolases (GHs), like cellobiohydrolase Cel7A of Trichoderma reesei (TrCel7A) are key components of efficient enzyme systems. TrCel7A consists of a catalytic domain (CD) and a smaller carbohydrate-binding module (CBM) connected through the glycosylated linker peptide. A tunnel-shaped active site rests in the CD and contains 10 glucose unit binding sites. The active site of TrCel7A is lined with four Trp residues with two of them, Trp-40 and Trp-38, in the substrate binding sites near the tunnel entrance. Although addressed in numerous studies the elucidation of the role of CBM and active site aromatics has been obscured by a complex multistep mechanism of processive GHs. Here we studied the role of the CBM-linker and Trp-38 of TrCel7A with respect to binding affinity, on- and off-rates, processivity, and synergism with endoglucanase. The CBM-linker increased the on-rate and substrate affinity of the enzyme. The Trp-38 to Ala substitution resulted in increased off-rates and decreased processivity. The effect of the Trp-38 to Ala substitution on on-rates was strongly dependent on the presence of the CBM-linker. This compensation between CBM-linker and Trp-38 indicates synergism between CBM-linker and CD in feeding the cellulose chain into the active site. The inter-domain synergism was pre-requisite for the efficient degradation of cellulose in the presence of endoglucanase. PMID:27780868

The spatial proximity effect of beta-glucosidase and cellulosomes on cellulose degradation.

PubMed

Li, Xiaoyi; Xiao, Yan; Feng, Yingang; Li, Bin; Li, Wenli; Cui, Qiu

2018-08-01

Low-cost saccharification is one of the key bottlenecks hampering the further application of lignocellulosic biomass. Clostridium thermocellum is a naturally ideal cellulose degrading bacterium armed with cellulosomes, which are multienzyme complexes that are capable of efficiently degrading cellulose. However, under controlled condition, the inhibition effect of hydrolysate cellobiose severely restricts the hydrolytic ability of cellulosomes. Although the addition of beta-glucosidase (Bgl) could effectively relieve this inhibition, the spatial proximity effect of Bgl and cellulosomes on cellulose degradation is still unclear. To address this issue, free Bgl from Caldicellulosiruptor sp. F32 (CaBglA), carbohydrate-binding module (CBM) fused CaBglA (CaBglA-CBM) and cellulosomal type II cohesin module (CohII) fused to CaBglA (CaBglA-CohII) were successfully constructed, and their enzymatic activities, binding abilities and saccharification efficiencies were systematically investigated in vitro and in vivo. In vivo, with the adjacency of CaBglA to cellulosomes, the saccharification efficiency of microcrystalline cellulose increased from 40% to 50%. For the pretreated wheat straw, the degradation rate of the combination of cells and the CaBglA-CohII or the CaBglA-CBM was as efficient as that of the free CaBglA (approximately 60%). This study demonstrated that the proximity of CaBglA to cellulosomes had a positive effect on microcrystalline cellulose but not on pretreated wheat straw, which may result from the nonproductive adsorption of lignin and the decreased thermostability of CaBglA-CBM and CaBglA-CohII compared to that of CaBglA. The above results will contribute to the design of cost-effective Bgls for industrial cellulose degradation. Copyright © 2018. Published by Elsevier Inc.

Amino Groups of Chitosan Are Crucial for Binding to a Family 32 Carbohydrate Binding Module of a Chitosanase from Paenibacillus elgii*

PubMed Central

Das, Subha Narayan; Wagenknecht, Martin; Nareddy, Pavan Kumar; Bhuvanachandra, Bhoopal; Niddana, Ramana; Balamurugan, Rengarajan; Swamy, Musti J.; Moerschbacher, Bruno M.; Podile, Appa Rao

2016-01-01

We report here the role and mechanism of specificity of a family 32 carbohydrate binding module (CBM32) of a glycoside hydrolase family 8 chitosanase from Paenibacillus elgii (PeCsn). Both the activity and mode of action of PeCsn toward soluble chitosan polymers were not different with/without the CBM32 domain of P. elgii (PeCBM32). The decreased activity of PeCsn without PeCBM32 on chitosan powder suggested that PeCBM32 increases the relative concentration of enzyme on the substrate and thereby enhanced enzymatic activity. PeCBM32 specifically bound to polymeric and oligomeric chitosan and showed very weak binding to chitin and cellulose. In isothermal titration calorimetry, the binding stoichiometry of 2 and 1 for glucosamine monosaccharide (GlcN) and disaccharide (GlcN)2, respectively, was indicative of two binding sites in PeCBM32. A three-dimensional model-guided site-directed mutagenesis and the use of defined disaccharides varying in the pattern of acetylation suggested that the amino groups of chitosan and the polar residues Glu-16 and Glu-38 of PeCBM32 play a crucial role for the observed binding. The specificity of CBM32 has been further elucidated by a generated fusion protein PeCBM32-eGFP that binds to the chitosan exposing endophytic infection structures of Puccinia graminis f. sp. tritici. Phylogenetic analysis showed that CBM32s appended to chitosanases are highly conserved across different chitosanase families suggesting their role in chitosan recognition and degradation. We have identified and characterized a chitosan-specific CBM32 useful for in situ staining of chitosans in the fungal cell wall during plant-fungus interaction. PMID:27405759

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.

Sugar-binding sites on the surface of the carbohydrate-binding module of CBH I from Trichoderma reesei.

PubMed

Tavagnacco, Letizia; Mason, Philip E; Schnupf, Udo; Pitici, Felicia; Zhong, Linghao; Himmel, Michael E; Crowley, Michael; Cesàro, Attilio; Brady, John W

2011-05-01

Molecular dynamics simulations were carried out for a system consisting of the carbohydrate-binding module (CBM) of the cellulase CBH I from Trichoderma reesei (Hypocrea jecorina) in a concentrated solution of β-D-glucopyranose, to determine whether there is any tendency for the sugar molecules to bind to the CBM. In spite of the general tendency of glucose to behave as an osmolyte, a marked tendency for the sugar molecules to bind to the protein was observed. However, the glucose molecules tended to bind only to specific sites on the protein. As expected, the hydrophobic face of the sugar molecules, comprising the axial H1, H3, and H5 aliphatic protons, tended to adhere to the flat faces of the three tyrosine side chains on the planar binding surface of the CBM. However, a significant tendency to bind to a groove-like feature on the upper surface of the CBM was also observed. These results would not be inconsistent with a model of the mechanism for this globular domain in which the cellodextrin chain being removed from the surface of crystalline cellulose passes over the upper surface of the CBM, presumably then available for hydrolysis in the active site tunnel of this processive cellulase. Copyright © 2011 Elsevier Ltd. All rights reserved.

Glycosylated linkers in multimodular lignocellulose-degrading enzymes dynamically bind to cellulose

PubMed Central

Payne, Christina M.; Resch, Michael G.; Chen, Liqun; Crowley, Michael F.; Himmel, Michael E.; Taylor, Larry E.; Sandgren, Mats; Ståhlberg, Jerry; Stals, Ingeborg; Tan, Zhongping; Beckham, Gregg T.

2013-01-01

Plant cell-wall polysaccharides represent a vast source of food in nature. To depolymerize polysaccharides to soluble sugars, many organisms use multifunctional enzyme mixtures consisting of glycoside hydrolases, lytic polysaccharide mono-oxygenases, polysaccharide lyases, and carbohydrate esterases, as well as accessory, redox-active enzymes for lignin depolymerization. Many of these enzymes that degrade lignocellulose are multimodular with carbohydrate-binding modules (CBMs) and catalytic domains connected by flexible, glycosylated linkers. These linkers have long been thought to simply serve as a tether between structured domains or to act in an inchworm-like fashion during catalytic action. To examine linker function, we performed molecular dynamics (MD) simulations of the Trichoderma reesei Family 6 and Family 7 cellobiohydrolases (TrCel6A and TrCel7A, respectively) bound to cellulose. During these simulations, the glycosylated linkers bind directly to cellulose, suggesting a previously unknown role in enzyme action. The prediction from the MD simulations was examined experimentally by measuring the binding affinity of the Cel7A CBM and the natively glycosylated Cel7A CBM-linker. On crystalline cellulose, the glycosylated linker enhances the binding affinity over the CBM alone by an order of magnitude. The MD simulations before and after binding of the linker also suggest that the bound linker may affect enzyme action due to significant damping in the enzyme fluctuations. Together, these results suggest that glycosylated linkers in carbohydrate-active enzymes, which are intrinsically disordered proteins in solution, aid in dynamic binding during the enzymatic deconstruction of plant cell walls. PMID:23959893

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 and the surface energy of lignin that facilitates the LW forces should be a priori to avoid nonproductive binding of cellulase to kraft lignin.

Processive Degradation of Crystalline Cellulose by a Multimodular Endoglucanase via a Wirewalking Mode.

PubMed

Zhang, Kun-Di; Li, Wen; Wang, Ye-Fei; Zheng, Yan-Lin; Tan, Fang-Cheng; Ma, Xiao-Qing; Yao, Li-Shan; Bayer, Edward A; Wang, Lu-Shan; Li, Fu-Li

2018-05-14

Processive hydrolysis of crystalline cellulose by cellulases is a critical step for lignocellulose deconstruction. The classic Trichoderma reesei exoglucanase TrCel7A, which has a closed active-site tunnel, starts each processive run by threading the tunnel with a cellulose chain. Loop regions are necessary for tunnel conformation, resulting in weak thermostability of fungal exoglucanases. However, endoglucanase CcCel9A, from the thermophilic bacterium Clostridium cellulosi, comprises a glycoside hydrolase (GH) family 9 module with an open cleft and five carbohydrate-binding modules (CBMs) and hydrolyzes crystalline cellulose processively. How CcCel9A and other similar GH9 enzymes bind to the smooth surface of crystalline cellulose to achieve processivity is still unknown. Our results demonstrate that the C-terminal CBM3b and three CBMX2s enhance productive adsorption to cellulose, while the CBM3c adjacent to the GH9 is tightly bound to 11 glucosyl units, thereby extending the catalytic cleft to 17 subsites, which facilitates decrystallization by forming a supramodular binding surface. In the open cleft, the strong interaction forces between substrate-binding subsites and glucosyl rings enable cleavage of the hydrogen bonds and extraction of a single cellulose chain. In addition, subsite -4 is capable of drawing the chain to its favored location. Cellotetraose is released from the open cleft as the initial product to achieve high processivity, which is further hydrolyzed to cellotriose, cellobiose and glucose by the catalytic cleft of the endoglucanase. On this basis, we propose a wirewalking mode for processive degradation of crystalline cellulose by an endoglucanase, which provides insights for rational design of industrial cellulases.

Chimeric proteins combining phosphatase and cellulose-binding activities: proof-of-concept and application in the hydrolysis of paraoxon.

PubMed

Gonçalves, Larissa M; Chaimovich, Hernan; Cuccovia, Iolanda M; Marana, Sandro R

2014-05-01

Phosphatases for organophosphate degradation and carbohydrate-binding domains (CBMs) have potential biotechnological applications. As a proof-of-concept, a soluble chimeric protein that combines acid phosphatase (AppA) from Escherichia coli and a CBM from Xanthomonas axonopodis pv. citri (AppA-CBM) was produced in E.coli. AppACBM adsorbed in microcrystalline cellulose Avicel PH101 catalyzed the hydrolysis of p-nitrophenyl phosphate (PNPP). The binding to microcrystalline cellulose displayed saturation behavior with an apparent binding constant (Kb) of 22 ± 5 mg and a maximum binding (Bmax) of 1.500 ± 0.001 enzyme units. Binding was highest at pH 2.5 and decreased above pH 6.5, as previously observed for family 2 CBMs. The Km values for PNPP of AppA-CBM and native AppA were identical (2.7 mM). To demonstrate that this strategy for protein engineering has practical applications and is largely functional, even for phosphatases exhibiting diverse folds, a chimeric protein combining human paraoxonase 1 (hPON1) and the CBM was produced. Both PON1-CBM and hPON1 had identical K_m values for paraoxon (1.3 mM). Additionally, hPON1 bound to microcrystalline cellulose with a Kb of 27 ± 3 mg, the same as that observed for AppA-CBM. These data show that the phosphatase domains are as functional in both of the chimeric proteins as they are in the native enzymes and that the CBM domain maintains the same cellulose affinity. Therefore, the engineering of chimeric proteins combining domains of phosphatases and CBMs is fully feasible, resulting in chimeric enzymes that exhibit potential for OP detoxification.

Characterization of the Carbohydrate Binding Module 18 gene family in the amphibian pathogen Batrachochytrium dendrobatidis.

PubMed

Liu, Peng; Stajich, Jason E

2015-04-01

Batrachochytrium dendrobatidis (Bd) is the causative agent of chytridiomycosis responsible for worldwide decline in amphibian populations. Previous analysis of the Bd genome revealed a unique expansion of the carbohydrate-binding module family 18 (CBM18) predicted to be a sub-class of chitin recognition domains. CBM expansions have been linked to the evolution of pathogenicity in a variety of fungal species by protecting the fungus from the host. Based on phylogenetic analysis and presence of additional protein domains, the gene family can be classified into 3 classes: Tyrosinase-, Deacetylase-, and Lectin-like. Examination of the mRNA expression levels from sporangia and zoospores of nine of the cbm18 genes found that the Lectin-like genes had the highest expression while the Tyrosinase-like genes showed little expression, especially in zoospores. Heterologous expression of GFP-tagged copies of four CBM18 genes in Saccharomyces cerevisiae demonstrated that two copies containing secretion signal peptides are trafficked to the cell boundary. The Lectin-like genes cbm18-ll1 and cbm18-ll2 co-localized with the chitinous cell boundaries visualized by staining with calcofluor white. In vitro assays of the full length and single domain copies from CBM18-LL1 demonstrated chitin binding and no binding to cellulose or xylan. Expressed CBM18 domain proteins were demonstrated to protect the fungus, Trichoderma reeseii, in vitro against hydrolysis from exogenously added chitinase, likely by binding and limiting exposure of fungal chitin. These results demonstrate that cbm18 genes can play a role in fungal defense and expansion of their copy number may be an important pathogenicity factor of this emerging infectious disease of amphibians. Copyright © 2015 Elsevier Inc. All rights reserved.

In situ imaging of single carbohydrate-binding modules on cellulose microfibrils.

PubMed

Dagel, Daryl J; Liu, Yu-San; Zhong, Lanlan; Luo, Yonghua; Himmel, Michael E; Xu, Qi; Zeng, Yining; Ding, Shi-You; Smith, Steve

2011-02-03

The low efficiency of enzymes used in the bioprocessing of biomass for biofuels is one of the primary bottlenecks that must be overcome to make lignocellulosic biofuels cost-competitive. One of the rate-limiting factors is the accessibility of the cellulase enzymes to insoluble cellulolytic substrates, facilitated by surface absorption of the carbohydrate-binding modules (CBMs), a component of most cellulase systems. Despite their importance, reports of direct observation of CBM function and activity using microscopic methods are still uncommon. Here, we examine the site-specific binding of individual CBMs to crystalline cellulose in an aqueous environment, using the single molecule fluorescence method known as Defocused Orientation and Position Imaging (DOPI). Systematic orientations were observed that are consistent with the CBMs binding to the two opposite hydrophobic faces of the cellulose microfibril, with a well-defined orientation relative to the fiber axis. The approach provides in situ physical evidence indicating the CBMs bind with a well-defined orientation on those planes, thus supporting a binding mechanism driven by chemical and structural recognition of the cellulose surface.

A biomolecular recognition approach for the functionalization of cellulose with gold nanoparticles.

PubMed

Almeida, A; Rosa, A M M; Azevedo, A M; Prazeres, D M F

2017-09-01

Materials with new and improved functionalities can be obtained by modifying cellulose with gold nanoparticles (AuNPs) via the in situ reduction of a gold precursor or the deposition or covalent immobilization of pre-synthesized AuNPs. Here, we present an alternative biomolecular recognition approach to functionalize cellulose with biotin-AuNPs that relies on a complex of 2 recognition elements: a ZZ-CBM3 fusion that combines a carbohydrate-binding module (CBM) with the ZZ fragment of the staphylococcal protein A and an anti-biotin antibody. Paper and cellulose microparticles with AuNPs immobilized via the ZZ-CBM3:anti-biotin IgG supramolecular complex displayed an intense red color, whereas essentially no color was detected when AuNPs were deposited over the unmodified materials. Scanning electron microscopy analysis revealed a homogeneous distribution of AuNPs when immobilized via ZZ-CBM3:anti-biotin IgG complexes and aggregation of AuNPs when deposited over paper, suggesting that color differences are due to interparticle plasmon coupling effects. The approach could be used to functionalize paper substrates and cellulose nanocrystals with AuNPs. More important, however, is the fact that the occurrence of a biomolecular recognition event between the CBM-immobilized antibody and its specific, AuNP-conjugated antigen is signaled by red color. This opens up the way for the development of simple and straightforward paper/cellulose-based tests where detection of a target analyte can be made by direct use of color signaling. Copyright © 2017 John Wiley & Sons, Ltd.

Versatile derivatives of carbohydrate-binding modules for imaging of complex carbohydrates approaching the molecular level of resolution.

PubMed

Ding, Shi-You; Xu, Qi; Ali, Mursheda K; Baker, John O; Bayer, Edward A; Barak, Yoav; Lamed, Raphael; Sugiyama, Junji; Rumbles, Garry; Himmel, Michael E

2006-10-01

The innate binding specificity of different carbohydrate-binding modules (CBMs) offers a versatile approach for mapping the chemistry and structure of surfaces that contain complex carbohydrates. We have employed the distinct recognition properties of a double His-tagged recombinant CBM tagged with semiconductor quantum dots for direct imaging of crystalline cellulose at the molecular level of resolution, using transmission and scanning transmission electron microscopy. In addition, three different types of CBMs from families 3, 6, and 20 that exhibit different carbohydrate specificities were each fused with either green fluorescent protein (GFP) or red fluorescent protein (RFP) and employed for double-labeling fluorescence microscopy studies of primary cell walls and various mixtures of complex carbohydrate target molecules. CBM probes can be used for characterizing both native complex carbohydrates and engineered biomaterials.

Growth modulation effects of CBM2a under the control of AtEXP4 and CaMV35S promoters in Arabidopsis thaliana, Nicotiana tabacum and Eucalyptus camaldulensis.

PubMed

Keadtidumrongkul, Pornthep; Suttangkakul, Anongpat; Pinmanee, Phitsanu; Pattana, Kanokwan; Kittiwongwattana, Chokchai; Apisitwanich, Somsak; Vuttipongchaikij, Supachai

2017-08-01

The expression of cell-wall-targeted Carbohydrate Binding Modules (CBMs) can alter cell wall properties and modulate growth and development in plants such as tobacco and potato. CBM2a identified in xylanase 10A from Cellulomonas fimi is of particular interest for its ability to bind crystalline cellulose. However, its potential for promoting plant growth has not been explored. In this work, we tested the ability of CBM2a to promote growth when expressed using both CaMV35S and a vascular tissue-specific promoter derived from Arabidopsis expansin4 (AtEXP4) in three plant species: Arabidopsis, Nicotiana tabacum and Eucalyptus camaldulensis. In Arabidopsis, the expression of AtEXP4pro:CBM2a showed trends for growth promoting effects including the increase of root and hypocotyl lengths and the enlargements of the vascular xylem area, fiber cells and vessel cells. However, in N. tabacum, the expression of CBM2a under the control of either CaMV35S or AtEXP4 promoter resulted in subtle changes in the plant growth, and the thickness of secondary xylem and vessel and fiber cell sizes were generally reduced in the transgenic lines with AtEXP4pro:CBM2a. In Eucalyptus, while transgenics expressing CaMV35S:CBM2a showed very subtle changes compared to wild type, those transgenics with AtEXP4pro:CBM2a showed increases in plant height, enlargement of xylem areas and xylem fiber and vessel cells. These data provide comparative effects of expressing CBM2a protein in different plant species, and this finding can be applied for plant biomass improvement.

Origin, evolution, and divergence of plant class C GH9 endoglucanases.

PubMed

Kundu, Siddhartha; Sharma, Rita

2018-05-30

Glycoside hydrolases of the GH9 family encode cellulases that predominantly function as endoglucanases and have wide applications in the food, paper, pharmaceutical, and biofuel industries. The partitioning of plant GH9 endoglucanases, into classes A, B, and C, is based on the differential presence of transmembrane, signal peptide, and the carbohydrate binding module (CBM49). There is considerable debate on the distribution and the functions of these enzymes which may vary in different organisms. In light of these findings we examined the origin, emergence, and subsequent divergence of plant GH9 endoglucanases, with an emphasis on elucidating the role of CBM49 in the digestion of crystalline cellulose by class C members. Since, the digestion of crystalline cellulose mandates the presence of a well-defined set of aromatic and polar amino acids and/or an attributable domain that can mediate this conversion, we hypothesize a vertical mode of transfer of genes that could favour the emergence of class C like GH9 endoglucanase activity in land plants from potentially ancestral non plant taxa. We demonstrated the concomitant occurrence of a GH9 domain with CBM49 and other homologous carbohydrate binding modules, in putative endoglucanase sequences from several non-plant taxa. In the absence of comparable full length CBMs, we have characterized several low strength patterns that could approximate the CBM49, thereby, extending support for digestion of crystalline cellulose to other segments of the protein. We also provide data suggestive of the ancestral role of putative class C GH9 endoglucanases in land plants, which includes detailed phylogenetics and the presence and subsequent loss of CBM49, transmembrane, and signal peptide regions in certain populations of early land plants. These findings suggest that classes A and B of modern vascular land plants may have emerged by diverging directly from CBM49 encompassing putative class C enzymes. Our detailed phylogenetic and bioinformatics analysis of putative GH9 endoglucanase sequences across major taxa suggests that plant class C enzymes, despite their recent discovery, could function as the last common ancestor of classes A and B. Additionally, research into their ability to digest or inter-convert crystalline and amorphous forms of cellulose could make them lucrative candidates for engineering biofuel feedstock.

Chimeric cellulase matrix for investigating intramolecular synergism between non-hydrolytic disruptive functions of carbohydrate-binding modules and catalytic hydrolysis.

PubMed

Wang, Yuguo; Tang, Rentao; Tao, Jin; Wang, Xiaonan; Zheng, Baisong; Feng, Yan

2012-08-24

The conversion of renewable cellulosic biomass is of considerable interest for the production of biofuels and materials. The bottleneck in the efficient conversion is the compactness and resistance of crystalline cellulose. Carbohydrate-binding modules (CBMs), which disrupt crystalline cellulose via non-hydrolytic mechanisms, are expected to overcome this bottleneck. However, the lack of convenient methods for quantitative analysis of the disruptive functions of CBMs have hindered systematic studies and molecular modifications. Here we established a practical and systematic platform for quantifying and comparing the non-hydrolytic disruptive activities of CBMs via the synergism of CBMs and a catalytic module within designed chimeric cellulase molecules. Bioinformatics and computational biology were also used to provide a deeper understanding. A convenient vector was constructed to serve as a cellulase matrix into which heterologous CBM sequences can be easily inserted. The resulting chimeric cellulases were suitable for studying disruptive functions, and their activities quantitatively reflected the disruptive functions of CBMs on crystalline cellulose. In addition, this cellulase matrix can be used to construct novel chimeric cellulases with high hydrolytic activities toward crystalline cellulose.

Characterization of a Cellulomonas fimi exoglucanase/xylanase-endoglucanase gene fusion which improves microbial degradation of cellulosic biomass.

PubMed

Duedu, Kwabena O; French, Christopher E

2016-11-01

Effective degradation of cellulose requires multiple classes of enzyme working together. However, naturally occurring cellulases with multiple catalytic domains seem to be rather rare in known cellulose-degrading organisms. A fusion protein made from Cellulomonas fimi exo- and endo- glucanases, Cex and CenA which improves breakdown of cellulose is described. A homologous carbohydrate binding module (CBM-2) present in both glucanases was fused to give a fusion protein CxnA. CxnA or unfused constructs (Cex+CenA, Cex, or CenA) were expressed in Escherichia coli and Citrobacter freundii. The latter recombinant strains were cultured at the expense of cellulose filter paper. The expressed CxnA had both exo- and endo- glucanase activities. It was also exported to the supernatant as were the non-fused proteins. In addition, the hybrid CBM from the fusion could bind to microcrystalline cellulose. Growth of C. freundii expressing CxnA was superior to that of cells expressing the unfused proteins. Physical degradation of filter paper was also faster with the cells expressing fusion protein than the other constructs. Our results show that fusion proteins with multiple catalytic domains can improve the efficiency of cellulose degradation. Such fusion proteins could potentially substitute cloning of multiple enzymes as well as improving product yields. Copyright © 2016 Elsevier Inc. All rights reserved.

Probing of exopolysaccharides with green fluorescence protein-labeled carbohydrate-binding module in Escherichia coli biofilms and flocs induced by bcsB overexpression.

PubMed

Nguyen, Minh Hong; Ojima, Yoshihiro; Sakka, Makiko; Sakka, Kazuo; Taya, Masahito

2014-10-01

Polysaccharides are major structural constituents to develop the three-dimensional architecture of Escherichia coli biofilms. In this study, confocal laser scanning microscopy was applied in combination with a fluorescent probe to analyze the location and arrangement of exopolysaccharide (EPSh) in microcolonies of E. coli K-12 derived strains, formed as biofilms on solid surfaces and flocs in the liquid phase. For this purpose, a novel fluorescent probe was constructed by conjugating a carbohydrate-binding module 3, from Paenibacillus curdlanolyticus, with the green fluorescence protein (GFP-CBM3). The GFP-CBM3 fused protein exhibited strong affinity to microcrystalline cellulose. Moreover, GFP-CBM3 specifically bound to cell-dense microcolonies in the E. coli biofilms, and to their flocs induced by bcsB overexpression. Therefore, the fused protein presents as a novel marker for EPSh produced by E. coli cells. Overexpression of bcsB was associated with abundant EPSh production and enhanced E. coli biofilm formation, which was similarly detectable by GFP-CBM3 probing. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Chimeric Cellulase Matrix for Investigating Intramolecular Synergism between Non-hydrolytic Disruptive Functions of Carbohydrate-binding Modules and Catalytic Hydrolysis*

PubMed Central

Wang, Yuguo; Tang, Rentao; Tao, Jin; Wang, Xiaonan; Zheng, Baisong; Feng, Yan

2012-01-01

The conversion of renewable cellulosic biomass is of considerable interest for the production of biofuels and materials. The bottleneck in the efficient conversion is the compactness and resistance of crystalline cellulose. Carbohydrate-binding modules (CBMs), which disrupt crystalline cellulose via non-hydrolytic mechanisms, are expected to overcome this bottleneck. However, the lack of convenient methods for quantitative analysis of the disruptive functions of CBMs have hindered systematic studies and molecular modifications. Here we established a practical and systematic platform for quantifying and comparing the non-hydrolytic disruptive activities of CBMs via the synergism of CBMs and a catalytic module within designed chimeric cellulase molecules. Bioinformatics and computational biology were also used to provide a deeper understanding. A convenient vector was constructed to serve as a cellulase matrix into which heterologous CBM sequences can be easily inserted. The resulting chimeric cellulases were suitable for studying disruptive functions, and their activities quantitatively reflected the disruptive functions of CBMs on crystalline cellulose. In addition, this cellulase matrix can be used to construct novel chimeric cellulases with high hydrolytic activities toward crystalline cellulose. PMID:22778256

Carbohydrate Recognition by an Architecturally Complex α-N-Acetylglucosaminidase from Clostridium perfringens

PubMed Central

Ficko-Blean, Elizabeth; Stuart, Christopher P.; Suits, Michael D.; Cid, Melissa; Tessier, Matthew; Woods, Robert J.; Boraston, Alisdair B.

2012-01-01

CpGH89 is a large multimodular enzyme produced by the human and animal pathogen Clostridium perfringens. The catalytic activity of this exo-α-d-N-acetylglucosaminidase is directed towards a rare carbohydrate motif, N-acetyl-β-d-glucosamine-α-1,4-d-galactose, which is displayed on the class III mucins deep within the gastric mucosa. In addition to the family 89 glycoside hydrolase catalytic module this enzyme has six modules that share sequence similarity to the family 32 carbohydrate-binding modules (CBM32s), suggesting the enzyme has considerable capacity to adhere to carbohydrates. Here we suggest that two of the modules, CBM32-1 and CBM32-6, are not functional as carbohydrate-binding modules (CBMs) and demonstrate that three of the CBMs, CBM32-3, CBM32-4, and CBM32-5, are indeed capable of binding carbohydrates. CBM32-3 and CBM32-4 have a novel binding specificity for N-acetyl-β-d-glucosamine-α-1,4-d-galactose, which thus complements the specificity of the catalytic module. The X-ray crystal structure of CBM32-4 in complex with this disaccharide reveals a mode of recognition that is based primarily on accommodation of the unique bent shape of this sugar. In contrast, as revealed by a series of X-ray crystal structures and quantitative binding studies, CBM32-5 displays the structural and functional features of galactose binding that is commonly associated with CBM family 32. The functional CBM32s that CpGH89 contains suggest the possibility for multivalent binding events and the partitioning of this enzyme to highly specific regions within the gastrointestinal tract. PMID:22479408

Structural and Functional Analysis of a Lytic Polysaccharide Monooxygenase Important for Efficient Utilization of Chitin in Cellvibrio japonicus*

PubMed Central

Forsberg, Zarah; Nelson, Cassandra E.; Dalhus, Bjørn; Mekasha, Sophanit; Loose, Jennifer S. M.; Crouch, Lucy I.; Røhr, Åsmund K.; Gardner, Jeffrey G.; Eijsink, Vincent G. H.; Vaaje-Kolstad, Gustav

2016-01-01

Cellvibrio japonicus is a Gram-negative soil bacterium that is primarily known for its ability to degrade plant cell wall polysaccharides through utilization of an extensive repertoire of carbohydrate-active enzymes. Several putative chitin-degrading enzymes are also found among these carbohydrate-active enzymes, such as chitinases, chitobiases, and lytic polysaccharide monooxygenases (LPMOs). In this study, we have characterized the chitin-active LPMO, CjLPMO10A, a tri-modular enzyme containing a catalytic family AA10 LPMO module, a family 5 chitin-binding module, and a C-terminal unclassified module of unknown function. Characterization of the latter module revealed tight and specific binding to chitin, thereby unraveling a new family of chitin-binding modules (classified as CBM73). X-ray crystallographic elucidation of the CjLPMO10A catalytic module revealed that the active site of the enzyme combines structural features previously only observed in either cellulose or chitin-active LPMO10s. Analysis of the copper-binding site by EPR showed a signal signature more similar to those observed for cellulose-cleaving LPMOs. The full-length LPMO shows no activity toward cellulose but is able to bind and cleave both α- and β-chitin. Removal of the chitin-binding modules reduced LPMO activity toward α-chitin compared with the full-length enzyme. Interestingly, the full-length enzyme and the individual catalytic LPMO module boosted the activity of an endochitinase equally well, also yielding similar amounts of oxidized products. Finally, gene deletion studies show that CjLPMO10A is needed by C. japonicus to obtain efficient growth on both purified chitin and crab shell particles. PMID:26858252

Structural and Functional Analysis of a Lytic Polysaccharide Monooxygenase Important for Efficient Utilization of Chitin in Cellvibrio japonicus.

PubMed

Forsberg, Zarah; Nelson, Cassandra E; Dalhus, Bjørn; Mekasha, Sophanit; Loose, Jennifer S M; Crouch, Lucy I; Røhr, Åsmund K; Gardner, Jeffrey G; Eijsink, Vincent G H; Vaaje-Kolstad, Gustav

2016-04-01

Cellvibrio japonicusis a Gram-negative soil bacterium that is primarily known for its ability to degrade plant cell wall polysaccharides through utilization of an extensive repertoire of carbohydrate-active enzymes. Several putative chitin-degrading enzymes are also found among these carbohydrate-active enzymes, such as chitinases, chitobiases, and lytic polysaccharide monooxygenases (LPMOs). In this study, we have characterized the chitin-active LPMO,CjLPMO10A, a tri-modular enzyme containing a catalytic family AA10 LPMO module, a family 5 chitin-binding module, and a C-terminal unclassified module of unknown function. Characterization of the latter module revealed tight and specific binding to chitin, thereby unraveling a new family of chitin-binding modules (classified as CBM73). X-ray crystallographic elucidation of theCjLPMO10A catalytic module revealed that the active site of the enzyme combines structural features previously only observed in either cellulose or chitin-active LPMO10s. Analysis of the copper-binding site by EPR showed a signal signature more similar to those observed for cellulose-cleaving LPMOs. The full-length LPMO shows no activity toward cellulose but is able to bind and cleave both α- and β-chitin. Removal of the chitin-binding modules reduced LPMO activity toward α-chitin compared with the full-length enzyme. Interestingly, the full-length enzyme and the individual catalytic LPMO module boosted the activity of an endochitinase equally well, also yielding similar amounts of oxidized products. Finally, gene deletion studies show thatCjLPMO10A is needed byC. japonicusto obtain efficient growth on both purified chitin and crab shell particles. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Predominant nonproductive substrate binding by fungal cellobiohydrolase I and implications for activity improvement.

PubMed

Rabinovich, Mikhail L; Melnik, Maria S; Herner, Mikhail L; Voznyi, Yakov V; Vasilchenko, Lilia G

2018-05-21

Enzymatic conversion of the most abundant renewable source of organic compounds, cellulose to fermentable sugars is attractive for production of green fuels and chemicals. The major component of industrial enzyme systems, cellobiohydrolase I from Hypocrea jecorina (Trichoderma reesei) (HjCel7A) processively splits disaccharide units from the reducing ends of tightly packed cellulose chains. HjCel7A consists of a catalytic domain (CD) and a carbohydrate-binding module (CBM) separated by a linker peptide. A tunnel-shaped substrate-binding site in the CD includes 9 subsites for β-D-glucose units, 7 of which (-7 to -1) precede the catalytic center. Low catalytic activity of Cel7A is the bottleneck and the primary target for improvement. Here it is shown for the first time that, in spite of much lower apparent k cat of HjCel7A at the hydrolysis of β-1,4-glucosidic linkages in the fluorogenic cellotetra- and -pentaose compared to the structurally related endoglucanase I (HjCel7B), the specificity constants (catalytic efficiency) k cat /K m for both enzymes are almost equal in these reactions. The observed activity difference appears from strong nonproductive substrate binding by HjCel7A, particularly significant for MU-β-cellotetraose (MUG 4 ). Interaction of substrates with the subsites -6 and -5 proximal to the non-conserved Gln101 residue in HjCel7A decreases K m,ap by >1500 times. HjCel7A can be nonproductively bound onto cellulose surface with K d ∼2-9 nM via CBM and CD that captures 6 terminal glucose units of cellulose chain. Decomposition of this nonproductive complex can determine the rate of cellulose conversion. MUG 4 is a promising substrate to select active cellobiohydrolase I variants with reduced nonproductive substrate binding. This article is protected by copyright. All rights reserved.

Distinct roles of N- and O-glycans in cellulase activity and stability

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

Amore, Antonella; Knott, Brandon C.; Supekar, Nitin T.

In nature, many microbes secrete mixtures of glycoside hydrolases, oxidoreductases, and accessory enzymes to deconstruct polysaccharides and lignin in plants. These enzymes are often decorated with N- and O-glycosylation, the roles of which have been broadly attributed to protection from proteolysis, as the extracellular milieu is an aggressive environment. Glycosylation has been shown to sometimes affect activity, but these effects are not fully understood. In this paper, we examine N- and O-glycosylation on a model, multimodular glycoside hydrolase family 7 cellobiohydrolase (Cel7A), which exhibits an O-glycosylated carbohydrate-binding module (CBM) and an O-glycosylated linker connected to an N- and O-glycosylated catalyticmore » domain (CD) - a domain architecture common to many biomass-degrading enzymes. We report consensus maps for Cel7A glycosylation that include glycan sites and motifs. Additionally, we examine the roles of glycans on activity, substrate binding, and thermal and proteolytic stability. N-glycan knockouts on the CD demonstrate that N-glycosylation has little impact on cellulose conversion or binding, but does have major stability impacts. O-glycans on the CBM have little impact on binding, proteolysis, or activity in the whole-enzyme context. However, linker O-glycans greatly impact cellulose conversion via their contribution to proteolysis resistance. Molecular simulations predict an additional role for linker O-glycans, namely that they are responsible for maintaining separation between ordered domains when Cel7A is engaged on cellulose, as models predict a-helix formation and decreased cellulose interaction for the nonglycosylated linker. In conclusion, this study reveals key roles for N- and O-glycosylation that are likely broadly applicable to other plant cell-wall-degrading enzymes.« less

Distinct roles of N- and O-glycans in cellulase activity and stability

DOE PAGES

Amore, Antonella; Knott, Brandon C.; Supekar, Nitin T.; ...

2017-12-11

In nature, many microbes secrete mixtures of glycoside hydrolases, oxidoreductases, and accessory enzymes to deconstruct polysaccharides and lignin in plants. These enzymes are often decorated with N- and O-glycosylation, the roles of which have been broadly attributed to protection from proteolysis, as the extracellular milieu is an aggressive environment. Glycosylation has been shown to sometimes affect activity, but these effects are not fully understood. In this paper, we examine N- and O-glycosylation on a model, multimodular glycoside hydrolase family 7 cellobiohydrolase (Cel7A), which exhibits an O-glycosylated carbohydrate-binding module (CBM) and an O-glycosylated linker connected to an N- and O-glycosylated catalyticmore » domain (CD) - a domain architecture common to many biomass-degrading enzymes. We report consensus maps for Cel7A glycosylation that include glycan sites and motifs. Additionally, we examine the roles of glycans on activity, substrate binding, and thermal and proteolytic stability. N-glycan knockouts on the CD demonstrate that N-glycosylation has little impact on cellulose conversion or binding, but does have major stability impacts. O-glycans on the CBM have little impact on binding, proteolysis, or activity in the whole-enzyme context. However, linker O-glycans greatly impact cellulose conversion via their contribution to proteolysis resistance. Molecular simulations predict an additional role for linker O-glycans, namely that they are responsible for maintaining separation between ordered domains when Cel7A is engaged on cellulose, as models predict a-helix formation and decreased cellulose interaction for the nonglycosylated linker. In conclusion, this study reveals key roles for N- and O-glycosylation that are likely broadly applicable to other plant cell-wall-degrading enzymes.« less

Biochemical and structural characterizations of two Dictyostelium cellobiohydrolases from the amoebozoa kingdom reveal a high level of conservation between distant phylogenetic trees of life

DOE PAGES

Hobdey, Sarah E.; Knott, Brandon C.; Momeni, Majid Haddad; ...

2016-04-01

Glycoside hydrolase family 7 (GH7) cellobiohydrolases (CBHs) are enzymes often employed in plant cell wall degradation across eukaryotic kingdoms of life, as they provide significant hydrolytic potential in cellulose turnover. To date, many fungal GH7 CBHs have been examined, yet many questions regarding structure-activity relationships in these important natural and commercial enzymes remain. Here, we present the crystal structures and a biochemical analysis of two GH7 CBHs from social amoeba: Dictyostelium discoideum Cel7A (DdiCel7A) and Dictyostelium purpureum Cel7A (DpuCel7A). DdiCel7A and DpuCel7A natively consist of a catalytic domain and do not exhibit a carbohydrate-binding module (CBM). The structures of DdiCel7Amore » and DpuCel7A, resolved to 2.1 Å and 2.7 Å, respectively, are homologous to those of other GH7 CBHs with an enclosed active-site tunnel. Two primary differences between the Dictyostelium CBHs and the archetypal model GH7 CBH, Trichoderma reesei Cel7A (TreCel7A), occur near the hydrolytic active site and the product-binding sites. To compare the activities of these enzymes with the activity of TreCel7A, the family 1 TreCel7A CBM and linker were added to the C terminus of each of the Dictyostelium enzymes, creating DdiCel7A CBM and DpuCel7A CBM, which were recombinantly expressed in T. reesei. DdiCel7A CBM and DpuCel7A CBM hydrolyzed Avicel, pretreated corn stover, and phosphoric acid-swollen cellulose as efficiently as TreCel7A when hydrolysis was compared at their temperature optima. The K i of cellobiose was significantly higher for DdiCel7A CBM and DpuCel7A CBM than for TreCel7A: 205, 130, and 29 μM, respectively. Finally, taken together, the present study highlights the remarkable degree of conservation of the activity of these key natural and industrial enzymes across quite distant phylogenetic trees of life.« less

Biochemical and structural characterizations of two Dictyostelium cellobiohydrolases from the amoebozoa kingdom reveal a high level of conservation between distant phylogenetic trees of life

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

Hobdey, Sarah E.; Knott, Brandon C.; Momeni, Majid Haddad

Glycoside hydrolase family 7 (GH7) cellobiohydrolases (CBHs) are enzymes often employed in plant cell wall degradation across eukaryotic kingdoms of life, as they provide significant hydrolytic potential in cellulose turnover. To date, many fungal GH7 CBHs have been examined, yet many questions regarding structure-activity relationships in these important natural and commercial enzymes remain. Here, we present the crystal structures and a biochemical analysis of two GH7 CBHs from social amoeba: Dictyostelium discoideum Cel7A (DdiCel7A) and Dictyostelium purpureum Cel7A (DpuCel7A). DdiCel7A and DpuCel7A natively consist of a catalytic domain and do not exhibit a carbohydrate-binding module (CBM). The structures of DdiCel7Amore » and DpuCel7A, resolved to 2.1 Å and 2.7 Å, respectively, are homologous to those of other GH7 CBHs with an enclosed active-site tunnel. Two primary differences between the Dictyostelium CBHs and the archetypal model GH7 CBH, Trichoderma reesei Cel7A (TreCel7A), occur near the hydrolytic active site and the product-binding sites. To compare the activities of these enzymes with the activity of TreCel7A, the family 1 TreCel7A CBM and linker were added to the C terminus of each of the Dictyostelium enzymes, creating DdiCel7A CBM and DpuCel7A CBM, which were recombinantly expressed in T. reesei. DdiCel7A CBM and DpuCel7A CBM hydrolyzed Avicel, pretreated corn stover, and phosphoric acid-swollen cellulose as efficiently as TreCel7A when hydrolysis was compared at their temperature optima. The K i of cellobiose was significantly higher for DdiCel7A CBM and DpuCel7A CBM than for TreCel7A: 205, 130, and 29 μM, respectively. Finally, taken together, the present study highlights the remarkable degree of conservation of the activity of these key natural and industrial enzymes across quite distant phylogenetic trees of life.« less

Unique aspects of fiber degradation by the ruminal ethanologen Ruminococcus albus 7 revealed by physiological and transcriptomic analysis

DOE PAGES

Christopherson, Melissa R.; Dawson, John A.; Stevenson, David M.; ...

2014-12-04

Bacteria in the genus Ruminococcus are ubiquitous members of the mammalian gastrointestinal tract. In particular, they are important in ruminants where they digest a wide range of plant cell wall polysaccharides. For example, Ruminococcus albus 7 is a primary cellulose degrader that produces acetate usable by its bovine host. Moreover, it is one of the few organisms that ferments cellulose to form ethanol at mesophilic temperatures in vitro. The mechanism of cellulose degradation by R. albus 7 is not well-defined and is thought to involve pilin-like proteins, unique carbohydrate-binding domains, a glycocalyx, and cellulosomes. We used a combination of comparativemore » genomics, fermentation analyses, and transcriptomics to further clarify the cellulolytic and fermentative potential of R. albus 7. A comparison of the R. albus 7 genome sequence against the genome sequences of related bacteria that either encode or do not encode cellulosomes revealed that R. albus 7 does not encode for most canonical cellulosomal components. Fermentation analysis of R. albus 7 revealed the ability to produce ethanol and acetate on a wide range of fibrous substrates in vitro. Global transcriptomic analysis of R. albus 7 grown at identical dilution rates on cellulose and cellobiose in a chemostat showed that this bacterium, when growing on cellulose, utilizes a carbohydrate-degrading strategy that involves increased transcription of the rare carbohydrate-binding module (CBM) family 37 domain and the tryptophan biosynthetic operon. Our data suggest that R. albus 7 does not use canonical cellulosomal components to degrade cellulose, but rather up-regulates the expression of CBM37-containing enzymes and tryptophan biosynthesis. This study contributes to a revised model of carbohydrate degradation by this key member of the rumen ecosystem.« less

Cloning, purification, crystallization and preliminary X-ray studies of a carbohydrate-binding module from family 64 (StX).

PubMed

Campos, Bruna Medeia; Liberato, Marcelo Vizona; Polikarpov, Igor; Zeri, Ana Carolina de Mattos; Squina, Fabio Marcio

2015-03-01

In recent years, biofuels have attracted great interest as a source of renewable energy owing to the growing global demand for energy, the dependence on fossil fuels, limited natural resources and environmental pollution. However, the cost-effective production of biofuels from plant biomass is still a challenge. In this context, the study of carbohydrate-binding modules (CBMs), which are involved in guiding the catalytic domains of glycoside hydrolases to polysaccharides, is crucial for enzyme development. Aiming at the structural and functional characterization of novel CBMs involved in plant polysaccharide deconstruction, an analysis of the CAZy database was performed and CBM family 64 was chosen owing to its capacity to bind with high specificity to microcrystalline cellulose and to the fact that is found in thermophilic microorganisms. In this communication, the CBM-encoding module named StX was expressed, purified and crystallized, and X-ray diffraction data were collected from native and derivatized crystals to 1.8 and 2.0 Å resolution, respectively. The crystals, which were obtained by the hanging-drop vapour-diffusion method, belonged to space group P3121, with unit-cell parameters a = b = 43.42, c = 100.96 Å for the native form. The phases were found using the single-wavelength anomalous diffraction method.

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.

Modular architecture of protein binding units for designing properties of cellulose nanomaterials.

PubMed

Malho, Jani-Markus; Arola, Suvi; Laaksonen, Päivi; Szilvay, Géza R; Ikkala, Olli; Linder, Markus B

2015-10-05

Molecular biomimetic models suggest that proteins in the soft matrix of nanocomposites have a multimodular architecture. Engineered proteins were used together with nanofibrillated cellulose (NFC) to show how this type of architecture leads to function. The proteins consist of two cellulose-binding modules (CBM) separated by 12-, 24-, or 48-mer linkers. Engineering the linkers has a considerable effects on the interaction between protein and NFC in both wet colloidal state and a dry film. The protein optionally incorporates a multimerizing hydrophobin (HFB) domain connected by another linker. The modular structure explains effects in the hydrated gel state, as well as the deformation of composite materials through stress distribution and crosslinking. Based on this work, strategies can be suggested for tuning the mechanical properties of materials through the coupling of protein modules and their interlinking architectures. © 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

RNA-Seq Analysis of the Expression of Genes Encoding Cell Wall Degrading Enzymes during Infection of Lupin (Lupinus angustifolius) by Phytophthora parasitica

PubMed Central

Blackman, Leila M.; Cullerne, Darren P.; Torreña, Pernelyn; Taylor, Jen; Hardham, Adrienne R.

2015-01-01

RNA-Seq analysis has shown that over 60% (12,962) of the predicted transcripts in the Phytophthora parasitica genome are expressed during the first 60 h of lupin root infection. The infection transcriptomes included 278 of the 431 genes encoding P. parasitica cell wall degrading enzymes. The transcriptome data provide strong evidence of global transcriptional cascades of genes whose encoded proteins target the main categories of plant cell wall components. A major cohort of pectinases is predominantly expressed early but as infection progresses, the transcriptome becomes increasingly dominated by transcripts encoding cellulases, hemicellulases, β-1,3-glucanases and glycoproteins. The most highly expressed P. parasitica carbohydrate active enzyme gene contains two CBM1 cellulose binding modules and no catalytic domains. The top 200 differentially expressed genes include β-1,4-glucosidases, β-1,4-glucanases, β-1,4-galactanases, a β-1,3-glucanase, an α-1,4-polygalacturonase, a pectin deacetylase and a pectin methylesterase. Detailed analysis of gene expression profiles provides clues as to the order in which linkages within the complex carbohydrates may come under attack. The gene expression profiles suggest that (i) demethylation of pectic homogalacturonan occurs before its deacetylation; (ii) cleavage of the backbone of pectic rhamnogalacturonan I precedes digestion of its side chains; (iii) early attack on cellulose microfibrils by non-catalytic cellulose-binding proteins and enzymes with auxiliary activities may facilitate subsequent attack by glycosyl hydrolases and enzymes containing CBM1 cellulose-binding modules; (iv) terminal hemicellulose backbone residues are targeted after extensive internal backbone cleavage has occurred; and (v) the carbohydrate chains on glycoproteins are degraded late in infection. A notable feature of the P. parasitica infection transcriptome is the high level of transcription of genes encoding enzymes that degrade β-1,3-glucanases during middle and late stages of infection. The results suggest that high levels of β-1,3-glucanases may effectively degrade callose as it is produced by the plant during the defence response. PMID:26332397

RNA-Seq Analysis of the Expression of Genes Encoding Cell Wall Degrading Enzymes during Infection of Lupin (Lupinus angustifolius) by Phytophthora parasitica.

PubMed

Blackman, Leila M; Cullerne, Darren P; Torreña, Pernelyn; Taylor, Jen; Hardham, Adrienne R

2015-01-01

RNA-Seq analysis has shown that over 60% (12,962) of the predicted transcripts in the Phytophthora parasitica genome are expressed during the first 60 h of lupin root infection. The infection transcriptomes included 278 of the 431 genes encoding P. parasitica cell wall degrading enzymes. The transcriptome data provide strong evidence of global transcriptional cascades of genes whose encoded proteins target the main categories of plant cell wall components. A major cohort of pectinases is predominantly expressed early but as infection progresses, the transcriptome becomes increasingly dominated by transcripts encoding cellulases, hemicellulases, β-1,3-glucanases and glycoproteins. The most highly expressed P. parasitica carbohydrate active enzyme gene contains two CBM1 cellulose binding modules and no catalytic domains. The top 200 differentially expressed genes include β-1,4-glucosidases, β-1,4-glucanases, β-1,4-galactanases, a β-1,3-glucanase, an α-1,4-polygalacturonase, a pectin deacetylase and a pectin methylesterase. Detailed analysis of gene expression profiles provides clues as to the order in which linkages within the complex carbohydrates may come under attack. The gene expression profiles suggest that (i) demethylation of pectic homogalacturonan occurs before its deacetylation; (ii) cleavage of the backbone of pectic rhamnogalacturonan I precedes digestion of its side chains; (iii) early attack on cellulose microfibrils by non-catalytic cellulose-binding proteins and enzymes with auxiliary activities may facilitate subsequent attack by glycosyl hydrolases and enzymes containing CBM1 cellulose-binding modules; (iv) terminal hemicellulose backbone residues are targeted after extensive internal backbone cleavage has occurred; and (v) the carbohydrate chains on glycoproteins are degraded late in infection. A notable feature of the P. parasitica infection transcriptome is the high level of transcription of genes encoding enzymes that degrade β-1,3-glucanases during middle and late stages of infection. The results suggest that high levels of β-1,3-glucanases may effectively degrade callose as it is produced by the plant during the defence response.

Complete Cellulase System in the Marine Bacterium Saccharophagus degradans Strain 2-40T

PubMed Central

Taylor, Larry E.; Henrissat, Bernard; Coutinho, Pedro M.; Ekborg, Nathan A.; Hutcheson, Steven W.; Weiner, Ronald M.

2006-01-01

Saccharophagus degradans strain 2-40 is a representative of an emerging group of marine complex polysaccharide (CP)-degrading bacteria. It is unique in its metabolic versatility, being able to degrade at least 10 distinct CPs from diverse algal, plant and invertebrate sources. The S. degradans genome has been sequenced to completion, and more than 180 open reading frames have been identified that encode carbohydrases. Over half of these are likely to act on plant cell wall polymers. In fact, there appears to be a full array of enzymes that degrade and metabolize plant cell walls. Genomic and proteomic analyses reveal 13 cellulose depolymerases complemented by seven accessory enzymes, including two cellodextrinases, three cellobiases, a cellodextrin phosphorylase, and a cellobiose phosphorylase. Most of these enzymes exhibit modular architecture, and some contain novel combinations of catalytic and/or substrate binding modules. This is exemplified by endoglucanase Cel5A, which has three internal family 6 carbohydrate binding modules (CBM6) and two catalytic modules from family five of glycosyl hydrolases (GH5) and by Cel6A, a nonreducing-end cellobiohydrolase from family GH6 with tandem CBM2s. This is the first report of a complete and functional cellulase system in a marine bacterium with a sequenced genome. PMID:16707677

Mutational Insights into the Roles of Amino Acid Residues in Ligand Binding for Two Closely Related Family 16 Carbohydrate Binding Modules

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

Su, Xiaoyun; Agarwal, Vinayak; Dodd, Dylan

2010-11-22

Carbohydrate binding modules (CBMs) are specialized proteins that bind to polysaccharides and oligosaccharides. Caldanaerobius polysaccharolyticus Man5ACBM16-1/CBM16-2 bind to glucose-, mannose-, and glucose/mannose-configured substrates. The crystal structures of the two proteins represent the only examples in CBM family 16, and studies that evaluate the roles of amino acid residues in ligand binding in this family are lacking. In this study, we probed the roles of amino acids (selected based on CBM16-1/ligand co-crystal structures) on substrate binding. Two tryptophan (Trp-20 and Trp-125) and two glutamine (Gln-81 and Gln-93) residues are shown to be critical in ligand binding. Additionally, several polar residues thatmore » flank the critical residues also contribute to ligand binding. The CBM16-1 Q121E mutation increased affinity for all substrates tested, whereas the Q21G and N97R mutants exhibited decreased substrate affinity. We solved CBM/substrate co-crystal structures to elucidate the molecular basis of the increased substrate binding by CBM16-1 Q121E. The Gln-121, Gln-21, and Asn-97 residues can be manipulated to fine-tune ligand binding by the Man5A CBMs. Surprisingly, none of the eight residues investigated was absolutely conserved in CBM family 16. Thus, the critical residues in the Man5A CBMs are either not essential for substrate binding in the other members of this family or the two CBMs are evolutionarily distinct from the members available in the current protein database. Man5A is dependent on its CBMs for robust activity, and insights from this study should serve to enhance our understanding of the interdependence of its catalytic and substrate binding modules.« less

Characterization of a novel swollenin from Penicillium oxalicum in facilitating enzymatic saccharification of cellulose

PubMed Central

2013-01-01

Background Plant expansins and fungal swollenin that can disrupt crystalline cellulose have great potential for applications in conversion of biomass. Recent studies have been mainly focused on Trichoderma reesei swollenin that show relatively low activity in the promotion of cellulosic hydrolysis. Our aim was to isolate a novel swollenin with greater disruptive activity, to establish an efficient way of producing recombinant swollenin, and to optimize the procedure using swollenin in facilitation of cellulosic hydrolysis. Results A novel gene encoding a swollenin-like protein, POSWOI, was isolated from the filamentous fungus Penicillium oxalicum by Thermal Asymmetric Interlaced PCR (TAIL-PCR). It consisted of a family 1 carbohydrate-binding module (CBM1) followed by a linker connected to a family 45 endoglucanase-like domain. Using the cellobiohydrolase I promoter, recombinant POSWOI was efficiently produced in T. reesei with a yield of 105 mg/L, and showed significant disruptive activity on crystalline cellulose. Simultaneous reaction with both POSWOI and cellulases enhanced the hydrolysis of crystalline cellulose Avicel by approximately 50%. Using a POSWOI-pretreatment procedure, cellulases can produce nearly twice as many reducing sugars as without pretreatment. The mechanism by which POSWOI facilitates the saccharification of cellulose was also studied using a cellulase binding assay. Conclusion We present a novel fungal swollenin with considerable disruptive activity on crystalline cellulose, and develop a better procedure for using swollenin in facilitating cellulosic hydrolysis. We thus provide a new approach for the effective bioconversion of cellulosic biomass. PMID:23688024

Effect of β-mannanase domain from Trichoderma reesei on its biochemical characters and synergistic hydrolysis of sugarcane bagasse.

PubMed

Ma, Lijuan; Ma, Qing; Cai, Rui; Zong, Zhiyou; Du, Liping; Guo, Gaojie; Zhang, Yingying; Xiao, Dongguang

2018-05-01

β-mannanase is a key enzyme for hydrolyzing mannan, a major constituent of hemicellulose, which is the second most abundant polysaccharide in nature. Different structural domains greatly affect its biochemical characters and catalytic efficiency. However, the effects of linker and carbohydrate-binding module (CBM) on β-mannanase from Trichoderma reesei (Man1) have not yet been fully described. The present study aimed to determine the influence of different domains on the expression efficiency, biochemical characteristics and hemicellulosic deconstruction of Man1. The expression efficiency was improved after truncating CBM. Activities of Man1 and Man1ΔCBM (CBM) in the culture supernatant after 168 h of induction were 34.5 and 42.9 IU mL -1 , although a value of only 0.36 IU mL -1 was detected for Man1ΔLCBM (lacking CBM and linker). Man1 showed higher thermostability than Man1ΔCBM at low temperature, whereas Man1ΔCBM had a higher specificity for galactomannan (K m  = 2.5 mg mL -1 ) than Man1 (K m  = 4.0 mg mL -1 ). Both Man1 and Man1ΔCBM could synergistically improve the hydrolysis of cellulose, galactomannan and pretreated sugarcane bagasse, with a 10-30% improvement of the reducing sugar yield. Linker and CBM domains were vital for mannanase activity and expression efficiency. CBM affected the thermostability and adsorption ability of Man1. The results obtained in the present study should help guide the rational design and directional modification of Man with respect to improving its catalytic efficiency. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Enhanced catalytic efficiency of endo-β-agarase I by fusion of carbohydrate-binding modules for agar prehydrolysis.

PubMed

Alkotaini, Bassam; Han, Nam Soo; Kim, Beom Soo

2016-11-01

Recently, Microbulbifer thermotolerans JAMB-A94 endo-β-agarase I was expressed as catalytic domain (GH16) without a carbohydrate-binding module (CBM). In this study, we successfully constructed different fusions of GH16 with its original CBM6 and CBM13 derived from Catenovulum agarivorans. The optimum temperature and pH for fusions GH16-CBM6, GH16-CBM13, GH16-CBM6-CBM13 and GH16-CBM13-CBM6 were similar to GH16, at 55°C and pH 7. All the constructed fusions significantly enhanced the GH16 affinity (Km) and the catalytic efficiency (Kcat/Km) toward agar. Among them, GH16-CBM6-CBM13 exhibited the highest agarolytic activity, for which Km decreased from 3.67 to 2.11mg/mL and Kcat/Km increased from 98.6 (mg/mL) -1 sec -1 to 400.6 (mg/mL) -1 sec -1 . Moreover, all fusions selectively increased GH16 binding ability to agar, in which the highest binding ability of 95% was obtained with fusion GH16-CBM6-CBM13. Melted agar was prehydrolyzed with GH16-CBM6-CBM13, resulting in a degree of liquefaction of 45.3% and reducing sugar yield of 14.2%. Further addition of Saccharophagus degradans agarolytic enzymes resulted in mono-sugar yields of 35.4% for galactose and 31.5% for 3,6-anhydro-l-galactose. There was no pH neutralization step required and no 5-hydroxymethylfurfural detected, suggesting the potential of a new enzymatic prehydrolysis process for efficient production of bio-products such as biofuels. Copyright © 2016 Elsevier Inc. All rights reserved.

Construction of Engineered Bifunctional Enzymes and Their Overproduction in Aspergillus niger for Improved Enzymatic Tools To Degrade Agricultural By-Products

PubMed Central

Levasseur, Anthony; Navarro, David; Punt, Peter J.; Belaïch, Jean-Pierre; Asther, Marcel; Record, Eric

2005-01-01

Two chimeric enzymes, FLX and FLXLC, were designed and successfully overproduced in Aspergillus niger. FLX construct is composed of the sequences encoding the feruloyl esterase A (FAEA) fused to the endoxylanase B (XYNB) of A. niger. A C-terminal carbohydrate-binding module (CBM family 1) was grafted to FLX, generating the second hybrid enzyme, FLXLC. Between each partner, a hyperglycosylated linker was included to stabilize the constructs. Hybrid proteins were purified to homogeneity, and molecular masses were estimated to be 72 and 97 kDa for FLX and FLXLC, respectively. Integrity of hybrid enzymes was checked by immunodetection that showed a single form by using antibodies raised against FAEA and polyhistidine tag. Physicochemical properties of each catalytic module of the bifunctional enzymes corresponded to those of the free enzymes. In addition, we verified that FLXLC exhibited an affinity for microcrystalline cellulose (Avicel) with binding parameters corresponding to a Kd of 9.9 × 10−8 M for the dissociation constant and 0.98 μmol/g Avicel for the binding capacity. Both bifunctional enzymes were investigated for their capacity to release ferulic acid from natural substrates: corn and wheat brans. Compared to free enzymes FAEA and XYNB, a higher synergistic effect was obtained by using FLX and FLXLC for both substrates. Moreover, the release of ferulic acid from corn bran was increased by using FLXLC rather than FLX. This result confirms a positive role of the CBM. In conclusion, these results demonstrated that the fusion of naturally free cell wall hydrolases and an A. niger-derived CBM onto bifunctional enzymes enables the increase of the synergistic effect on the degradation of complex substrates. PMID:16332795

Unravelling the specificity and mechanism of sialic acid recognition by the gut symbiont Ruminococcus gnavus.

PubMed

Owen, C David; Tailford, Louise E; Monaco, Serena; Šuligoj, Tanja; Vaux, Laura; Lallement, Romane; Khedri, Zahra; Yu, Hai; Lecointe, Karine; Walshaw, John; Tribolo, Sandra; Horrex, Marc; Bell, Andrew; Chen, Xi; Taylor, Gary L; Varki, Ajit; Angulo, Jesus; Juge, Nathalie

2017-12-19

Ruminococcus gnavus is a human gut symbiont wherein the ability to degrade mucins is mediated by an intramolecular trans-sialidase (RgNanH). RgNanH comprises a GH33 catalytic domain and a sialic acid-binding carbohydrate-binding module (CBM40). Here we used glycan arrays, STD NMR, X-ray crystallography, mutagenesis and binding assays to determine the structure and function of RgNanH_CBM40 (RgCBM40). RgCBM40 displays the canonical CBM40 β-sandwich fold and broad specificity towards sialoglycans with millimolar binding affinity towards α2,3- or α2,6-sialyllactose. RgCBM40 binds to mucus produced by goblet cells and to purified mucins, providing direct evidence for a CBM40 as a novel bacterial mucus adhesin. Bioinformatics data show that RgCBM40 canonical type domains are widespread among Firmicutes. Furthermore, binding of R. gnavus ATCC 29149 to intestinal mucus is sialic acid mediated. Together, this study reveals novel features of CBMs which may contribute to the biogeography of symbiotic bacteria in the gut.

Functional Analysis of the Glucan Degradation Locus in Caldicellulosiruptor bescii Reveals Essential Roles of Component Glycoside Hydrolases in Plant Biomass Deconstruction

PubMed Central

Conway, Jonathan M.; McKinley, Bennett S.; Seals, Nathaniel L.; Hernandez, Diana; Khatibi, Piyum A.; Poudel, Suresh; Giannone, Richard J.; Hettich, Robert L.; Williams-Rhaesa, Amanda M.; Lipscomb, Gina L.; Adams, Michael W. W.

2017-01-01

ABSTRACT The ability to hydrolyze microcrystalline cellulose is an uncommon feature in the microbial world, but it can be exploited for conversion of lignocellulosic feedstocks into biobased fuels and chemicals. Understanding the physiological and biochemical mechanisms by which microorganisms deconstruct cellulosic material is key to achieving this objective. The glucan degradation locus (GDL) in the genomes of extremely thermophilic Caldicellulosiruptor species encodes polysaccharide lyases (PLs), unique cellulose binding proteins (tāpirins), and putative posttranslational modifying enzymes, in addition to multidomain, multifunctional glycoside hydrolases (GHs), thereby representing an alternative paradigm for plant biomass degradation compared to fungal or cellulosomal systems. To examine the individual and collective in vivo roles of the glycolytic enzymes, the six GH genes in the GDL of Caldicellulosiruptor bescii were systematically deleted, and the extents to which the resulting mutant strains could solubilize microcrystalline cellulose (Avicel) and plant biomass (switchgrass or poplar) were examined. Three of the GDL enzymes, Athe_1867 (CelA) (GH9-CBM3-CBM3-CBM3-GH48), Athe_1859 (GH5-CBM3-CBM3-GH44), and Athe_1857 (GH10-CBM3-CBM3-GH48), acted synergistically in vivo and accounted for 92% of naked microcrystalline cellulose (Avicel) degradation. However, the relative importance of the GDL GHs varied for the plant biomass substrates tested. Furthermore, mixed cultures of mutant strains showed that switchgrass solubilization depended on the secretome-bound enzymes collectively produced by the culture, not on the specific strain from which they came. These results demonstrate that certain GDL GHs are primarily responsible for the degradation of microcrystalline cellulose-containing substrates by C. bescii and provide new insights into the workings of a novel microbial mechanism for lignocellulose utilization. IMPORTANCE The efficient and extensive degradation of complex polysaccharides in lignocellulosic biomass, particularly microcrystalline cellulose, remains a major barrier to its use as a renewable feedstock for the production of fuels and chemicals. Extremely thermophilic bacteria from the genus Caldicellulosiruptor rapidly degrade plant biomass to fermentable sugars at temperatures of 70 to 78°C, although the specific mechanism by which this occurs is not clear. Previous comparative genomic studies identified a genomic locus found only in certain Caldicellulosiruptor species that was hypothesized to be mainly responsible for microcrystalline cellulose degradation. By systematically deleting genes in this locus in Caldicellulosiruptor bescii, the nuanced, substrate-specific in vivo roles of glycolytic enzymes in deconstructing crystalline cellulose and plant biomasses could be discerned. The results here point to synergism of three multidomain cellulases in C. bescii, working in conjunction with the aggregate secreted enzyme inventory, as the key to the plant biomass degradation ability of this extreme thermophile. PMID:28986379

Structure of a Thermobifida fusca lytic polysaccharide monooxygenase and mutagenesis of key residues

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

Kruer-Zerhusen, Nathan; Alahuhta, Markus; Lunin, Vladimir V.

Auxiliary activity (AA) enzymes are produced by numerous bacterial and fungal species to assist in the degradation of biomass. These enzymes are abundant but have yet to be fully characterized. Here, we report the X-ray structure of Thermobifida fusca AA10A (TfAA10A), investigate mutational characterization of key surface residues near its active site, and explore the importance of the various domains of Thermobifida fusca AA10B (TfAA10B). The structure of TfAA10A is similar to other bacterial LPMOs (lytic polysaccharide monooxygenases), including signs of photo-reduction and a distorted active site, with mixed features showing both type I and II copper coordination. The pointmore » mutation experiments of TfAA10A show that Trp82 and Asn83 are needed for binding, but only Trp82 affects activity. The TfAA10B domain truncation mutants reveal that CBM2 is crucial for the binding of substrate, but that the X1 module does not affect binding or activity. In TfAA10A, Trp82 and Asn83 are needed for binding, but only Trp82 affects activity. The TfAA10B domain truncation mutants reveal that CBM2 is crucial for substrate binding, but that the X1 module does not affect binding or activity. The structure of TfAA10A is similar to other bacterial lytic polysaccharide monooxygenases with mixed features showing both type I and II copper coordination. The role of LPMOs and the variability of abundance in genomes are not fully explored. LPMOs likely perform initial attacks into crystalline cellulose to allow larger processive cellulases to bind and attack, but the precise nature of their synergistic behavior remains to be definitively characterized.« less

Structure of a Thermobifida fusca lytic polysaccharide monooxygenase and mutagenesis of key residues

DOE PAGES

Kruer-Zerhusen, Nathan; Alahuhta, Markus; Lunin, Vladimir V.; ...

2017-11-30

Auxiliary activity (AA) enzymes are produced by numerous bacterial and fungal species to assist in the degradation of biomass. These enzymes are abundant but have yet to be fully characterized. Here, we report the X-ray structure of Thermobifida fusca AA10A (TfAA10A), investigate mutational characterization of key surface residues near its active site, and explore the importance of the various domains of Thermobifida fusca AA10B (TfAA10B). The structure of TfAA10A is similar to other bacterial LPMOs (lytic polysaccharide monooxygenases), including signs of photo-reduction and a distorted active site, with mixed features showing both type I and II copper coordination. The pointmore » mutation experiments of TfAA10A show that Trp82 and Asn83 are needed for binding, but only Trp82 affects activity. The TfAA10B domain truncation mutants reveal that CBM2 is crucial for the binding of substrate, but that the X1 module does not affect binding or activity. In TfAA10A, Trp82 and Asn83 are needed for binding, but only Trp82 affects activity. The TfAA10B domain truncation mutants reveal that CBM2 is crucial for substrate binding, but that the X1 module does not affect binding or activity. The structure of TfAA10A is similar to other bacterial lytic polysaccharide monooxygenases with mixed features showing both type I and II copper coordination. The role of LPMOs and the variability of abundance in genomes are not fully explored. LPMOs likely perform initial attacks into crystalline cellulose to allow larger processive cellulases to bind and attack, but the precise nature of their synergistic behavior remains to be definitively characterized.« less

Characterization of the cellulosomal scaffolding protein CbpC from Clostridium cellulovorans 743B.

PubMed

Nakajima, Daichi; Shibata, Toshiyuki; Tanaka, Reiji; Kuroda, Kouichi; Ueda, Mitsuyoshi; Miyake, Hideo

2017-10-01

Clostridium cellulovorans 743B, an anaerobic and mesophilic bacterium, produces an extracellular enzyme complex called the cellulosome on the cell surface. Recently, we have reported the whole genome sequence of C. cellulovorans, which revealed that a total of 4 cellulosomal scaffolding proteins: CbpA, HbpA, CbpB, and CbpC were encoded in the C. cellulovorans genome. In particular, cbpC encoded a 429-residue polypeptide that includes a carbohydrate-binding module (CBM), an S-layer homology module, and a cohesin. CbpC was also detected in the culture supernatant of C. cellulovorans. Genomic DNA coding for CbpC was subcloned into a pET-22b+ vector in order to express and produce the recombinant protein in Escherichia coli BL21(DE3). Measurement of CbpC adsorption to crystalline cellulose indicated a dissociation constant of 0.60 μM, which is a similar to that of CBM from CbpA. We also subcloned the region encoding xylanase B (XynB) with the dockerin from C. cellulovorans and analyzed the interaction between XynB and CbpC by GST pull-down assay. It was observed that GST-CbpC assembles with XynB to form a minimal cellulosome. The activity of XynB against rice straw tended to be increased in the presence of CbpC. These results showed a synergistic effect on rice straw as a representative cellulosic biomass through the formation of a minimal cellulosome containing XynB bound to CbpC. Thus, our findings provide a foundation for the development of cellulosic biomass saccharification using a minimal cellulosome. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Discovery and characterization of a thermostable two-domain GH6 endoglucanase from a compost metagenome.

PubMed

Jensen, Marianne S; Fredriksen, Lasse; MacKenzie, Alasdair K; Pope, Phillip B; Leiros, Ingar; Chylenski, Piotr; Williamson, Adele K; Christopeit, Tony; Østby, Heidi; Vaaje-Kolstad, Gustav; Eijsink, Vincent G H

2018-01-01

Enzymatic depolymerization of recalcitrant polysaccharides plays a key role in accessing the renewable energy stored within lignocellulosic biomass, and natural biodiversities may be explored to discover microbial enzymes that have evolved to conquer this task in various environments. Here, a metagenome from a thermophilic microbial community was mined to yield a novel, thermostable cellulase, named mgCel6A, with activity on an industrial cellulosic substrate (sulfite-pulped Norway spruce) and a glucomannanase side activity. The enzyme consists of a glycoside hydrolase family 6 catalytic domain (GH6) and a family 2 carbohydrate binding module (CBM2) that are connected by a linker rich in prolines and threonines. MgCel6A exhibited maximum activity at 85°C and pH 5.0 on carboxymethyl cellulose (CMC), but in prolonged incubations with the industrial substrate, the highest yields were obtained at 60°C, pH 6.0. Differential scanning calorimetry (DSC) indicated a Tm(app) of 76°C. Both functional data and the crystal structure, solved at 1.88 Å resolution, indicate that mgCel6A is an endoglucanase. Comparative studies with a truncated variant of the enzyme showed that the CBM increases substrate binding, while not affecting thermal stability. Importantly, at higher substrate concentrations the full-length enzyme was outperformed by the catalytic domain alone, underpinning previous suggestions that CBMs may be less useful in high-consistency bioprocessing.

Carbohydrate-binding module 74 is a novel starch-binding domain associated with large and multidomain α-amylase enzymes.

PubMed

Valk, Vincent; Lammerts van Bueren, Alicia; van der Kaaij, Rachel M; Dijkhuizen, Lubbert

2016-06-01

Microbacterium aurum B8.A is a bacterium that originates from a potato starch-processing plant and employs a GH13 α-amylase (MaAmyA) enzyme that forms pores in potato starch granules. MaAmyA is a large and multi-modular protein that contains a novel domain at its C terminus (Domain 2). Deletion of Domain 2 from MaAmyA did not affect its ability to degrade starch granules but resulted in a strong reduction in granular pore size. Here, we separately expressed and purified this Domain 2 in Escherichia coli and determined its likely function in starch pore formation. Domain 2 independently binds amylose, amylopectin, and granular starch but does not have any detectable catalytic (hydrolytic or oxidizing) activity on α-glucan substrates. Therefore, we propose that this novel starch-binding domain is a new carbohydrate-binding module (CBM), the first representative of family CBM74 that assists MaAmyA in efficient pore formation in starch granules. Protein sequence-based BLAST searches revealed that CBM74 occurs widespread, but in bacteria only, and is often associated with large and multi-domain α-amylases containing family CBM25 or CBM26 domains. CBM74 may specifically function in binding to granular starches to enhance the capability of α-amylase enzymes to degrade resistant starches (RSs). Interestingly, the majority of family CBM74 representatives are found in α-amylases originating from human gut-associated Bifidobacteria, where they may assist in resistant starch degradation. The CBM74 domain thus may have a strong impact on the efficiency of RS digestion in the mammalian gastrointestinal tract. © 2016 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

A synthetic biology approach for evaluating the functional contribution of designer cellulosome components to deconstruction of cellulosic substrates

PubMed Central

2013-01-01

Background Select cellulolytic bacteria produce multi-enzymatic cellulosome complexes that bind to the plant cell wall and catalyze its efficient degradation. The multi-modular interconnecting cellulosomal subunits comprise dockerin-containing enzymes that bind cohesively to cohesin-containing scaffoldins. The organization of the modules into functional polypeptides is achieved by intermodular linkers of different lengths and composition, which provide flexibility to the complex and determine its overall architecture. Results Using a synthetic biology approach, we systematically investigated the spatial organization of the scaffoldin subunit and its effect on cellulose hydrolysis by designing a combinatorial library of recombinant trivalent designer scaffoldins, which contain a carbohydrate-binding module (CBM) and 3 divergent cohesin modules. The positions of the individual modules were shuffled into 24 different arrangements of chimaeric scaffoldins. This basic set was further extended into three sub-sets for each arrangement with intermodular linkers ranging from zero (no linkers), 5 (short linkers) and native linkers of 27–35 amino acids (long linkers). Of the 72 possible scaffoldins, 56 were successfully cloned and 45 of them expressed, representing 14 full sets of chimaeric scaffoldins. The resultant 42-component scaffoldin library was used to assemble designer cellulosomes, comprising three model C. thermocellum cellulases. Activities were examined using Avicel as a pure microcrystalline cellulose substrate and pretreated cellulose-enriched wheat straw as a model substrate derived from a native source. All scaffoldin combinations yielded active trivalent designer cellulosome assemblies on both substrates that exceeded the levels of the free enzyme systems. A preferred modular arrangement for the trivalent designer scaffoldin was not observed for the three enzymes used in this study, indicating that they could be integrated at any position in the designer cellulosome without significant effect on cellulose-degrading activity. Designer cellulosomes assembled with the long-linker scaffoldins achieved higher levels of activity, compared to those assembled with short-and no-linker scaffoldins. Conclusions The results demonstrate the robustness of the cellulosome system. Long intermodular scaffoldin linkers are preferable, thus leading to enhanced degradation of cellulosic substrates, presumably due to the increased flexibility and spatial positioning of the attached enzymes in the complex. These findings provide a general basis for improved designer cellulosome systems as a platform for bioethanol production. PMID:24341331

Functional analysis of the Glucan Degradation Locus (GDL) in Caldicellulosiruptor bescii reveals essential roles of component glycoside hydrolases in plant biomass deconstruction.

PubMed

Conway, Jonathan M; McKinley, Bennett S; Seals, Nathaniel L; Hernandez, Diana; Khatibi, Piyum A; Poudel, Suresh; Giannone, Richard J; Hettich, Robert L; Williams-Rhaesa, Amanda M; Lipscomb, Gina L; Adams, Michael W W; Kelly, Robert M

2017-10-06

The ability to hydrolyze microcrystalline cellulose is an uncommon feature in the microbial world, but one that can be exploited for conversion of lignocellulosic feedstocks into bio-based fuels and chemicals. Understanding the physiological and biochemical mechanisms by which microorganisms deconstruct cellulosic material is key to achieving this objective. The Glucan Degradation Locus (GDL) in the genomes of extremely thermophilic Caldicellulosiruptor species encodes polysaccharide lyases (PLs), unique cellulose binding proteins (tāpirins), and putative post-translational modifying enzymes, in addition to multi-domain, multi-functional glycoside hydrolases (GHs), thereby representing an alternative paradigm for plant biomass degradation, as compared to fungal or cellulosomal systems. To examine the individual and collective in vivo roles of the glycolytic enzymes, the six GHs in the GDL of Caldicellulosiruptor bescii were systematically deleted, and the extent to which the resulting mutant strains could solubilize microcrystalline cellulose (Avicel) and plant biomasses (switchgrass or poplar) was examined. Three of the GDL enzymes, Athe_1867 (CelA) (GH9-CBM3-CBM3-CBM3-GH48), Athe_1859 (GH5-CBM3-CBM3-GH44), and Athe_1857 (GH10-CBM3-CBM3-GH48), acted synergistically in vivo and accounted for 92% of naked microcellulose (Avicel) degradation. However, the relative importance of the GDL GHs varied for the plant biomass substrates tested. Furthermore, mixed cultures of mutant strains showed switchgrass solubilization depended on the secretome-bound enzymes collectively produced by the culture and not on the specific strain from which they came. These results demonstrate that certain GDL GHs are primarily responsible for the degradation of microcrystalline-containing substrates by C. bescii and provide new insights into the workings of a novel microbial mechanism for lignocellulose utilization. Importance The efficient and extensive degradation of complex polysaccharides in lignocellulosic biomass, particularly microcrystalline cellulose, remains a major barrier to its use as a renewable feedstock for the production of fuels and chemicals. Extremely thermophilic bacteria from the genus Caldicellulosiruptor rapidly degrade plant biomass to fermentable sugars at temperatures between 70-78°C, although the specific mechanism by which this occurs is not clear. Previous comparative genomic studies identified a genomic locus found only in certain Caldicellulosiruptor species that was hypothesized to be mainly responsible for microcrystalline cellulose degradation. By systematically deleting genes in this locus in Caldicellulosiruptor bescii , the nuanced, substrate-specific, in vivo roles of glycolytic enzymes in deconstructing crystalline cellulose and plant biomasses could be discerned. The results here point to synergism of three multi-domain cellulases in C. bescii , working in conjunction with the aggregate, secreted enzyme inventory, as the key to the plant biomass degradation ability by this extreme thermophile. Copyright © 2017 American Society for Microbiology.

Impact of orientation of carbohydrate binding modules family 22 and 6 on the catalytic activity of Thermotoga maritima xylanase XynB.

PubMed

Tajwar, Razia; Shahid, Saher; Zafar, Rehan; Akhtar, Muhammad Waheed

2017-11-01

Xylanase XynB of the hyperthermophile Thermotoga maritima, which belongs to glycoside hydrolase family 10 (GH10), does not have an associated carbohydrate binding module (CBM) in the native state. CBM6 and CBM22 from a thermophile Clostridium thermocellum were fused to the catalytic domain of XynB (XynB-C) to determine the effects on activity and other properties. XynB-B22C and XynB-CB22, produced by fusing CBM22 to the N- and C-terminal of XynB-C, showed 1.7- and 3.24-fold increase in activity against the insoluble birchwood xylan, respectively. Similarly, CBM6 when attached to the C-terminal of XynB-C resulted in 2.0-fold increase in activity, whereas its attachment to the N-terminal did not show any increase of activity. XynB-B22C and XynB-CB22 retained all the activity, whereas XynB-B6C and XynB-CB6 lost 17 and 11% of activity, respectively, at 60°C for 4h. Thermostability data and the secondary structure contents obtained by molecular modelling are in agreement with the data from circular dichroism analysis. Molecular modelling analysis showed that the active site residues of the catalytic domain and the binding residues of CBM6 and CBM22 were located on the surface of molecule, except XynB-B6C, where the binding residues were found somewhat buried. In the case of XynB-CB22, the catalytic and the binding residues seem to be located favorably adjacent to each other, thus showing higher increase in activity. This study shows that the active site residues of the catalytic domain and the binding residues of the CBM are arranged in a unique fashion, not reported before. Copyright © 2017 Elsevier Inc. All rights reserved.

Novel characteristics of a carbohydrate-binding module 20 from hyperthermophilic bacterium.

PubMed

Oh, Il-Nam; Jane, Jay-Lin; Wang, Kan; Park, Jong-Tae; Park, Kwan-Hwa

2015-03-01

In this study, a gene fragment coding carbohydrate-binding module 20 (CBM20) in the amylopullulanase (APU) gene was cloned from the hyperthermophilic bacteria Thermoanaerobacter pseudoethanolicus 39E and expressed in Escherichia coli. The protein, hereafter Tp39E, possesses very low sequence similarity with the CBM20s previously reported and has no starch binding site 2. Tp39E did not demonstrate thermal denaturation at 50 °C; however, thermal unfolding of the protein was observed at 59.5 °C. A binding assay with Tp39E was conducted using various soluble and insoluble substrates, and starch was the best binding polysaccharide. Intriguingly, Tp39E bound, to a lesser extent, to soluble and insoluble xylan as well. The dissociation constant (K d) and the maximum specific binding (B max) of Tp39E to corn starch granules were 0.537 μM and 5.79 μM/g, respectively, at pH 5.5 and 20 °C. 99APU1357 with a Tp39E domain exhibited 2.2-fold greater activity than a CBM20-truncation mutant when starch granules were the substrate. Tp39E was an independently thermostable CBM and had a considerable effect on APU activity in the hydrolysis of insoluble substrates.

Biochemical properties and atomic resolution structure of a proteolytically processed β-mannanase from cellulolytic Streptomyces sp. SirexAA-E.

PubMed

Takasuka, Taichi E; Acheson, Justin F; Bianchetti, Christopher M; Prom, Ben M; Bergeman, Lai F; Book, Adam J; Currie, Cameron R; Fox, Brian G

2014-01-01

β-Mannanase SACTE_2347 from cellulolytic Streptomyces sp. SirexAA-E is abundantly secreted into the culture medium during growth on cellulosic materials. The enzyme is composed of domains from the glycoside hydrolase family 5 (GH5), fibronectin type-III (Fn3), and carbohydrate binding module family 2 (CBM2). After secretion, the enzyme is proteolyzed into three different, catalytically active variants with masses of 53, 42 and 34 kDa corresponding to the intact protein, loss of the CBM2 domain, or loss of both the Fn3 and CBM2 domains. The three variants had identical N-termini starting with Ala51, and the positions of specific proteolytic reactions in the linker sequences separating the three domains were identified. To conduct biochemical and structural characterizations, the natural proteolytic variants were reproduced by cloning and heterologously expressed in Escherichia coli. Each SACTE_2347 variant hydrolyzed only β-1,4 mannosidic linkages, and also reacted with pure mannans containing partial galactosyl- and/or glucosyl substitutions. Examination of the X-ray crystal structure of the GH5 domain of SACTE_2347 suggests that two loops adjacent to the active site channel, which have differences in position and length relative to other closely related mannanases, play a role in producing the observed substrate selectivity.

A metagenome-derived thermostable β-glucanase with an unusual module architecture which defines the new glycoside hydrolase family GH148.

PubMed

Angelov, Angel; Pham, Vu Thuy Trang; Übelacker, Maria; Brady, Silja; Leis, Benedikt; Pill, Nicole; Brolle, Judith; Mechelke, Matthias; Moerch, Matthias; Henrissat, Bernard; Liebl, Wolfgang

2017-12-11

The discovery of novel and robust enzymes for the breakdown of plant biomass bears tremendous potential for the development of sustainable production processes in the rapidly evolving new bioeconomy. By functional screening of a metagenomic library from a volcano soil sample a novel thermostable endo-β-glucanase (EngU) which is unusual with regard to its module architecture and cleavage specificity was identified. Various recombinant EngU variants were characterized. Assignment of EngU to an existing glycoside hydrolase (GH) family was not possible. Two regions of EngU showed weak sequence similarity to proteins of the GH clan GH-A, and acidic residues crucial for catalytic activity of EngU were identified by mutation. Unusual, a carbohydrate-binding module (CBM4) which displayed binding affinity for β-glucan, lichenin and carboxymethyl-cellulose was found as an insertion between these two regions. EngU hydrolyzed β-1,4 linkages in carboxymethyl-cellulose, but displayed its highest activity with mixed linkage (β-1,3-/β-1,4-) glucans such as barley β-glucan and lichenin, where in contrast to characterized lichenases cleavage occurred predominantly at the β-1,3 linkages of C4-substituted glucose residues. EngU and numerous related enzymes with previously unknown function represent a new GH family of biomass-degrading enzymes within the GH-A clan. The name assigned to the new GH family is GH148.

Cellulosome-based, Clostridium-derived multi-functional enzyme complexes for advanced biotechnology tool development: advances and applications.

PubMed

Hyeon, Jeong Eun; Jeon, Sang Duck; Han, Sung Ok

2013-11-01

The cellulosome is one of nature's most elegant and elaborate nanomachines and a key biological and biotechnological macromolecule that can be used as a multi-functional protein complex tool. Each protein module in the cellulosome system is potentially useful in an advanced biotechnology application. The high-affinity interactions between the cohesin and dockerin domains can be used in protein-based biosensors to improve both sensitivity and selectivity. The scaffolding protein includes a carbohydrate-binding module (CBM) that attaches strongly to cellulose substrates and facilitates the purification of proteins fused with the dockerin module through a one-step CBM purification method. Although the surface layer homology (SLH) domain of CbpA is not present in other strains, replacement of the cell surface anchoring domain allows a foreign protein to be displayed on the surface of other strains. The development of a hydrolysis enzyme complex is a useful strategy for consolidated bioprocessing (CBP), enabling microorganisms with biomass hydrolysis activity. Thus, the development of various configurations of multi-functional protein complexes for use as tools in whole-cell biocatalyst systems has drawn considerable attention as an attractive strategy for bioprocess applications. This review provides a detailed summary of the current achievements in Clostridium-derived multi-functional complex development and the impact of these complexes in various areas of biotechnology. Copyright © 2013 Elsevier Inc. All rights reserved.

AMP-Activated Protein Kinase β-Subunit Requires Internal Motion for Optimal Carbohydrate Binding

PubMed Central

Bieri, Michael; Mobbs, Jesse I.; Koay, Ann; Louey, Gavin; Mok, Yee-Foong; Hatters, Danny M.; Park, Jong-Tae; Park, Kwan-Hwa; Neumann, Dietbert; Stapleton, David; Gooley, Paul R.

2012-01-01

AMP-activated protein kinase interacts with oligosaccharides and glycogen through the carbohydrate-binding module (CBM) containing the β-subunit, for which there are two isoforms (β1 and β2). Muscle-specific β2-CBM, either as an isolated domain or in the intact enzyme, binds carbohydrates more tightly than the ubiquitous β1-CBM. Although residues that contact carbohydrate are strictly conserved, an additional threonine in a loop of β2-CBM is concurrent with an increase in flexibility in β2-CBM, which may account for the affinity differences between the two isoforms. In contrast to β1-CBM, unbound β2-CBM showed microsecond-to-millisecond motion at the base of a β-hairpin that contains residues that make critical contacts with carbohydrate. Upon binding to carbohydrate, similar microsecond-to-millisecond motion was observed in this β-hairpin and the loop that contains the threonine insertion. Deletion of the threonine from β2-CBM resulted in reduced carbohydrate affinity. Although motion was retained in the unbound state, a significant loss of motion was observed in the bound state of the β2-CBM mutant. Insertion of a threonine into the background of β1-CBM resulted in increased ligand affinity and flexibility in these loops when bound to carbohydrate. However, these mutations indicate that the additional threonine is not solely responsible for the differences in carbohydrate affinity and protein dynamics. Nevertheless, these results suggest that altered protein dynamics may contribute to differences in the ligand affinity of the two naturally occurring CBM isoforms. PMID:22339867

Family 46 Carbohydrate-binding Modules Contribute to the Enzymatic Hydrolysis of Xyloglucan and β-1,3-1,4-Glucans through Distinct Mechanisms.

PubMed

Venditto, Immacolata; Najmudin, Shabir; Luís, Ana S; Ferreira, Luís M A; Sakka, Kazuo; Knox, J Paul; Gilbert, Harry J; Fontes, Carlos M G A

2015-04-24

Structural carbohydrates comprise an extraordinary source of energy that remains poorly utilized by the biofuel sector as enzymes have restricted access to their substrates within the intricacy of plant cell walls. Carbohydrate active enzymes (CAZYmes) that target recalcitrant polysaccharides are modular enzymes containing noncatalytic carbohydrate-binding modules (CBMs) that direct enzymes to their cognate substrate, thus potentiating catalysis. In general, CBMs are functionally and structurally autonomous from their associated catalytic domains from which they are separated through flexible linker sequences. Here, we show that a C-terminal CBM46 derived from BhCel5B, a Bacillus halodurans endoglucanase, does not interact with β-glucans independently but, uniquely, acts cooperatively with the catalytic domain of the enzyme in substrate recognition. The structure of BhCBM46 revealed a β-sandwich fold that abuts onto the region of the substrate binding cleft upstream of the active site. BhCBM46 as a discrete entity is unable to bind to β-glucans. Removal of BhCBM46 from BhCel5B, however, abrogates binding to β-1,3-1,4-glucans while substantially decreasing the affinity for decorated β-1,4-glucan homopolymers such as xyloglucan. The CBM46 was shown to contribute to xyloglucan hydrolysis only in the context of intact plant cell walls, but it potentiates enzymatic activity against purified β-1,3-1,4-glucans in solution or within the cell wall. This report reveals the mechanism by which a CBM can promote enzyme activity through direct interaction with the substrate or by targeting regions of the plant cell wall where the target glucan is abundant. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Evidence that family 35 carbohydrate binding modules display conserved specificity but divergent function

PubMed Central

Montanier, Cedric; van Bueren, Alicia Lammerts; Dumon, Claire; Flint, James E.; Correia, Marcia A.; Prates, Jose A.; Firbank, Susan J.; Lewis, Richard J.; Grondin, Gilles G.; Ghinet, Mariana G.; Gloster, Tracey M.; Herve, Cecile; Knox, J. Paul; Talbot, Brian G.; Turkenburg, Johan P.; Kerovuo, Janne; Brzezinski, Ryszard; Fontes, Carlos M. G. A.; Davies, Gideon J.; Boraston, Alisdair B.; Gilbert, Harry J.

2009-01-01

Enzymes that hydrolyze complex carbohydrates play important roles in numerous biological processes that result in the maintenance of marine and terrestrial life. These enzymes often contain noncatalytic carbohydrate binding modules (CBMs) that have important substrate-targeting functions. In general, there is a tight correlation between the ligands recognized by bacterial CBMs and the substrate specificity of the appended catalytic modules. Through high-resolution structural studies, we demonstrate that the architecture of the ligand binding sites of 4 distinct family 35 CBMs (CBM35s), appended to 3 plant cell wall hydrolases and the exo-β-d-glucosaminidase CsxA, which contributes to the detoxification and metabolism of an antibacterial fungal polysaccharide, is highly conserved and imparts specificity for glucuronic acid and/or Δ4,5-anhydrogalaturonic acid (Δ4,5-GalA). Δ4,5-GalA is released from pectin by the action of pectate lyases and as such acts as a signature molecule for plant cell wall degradation. Thus, the CBM35s appended to the 3 plant cell wall hydrolases, rather than targeting the substrates of the cognate catalytic modules, direct their appended enzymes to regions of the plant that are being actively degraded. Significantly, the CBM35 component of CsxA anchors the enzyme to the bacterial cell wall via its capacity to bind uronic acid sugars. This latter observation reveals an unusual mechanism for bacterial cell wall enzyme attachment. This report shows that the biological role of CBM35s is not dictated solely by their carbohydrate specificities but also by the context of their target ligands. PMID:19218457

Cloning, purification, crystallization and preliminary X-ray studies of a carbohydrate-binding module (CBM_E1) derived from sugarcane soil metagenome.

PubMed

Campos, Bruna Medeia; Alvarez, Thabata Maria; Liberato, Marcelo Vizona; Polikarpov, Igor; Gilbert, Harry J; Zeri, Ana Carolina de Mattos; Squina, Fabio Marcio

2014-09-01

In recent years, owing to the growing global demand for energy, dependence on fossil fuels, limited natural resources and environmental pollution, biofuels have attracted great interest as a source of renewable energy. However, the production of biofuels from plant biomass is still considered to be an expensive technology. In this context, the study of carbohydrate-binding modules (CBMs), which are involved in guiding the catalytic domains of glycoside hydrolases for polysaccharide degradation, is attracting growing attention. Aiming at the identification of new CBMs, a sugarcane soil metagenomic library was analyzed and an uncharacterized CBM (CBM_E1) was identified. In this study, CBM_E1 was expressed, purified and crystallized. X-ray diffraction data were collected to 1.95 Å resolution. The crystals, which were obtained by the sitting-drop vapour-diffusion method, belonged to space group I23, with unit-cell parameters a = b = c = 88.07 Å.

Reassembly and co-crystallization of a family 9 processive endoglucanase from its component parts: structural and functional significance of the intermodular linker

PubMed Central

Petkun, Svetlana; Rozman Grinberg, Inna; Lamed, Raphael; Jindou, Sadanari; Burstein, Tal; Yaniv, Oren; Shoham, Yuval; Shimon, Linda J.W.; Frolow, Felix

2015-01-01

Non-cellulosomal processive endoglucanase 9I (Cel9I) from Clostridium thermocellum is a modular protein, consisting of a family-9 glycoside hydrolase (GH9) catalytic module and two family-3 carbohydrate-binding modules (CBM3c and CBM3b), separated by linker regions. GH9 does not show cellulase activity when expressed without CBM3c and CBM3b and the presence of the CBM3c was previously shown to be essential for endoglucanase activity. Physical reassociation of independently expressed GH9 and CBM3c modules (containing linker sequences) restored 60–70% of the intact Cel9I endocellulase activity. However, the mechanism responsible for recovery of activity remained unclear. In this work we independently expressed recombinant GH9 and CBM3c with and without their interconnecting linker in Escherichia coli. We crystallized and determined the molecular structure of the GH9/linker-CBM3c heterodimer at a resolution of 1.68 Å to understand the functional and structural importance of the mutual spatial orientation of the modules and the role of the interconnecting linker during their re-association. Enzyme activity assays and isothermal titration calorimetry were performed to study and compare the effect of the linker on the re-association. The results indicated that reassembly of the modules could also occur without the linker, albeit with only very low recovery of endoglucanase activity. We propose that the linker regions in the GH9/CBM3c endoglucanases are important for spatial organization and fixation of the modules into functional enzymes. PMID:26401442

Biochemical Properties and Atomic Resolution Structure of a Proteolytically Processed β-Mannanase from Cellulolytic Streptomyces sp. SirexAA-E

PubMed Central

Takasuka, Taichi E.; Acheson, Justin F.; Bianchetti, Christopher M.; Prom, Ben M.; Bergeman, Lai F.; Book, Adam J.; Currie, Cameron R.; Fox, Brian G.

2014-01-01

β-mannanase SACTE_2347 from cellulolytic Streptomyces sp. SirexAA-E is abundantly secreted into the culture medium during growth on cellulosic materials. The enzyme is composed of domains from the glycoside hydrolase family 5 (GH5), fibronectin type-III (Fn3), and carbohydrate binding module family 2 (CBM2). After secretion, the enzyme is proteolyzed into three different, catalytically active variants with masses of 53, 42 and 34 kDa corresponding to the intact protein, loss of the CBM2 domain, or loss of both the Fn3 and CBM2 domains. The three variants had identical N-termini starting with Ala51, and the positions of specific proteolytic reactions in the linker sequences separating the three domains were identified. To conduct biochemical and structural characterizations, the natural proteolytic variants were reproduced by cloning and heterologously expressed in Escherichia coli. Each SACTE_2347 variant hydrolyzed only β-1,4 mannosidic linkages, and also reacted with pure mannans containing partial galactosyl- and/or glucosyl substitutions. Examination of the X-ray crystal structure of the GH5 domain of SACTE_2347 suggests that two loops adjacent to the active site channel, which have differences in position and length relative to other closely related mannanases, play a role in producing the observed substrate selectivity. PMID:24710170

Unique carbohydrate binding platforms employed by the glucan phosphatases

PubMed Central

MEEKINS, David A.; GENTRY, Matthew S.

2016-01-01

Glucan phosphatases are a family of enzymes that are functionally conserved at the enzymatic level in animals and plants. These enzymes bind and dephosphorylate glycogen in animals and starch in plants. While the enzymatic function is conserved, the glucan phosphatases employ distinct mechanisms to bind and dephosphorylate glycogen or starch. The founding member of the family is a bimodular human protein called laforin that is comprised of a carbohydrate binding module 20 (CBM20) followed by a dual specificity phosphatase domain. Plants contain two glucan phosphatases: Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2). SEX4 contains a chloroplast targeting peptide, dual specificity phosphatase (DSP) domain, a CBM45, and a carboxy-terminal motif. LSF2 is comprised of simply a chloroplast targeting peptide, DSP domain, and carboxy-terminal motif. SEX4 employs an integrated DSP-CBM glucan-binding platform to engage and dephosphorylate starch. LSF2 lacks a CBM and instead utilizes two surface binding sites to bind and dephosphorylate starch. Laforin is a dimeric protein in solution and it utilizes a tetramodular architecture and cooperativity to bind and dephosphorylate glycogen. This chapter describes the biological role of glucan phosphatases in glycogen and starch metabolism and compares and contrasts their ability to bind and dephosphorylate glucans. PMID:27147465

Conformational analysis of the Streptococcus pneumoniae hyaluronate lyase and characterization of its hyaluronan-specific carbohydrate-binding module.

PubMed

Suits, Michael D L; Pluvinage, Benjamin; Law, Adrienne; Liu, Yan; Palma, Angelina S; Chai, Wengang; Feizi, Ten; Boraston, Alisdair B

2014-09-26

For a subset of pathogenic microorganisms, including Streptococcus pneumoniae, the recognition and degradation of host hyaluronan contributes to bacterial spreading through the extracellular matrix and enhancing access to host cell surfaces. The hyaluronate lyase (Hyl) presented on the surface of S. pneumoniae performs this role. Using glycan microarray screening, affinity electrophoresis, and isothermal titration calorimetry we show that the N-terminal module of Hyl is a hyaluronan-specific carbohydrate-binding module (CBM) and the founding member of CBM family 70. The 1.2 Å resolution x-ray crystal structure of CBM70 revealed it to have a β-sandwich fold, similar to other CBMs. The electrostatic properties of the binding site, which was identified by site-directed mutagenesis, are distinct from other CBMs and complementary to its acidic ligand, hyaluronan. Dynamic light scattering and solution small angle x-ray scattering revealed the full-length Hyl protein to exist as a monomer/dimer mixture in solution. Through a detailed analysis of the small angle x-ray scattering data, we report the pseudoatomic solution structures of the monomer and dimer forms of the full-length multimodular Hyl. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Neutron Reflectometry and QCM-D Study of the Interaction of Cellulase Enzymes with Films of Amorphous Cellulose

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

Halbert, Candice E; Ankner, John Francis; Kent, Michael S

2011-01-01

Improving the efficiency of enzymatic hydrolysis of cellulose is one of the key technological hurdles to reduce the cost of producing ethanol and other transportation fuels from lignocellulosic material. A better understanding of how soluble enzymes interact with insoluble cellulose will aid in the design of more efficient enzyme systems. We report a study involving neutron reflectometry (NR) and quartz crystal microbalance with dissipation (QCM-D) of the interaction of a commercial fungal enzyme extract (T. viride), two purified endoglucanses from thermophilic bacteria (Cel9A from A. acidocaldarius and Cel5A from T. maritima), and a mesophilic fungal endoglucanase (Cel45A from H. insolens)more » with amorphous cellulose films. The use of amorphous cellulose is motivated by the promise of ionic liquid pretreatment as a second generation technology that disrupts the native crystalline structure of cellulose. NR reveals the profile of water through the film at nm resolution, while QCM-D provides changes in mass and film stiffness. At 20 oC and 0.3 mg/ml, the T. viride cocktail rapidly digested the entire film, beginning from the surface followed by activity throughout the bulk of the film. For similar conditions, Cel9A and Cel5A were active for only a short period of time and only at the surface of the film, with Cel9A releasing 40 from the ~ 700 film and Cel5A resulting in only a slight roughening/swelling effect at the surface. Subsequent elevation of the temperature to the Topt in each case resulted in a very limited increase in activity, corresponding to the loss of an additional 60 from the film for Cel9A and 20 from the film for Cel5A, and very weak penetration into and digestion within the bulk of the film, before the activity again ceased. The results for Cel9A and Cel5A contrast sharply with results for Cel45A where very rapid and extensive penetration and digestion within the bulk of the film was observed at 20 C. We speculate that the large differences are due to the use of the thermophilic enzymes far below their optimal temperatures and also the presence of a cellulose binding module (CBM) on Cel45A while the thermophilic enzymes lack a CBM.« less

The carbohydrate-binding module (CBM)-like sequence is crucial for rice CWA1/BC1 function in proper assembly of secondary cell wall materials.

PubMed

Sato, Kanna; Ito, Sachiko; Fujii, Takeo; Suzuki, Ryu; Takenouchi, Sachi; Nakaba, Satoshi; Funada, Ryo; Sano, Yuzou; Kajita, Shinya; Kitano, Hidemi; Katayama, Yoshihiro

2010-11-01

We recently reported that the cwa1 mutation disturbed the deposition and assembly of secondary cell wall materials in the cortical fiber of rice internodes. Genetic analysis revealed that cwa1 is allelic to bc1, which encodes glycosylphosphatidylinositol (GPI)-anchored COBRA-like protein with the highest homology to Arabidopsis COBRA-like 4 (COBL4) and maize Brittle Stalk 2 (Bk2). Our results suggested that CWA1/BC1 plays a role in assembling secondary cell wall materials at appropriate sites, enabling synthesis of highly ordered secondary cell wall structure with solid and flexible internodes in rice. The N-terminal amino acid sequence of CWA1/BC1, as well as its orthologs (COBL4, Bk2) and other BC1-like proteins in rice, shows weak similarity to a family II carbohydrate-binding module (CBM2) of several bacterial cellulases. To investigate the importance of the CBM-like sequence of CWA1/BC1 in the assembly of secondary cell wall materials, Trp residues in the CBM-like sequence, which is important for carbohydrate binding, were substituted for Val residues and introduced into the cwa1 mutant. CWA1/BC1 with the mutated sequence did not complement the abnormal secondary cell walls seen in the cwa1 mutant, indicating that the CBM-like sequence is essential for the proper function of CWA1/BC1, including assembly of secondary cell wall materials.

Crystal Structure of Chitinase ChiW from Paenibacillus sp. str. FPU-7 Reveals a Novel Type of Bacterial Cell-Surface-Expressed Multi-Modular Enzyme Machinery

PubMed Central

Itoh, Takafumi; Hibi, Takao; Suzuki, Fumiko; Sugimoto, Ikumi; Fujiwara, Akihiro; Inaka, Koji; Tanaka, Hiroaki; Ohta, Kazunori; Fujii, Yutaka; Taketo, Akira; Kimoto, Hisashi

2016-01-01

The Gram-positive bacterium Paenibacillus sp. str. FPU-7 effectively hydrolyzes chitin by using a number of chitinases. A unique chitinase with two catalytic domains, ChiW, is expressed on the cell surface of this bacterium and has high activity towards various chitins, even crystalline chitin. Here, the crystal structure of ChiW at 2.1 Å resolution is presented and describes how the enzyme degrades chitin on the bacterial cell surface. The crystal structure revealed a unique multi-modular architecture composed of six domains to function efficiently on the cell surface: a right-handed β-helix domain (carbohydrate-binding module family 54, CBM-54), a Gly-Ser-rich loop, 1st immunoglobulin-like (Ig-like) fold domain, 1st β/α-barrel catalytic domain (glycoside hydrolase family 18, GH-18), 2nd Ig-like fold domain and 2nd β/α-barrel catalytic domain (GH-18). The structure of the CBM-54, flexibly linked to the catalytic region of ChiW, is described here for the first time. It is similar to those of carbohydrate lyases but displayed no detectable carbohydrate degradation activities. The CBM-54 of ChiW bound to cell wall polysaccharides, such as chin, chitosan, β-1,3-glucan, xylan and cellulose. The structural and biochemical data obtained here also indicated that the enzyme has deep and short active site clefts with endo-acting character. The affinity of CBM-54 towards cell wall polysaccharides and the degradation pattern of the catalytic domains may help to efficiently decompose the cell wall chitin through the contact surface. Furthermore, we clarify that other Gram-positive bacteria possess similar cell-surface-expressed multi-modular enzymes for cell wall polysaccharide degradation. PMID:27907169

Crystallization and preliminary crystallographic studies of a novel noncatalytic carbohydrate-binding module from the Ruminococcus flavefaciens cellulosome.

PubMed

Venditto, Immacolata; Goyal, Arun; Thompson, Andrew; Ferreira, Luis M A; Fontes, Carlos M G A; Najmudin, Shabir

2015-01-01

Microbial degradation of the plant cell wall is a fundamental biological process with considerable industrial importance. Hydrolysis of recalcitrant polysaccharides is orchestrated by a large repertoire of carbohydrate-active enzymes that display a modular architecture in which a catalytic domain is connected via linker sequences to one or more noncatalytic carbohydrate-binding modules (CBMs). CBMs direct the appended catalytic modules to their target substrates, thus potentiating catalysis. The genome of the most abundant ruminal cellulolytic bacterium, Ruminococcus flavefaciens strain FD-1, provides an opportunity to discover novel cellulosomal proteins involved in plant cell-wall deconstruction. It encodes a modular protein comprising a glycoside hydrolase family 9 catalytic module (GH9) linked to two unclassified tandemly repeated CBMs (termed CBM-Rf6A and CBM-Rf6B) and a C-terminal dockerin. The novel CBM-Rf6A from this protein has been crystallized and data were processed for the native and a selenomethionine derivative to 1.75 and 1.5 Å resolution, respectively. The crystals belonged to orthorhombic and cubic space groups, respectively. The structure was solved by a single-wavelength anomalous dispersion experiment using the CCP4 program suite and SHELXC/D/E.

Molecular characterization of a family 5 glycoside hydrolase suggests an induced-fit enzymatic mechanism.

PubMed

Liberato, Marcelo V; Silveira, Rodrigo L; Prates, Érica T; de Araujo, Evandro A; Pellegrini, Vanessa O A; Camilo, Cesar M; Kadowaki, Marco A; Neto, Mario de O; Popov, Alexander; Skaf, Munir S; Polikarpov, Igor

2016-04-01

Glycoside hydrolases (GHs) play fundamental roles in the decomposition of lignocellulosic biomaterials. Here, we report the full-length structure of a cellulase from Bacillus licheniformis (BlCel5B), a member of the GH5 subfamily 4 that is entirely dependent on its two ancillary modules (Ig-like module and CBM46) for catalytic activity. Using X-ray crystallography, small-angle X-ray scattering and molecular dynamics simulations, we propose that the C-terminal CBM46 caps the distal N-terminal catalytic domain (CD) to establish a fully functional active site via a combination of large-scale multidomain conformational selection and induced-fit mechanisms. The Ig-like module is pivoting the packing and unpacking motions of CBM46 relative to CD in the assembly of the binding subsite. This is the first example of a multidomain GH relying on large amplitude motions of the CBM46 for assembly of the catalytically competent form of the enzyme.

Molecular characterization of a family 5 glycoside hydrolase suggests an induced-fit enzymatic mechanism

NASA Astrophysics Data System (ADS)

Liberato, Marcelo V.; Silveira, Rodrigo L.; Prates, Érica T.; de Araujo, Evandro A.; Pellegrini, Vanessa O. A.; Camilo, Cesar M.; Kadowaki, Marco A.; Neto, Mario De O.; Popov, Alexander; Skaf, Munir S.; Polikarpov, Igor

2016-04-01

Glycoside hydrolases (GHs) play fundamental roles in the decomposition of lignocellulosic biomaterials. Here, we report the full-length structure of a cellulase from Bacillus licheniformis (BlCel5B), a member of the GH5 subfamily 4 that is entirely dependent on its two ancillary modules (Ig-like module and CBM46) for catalytic activity. Using X-ray crystallography, small-angle X-ray scattering and molecular dynamics simulations, we propose that the C-terminal CBM46 caps the distal N-terminal catalytic domain (CD) to establish a fully functional active site via a combination of large-scale multidomain conformational selection and induced-fit mechanisms. The Ig-like module is pivoting the packing and unpacking motions of CBM46 relative to CD in the assembly of the binding subsite. This is the first example of a multidomain GH relying on large amplitude motions of the CBM46 for assembly of the catalytically competent form of the enzyme.

CelF of Orpinomyces PC-2 has an intron and encodes a cellulase (CelF) containing a carbohydrate-binding module.

PubMed

Chen, Huizhong; Li, Xin-Liang; Blum, David L; Ximenes, Eduardo A; Ljungdahl, Lars G

2003-01-01

A cDNA, designated celF, encoding a cellulase (CelF) was isolated from the anaerobic fungus Orpinomyces PC-2. The open reading frame contains regions coding for a signal peptide, a carbohydrate-binding module (CBM), a linker, and a catalytic domain. The catalytic domain was homologous to those of CelA and CelC of the same fungus and to that of the Neocallimastix patriciarum CELA, but CelF lacks a docking domain, characteristic for enzymes of cellulosomes. It was also homologous to the cellobiohydrolase IIs and endoglucanases of aerobic organisms. The gene has a 111-bp intron, located within the CBM-coding region. Some biochemical properties of the purified recombinant enzyme are described.

Comparative Metagenomics of Cellulose- and Poplar Hydrolysate-Degrading Microcosms from Gut Microflora of the Canadian Beaver (Castor canadensis) and North American Moose (Alces americanus) after Long-Term Enrichment

PubMed Central

Wong, Mabel T.; Wang, Weijun; Couturier, Marie; Razeq, Fakhria M.; Lombard, Vincent; Lapebie, Pascal; Edwards, Elizabeth A.; Terrapon, Nicolas; Henrissat, Bernard; Master, Emma R.

2017-01-01

To identify carbohydrate-active enzymes (CAZymes) that might be particularly relevant for wood fiber processing, we performed a comparative metagenomic analysis of digestive systems from Canadian beaver (Castor canadensis) and North American moose (Alces americanus) following 3 years of enrichment on either microcrystalline cellulose or poplar hydrolysate. In total, 9,386 genes encoding CAZymes and carbohydrate-binding modules (CBMs) were identified, with up to half predicted to originate from Firmicutes, Bacteroidetes, Chloroflexi, and Proteobacteria phyla, and up to 17% from unknown phyla. Both PCA and hierarchical cluster analysis distinguished the annotated glycoside hydrolase (GH) distributions identified herein, from those previously reported for grass-feeding mammals and herbivorous foragers. The CAZyme profile of moose rumen enrichments also differed from a recently reported moose rumen metagenome, most notably by the absence of GH13-appended dockerins. Consistent with substrate-driven convergence, CAZyme profiles from both poplar hydrolysate-fed cultures differed from cellulose-fed cultures, most notably by increased numbers of unique sequences belonging to families GH3, GH5, GH43, GH53, and CE1. Moreover, pairwise comparisons of moose rumen enrichments further revealed higher counts of GH127 and CE15 families in cultures fed with poplar hydrolysate. To expand our scope to lesser known carbohydrate-active proteins, we identified and compared multi-domain proteins comprising both a CBM and domain of unknown function (DUF) as well as proteins with unknown function within the 416 predicted polysaccharide utilization loci (PULs). Interestingly, DUF362, identified in iron–sulfur proteins, was consistently appended to CBM9; on the other hand, proteins with unknown function from PULs shared little identity unless from identical PULs. Overall, this study sheds new light on the lignocellulose degrading capabilities of microbes originating from digestive systems of mammals known for fiber-rich diets, and highlights the value of enrichment to select new CAZymes from metagenome sequences for future biochemical characterization. PMID:29326667

Fluorescence correlation spectroscopy study of the complexation of DNA hybrids, IgG antibody, and a chimeric protein of IgG-binding ZZ domains fused with a carbohydrate binding module.

PubMed

Rosa, A M M; Prazeres, D M F; Paulo, P M R

2017-06-28

Fluorescence correlation spectroscopy (FCS) was used to characterize the molecular interactions between the four components of a DNA recognition system. A fluorescent DNA probe was used to assess: (i) the hybridization with a complementary biotin-labeled target, (ii) the complexation of the resulting hybrid and an anti-biotin antibody, and (iii) the binding of the latter complex to a ZZ-CBM fusion protein that combines small synthetic IgG Fc-binding Z domains with a carbohydrate binding module (CBM). These binding interactions were monitored by exposing the fluorescent DNA probe to different amounts and combinations of the other molecules in solution. Through the analysis of FCS autocorrelation curves, an association constant (K a ) of 2.9 × 10 7 M -1 was estimated for DNA·DNA hybridization, and the presence of (non-) complementary target DNA in solution could be discriminated. The specific capture of biotinylated DNA hybrids by anti-biotin IgG was verified, with an apparent K a of 2.5 × 10 6 M -1 . The increment in the diffusion time measured when the DNA·DNA:antibody complexes were in contact with the ZZ-CBM fusion protein suggested that the binding occurs at a stoichiometric ratio of DNA/antibody complex to fusion larger than 1 : 1. The FCS-derived information obtained is useful to gain insight into molecular interactions involved in diagnostic assays.

The Copper Active Site of CBM33 Polysaccharide Oxygenases

PubMed Central

2013-01-01

The capacity of metal-dependent fungal and bacterial polysaccharide oxygenases, termed GH61 and CBM33, respectively, to potentiate the enzymatic degradation of cellulose opens new possibilities for the conversion of recalcitrant biomass to biofuels. GH61s have already been shown to be unique metalloenzymes containing an active site with a mononuclear copper ion coordinated by two histidines, one of which is an unusual τ-N-methylated N-terminal histidine. We now report the structural and spectroscopic characterization of the corresponding copper CBM33 enzymes. CBM33 binds copper with high affinity at a mononuclear site, significantly stabilizing the enzyme. X-band EPR spectroscopy of Cu(II)-CBM33 shows a mononuclear type 2 copper site with the copper ion in a distorted axial coordination sphere, into which azide will coordinate as evidenced by the concomitant formation of a new absorption band in the UV/vis spectrum at 390 nm. The enzyme’s three-dimensional structure contains copper, which has been photoreduced to Cu(I) by the incident X-rays, confirmed by X-ray absorption/fluorescence studies of both aqueous solution and intact crystals of Cu-CBM33. The single copper(I) ion is ligated in a T-shaped configuration by three nitrogen atoms from two histidine side chains and the amino terminus, similar to the endogenous copper coordination geometry found in fungal GH61. PMID:23540833

Lipopeptide produced from Bacillus sp. W112 improves the hydrolysis of lignocellulose by specifically reducing non-productive binding of cellulases with and without CBMs.

PubMed

Liu, Jiawen; Zhu, Ning; Yang, Jinshui; Yang, Yi; Wang, Ruonan; Liu, Liang; Yuan, Hongli

2017-01-01

Surfactants have attracted increasing interest for their capability to improve the enzymatic hydrolysis of lignocellulosic biomass. Compared to chemical surfactants, biosurfactants have a broader prospect for industrial applications because they are more environmentally friendly and more effective in some researches. Commercial cellulase preparations are mainly composed of endoglucanases (EGs) and cellobiohydrolases (CBHs) that possess carbohydrate-binding modules (CBMs). However, the effects of lipopeptide-type biosurfactants on enzymatic saccharification of lignocellulose and adsorption behaviors of cellulases with CBMs remain unclear. In this study, we found that Bacillus sp. W112 could produce a lipopeptide-type biosurfactant from untreated biomass, such as wheat bran and Jerusalem artichoke tuber. The lipopeptide could enhance the enzymatic hydrolysis of dilute acid pretreated Giant Juncao grass (DA-GJG) by fungal and bacterial enzymes. The enhancement increased over a range of temperatures from 30 to 50 °C. Lipopeptide was shown to be more effective in promoting DA-GJG saccharification than chemical surfactants at low dosages, with a best stimulatory degree of 20.8% at 2% loading of the substrates (w/w). Lipopeptide increased the thermostability of EG and CBH in commercial cellulase cocktails. Moreover, the dual effects of lipopeptide on the adsorption behaviors of cellulases were found. It specifically lowered the non-productive binding of cellulases to lignin and increased the binding of cellulases to cellulose. In addition, we investigated the influence of lipopeptide on the adsorption behaviors of CBHs with CBMs for the first time. Our results showed that lipopeptide reduced the adsorption of CBM-deleted CBH to DA-GJG to a greater extent than that of intact CBH while the non-productive binding of intact CBH to lignin was reduced more, indicating that lipopeptide decreased the binding of CBMs onto lignin but not their combination with cellulose. In this study, we found that lipopeptide from Bacillus sp. W112 promoted the enzymatic hydrolysis of DA-GJG at relative low loadings. The stimulatory effect could be attributed to increasing the cellulase thermostability, reducing non-productive adsorption of cellulases with CBMs caused by lignin and enhancing the binding of cellulases to cellulose.

Overexpression of the carbohydrate binding module from Solanum lycopersicum expansin 1 (Sl-EXP1) modifies tomato fruit firmness and Botrytis cinerea susceptibility.

PubMed

Perini, M A; Sin, I N; Villarreal, N M; Marina, M; Powell, A L T; Martínez, G A; Civello, P M

2017-04-01

Firmness, one of the major determinants of postharvest quality and shelf life of fruits is determined by the mechanical resistance imposed by the plant cell wall. Expansins (EXP) are involved in the non-hydrolytic metabolic disassembly of plant cell walls, particularly in processes where relaxation of the wall is necessary, such as fruit development and ripening. As many carbohydrate-associated proteins, expansins have a putative catalytic domain and a carbohydrate-binding module (CBM). Several strategies have been pursued to control the loss of fruit firmness during storage. Most of the approaches have been to suppress the expression of key enzymes involved in the cell wall metabolism, but this is the first time that a CBM was overexpressed in a fruit aimed to control cell wall degradation and fruit softening. We report the constitutive overexpression of the CBM of Solanum lycopersicum expansin 1 (CBM-SlExp1) in the cell wall of tomato plants, and its effects on plant and fruit phenotype. Overexpression of CBM-SlExp1 increased the mechanical resistance of leaves, whereas it did not modify plant growth and general phenotype. However, transgenic plants showed delayed softening and firmer fruits. In addition, fruits were less susceptible to Botrytis cinerea infection, and the "in vitro" growth of the fungus on media containing AIR from the pericarp of transgenic fruits was lower than controls. The possibility of overexpressing a CBM of a fruit-specific expansin to control cell wall degradation and fruit softening is discussed. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Characterization of Protein-Carbohydrate Interactions by NMR Spectroscopy.

PubMed

Grondin, Julie M; Langelaan, David N; Smith, Steven P

2017-01-01

Solution-state nuclear magnetic resonance (NMR) spectroscopy can be used to monitor protein-carbohydrate interactions. Two-dimensional 1 H- 15 N heteronuclear single quantum coherence (HSQC)-based techniques described in this chapter can be used quickly and effectively to screen a set of possible carbohydrate binding partners, to quantify the dissociation constant (K d ) of any identified interactions, and to map the carbohydrate binding site on the structure of the protein. Here, we describe the titration of a family 32 carbohydrate binding module from Clostridium perfringens (CpCBM32) with the monosaccharide N-acetylgalactosamine (GalNAc), in which we calculate the apparent dissociation of the interaction, and map the GalNAc binding site onto the structure of CpCBM32.

Cell wall targeted in planta iron accumulation enhances biomass conversion and seed iron concentration in Arabidopsis and rice

DOE PAGES

Yang, Haibing; Wei, Hui; Ma, Guojie; ...

2016-04-07

Conversion of nongrain biomass into liquid fuel is a sustainable approach to energy demands as global population increases. Previously, we showed that iron can act as a catalyst to enhance the degradation of lignocellulosic biomass for biofuel production. However, direct addition of iron catalysts to biomass pretreatment is diffusion-limited, would increase the cost and complexity of biorefinery unit operations and may have deleterious environmental impacts. Here, we show a new strategy for in planta accumulation of iron throughout the volume of the cell wall where iron acts as a catalyst in the deconstruction of lignocellulosic biomass. We engineered CBM-IBP fusionmore » polypeptides composed of a carbohydrate-binding module family 11 (CBM11) and an iron-binding peptide (IBP) for secretion into Arabidopsis and rice cell walls. CBM-IBP transformed Arabidopsis and rice plants show significant increases in iron accumulation and biomass conversion compared to respective controls. Further, CBM-IBP rice shows a 35% increase in seed iron concentration and a 40% increase in seed yield in greenhouse experiments. In conclusion, CBM-IBP rice potentially could be used to address iron deficiency, the most common and widespread nutritional disorder according to the World Health Organization.« less

Cell wall targeted in planta iron accumulation enhances biomass conversion and seed iron concentration in Arabidopsis and rice

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

Yang, Haibing; Wei, Hui; Ma, Guojie

Conversion of nongrain biomass into liquid fuel is a sustainable approach to energy demands as global population increases. Previously, we showed that iron can act as a catalyst to enhance the degradation of lignocellulosic biomass for biofuel production. However, direct addition of iron catalysts to biomass pretreatment is diffusion-limited, would increase the cost and complexity of biorefinery unit operations and may have deleterious environmental impacts. Here, we show a new strategy for in planta accumulation of iron throughout the volume of the cell wall where iron acts as a catalyst in the deconstruction of lignocellulosic biomass. We engineered CBM-IBP fusionmore » polypeptides composed of a carbohydrate-binding module family 11 (CBM11) and an iron-binding peptide (IBP) for secretion into Arabidopsis and rice cell walls. CBM-IBP transformed Arabidopsis and rice plants show significant increases in iron accumulation and biomass conversion compared to respective controls. Further, CBM-IBP rice shows a 35% increase in seed iron concentration and a 40% increase in seed yield in greenhouse experiments. CBM-IBP rice potentially could be used to address iron deficiency, the most common and widespread nutritional disorder according to the World Health Organization.« less

Cell wall targeted in planta iron accumulation enhances biomass conversion and seed iron concentration in Arabidopsis and rice

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

Yang, Haibing; Wei, Hui; Ma, Guojie

Conversion of nongrain biomass into liquid fuel is a sustainable approach to energy demands as global population increases. Previously, we showed that iron can act as a catalyst to enhance the degradation of lignocellulosic biomass for biofuel production. However, direct addition of iron catalysts to biomass pretreatment is diffusion-limited, would increase the cost and complexity of biorefinery unit operations and may have deleterious environmental impacts. Here, we show a new strategy for in planta accumulation of iron throughout the volume of the cell wall where iron acts as a catalyst in the deconstruction of lignocellulosic biomass. We engineered CBM-IBP fusionmore » polypeptides composed of a carbohydrate-binding module family 11 (CBM11) and an iron-binding peptide (IBP) for secretion into Arabidopsis and rice cell walls. CBM-IBP transformed Arabidopsis and rice plants show significant increases in iron accumulation and biomass conversion compared to respective controls. Further, CBM-IBP rice shows a 35% increase in seed iron concentration and a 40% increase in seed yield in greenhouse experiments. In conclusion, CBM-IBP rice potentially could be used to address iron deficiency, the most common and widespread nutritional disorder according to the World Health Organization.« less

Specific characteristics of family 45 endoglucanases from Mucorales in the use of textiles and laundry.

PubMed

Shimonaka, Atsushi; Koga, Jinichiro; Baba, Yuko; Nishimura, Tomoko; Murashima, Koichiro; Kubota, Hidetoshi; Kono, Toshiaki

2006-04-01

We examined the characteristics of family 45 endoglucanases (glycoside hydrolases family 45; GH45) from Mucorales belonging to Zygomycota in the use of textiles and laundry. The defibrillation activities on lyocell fabric of family 45 endoglucanases from Mucorales, such as RCE1 and RCE2 from Rhizopus oryzae, MCE1 and MCE2 from Mucor circinelloides, and PCE1 from Phycomyces nitens, were much higher than those of the other family 45 endoglucanases. By contrast, family 45 endoglucanases from Mucorales were less resistant to anionic surfactant and oxidizing agent, main components in detergents, than the other family 45 endoglucanases. RCE1 consists of two distinct modules, a catalytic module and a carbohydrate-binding module family 1 (CBM1), and these common specific characteristics were considered to due to the catalytic module, but not to the CBM1.

Cell wall targeted in planta iron accumulation enhances biomass conversion and seed iron concentration in Arabidopsis and rice.

PubMed

Yang, Haibing; Wei, Hui; Ma, Guojie; Antunes, Mauricio S; Vogt, Stefan; Cox, Joseph; Zhang, Xiao; Liu, Xiping; Bu, Lintao; Gleber, S Charlotte; Carpita, Nicholas C; Makowski, Lee; Himmel, Michael E; Tucker, Melvin P; McCann, Maureen C; Murphy, Angus S; Peer, Wendy A

2016-10-01

Conversion of nongrain biomass into liquid fuel is a sustainable approach to energy demands as global population increases. Previously, we showed that iron can act as a catalyst to enhance the degradation of lignocellulosic biomass for biofuel production. However, direct addition of iron catalysts to biomass pretreatment is diffusion-limited, would increase the cost and complexity of biorefinery unit operations and may have deleterious environmental impacts. Here, we show a new strategy for in planta accumulation of iron throughout the volume of the cell wall where iron acts as a catalyst in the deconstruction of lignocellulosic biomass. We engineered CBM-IBP fusion polypeptides composed of a carbohydrate-binding module family 11 (CBM11) and an iron-binding peptide (IBP) for secretion into Arabidopsis and rice cell walls. CBM-IBP transformed Arabidopsis and rice plants show significant increases in iron accumulation and biomass conversion compared to respective controls. Further, CBM-IBP rice shows a 35% increase in seed iron concentration and a 40% increase in seed yield in greenhouse experiments. CBM-IBP rice potentially could be used to address iron deficiency, the most common and widespread nutritional disorder according to the World Health Organization. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Influence of a family 29 carbohydrate binding module on the recombinant production of galactose oxidase in Pichia pastoris

PubMed Central

Mollerup, Filip; Master, Emma

2015-01-01

Herein, we report the extracellular expression of carbohydrate active fusion enzymes in Pichia pastoris. Particularly, CBM29-1-2 from Piromyces equi was separately fused to the N- and C-terminus of galactose 6-oxidase (GaO, D-galactose: oxygen 6-oxidoreductase, EC 1.1.13.9, CAZy family AA5) from Fusarium graminearum, generating CBM29-GaO and GaO-CBM29, respectively. P. pastoris was transformed with expression vectors encoding GaO, CBM29-GaO and GaO-CBM29, and the fusion proteins were expressed in both shake-flask and 2L bioreactor systems. Volumetric production yields and specific GaO activity increased when expression was performed in a bioreactor system compared to shake-flask cultivation. This was observed for both CBM29-GaO and GaO-CBM29, and is consistent with previous reports of GaO expression in P. pastoris (Spadiut et al., 2010; Anasontzis et al., 2014) [1], [2]. Fusion of CBM29 to the C-terminal of GaO (GaO-CBM29) resulted in a stable uniform protein at the expected calculated size (107 kDa) when analyzed with SDS-PAGE. By comparison, the expression of the N-terminal fusion protein (CBM29-GaO) was low, and two truncated versions of CBM29-GaO were coexpressed with the full-sized protein. Despite differences in protein yield, the specific GaO activity on galactose was not affected by CBM29 fusion to either the N- or C-terminus of the enzyme. A detailed description of the catalytic and physiochemical properties of CBM29-GaO and GaO-CBM29 is available in the parent publication (Mollerup et al., 2015) [3]. PMID:26858983

Enhanced cellulase recovery without β-glucosidase supplementation for cellulosic ethanol production using an engineered strain and surfactant.

PubMed

Huang, Renliang; Guo, Hong; Su, Rongxin; Qi, Wei; He, Zhimin

2017-03-01

Recycling cellulases by substrate adsorption is a promising strategy for reducing the enzyme cost of cellulosic ethanol production. However, β-glucosidase has no carbohydrate-binding module (CBM). Thus, additional enzymes are required in each cycle to achieve a high ethanol yield. In this study, we report a new method of recycling cellulases without β-glucosidase supplementation using lignocellulosic substrate, an engineered strain expressing β-glucosidase and Tween 80. The cellulases and Tween 80 were added to an aqueous suspension of diluted sulfuric acid/ammonia-treated corncobs in a simultaneous saccharification and fermentation (SSF) process for ethanol production. Subsequently, the addition of fresh pretreated corncobs to the fermentation liquor and remaining solid residue provided substrates with absorbed cellulases for the next SSF cycle. This method provided excellent ethanol production in three successive SSF cycles without requiring the addition of new cellulases. For a 10% (w/v) solid loading, a cellulase dosage of 30 filter paper units (FPU)/g cellulose, 0.5% Tween 80, and 2 g/L of the engineered strain, approximately 90% of the initial ethanol concentration from the first SSF process was obtained in the next two SSF processes, with a total ethanol production of 306.27 g/kg corncobs and an enzyme productivity of 0.044 g/FPU. Tween 80 played an important role in enhancing cellulase recovery. This new enzyme recycling method is more efficient and practical than other reported methods. Biotechnol. Bioeng. 2017;114: 543-551. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Species-specific chitin-binding module 18 expansion in the amphibian pathogen Batrachochytrium dendrobatidis.

PubMed

Abramyan, John; Stajich, Jason E

2012-01-01

Batrachochytrium dendrobatidis is the causative agent of chytridiomycosis, which is considered one of the driving forces behind the worldwide decline in populations of amphibians. As a member of the phylum Chytridiomycota, B. dendrobatidis has diverged significantly to emerge as the only pathogen of adult vertebrates. Such shifts in lifestyle are generally accompanied by various degrees of genomic modifications, yet neither its mode of pathogenicity nor any factors associated with it have ever been identified. Presented here is the identification and characterization of a unique expansion of the carbohydrate-binding module family 18 (CBM18), specific to B. dendrobatidis. CBM (chitin-binding module) expansions have been likened to the evolution of pathogenicity in a variety of fungus species, making this expanded group a prime candidate for the identification of potential pathogenicity factors. Furthermore, the CBM18 expansions are confined to three categories of genes, each having been previously implicated in host-pathogen interactions. These correlations highlight this specific domain expansion as a potential key player in the mode of pathogenicity in this unique fungus. The expansion of CBM18 in B. dendrobatidis is exceptional in its size and diversity compared to other pathogenic species of fungi, making this genomic feature unique in an evolutionary context as well as in pathogenicity. Amphibian populations are declining worldwide at an unprecedented rate. Although various factors are thought to contribute to this phenomenon, chytridiomycosis has been identified as one of the leading causes. This deadly fungal disease is cause by Batrachochytrium dendrobatidis, a chytrid fungus species unique in its pathogenicity and, furthermore, its specificity to amphibians. Despite more than two decades of research, the biology of this fungus species and its deadly interaction with amphibians had been notoriously difficult to unravel. Due to the alarming rate of worldwide spread and associated decline in amphibian populations, it is imperative to incorporate novel genomic and genetic techniques into the study of this species. In this study, we present the first reported potential pathogenicity factors in B. dendrobatidis. In silico studies such as this allow us to identify putative targets for more specific molecular analyses, furthering our hope for the control of this pathogen.

Evaluation of secretome of highly efficient lignocellulolytic Penicillium sp. Dal 5 isolated from rhizosphere of conifers.

PubMed

Rai, Rohit; Kaur, Baljit; Singh, Surender; Di Falco, Macros; Tsang, Adrian; Chadha, B S

2016-09-01

Penicillium sp. (Dal 5) isolated from rhizosphere of conifers from Dalhousie (Himachal Pradesh, India) was found to be an efficient cellulolytic strain. The culture under shake flask on CWR (cellulose, wheat bran and rice straw) medium produced appreciably higher levels of endoglucanase (35.69U/ml), β-glucosidase (4.20U/ml), cellobiohydrolase (2.86U/ml), FPase (1.2U/ml) and xylanase (115U/ml) compared to other Penicillium strains reported in literature. The mass spectroscopy analysis of Penicillium sp. Dal 5 secretome identified 108 proteins constituting an array of CAZymes including glycosyl hydrolases (GH) belonging to 24 different families, polysaccharide lyases (PL), carbohydrate esterases (CE), lytic polysaccharide mono-oxygenases (LPMO) in addition to swollenin and a variety of carbohydrate binding modules (CBM) indicating an elaborate genetic potential of this strain for hydrolysis of lignocellulosics. Further, the culture extract was evaluated for hydrolysis of alkali treated rice straw, wheat straw, bagasse and corn cob at 10% substrate loading rate. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cloning of novel cellulases from cellulolytic fungi: heterologous expression of a family 5 glycoside hydrolase from Trametes versicolor in Pichia pastoris.

PubMed

Salinas, Alejandro; Vega, Marcela; Lienqueo, María Elena; Garcia, Alejandro; Carmona, Rene; Salazar, Oriana

2011-12-10

Total cDNA isolated from cellulolytic fungi cultured in cellulose was examined for the presence of sequences encoding for endoglucanases. Novel sequences encoding for glycoside hydrolases (GHs) were identified in Fusarium oxysporum, Ganoderma applanatum and Trametes versicolor. The cDNA encoding for partial sequences of GH family 61 cellulases from F. oxysporum and G. applanatum shares 58 and 68% identity with endoglucanases from Glomerella graminicola and Laccaria bicolor, respectively. A new GH family 5 endoglucanase from T. versicolor was also identified. The cDNA encoding for the mature protein was completely sequenced. This enzyme shares 96% identity with Trametes hirsuta endoglucanase and 22% with Trichoderma reesei endoglucanase II (EGII). The enzyme, named TvEG, has N-terminal family 1 carbohydrate binding module (CBM1). The full length cDNA was cloned into the pPICZαB vector and expressed as an active, extracellular enzyme in the methylotrophic yeast Pichia pastoris. Preliminary studies suggest that T. versicolor could be useful for lignocellulose degradation. Copyright © 2011 Elsevier Inc. All rights reserved.

Isolation, molecular cloning and expression of cellobiohydrolase B (CbhB) from Aspergillus niger in Escherichia coli

NASA Astrophysics Data System (ADS)

Woon, J. S. K.; Murad, A. M. A.; Abu Bakar, F. D.

2015-09-01

A cellobiohydrolase B (CbhB) from Aspergillus niger ATCC 10574 was cloned and expressed in E. coli. CbhB has an open reading frame of 1611 bp encoding a putative polypeptide of 536 amino acids. Analysis of the encoded polypeptide predicted a molecular mass of 56.2 kDa, a cellulose binding module (CBM) and a catalytic module. In order to obtain the mRNA of cbhB, total RNA was extracted from A. niger cells induced by 1% Avicel. First strand cDNA was synthesized from total RNA via reverse transcription. The full length cDNA of cbhB was amplified by PCR and cloned into the cloning vector, pGEM-T Easy. A comparison between genomic DNA and cDNA sequences of cbhB revealed that the gene is intronless. Upon the removal of the signal peptide, the cDNA of cbhB was cloned into the expression vector pET-32b. However, the recombinant CbhB was expressed in Escherichia coli Origami DE3 as an insoluble protein. A homology model of CbhB predicted the presence of nine disulfide bonds in the protein structure which may have contributed to the improper folding of the protein and thus, resulting in inclusion bodies in E. coli.

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

Vermaas, Josh V.; Petridis, Loukas; Qi, Xianghong

The conversion of plant biomass to ethanol via enzymatic cellulose hydrolysis offers a potentially sustainable route to biofuel production. However, the inhibition of enzymatic activity in pretreated biomass by lignin severely limits the efficiency of this process. By performing atomic-detail molecular dynamics simulation of a biomass model containing cellulose, lignin, and cellulases (TrCel7A), we elucidate detailed lignin inhibition mechanisms. We find that lignin binds preferentially both to the elements of cellulose to which the cellulases also preferentially bind (the hydrophobic faces) and also to the specific residues on the cellulose-binding module of the cellulase that are critical for cellulose bindingmore » of TrCel7A (Y466, Y492, and Y493). In conclusion, lignin thus binds exactly where for industrial purposes it is least desired, providing a simple explanation of why hydrolysis yields increase with lignin removal.« less

Fragments of Bacterial Endoglycosidase S and Immunoglobulin G Reveal Subdomains of Each That Contribute to Deglycosylation*

PubMed Central

Dixon, Emma V.; Claridge, Jolyon K.; Harvey, David J.; Baruah, Kavitha; Yu, Xiaojie; Vesiljevic, Snezana; Mattick, Susan; Pritchard, Laura K.; Krishna, Benjamin; Scanlan, Christopher N.; Schnell, Jason R.; Higgins, Matthew K.; Zitzmann, Nicole; Crispin, Max

2014-01-01

Endoglycosidase S (EndoS) is a glycoside-hydrolase secreted by the bacterium Streptococcus pyogenes. EndoS preferentially hydrolyzes the N-linked glycans from the Fc region of IgG during infection. This hydrolysis impedes Fc functionality and contributes to the immune evasion strategy of S. pyogenes. Here, we investigate the mechanism of human serum IgG deactivation by EndoS. We expressed fragments of IgG1 and demonstrated that EndoS was catalytically active against all of them including the isolated CH2 domain of the Fc domain. Similarly, we sought to investigate which domains within EndoS could contribute to activity. Bioinformatics analysis of the domain organization of EndoS confirmed the previous predictions of a chitinase domain and leucine-rich repeat but also revealed a putative carbohydrate binding module (CBM) followed by a C-terminal region. Using expressed fragments of EndoS, circular dichroism of the isolated CBM, and a CBM-C-terminal region fusion revealed folded domains dominated by β sheet and α helical structure, respectively. Nuclear magnetic resonance analysis of the CBM with monosaccharides was suggestive of carbohydrate binding functionality. Functional analysis of truncations of EndoS revealed that, whereas the C-terminal of EndoS is dispensable for activity, its deletion impedes the hydrolysis of IgG glycans. PMID:24668806

Mechanism of lignin inhibition of enzymatic biomass deconstruction

DOE PAGES

Vermaas, Josh V.; Petridis, Loukas; Qi, Xianghong; ...

2015-12-01

The conversion of plant biomass to ethanol via enzymatic cellulose hydrolysis offers a potentially sustainable route to biofuel production. However, the inhibition of enzymatic activity in pretreated biomass by lignin severely limits the efficiency of this process. By performing atomic-detail molecular dynamics simulation of a biomass model containing cellulose, lignin, and cellulases (TrCel7A), we elucidate detailed lignin inhibition mechanisms. We find that lignin binds preferentially both to the elements of cellulose to which the cellulases also preferentially bind (the hydrophobic faces) and also to the specific residues on the cellulose-binding module of the cellulase that are critical for cellulose bindingmore » of TrCel7A (Y466, Y492, and Y493). In conclusion, lignin thus binds exactly where for industrial purposes it is least desired, providing a simple explanation of why hydrolysis yields increase with lignin removal.« less

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.

Hydrolysis of model cellulose films by cellulosomes: Extension of quartz crystal microbalance techniques to multienzymatic complexes

USDA-ARS?s Scientific Manuscript database

Clostridium thermocellum, a well-studied cellulolytic bacterium, produces highly active cellulases in the form of cellulosomes. The ability of the cellulose binding module within the cellulosome to adhere C. thermocellum cells to the cellulosic substrate is considered to contribute to its high cellu...

Genomewide analysis of polysaccharides degrading enzymes in 11 white- and brown-rot Polyporales provides insight into mechanisms of wood decay

Treesearch

Chiaki Hori; Jill Gaskell; Kiyohiko Igarashi; Masahiro Samejima; David Hibbett; Bernard Henrissat; Dan Cullen

2013-01-01

To degrade the polysaccharides, wood-decay fungi secrete a variety of glycoside hydrolases (GHs) and carbohydrate esterases (CEs) classified into various sequence-based families of carbohydrate-active enzymes (CAZys) and their appended carbohydrate-binding modules (CBM). Oxidative enzymes, such as cellobiose dehydrogenase (CDH) and lytic polysaccharide monooxygenase (...

Development of the geometry database for the CBM experiment

NASA Astrophysics Data System (ADS)

Akishina, E. P.; Alexandrov, E. I.; Alexandrov, I. N.; Filozova, I. A.; Friese, V.; Ivanov, V. V.

2018-01-01

The paper describes the current state of the Geometry Database (Geometry DB) for the CBM experiment. The main purpose of this database is to provide convenient tools for: (1) managing the geometry modules; (2) assembling various versions of the CBM setup as a combination of geometry modules and additional files. The CBM users of the Geometry DB may use both GUI (Graphical User Interface) and API (Application Programming Interface) tools for working with it.

Isolation, molecular cloning and expression of cellobiohydrolase B (CbhB) from Aspergillus niger in Escherichia coli

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

Woon, J. S. K., E-mail: jameswoon@siswa.ukm.edu.my; Murad, A. M. A., E-mail: munir@ukm.edu.my; Abu Bakar, F. D., E-mail: fabyff@ukm.edu.my

A cellobiohydrolase B (CbhB) from Aspergillus niger ATCC 10574 was cloned and expressed in E. coli. CbhB has an open reading frame of 1611 bp encoding a putative polypeptide of 536 amino acids. Analysis of the encoded polypeptide predicted a molecular mass of 56.2 kDa, a cellulose binding module (CBM) and a catalytic module. In order to obtain the mRNA of cbhB, total RNA was extracted from A. niger cells induced by 1% Avicel. First strand cDNA was synthesized from total RNA via reverse transcription. The full length cDNA of cbhB was amplified by PCR and cloned into the cloning vector, pGEM-Tmore » Easy. A comparison between genomic DNA and cDNA sequences of cbhB revealed that the gene is intronless. Upon the removal of the signal peptide, the cDNA of cbhB was cloned into the expression vector pET-32b. However, the recombinant CbhB was expressed in Escherichia coli Origami DE3 as an insoluble protein. A homology model of CbhB predicted the presence of nine disulfide bonds in the protein structure which may have contributed to the improper folding of the protein and thus, resulting in inclusion bodies in E. coli.« less

Functional characterization of two distinct xyoglucanases from rumenal microbes

USDA-ARS?s Scientific Manuscript database

Xyloglucans are known to function by binding to cellulose microfibrils, crosslinking adjacent fibers forming cellulose-XG networks important for modulation of rigidity and extensibility of the primary cell wall of plants. Enzymatic hydrolysis and modification of xyloglucans has received considerabl...

Exploring the structural insights on human laforin mutation K87A in Lafora disease--a molecular dynamics study.

PubMed

Srikumar, P S; Rohini, K

2013-10-01

Lafora disease (LD) is an autosomal recessive, progressive form of myoclonus epilepsy which affects worldwide. LD occurs mainly in countries like southern Europe, northern Africa, South India, and in the Middle East. LD occurs with its onset mainly in teenagers and leads to decline and death within 2 to 10 years. The genes EPM2A and EPM2B are commonly involved in 90 % of LD cases. EPM2A codes for protein laforin which contains an amino terminal carbohydrate binding module (CBM) belonging to the CBM20 family and a carboxy terminal dual specificity phosphatase domain. Mutations in laforin are found to abolish glycogen binding and have been reported in wet lab methods. In order to investigate on structural insights on laforin mutation K81A, we performed molecular dynamics (MD) simulation studies for native and mutant protein. MD simulation results showed loss of stability due to mutation K87A which confirmed the structural reason for conformational changes observed in laforin. The conformational change of mutant laforin was confirmed by analysis using root mean square deviation, root mean square fluctuation, solvent accessibility surface area, radius of gyration, hydrogen bond, and principle component analysis. Our results identified that the flexibility of K87A mutated laforin structure, with replacement of acidic amino acid to aliphatic amino acid in functional CBM domain, have more impact in abolishing glycogen binding that favors LD.

A novel GH10 xylanase from Penicillium sp. accelerates saccharification of alkaline-pretreated bagasse by an enzyme from recombinant Trichoderma reesei expressing Aspergillus β-glucosidase.

PubMed

Shibata, Nozomu; Suetsugu, Mari; Kakeshita, Hiroshi; Igarashi, Kazuaki; Hagihara, Hiroshi; Takimura, Yasushi

2017-01-01

Trichoderma reesei is considered a candidate fungal enzyme producer for the economic saccharification of cellulosic biomass. However, performance of the saccharifying enzymes produced by T. reesei is insufficient. Therefore, many attempts have been made to improve its performance by heterologous protein expression. In this study, to increase the conversion efficiency of alkaline-pretreated bagasse to sugars, we conducted screening of biomass-degrading enzymes that showed synergistic effects with enzyme preparations produced by recombinant T. reesei . Penicillium sp. strain KSM-F532 produced the most effective enzyme to promote the saccharification of alkaline-pretreated bagasse. Biomass-degrading enzymes from strain KSM-F532 were fractionated and analyzed, and a xylanase, named PspXyn10, was identified. The amino acid sequence of PspXyn10 was determined by cDNA analysis: the enzyme shows a modular structure consisting of glycoside hydrolase family 10 (GH10) and carbohydrate-binding module family 1 (CBM1) domains. Purified PspXyn10 was prepared from the supernatant of a recombinant T. reesei strain. The molecular weight of PspXyn10 was estimated to be 55 kDa, and its optimal temperature and pH for xylanase activity were 75 °C and pH 4.5, respectively. More than 80% of the xylanase activity was maintained at 65 °C for 10 min. With beechwood xylan as the substrate, the enzyme had a K m of 2.2 mg/mL and a V max of 332 μmol/min/mg. PspXyn10ΔCBM, which lacked the CBM1 domain, was prepared by limited proteolysis. PspXyn10ΔCBM showed increased activity against soluble xylan, but decreased saccharification efficiency of alkaline-pretreated bagasse. This result indicated that the CBM1 domain of PspXyn10 contributes to the enhancement of the saccharification efficiency of alkaline-pretreated bagasse. A recombinant T. reesei strain, named X2PX10, was constructed from strain X3AB1. X3AB1 is an Aspergillus aculeatus β-glucosidase-expressing T. reesei PC-3-7. X2PX10 also expressed PspXyn10 under the control of the xyn2 promoter. An enzyme preparation from X2PX10 showed almost the same saccharification efficiency of alkaline-pretreated bagasse at half the enzyme dosage as that used for an enzyme preparation from X3AB1. Our results suggest that PspXyn10 promotes the saccharification of alkaline-pretreated bagasse more efficiently than TrXyn3, a GH10 family xylanase from T. reesei , and that the PspXyn10-expressing strain is suitable for enzyme production for biomass saccharification.

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.

Mining of Novel Thermo-Stable Cellulolytic Genes from a Thermophilic Cellulose-Degrading Consortium by Metagenomics

PubMed Central

Xia, Yu; Ju, Feng; Fang, Herbert H. P.; Zhang, Tong

2013-01-01

In this study, metagenomics was applied to characterize the microbial community and to discover carbohydrate-active genes of an enriched thermophilic cellulose-degrading sludge. The 16S analysis showed that the sludge microbiome was dominated by genus of cellulolytic Clostridium and methanogenesis Methanothermobacter. In order to retrieve genes from the metagenome, de novo assembly of the 11,930,760 Illumina 100 bp paired-end reads (totally 1.2 Gb) was carried out. 75% of all reads was utilized in the de novo assembly. 31,499 ORFs (Open Reading Frame) with an average length of 852 bp were predicted from the assembly; and 64% of these ORFs were predicted to present full-length genes. Based on the Hidden Markol Model, 253 of the predicted thermo-stable genes were identified as putatively carbohydrate-active. Among them the relative dominance of GH9 (Glycoside Hydrolase) and corresponding CBM3 (Carbohydrate Binding Module) revealed a cellulosome-based attached metabolism of polysaccharide in the thermophilic sludge. The putative carbohydrate-active genes ranged from 20% to 100% amino acid sequence identity to known proteins in NCBI nr database, with half of them showed less than 50% similarity. In addition, the coverage of the genes (in terms of ORFs) identified in the sludge were developed into three clear trends (112×, 29× and 8×) in which 85% of the high coverage trend (112×) mainly consisted of phylum of Firmicutes while 49.3% of the 29× trend was affiliated to the phylum of Chloroflexi. PMID:23341999

Cerebral CBM1 neuron contributes to synaptic modulation appearing during rejection of seaweed in Aplysia kurodai.

PubMed

Narusuye, Kenji; Nagahama, Tatsumi

2002-11-01

The Japanese species Aplysia kurodai feeds well on Ulva but rejects Gelidium with distinctive rhythmic patterned movements of the jaws and radula. We have previously shown that the patterned jaw movements during the rejection of Gelidium might be caused by long-lasting suppression of the monosynaptic transmission from the multiaction MA neurons to the jaw-closing (JC) motor neurons in the buccal ganglia and that the modulation might be directly produced by some cerebral neurons. In the present paper, we have identified a pair of catecholaminergic neurons (CBM1) in bilateral cerebral M clusters. The CBM1, probably equivalent to CBI-1 in A. californica, simultaneously produced monosynaptic excitatory postsynaptic potentials (EPSPs) in the MA and JC neurons. Firing of the CBM1 reduced the size of the inhibitory postsynaptic currents (IPSCs) in the JC neuron, evoked by the MA spikes, for >100 s. Moreover, the application of dopamine mimicked the CBM1 modulatory effects and pretreatment with a D1 antagonist, SCH23390, blocked the modulatory effects induced by dopamine. It could also largely block the modulatory effects induced by the CBM1 firing. These results suggest that the CBM1 may directly modulate the synaptic transmission by releasing dopamine. Moreover, we explored the CBM1 spike activity induced by taste stimulation of the animal lips with seaweed extracts by the use of calcium imaging. The calcium-sensitive dye, Calcium Green-1, was iontophoretically loaded into a cell body of the CBM1 using a microelectrode. Application of either Ulva or Gelidium extract to the lips increased the fluorescence intensity, but the Gelidium extract always induced a larger change in fluorescence compared with the Ulva extract, although the solution used induced the maximum spike responses of the CBM1 for each of the seaweed extracts. When the firing frequency of the CBM1 activity after taste stimulation was estimated, the Gelidium extract induced a spike activity of ~30 spikes/s while the Ulva extract induced an activity of ~20 spikes/s, consistent with the effective firing frequency (>25 spikes/s) for the synaptic modulation. These results suggest that the CBM1 may be one of the cerebral neurons contributing to the modulation of the basic feeding circuits for rejection induced by the taste of seaweeds such as Gelidium.

Determination of optimal biomass pretreatment strategies for biofuel production: investigation of relationships between surface-exposed polysaccharides and their enzymatic conversion using carbohydrate-binding modules.

PubMed

Khatri, Vinay; Meddeb-Mouelhi, Fatma; Adjallé, Kokou; Barnabé, Simon; Beauregard, Marc

2018-01-01

Pretreatment of lignocellulosic biomass (LCB) is a key step for its efficient bioconversion into ethanol. Determining the best pretreatment and its parameters requires monitoring its impacts on the biomass material. Here, we used fluorescent protein-tagged carbohydrate-binding modules method (FTCM)-depletion assay to study the relationship between surface-exposed polysaccharides and enzymatic hydrolysis of LCB. Our results indicated that alkali extrusion pretreatment led to the highest hydrolysis rates for alfalfa stover, cattail stems and flax shives, despite its lower lignin removal efficiency compared to alkali pretreatment. Corn crop residues were more sensitive to alkali pretreatments, leading to higher hydrolysis rates. A clear relationship was consistently observed between total surface-exposed cellulose detected by the FTCM-depletion assay and biomass enzymatic hydrolysis. Comparison of bioconversion yield and total composition analysis (by NREL/TP-510-42618) of LCB prior to or after pretreatments did not show any close relationship. Lignin removal efficiency and total cellulose content (by NREL/TP-510-42618) led to an unreliable prediction of enzymatic polysaccharide hydrolysis. Fluorescent protein-tagged carbohydrate-binding modules method (FTCM)-depletion assay provided direct evidence that cellulose exposure is the key determinant of hydrolysis yield. The clear and robust relationships that were observed between the cellulose accessibility by FTCM probes and enzymatic hydrolysis rates change could be evolved into a powerful prediction tool that might help develop optimal biomass pretreatment strategies for biofuel production.

Solution conformation of a cohesin module and its scaffoldin linker from a prototypical cellulosome.

PubMed

Galera-Prat, Albert; Pantoja-Uceda, David; Laurents, Douglas V; Carrión-Vázquez, Mariano

2018-04-15

Bacterial cellulases are drawing increased attention as a means to obtain plentiful chemical feedstocks and fuels from renewable lignocellulosic biomass sources. Certain bacteria deploy a large extracellular multi-protein complex, called the cellulosome, to degrade cellulose. Scaffoldin, a key non-catalytic cellulosome component, is a large protein containing a cellulose-specific carbohydrate-binding module and several cohesin modules which bind and organize the hydrolytic enzymes. Despite the importance of the structure and protein/protein interactions of the cohesin module in the cellulosome, its structure in solution has remained unknown to date. Here, we report the backbone 1 H, 13 C and 15 N NMR assignments of the Cohesin module 5 from the highly stable and active cellulosome from Clostridium thermocellum. These data reveal that this module adopts a tightly packed, well folded and rigid structure in solution. Furthermore, since in scaffoldin, the cohesin modules are connected by linkers we have also characterized the conformation of a representative linker segment using NMR spectroscopy. Analysis of its chemical shift values revealed that this linker is rather stiff and tends to adopt extended conformations. This suggests that the scaffoldin linkers act to minimize interactions between cohesin modules. These results pave the way towards solution studies on cohesin/dockerin's fascinating dual-binding mode. Copyright © 2018 Elsevier Inc. All rights reserved.

Fusion of Dioxygenase and Lignin-binding Domains in a Novel Secreted Enzyme from Cellulolytic Streptomyces sp. SirexAA-E*

PubMed Central

Bianchetti, Christopher M.; Harmann, Connor H.; Takasuka, Taichi E.; Hura, Gregory L.; Dyer, Kevin; Fox, Brian G.

2013-01-01

Streptomyces sp. SirexAA-E is a highly cellulolytic bacterium isolated from an insect/microbe symbiotic community. When grown on lignin-containing biomass, it secretes SACTE_2871, an aromatic ring dioxygenase domain fused to a family 5/12 carbohydrate-binding module (CBM 5/12). Here we present structural and catalytic studies of this novel fusion enzyme, thus providing insight into its function. The dioxygenase domain has the core β-sandwich fold typical of this enzyme family but lacks a dimerization domain observed in other intradiol dioxygenases. Consequently, the x-ray structure shows that the enzyme is monomeric and the Fe(III)-containing active site is exposed to solvent in a shallow depression on a planar surface. Purified SACTE_2871 catalyzes the O2-dependent intradiol cleavage of catechyl compounds from lignin biosynthetic pathways, but not their methylated derivatives. Binding studies show that SACTE_2871 binds synthetic lignin polymers and chitin through the interactions of the CBM 5/12 domain, representing a new binding specificity for this fold-family. Based on its unique structural features and functional properties, we propose that SACTE_2871 contributes to the invasive nature of the insect/microbial community by destroying precursors needed by the plant for de novo lignin biosynthesis as part of its natural wounding response. PMID:23653358

Discrete and structurally unique proteins (tāpirins) mediate attachment of extremely thermophilic Caldicellulosiruptor species to cellulose.

PubMed

Blumer-Schuette, Sara E; Alahuhta, Markus; Conway, Jonathan M; Lee, Laura L; Zurawski, Jeffrey V; Giannone, Richard J; Hettich, Robert L; Lunin, Vladimir V; Himmel, Michael E; Kelly, Robert M

2015-04-24

A variety of catalytic and noncatalytic protein domains are deployed by select microorganisms to deconstruct lignocellulose. These extracellular proteins are used to attach to, modify, and hydrolyze the complex polysaccharides present in plant cell walls. Cellulolytic enzymes, often containing carbohydrate-binding modules, are key to this process; however, these enzymes are not solely responsible for attachment. Few mechanisms of attachment have been discovered among bacteria that do not form large polypeptide structures, called cellulosomes, to deconstruct biomass. In this study, bioinformatics and proteomics analyses identified unique, discrete, hypothetical proteins ("tāpirins," origin from Māori: to join), not directly associated with cellulases, that mediate attachment to cellulose by species in the noncellulosomal, extremely thermophilic bacterial genus Caldicellulosiruptor. Two tāpirin genes are located directly downstream of a type IV pilus operon in strongly cellulolytic members of the genus, whereas homologs are absent from the weakly cellulolytic Caldicellulosiruptor species. Based on their amino acid sequence, tāpirins are specific to these extreme thermophiles. Tāpirins are also unusual in that they share no detectable protein domain signatures with known polysaccharide-binding proteins. Adsorption isotherm and trans vivo analyses demonstrated the carbohydrate-binding module-like affinity of the tāpirins for cellulose. Crystallization of a cellulose-binding truncation from one tāpirin indicated that these proteins form a long β-helix core with a shielded hydrophobic face. Furthermore, they are structurally unique and define a new class of polysaccharide adhesins. Strongly cellulolytic Caldicellulosiruptor species employ tāpirins to complement substrate-binding proteins from the ATP-binding cassette transporters and multidomain extracellular and S-layer-associated glycoside hydrolases to process the carbohydrate content of lignocellulose. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Discrete and Structurally Unique Proteins (Tāpirins) Mediate Attachment of Extremely Thermophilic Caldicellulosiruptor Species to Cellulose*

PubMed Central

Blumer-Schuette, Sara E.; Alahuhta, Markus; Conway, Jonathan M.; Lee, Laura L.; Zurawski, Jeffrey V.; Giannone, Richard J.; Hettich, Robert L.; Lunin, Vladimir V.; Himmel, Michael E.; Kelly, Robert M.

2015-01-01

A variety of catalytic and noncatalytic protein domains are deployed by select microorganisms to deconstruct lignocellulose. These extracellular proteins are used to attach to, modify, and hydrolyze the complex polysaccharides present in plant cell walls. Cellulolytic enzymes, often containing carbohydrate-binding modules, are key to this process; however, these enzymes are not solely responsible for attachment. Few mechanisms of attachment have been discovered among bacteria that do not form large polypeptide structures, called cellulosomes, to deconstruct biomass. In this study, bioinformatics and proteomics analyses identified unique, discrete, hypothetical proteins (“tāpirins,” origin from Māori: to join), not directly associated with cellulases, that mediate attachment to cellulose by species in the noncellulosomal, extremely thermophilic bacterial genus Caldicellulosiruptor. Two tāpirin genes are located directly downstream of a type IV pilus operon in strongly cellulolytic members of the genus, whereas homologs are absent from the weakly cellulolytic Caldicellulosiruptor species. Based on their amino acid sequence, tāpirins are specific to these extreme thermophiles. Tāpirins are also unusual in that they share no detectable protein domain signatures with known polysaccharide-binding proteins. Adsorption isotherm and trans vivo analyses demonstrated the carbohydrate-binding module-like affinity of the tāpirins for cellulose. Crystallization of a cellulose-binding truncation from one tāpirin indicated that these proteins form a long β-helix core with a shielded hydrophobic face. Furthermore, they are structurally unique and define a new class of polysaccharide adhesins. Strongly cellulolytic Caldicellulosiruptor species employ tāpirins to complement substrate-binding proteins from the ATP-binding cassette transporters and multidomain extracellular and S-layer-associated glycoside hydrolases to process the carbohydrate content of lignocellulose. PMID:25720489

Enhanced xyloglucan-specific endo-β-1,4-glucanase efficiency in an engineered CBM44-XegA chimera.

PubMed

Furtado, Gilvan P; Santos, Camila R; Cordeiro, Rosa L; Ribeiro, Lucas F; de Moraes, Luiz A B; Damásio, André R L; Polizeli, Maria de Lourdes T M; Lourenzoni, Marcos R; Murakami, Mário T; Ward, Richard J

2015-06-01

Xyloglucan-specific endo-β-1,4-glucanases (Xegs, EC 3.2.1.151) exhibit high catalytic specificity for β-1,4 linkages of xyloglucan, a branched hemicellulosic polysaccharide abundant in dicot primary cell walls and present in many monocot species. In nature, GH12 Xegs are not associated with carbohydrate-binding modules (CBMs), and here, we have investigated the effect of the fusion of the xyloglucan-specific CBM44 on the structure and function of a GH12 Xeg from Aspergillus niveus (XegA). This fusion presented enhanced catalytic properties and conferred superior thermal stability on the XegA. An increased k cat (chimera, 177.03 s(-1); XegA, 144.31 s(-1)) and reduced KM (chimera, 1.30 mg mL(-1); XegA, 1.50 mg mL(-1)) resulted in a 1.3-fold increase in catalytic efficiency of the chimera over the parental XegA. Although both parental and chimeric enzymes presented catalytic optima at pH 5.5 and 60 °C, the thermostabilitiy of the chimera at 60 °C was greater than the parental XegA. Moreover, the crystallographic structure of XegA together with small-angle X-ray scattering (SAXS) and molecular dynamics simulations revealed that the spatial arrangement of the domains in the chimeric enzyme resulted in the formation of an extended binding cleft that may explain the improved kinetic properties of the CBM44-XegA chimera.

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.

SACCHARIS: an automated pipeline to streamline discovery of carbohydrate active enzyme activities within polyspecific families and de novo sequence datasets.

PubMed

Jones, Darryl R; Thomas, Dallas; Alger, Nicholas; Ghavidel, Ata; Inglis, G Douglas; Abbott, D Wade

2018-01-01

Deposition of new genetic sequences in online databases is expanding at an unprecedented rate. As a result, sequence identification continues to outpace functional characterization of carbohydrate active enzymes (CAZymes). In this paradigm, the discovery of enzymes with novel functions is often hindered by high volumes of uncharacterized sequences particularly when the enzyme sequence belongs to a family that exhibits diverse functional specificities (i.e., polyspecificity). Therefore, to direct sequence-based discovery and characterization of new enzyme activities we have developed an automated in silico pipeline entitled: Sequence Analysis and Clustering of CarboHydrate Active enzymes for Rapid Informed prediction of Specificity (SACCHARIS). This pipeline streamlines the selection of uncharacterized sequences for discovery of new CAZyme or CBM specificity from families currently maintained on the CAZy website or within user-defined datasets. SACCHARIS was used to generate a phylogenetic tree of a GH43, a CAZyme family with defined subfamily designations. This analysis confirmed that large datasets can be organized into sequence clusters of manageable sizes that possess related functions. Seeding this tree with a GH43 sequence from Bacteroides dorei DSM 17855 (BdGH43b, revealed it partitioned as a single sequence within the tree. This pattern was consistent with it possessing a unique enzyme activity for GH43 as BdGH43b is the first described α-glucanase described for this family. The capacity of SACCHARIS to extract and cluster characterized carbohydrate binding module sequences was demonstrated using family 6 CBMs (i.e., CBM6s). This CBM family displays a polyspecific ligand binding profile and contains many structurally determined members. Using SACCHARIS to identify a cluster of divergent sequences, a CBM6 sequence from a unique clade was demonstrated to bind yeast mannan, which represents the first description of an α-mannan binding CBM. Additionally, we have performed a CAZome analysis of an in-house sequenced bacterial genome and a comparative analysis of B. thetaiotaomicron VPI-5482 and B. thetaiotaomicron 7330, to demonstrate that SACCHARIS can generate "CAZome fingerprints", which differentiate between the saccharolytic potential of two related strains in silico. Establishing sequence-function and sequence-structure relationships in polyspecific CAZyme families are promising approaches for streamlining enzyme discovery. SACCHARIS facilitates this process by embedding CAZyme and CBM family trees generated from biochemically to structurally characterized sequences, with protein sequences that have unknown functions. In addition, these trees can be integrated with user-defined datasets (e.g., genomics, metagenomics, and transcriptomics) to inform experimental characterization of new CAZymes or CBMs not currently curated, and for researchers to compare differential sequence patterns between entire CAZomes. In this light, SACCHARIS provides an in silico tool that can be tailored for enzyme bioprospecting in datasets of increasing complexity and for diverse applications in glycobiotechnology.

A thermophilic alkalophilic α-amylase from Bacillus sp. AAH-31 shows a novel domain organization among glycoside hydrolase family 13 enzymes.

PubMed

Saburi, Wataru; Morimoto, Naoki; Mukai, Atsushi; Kim, Dae Hoon; Takehana, Toshihiko; Koike, Seiji; Matsui, Hirokazu; Mori, Haruhide

2013-01-01

α-Amylases (EC 3.2.1.1) hydrolyze internal α-1,4-glucosidic linkages of starch and related glucans. Bacillus sp. AAH-31 produces an alkalophilic thermophilic α-amylase (AmyL) of higher molecular mass, 91 kDa, than typical bacterial α-amylases. In this study, the AmyL gene was cloned to determine its primary structure, and the recombinant enzyme, produced in Escherichia coli, was characterized. AmyL shows no hydrolytic activity towards pullulan, but the central region of AmyL (Gly395-Asp684) was similar to neopullulanase-like α-amylases. In contrast to known neopullulanase-like α-amylases, the N-terminal region (Gln29-Phe102) of AmyL was similar to carbohydrate-binding module family 20 (CBM20), which is involved in the binding of enzymes to starch granules. Recombinant AmyL showed more than 95% of its maximum activity in a pH range of 8.2-10.5, and was stable below 65 °C and from pH 6.4 to 11.9. The kcat values for soluble starch, γ-cyclodextrin, and maltotriose were 103 s(-1), 67.6 s(-1), and 5.33 s(-1), respectively, and the Km values were 0.100 mg/mL, 0.348 mM, and 2.06 mM, respectively. Recombinant AmyL did not bind to starch granules. But the substitution of Trp45 and Trp84, conserved in site 1 of CBM20, with Ala reduced affinity to soluble starch, while the mutations did not affect affinity for oligosaccharides. Substitution of Trp61, conserved in site 2 of CBM20, with Ala enhanced hydrolytic activity towards soluble starch, indicating that site 2 of AmyL does not contribute to binding to soluble long-chain substrates.

Computational studies of the binding profile of phosphoinositide PtdIns (3,4,5) P3 with the pleckstrin homology domain of an oomycete cellulose synthase

PubMed Central

Kuang, Guanglin; Bulone, Vincent; Tu, Yaoquan

2016-01-01

Saprolegnia monoica is a model organism to investigate Saprolegnia parasitica, an important oomycete which causes considerable loss in aquaculture every year. S. monoica contains cellulose synthases vital for oomycete growth. However, the molecular mechanism of the cellulose biosynthesis process in the oomycete growth is still poorly understood. Some cellulose synthases of S. monoica, such as SmCesA2, are found to contain a plecsktrin homology (PH) domain, which is a protein module widely found in nature and known to bind to phosphoinositides, a class of signaling compounds involved in many biological processes. Understanding the molecular interactions between the PH domain and phosphoinositides would help to unravel the cellulose biosynthesis process of oomycetes. In this work, the binding profile of PtdIns (3,4,5) P3, a typical phosphoinositide, with SmCesA2-PH was studied by molecular docking, molecular dynamics and metadynamics simulations. PtdIns (3,4,5) P3 is found to bind at a specific site located at β1, β2 and β1-β2 loop of SmCesA2-PH. The high affinity of PtdIns (3,4,5) P3 to SmCesA2-PH is contributed by the free phosphate groups, which have electrostatic and hydrogen-bond interactions with Lys88, Lys100 and Arg102 in the binding site. PMID:26857031

Discrete and Structurally Unique Proteins (T$$\\bar{a}$$pirins) Mediate Attachment of Extremely Thermophilic Caldicellulosiruptor Species to Cellulose

DOE PAGES

Blumer-Schuette, S. E.; Alahuhta, M.; Conway, J. M.; ...

2015-04-24

A variety of catalytic and noncatalytic protein domains are deployed by select microorganisms to deconstruct lignocellulose. These extracellular proteins are used to attach to, modify, and hydrolyze the complex polysaccharides present in plant cell walls. Cellulolytic enzymes, often containing carbohydrate-binding modules, are key to this process; however, these enzymes are not solely responsible for attachment. Few mechanisms of attachment have been discovered among bacteria that do not form large polypeptide structures, called cellulosomes, to deconstruct biomass. In this study, bioinformatics and proteomics analyses identified unique, discrete, hypothetical proteins (“tmore » $$\\bar{a}$$pirins,” origin from M$$\\bar{a}$$ori: to join), not directly associated with cellulases, that mediate attachment to cellulose by species in the noncellulosomal, extremely thermophilic bacterial genus Caldicellulosiruptor. Two t$$\\bar{a}$$pirin genes are located directly downstream of a type IV pilus operon in strongly cellulolytic members of the genus, whereas homologs are absent from the weakly cellulolytic Caldicellulosiruptor species. Based on their amino acid sequence, t$$\\bar{a}$$pirins are specific to these extreme thermophiles. T$$\\bar{a}$$pirins are also unusual in that they share no detectable protein domain signatures with known polysaccharide-binding proteins. Adsorption isotherm and trans vivo analyses demonstrated the carbohydrate-binding module-like affinity of the t$$\\bar{a}$$pirins for cellulose. Crystallization of a cellulose-binding truncation from one t$$\\bar{a}$$pirin indicated that these proteins form a long β-helix core with a shielded hydrophobic face. In addition, they are structurally unique and define a new class of polysaccharide adhesins. Strongly cellulolytic Caldicellulosiruptor species employ t$$\\bar{a}$$pirins to complement substrate-binding proteins from the ATP-binding cassette transporters and multidomain extracellular and S-layer-associated glycoside hydrolases to process the carbohydrate content of lignocellulose.« less

Discrete and Structurally Unique Proteins (T$$\\bar{a}$$pirins) Mediate Attachment of Extremely Thermophilic Caldicellulosiruptor Species to Cellulose

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

Blumer-Schuette, S. E.; Alahuhta, M.; Conway, J. M.

A variety of catalytic and noncatalytic protein domains are deployed by select microorganisms to deconstruct lignocellulose. These extracellular proteins are used to attach to, modify, and hydrolyze the complex polysaccharides present in plant cell walls. Cellulolytic enzymes, often containing carbohydrate-binding modules, are key to this process; however, these enzymes are not solely responsible for attachment. Few mechanisms of attachment have been discovered among bacteria that do not form large polypeptide structures, called cellulosomes, to deconstruct biomass. In this study, bioinformatics and proteomics analyses identified unique, discrete, hypothetical proteins (“tmore » $$\\bar{a}$$pirins,” origin from M$$\\bar{a}$$ori: to join), not directly associated with cellulases, that mediate attachment to cellulose by species in the noncellulosomal, extremely thermophilic bacterial genus Caldicellulosiruptor. Two t$$\\bar{a}$$pirin genes are located directly downstream of a type IV pilus operon in strongly cellulolytic members of the genus, whereas homologs are absent from the weakly cellulolytic Caldicellulosiruptor species. Based on their amino acid sequence, t$$\\bar{a}$$pirins are specific to these extreme thermophiles. T$$\\bar{a}$$pirins are also unusual in that they share no detectable protein domain signatures with known polysaccharide-binding proteins. Adsorption isotherm and trans vivo analyses demonstrated the carbohydrate-binding module-like affinity of the t$$\\bar{a}$$pirins for cellulose. Crystallization of a cellulose-binding truncation from one t$$\\bar{a}$$pirin indicated that these proteins form a long β-helix core with a shielded hydrophobic face. In addition, they are structurally unique and define a new class of polysaccharide adhesins. Strongly cellulolytic Caldicellulosiruptor species employ t$$\\bar{a}$$pirins to complement substrate-binding proteins from the ATP-binding cassette transporters and multidomain extracellular and S-layer-associated glycoside hydrolases to process the carbohydrate content of lignocellulose.« less

Laccase/HBT and laccase-CBM/HBT treatment of softwood kraft pulp: impact on pulp bleachability and physical properties.

PubMed

Ravalason, Holy; Bertaud, Frédérique; Herpoël-Gimbert, Isabelle; Meyer, Valérie; Ruel, Katia; Joseleau, Jean-Paul; Grisel, Sacha; Olivé, Caroline; Sigoillot, Jean-Claude; Petit-Conil, Michel

2012-10-01

Pycnoporus cinnabarinus laccase and a chimeric laccase-CBM were applied in softwood kraft pulp biobleaching in the presence of 1-hydroxybenzotriazole (HBT). The presence of CBM could enhance the laccase biobleaching potential as a decrease in the enzymatic charge and chlorine dioxide consumption, as well as an increase in pulp brightness were observed. Laccase/HBT treatment could be improved by increasing oxygen pressure from 1 to 3bar and pulp consistency from 5% to 10%. Conversely, under the same conditions, no improvement of laccase-CBM/HBT treatment was observed, indicating a different behavior of both systems. However, laccase-CBM/HBT treatment led to a better preservation of pulp properties. This effect was probably due to fiber surface modifications involving the action of the CBM. Transmission electron microscopy examination of pulp fibers indicated a retention of laccase-CBM inside the pulp fibers due to CBM binding and an increased external microfibrillation of the fibers due to enzymatic treatments. Copyright © 2012 Elsevier Ltd. All rights reserved.

Cellulose and hemicellulose decomposition by forest soil bacteria proceeds by the action of structurally variable enzymatic systems

PubMed Central

López-Mondéjar, Rubén; Zühlke, Daniela; Becher, Dörte; Riedel, Katharina; Baldrian, Petr

2016-01-01

Evidence shows that bacteria contribute actively to the decomposition of cellulose and hemicellulose in forest soil; however, their role in this process is still unclear. Here we performed the screening and identification of bacteria showing potential cellulolytic activity from litter and organic soil of a temperate oak forest. The genomes of three cellulolytic isolates previously described as abundant in this ecosystem were sequenced and their proteomes were characterized during the growth on plant biomass and on microcrystalline cellulose. Pedobacter and Mucilaginibacter showed complex enzymatic systems containing highly diverse carbohydrate-active enzymes for the degradation of cellulose and hemicellulose, which were functionally redundant for endoglucanases, β-glucosidases, endoxylanases, β-xylosidases, mannosidases and carbohydrate-binding modules. Luteibacter did not express any glycosyl hydrolases traditionally recognized as cellulases. Instead, cellulose decomposition was likely performed by an expressed GH23 family protein containing a cellulose-binding domain. Interestingly, the presence of plant lignocellulose as well as crystalline cellulose both trigger the production of a wide set of hydrolytic proteins including cellulases, hemicellulases and other glycosyl hydrolases. Our findings highlight the extensive and unexplored structural diversity of enzymatic systems in cellulolytic soil bacteria and indicate the roles of multiple abundant bacterial taxa in the decomposition of cellulose and other plant polysaccharides. PMID:27125755

Human Chitotriosidase Is an Endo-Processive Enzyme

PubMed Central

Sørlie, Morten; Väljamäe, Priit

2017-01-01

Human chitotriosidase (HCHT) is involved in immune response to chitin-containing pathogens in humans. The enzyme is able to degrade chitooligosaccharides as well as crystalline chitin. The catalytic domain of HCHT is connected to the carbohydrate binding module (CBM) through a flexible hinge region. In humans, two active isoforms of HCHT are found–the full length enzyme and its truncated version lacking CBM and the hinge region. The active site architecture of HCHT is reminiscent to that of the reducing-end exo-acting processive chitinase ChiA from bacterium Serratia marcescens (SmChiA). However, the presence of flexible hinge region and occurrence of two active isoforms are reminiscent to that of non-processive endo-chitinase from S. marcescens, SmChiC. Although the studies on soluble chitin derivatives suggest the endo-character of HCHT, the mode of action of the enzyme on crystalline chitin is not known. Here, we made a thorough characterization of HCHT in terms of the mode of action, processivity, binding, and rate constants for the catalysis and dissociation using α-chitin as substrate. HCHT efficiently released the end-label from reducing-end labelled chitin and had also high probability (95%) of endo-mode initiation of processive run. These results qualify HCHT as an endo-processive enzyme. Processivity and the rate constant of dissociation of HCHT were found to be in-between those, characteristic to processive exo-enzymes, like SmChiA and randomly acting non-processive endo-enzymes, like SmChiC. Apart from increasing the affinity for chitin, CBM had no major effect on kinetic properties of HCHT. PMID:28129403

Contribution of the Interaction of Streptococcus mutans Serotype k Strains with Fibrinogen to the Pathogenicity of Infective Endocarditis

PubMed Central

Nomura, Ryota; Otsugu, Masatoshi; Naka, Shuhei; Teramoto, Noboru; Kojima, Ayuchi; Muranaka, Yoshinori; Matsumoto-Nakano, Michiyo; Ooshima, Takashi

2014-01-01

Streptococcus mutans, a pathogen responsible for dental caries, is occasionally isolated from the blood of patients with bacteremia and infective endocarditis (IE). Our previous study demonstrated that serotype k-specific bacterial DNA is frequently detected in S. mutans-positive heart valve specimens extirpated from IE patients. However, the reason for this frequent detection remains unknown. In the present study, we analyzed the virulence of IE from S. mutans strains, focusing on the characterization of serotype k strains, most of which are positive for the 120-kDa cell surface collagen-binding protein Cbm and negative for the 190-kDa protein antigen (PA) known as SpaP, P1, antigen I/II, and other designations. Fibrinogen-binding assays were performed with 85 clinical strains classified by Cbm and PA expression levels. The Cbm+/PA− group strains had significantly higher fibrinogen-binding rates than the other groups. Analysis of platelet aggregation revealed that SA31, a Cbm+/PA− strain, induced an increased level of aggregation in the presence of fibrinogen, while negligible aggregation was induced by the Cbm-defective isogenic mutant SA31CBD. A rat IE model with an artificial impairment of the aortic valve created using a catheter showed that extirpated heart valves in the SA31 group displayed a prominent vegetation mass not seen in those in the SA31CBD group. These findings could explain why Cbm+/PA− strains are highly virulent and are related to the development of IE, and the findings could also explain the frequent detection of serotype k DNA in S. mutans-positive heart valve clinical specimens. PMID:25287921

Contribution of the interaction of Streptococcus mutans serotype k strains with fibrinogen to the pathogenicity of infective endocarditis.

PubMed

Nomura, Ryota; Otsugu, Masatoshi; Naka, Shuhei; Teramoto, Noboru; Kojima, Ayuchi; Muranaka, Yoshinori; Matsumoto-Nakano, Michiyo; Ooshima, Takashi; Nakano, Kazuhiko

2014-12-01

Streptococcus mutans, a pathogen responsible for dental caries, is occasionally isolated from the blood of patients with bacteremia and infective endocarditis (IE). Our previous study demonstrated that serotype k-specific bacterial DNA is frequently detected in S. mutans-positive heart valve specimens extirpated from IE patients. However, the reason for this frequent detection remains unknown. In the present study, we analyzed the virulence of IE from S. mutans strains, focusing on the characterization of serotype k strains, most of which are positive for the 120-kDa cell surface collagen-binding protein Cbm and negative for the 190-kDa protein antigen (PA) known as SpaP, P1, antigen I/II, and other designations. Fibrinogen-binding assays were performed with 85 clinical strains classified by Cbm and PA expression levels. The Cbm(+)/PA(-) group strains had significantly higher fibrinogen-binding rates than the other groups. Analysis of platelet aggregation revealed that SA31, a Cbm(+)/PA(-) strain, induced an increased level of aggregation in the presence of fibrinogen, while negligible aggregation was induced by the Cbm-defective isogenic mutant SA31CBD. A rat IE model with an artificial impairment of the aortic valve created using a catheter showed that extirpated heart valves in the SA31 group displayed a prominent vegetation mass not seen in those in the SA31CBD group. These findings could explain why Cbm(+)/PA(-) strains are highly virulent and are related to the development of IE, and the findings could also explain the frequent detection of serotype k DNA in S. mutans-positive heart valve clinical specimens. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Atomic Oxygen Effects on Seal Leakage

NASA Technical Reports Server (NTRS)

Christensen, John R.; Underwood, Steve D.; Kamenetzky, Rachel R.; Vaughn, Jason A.

1999-01-01

Common Berthing Mechanism (CBM provides the structural interface between separate International Space Station (ISS) elements, such as the Laboratory and Node modules. The CBM consists of an active and a passive half that join together with structural bolts. The seal at this interface is the CBM-to-CBM molded seal. The CBM-to-CBM interface is assembled on orbit, thus the seals can be exposed to the space environment for up to 65 hours. Atomic Oxygen/Vacuum Ultraviolet radiation (AO/VUV) in space is a potential hazard to the seals. Testing was conducted to determine the effect on leakage of the CBM-to-CBM seal material exposed to AO/VUV. The sealing materials were S383 silicone and V835 fluorocarbon material. Control samples, which were not exposed to the AO/VUV environment, were used to ensure that ff any changes in leakage occurred, they could be attributed to the AO/VUV exposure. After exposure to the AO/VUV environment the leakage increase was dramatic for the fluorocarbon. This testing was a major contributing factor in selecting silicone as the CBM-to-CBM seal material.

Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes.

PubMed

Cockburn, Darrell; Wilkens, Casper; Dilokpimol, Adiphol; Nakai, Hiroyuki; Lewińska, Anna; Abou Hachem, Maher; Svensson, Birte

2016-01-01

Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites are identified, including the first SBS ever reported in a cellulase. This work demonstrates that combinations of these methods can be used as a part of routine enzyme characterization to identify new binding sites and advance the study of SBSs and CBMs, allowing them to be detected in the absence of structural data.

Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes

PubMed Central

Wilkens, Casper; Dilokpimol, Adiphol; Nakai, Hiroyuki; Lewińska, Anna; Abou Hachem, Maher; Svensson, Birte

2016-01-01

Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites are identified, including the first SBS ever reported in a cellulase. This work demonstrates that combinations of these methods can be used as a part of routine enzyme characterization to identify new binding sites and advance the study of SBSs and CBMs, allowing them to be detected in the absence of structural data. PMID:27504624

Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on Lactococcus lactis

PubMed Central

2010-01-01

Background The assembly and spatial organization of enzymes in naturally occurring multi-protein complexes is of paramount importance for the efficient degradation of complex polymers and biosynthesis of valuable products. The degradation of cellulose into fermentable sugars by Clostridium thermocellum is achieved by means of a multi-protein "cellulosome" complex. Assembled via dockerin-cohesin interactions, the cellulosome is associated with the cell surface during cellulose hydrolysis, forming ternary cellulose-enzyme-microbe complexes for enhanced activity and synergy. The assembly of recombinant cell surface displayed cellulosome-inspired complexes in surrogate microbes is highly desirable. The model organism Lactococcus lactis is of particular interest as it has been metabolically engineered to produce a variety of commodity chemicals including lactic acid and bioactive compounds, and can efficiently secrete an array of recombinant proteins and enzymes of varying sizes. Results Fragments of the scaffoldin protein CipA were functionally displayed on the cell surface of Lactococcus lactis. Scaffolds were engineered to contain a single cohesin module, two cohesin modules, one cohesin and a cellulose-binding module, or only a cellulose-binding module. Cell toxicity from over-expression of the proteins was circumvented by use of the nisA inducible promoter, and incorporation of the C-terminal anchor motif of the streptococcal M6 protein resulted in the successful surface-display of the scaffolds. The facilitated detection of successfully secreted scaffolds was achieved by fusion with the export-specific reporter staphylococcal nuclease (NucA). Scaffolds retained their ability to associate in vivo with an engineered hybrid reporter enzyme, E. coli β-glucuronidase fused to the type 1 dockerin motif of the cellulosomal enzyme CelS. Surface-anchored complexes exhibited dual enzyme activities (nuclease and β-glucuronidase), and were displayed with efficiencies approaching 104 complexes/cell. Conclusions We report the successful display of cellulosome-inspired recombinant complexes on the surface of Lactococcus lactis. Significant differences in display efficiency among constructs were observed and attributed to their structural characteristics including protein conformation and solubility, scaffold size, and the inclusion and exclusion of non-cohesin modules. The surface-display of functional scaffold proteins described here represents a key step in the development of recombinant microorganisms capable of carrying out a variety of metabolic processes including the direct conversion of cellulosic substrates into fuels and chemicals. PMID:20840763

Novel method for detection of glycogen in cells

PubMed Central

Segvich, Dyann M; DePaoli-Roach, Anna A; Roach, Peter J

2017-01-01

Abstract Glycogen, a branched polymer of glucose, functions as an energy reserve in many living organisms. Abnormalities in glycogen metabolism, usually excessive accumulation, can be caused genetically, most often through mutation of the enzymes directly involved in synthesis and degradation of the polymer leading to a variety of glycogen storage diseases (GSDs). Microscopic visualization of glycogen deposits in cells and tissues is important for the study of normal glycogen metabolism as well as diagnosis of GSDs. Here, we describe a method for the detection of glycogen using a renewable, recombinant protein which contains the carbohydrate-binding module (CBM) from starch-binding domain containing protein 1 (Stbd1). We generated a fusion protein containing glutathione S-transferase, a cMyc eptitope and the Stbd1CBM (GYSC) for use as a glycogen-binding probe, which can be detected with secondary antibodies against glutathione S-transferase or cMyc. By enzyme-linked immunosorbent assay, we demonstrate that GYSC binds glycogen and two other polymers of glucose, amylopectin and amylose. Immunofluorescence staining of cultured cells indicate a GYSC-specific signal that is co-localized with signals obtained with anti-glycogen or anti-glycogen synthase antibodies. GYSC-positive staining inside of lysosomes is observed in individual muscle fibers isolated from mice deficient in lysosomal enzyme acid alpha-glucosidase, a well-characterized model of GSD II (Pompe disease). Co-localized GYSC and glycogen signals are also found in muscle fibers isolated from mice deficient in malin, a model for Lafora disease. These data indicate that GYSC is a novel probe that can be used to study glycogen metabolism under normal and pathological conditions. PMID:28077463

Architectural plasticity of AMPK revealed by electron microscopy and X-ray crystallography

PubMed Central

Ouyang, Yan; Zhu, Li; Li, Yifang; Guo, Miaomiao; Liu, Yang; Cheng, Jin; Zhao, Jing; Wu, Yi

2016-01-01

Mammalian AMP-activated protein kinase (AMPK) acts as an important sensor of cellular energy homeostasis related with AMP/ADP to ATP ratio. The overall architecture of AMPK has been determined in either homotrimer or monomer form by electron microscopy (EM) and X-ray crystallography successively. Accordingly proposed models have consistently revealed a key role of the α subunit linker in sensing adenosine nucleoside binding on the γ subunit and mediating allosteric regulation of kinase domain (KD) activity, whereas there are vital differences in orienting N-terminus of α subunit and locating carbohydrate-binding module (CBM) of β subunit. Given that Mg2+, an indispensable cofactor of AMPK was present in the EM sample preparation buffer however absent when forming crystals, here we carried out further reconstructions without Mg2+ to expectably inspect if this ion may contribute to this difference. However, no essential alteration has been found in this study compared to our early work. Further analyses indicate that the intra-molecular movement of the KD and CBM are most likely due to the flexible linkage of the disordered linkers with the rest portion as well as a contribution from the plasticity in the inter-molecular assembly mode, which might ulteriorly reveal an architectural complication of AMPK. PMID:27063142

Computational Investigations of Trichoderma Reesei Cel7A Suggest New Routes for Enzyme Activity Improvements

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

Beckham, G. T.; Payne, C. M.; Bu, L.

2012-01-01

The Trichoderma reesei Family 7 cellulase (Cel7A) is a key industrial enzyme in the production of biofuels from lignocellulosic biomass. It is a multi-modular enzyme with a Family 1 carbohydrate-binding module, a flexible O-glycosylated linker, and a large catalytic domain. We have used simulation to elucidate new functions for the 3 sub-domains, which suggests new routes to increase the activity of this central enzyme. These findings include new roles for glycosylation, which we have shown can be used to tune the binding affinity. We have also examined the structures of the catalytically-active complex of Cel7A and its non-processive counterpart, Cel7B,more » engaged on cellulose, which suggests allosteric mechanisms involved in chain binding when these cellulases are complexed on cellulose. Our computational results also suggest that product inhibition varies significantly between Cel7A and Cel7B, and we offer a molecular-level explanation for this observation. Finally, we discuss simulations of the absolute and relative binding free energy of cellulose ligands and various mutations along the CD tunnel, which will affect processivity and the ability of Cel7A (and related enzymes) to digest cellulose. These results highlight new considerations in protein engineering for processive and non-processive cellulases for production of lignocellulosic biofuels.« less

The productive cellulase binding capacity of cellulosic substrates.

PubMed

Karuna, Nardrapee; Jeoh, Tina

2017-03-01

Cellulosic biomass is the most promising feedstock for renewable biofuel production; however, the mechanisms of the heterogeneous cellulose saccharification reaction are still unsolved. As cellulases need to bind isolated molecules of cellulose at the surface of insoluble cellulose fibrils or larger aggregated cellulose structures in order to hydrolyze glycosidic bonds, the "accessibility of cellulose to cellulases" is considered to be a reaction limiting property of cellulose. We have defined the accessibility of cellulose to cellulases as the productive binding capacity of cellulose, that is, the concentration of productive binding sites on cellulose that are accessible for binding and hydrolysis by cellulases. Productive cellulase binding to cellulose results in hydrolysis and can be quantified by measuring hydrolysis rates. In this study, we measured the productive Trichoderma reesei Cel7A (TrCel7A) binding capacity of five cellulosic substrates from different sources and processing histories. Swollen filter paper and bacterial cellulose had higher productive binding capacities of ∼6 µmol/g while filter paper, microcrystalline cellulose, and algal cellulose had lower productive binding capacities of ∼3 µmol/g. Swelling and regenerating filter paper using phosphoric acid increased the initial accessibility of the reducing ends to TrCel7A from 4 to 6 µmol/g. Moreover, this increase in initial productive binding capacity accounted in large part for the difference in the overall digestibility between filter paper and swollen filter paper. We further demonstrated that an understanding of how the productive binding capacity declines over the course of the hydrolysis reaction has the potential to predict overall saccharification time courses. Biotechnol. Bioeng. 2017;114: 533-542. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Choline Binding Proteins from Streptococcus pneumoniae: A Dual Role as Enzybiotics and Targets for the Design of New Antimicrobials

PubMed Central

Maestro, Beatriz; Sanz, Jesús M.

2016-01-01

Streptococcus pneumoniae (pneumococcus) is an important pathogen responsible for acute invasive and non-invasive infections such as meningitis, sepsis and otitis media, being the major cause of community-acquired pneumonia. The fight against pneumococcus is currently hampered both by insufficient vaccine coverage and by rising antimicrobial resistances to traditional antibiotics, making necessary the research on novel targets. Choline binding proteins (CBPs) are a family of polypeptides found in pneumococcus and related species, as well as in some of their associated bacteriophages. They are characterized by a structural organization in two modules: a functional module (FM), and a choline-binding module (CBM) that anchors the protein to the choline residues present in the cell wall through non-covalent interactions. Pneumococcal CBPs include cell wall hydrolases, adhesins and other virulence factors, all playing relevant physiological roles for bacterial viability and virulence. Moreover, many pneumococcal phages also make use of hydrolytic CBPs to fulfill their infectivity cycle. Consequently, CBPs may play a dual role for the development of novel antipneumococcal drugs, both as targets for inhibitors of their binding to the cell wall and as active cell lytic agents (enzybiotics). In this article, we review the current state of knowledge about host- and phage-encoded pneumococcal CBPs, with a special focus on structural issues, together with their perspectives for effective anti-infectious treatments. PMID:27314398

Readout electronics for CBM-TOF super module quality evaluation based on 10 Gbps ethernet

NASA Astrophysics Data System (ADS)

Jiang, D.; Cao, P.; Huang, X.; Zheng, J.; Wang, Q.; Li, B.; Li, J.; Liu, S.; An, Q.

2017-07-01

The Compressed Baryonic Matter-Time of Flight (CBM-TOF) wall uses high performance of Multi-gap Resistive Plate Chambers (MRPC) assembled in super modules to identify charged particles with high channel density and high measurement precision at high event rate. Electronics meet the challenge for reading data out from a super module at high speed of about 6 Gbps in real time. In this paper, the readout electronics for CBM-TOF super module quality evaluation is proposed based on 10 Gigabit Ethernet. The digitized TOF data from one super module will be concentrated at the front-end electronics residing on the side of the super module and transmitted to an extreme speed readout module (XSRM) housed in the backend crate through the PCI Express (PCIe) protocol via optic channels. Eventually, the XSRM transmits data to the data acquisition (DAQ) system through four 10 Gbps Ethernet ports in real time. This readout structure has advantages of high performance and expansibility. Furthermore, it is easy to operate. Test results on the prototype show that the overall data readout performance for each XSRM can reach up to 28.8 Gbps, which means XSRM can meet the requirement of reading data out from 4 super modules with 1280 channels in real time.

Genome mining and motif truncation of glycoside hydrolase family 5 endo-β-1,4-mannanase encoded by Aspergillus oryzae RIB40 for potential konjac flour hydrolysis or feed additive.

PubMed

Tang, Cun-Duo; Shi, Hong-Ling; Tang, Qing-Hai; Zhou, Jun-Shi; Yao, Lun-Guang; Jiao, Zhu-Jin; Kan, Yun-Chao

2016-11-01

Two novel glycosyl hydrolase family 5 (GH5) β-mannanases (AoMan5A and AoMan5B) were identified from Aspergillus oryzae RIB40 by genome mining. The AoMan5A contains a predicted family 1 carbohydrate binding module (CBM-1), located at its N-terminal. The AoMan5A, AoMan5B and truncated mutant AoMan5AΔCL (truncating the N-terminal CBM and linker of AoMan5A) were expressed retaining the N-terminus of the native protein in Pichia pastoris GS115 by pPIC9K M . The specific enzyme activity of the purified reAoMan5A, reAoMan5B and reAoMan5AΔCL towards locust bean gum at pH 3.6 and 40°C for 10min, was 8.3, 104.2 and 15.8U/mg, respectively. The temperature properties of the reAoMan5AΔCL were improved by truncating CBM. They can degrade the pretreated konjac flour and produce prebiotics. In addition, they had excellent stability under simulative gastric fluid and simulative prilling process. All these properties make these recombinant β-mannanases potential additives for use in the food and feed industries. Copyright © 2016. Published by Elsevier Inc.

Degradation of Granular Starch by the Bacterium Microbacterium aurum Strain B8.A Involves a Modular α-Amylase Enzyme System with FNIII and CBM25 Domains

PubMed Central

Eeuwema, Wieger; Sarian, Fean D.; van der Kaaij, Rachel M.

2015-01-01

The bacterium Microbacterium aurum strain B8.A, originally isolated from a potato plant wastewater facility, is able to degrade different types of starch granules. Here we report the characterization of an unusually large, multidomain M. aurum B8.A α-amylase enzyme (MaAmyA). MaAmyA is a 1,417-amino-acid (aa) protein with a predicted molecular mass of 148 kDa. Sequence analysis of MaAmyA showed that its catalytic core is a family GH13_32 α-amylase with the typical ABC domain structure, followed by a fibronectin (FNIII) domain, two carbohydrate binding modules (CBM25), and another three FNIII domains. Recombinant expression and purification yielded an enzyme with the ability to degrade wheat and potato starch granules by introducing pores. Characterization of various truncated mutants of MaAmyA revealed a direct relationship between the presence of CBM25 domains and the ability of MaAmyA to form pores in starch granules, while the FNIII domains most likely function as stable linkers. At the C terminus, MaAmyA carries a 300-aa domain which is uniquely associated with large multidomain amylases; its function remains to be elucidated. We concluded that M. aurum B8.A employs a multidomain enzyme system to initiate degradation of starch granules via pore formation. PMID:26187958

The deposition and characterization of starch in Brachypodium distachyon

PubMed Central

Tanackovic, Vanja; Svensson, Jan T.; Jensen, Susanne L.; Buléon, Alain; Blennow, Andreas

2014-01-01

Brachypodium distachyon is a non-domesticated cereal. Nonetheless, Brachypodium was recently introduced as a model plant for temperate cereals. This study compares grain starch metabolism in Brachypodium and barley (Hordeum vulgare). In Brachypodium, we identified and annotated 28 genes involved in starch metabolism and identified important motifs including transit peptides and putative carbohydrate-binding modules (CBMs) of the families CBM20, CBM45, CBM48, and CBM53. Starch content was markedly lower in Brachypodium grains (12%) compared to barley grains (47%). Brachypodium starch granules were doughnut shaped and bimodally distributed into distinct small B-type (2.5–10 µm) and very small C-type (0.5–2.5 µm) granules. Large A-type granules, typical of cereals, were absent. Starch-bound phosphate, important for starch degradation, was 2-fold lower in Brachypodium compared with barley indicating different requirements for starch mobilization. The amylopectin branch profiles were similar and the amylose content was only slightly higher compared with barley cv. Golden Promise. The crystallinity of Brachypodium starch granules was low (10%) compared to barley (20%) as determined by wide-angle X-ray scattering (WAXS) and molecular disorder was confirmed by differential scanning calorimetry (DSC). The expression profiles in grain for most genes were distinctly different for Brachypodium compared to barley, typically showing earlier decline during the course of development, which can explain the low starch content and differences in starch molecular structure and granule characteristics. High transitory starch levels were observed in leaves of Brachypodium (2.8% after 14h of light) compared to barley (1.9% after 14h of light). The data suggest important pre-domesticated features of cereals. PMID:25056772

Overexpression, crystallization and preliminary X-ray crystallographic analysis of glucuronoxylan xylanohydrolase (Xyn30A) from Clostridium thermocellum

PubMed Central

Verma, Anil Kumar; Goyal, Arun; Freire, Filipe; Bule, Pedro; Venditto, Immacolata; Brás, Joana L. A.; Santos, Helena; Cardoso, Vânia; Bonifácio, Cecília; Thompson, Andrew; Romão, Maria João; Prates, José A. M.; Ferreira, Luís M. A.; Fontes, Carlos M. G. A.; Najmudin, Shabir

2013-01-01

The modular carbohydrate-active enzyme belonging to glycoside hydrolase family 30 (GH30) from Clostridium thermocellum (CtXynGH30) is a cellulosomal protein which plays an important role in plant cell-wall degradation. The full-length CtXynGH30 contains an N-terminal catalytic module (Xyn30A) followed by a family 6 carbohydrate-binding module (CBM6) and a dockerin at the C-terminus. The recombinant protein has a molecular mass of 45 kDa. Preliminary structural characterization was carried out on Xyn30A crystallized in different conditions. All tested crystals belonged to space group P1 with one molecule in the asymmetric unit. Molecular replacement has been used to solve the Xyn30A structure. PMID:24316849

Hydrophobic kenaf nanocrystalline cellulose for the binding of curcumin.

PubMed

Zainuddin, Norhidayu; Ahmad, Ishak; Kargarzadeh, Hanieh; Ramli, Suria

2017-05-01

Nanocrystalline cellulose (NCC) extracted from lignocellulosic materials has been actively investigated as a drug delivery excipients due to its large surface area, high aspect ratio, and biodegradability. In this study, the hydrophobically modified NCC was used as a drug delivery excipient of hydrophobic drug curcumin. The modification of NCC with a cationic surfactant, cetyl trimethylammonium bromide (CTAB) was used to modulate the loading of hydrophobic drugs that would not normally bind to NCC. The FTIR, Elemental analysis, XRD, TGA, and TEM were used to confirm the modification of NCC with CTAB. The effect of concentration of CTAB on the binding efficiency of hydrophobic drug curcumin was investigated. The amounts of curcumin bound onto the CTAB-NCC nanoparticles were analyzed by UV-vis Spectrophotometric. The result showed that the modified CTAB-NCC bound a significant amount of curcumin, in a range from 80% to 96% curcumin added. Nevertheless, at higher concentration of CTAB resulted in lower binding efficiency. Copyright © 2017 Elsevier Ltd. All rights reserved.

Homologs of PROTEIN TARGETING TO STARCH Control Starch Granule Initiation in Arabidopsis Leaves[OPEN

PubMed Central

David, Laure C.; Abt, Melanie; Lu, Kuan-Jen

2017-01-01

The molecular mechanism that initiates the synthesis of starch granules is poorly understood. Here, we discovered two plastidial proteins involved in granule initiation in Arabidopsis thaliana leaves. Both contain coiled coils and a family-48 carbohydrate binding module (CBM48) and are homologs of the PROTEIN TARGETING TO STARCH (PTST) protein; thus, we named them PTST2 and PTST3. Chloroplasts in mesophyll cells typically contain five to seven granules, but remarkably, most chloroplasts in ptst2 mutants contained zero or one large granule. Chloroplasts in ptst3 had a slight reduction in granule number compared with the wild type, while those of the ptst2 ptst3 double mutant contained even fewer granules than ptst2. The ptst2 granules were larger but similar in morphology to wild-type granules, but those of the double mutant had an aberrant morphology. Immunoprecipitation showed that PTST2 interacts with STARCH SYNTHASE4 (SS4), which influences granule initiation and morphology. Overexpression of PTST2 resulted in chloroplasts containing many small granules, an effect that was dependent on the presence of SS4. Furthermore, isothermal titration calorimetry revealed that the CBM48 domain of PTST2, which is essential for its function, interacts with long maltooligosaccharides. We propose that PTST2 and PTST3 are critical during granule initiation, as they bind and deliver suitable maltooligosaccharide primers to SS4. PMID:28684429

Bioinspired assemblies of plant cell wall polymers unravel the affinity properties of carbohydrate-binding modules.

PubMed

Paës, Gabriel; von Schantz, Laura; Ohlin, Mats

2015-09-07

Lignocellulose-acting enzymes play a central role in the biorefinery of plant biomass to make fuels, chemicals and materials. These enzymes are often appended to carbohydrate binding modules (CBMs) that promote substrate targeting. When used in plant materials, which are complex assemblies of polymers, the binding properties of CBMs can be difficult to understand and predict, thus limiting the efficiency of enzymes. In order to gain more information on the binding properties of CBMs, some bioinspired model assemblies that contain some of the polymers and covalent interactions found in the plant cell walls have been designed. The mobility of three engineered CBMs has been investigated by FRAP in these assemblies, while varying the parameters related to the polymer concentration, the physical state of assemblies and the oligomerization state of CBMs. The features controlling the mobility of the CBMs in the assemblies have been quantified and hierarchized. We demonstrate that the parameters can have additional or opposite effects on mobility, depending on the CBM tested. We also find evidence of a relationship between the mobility of CBMs and their binding strength. Overall, bioinspired assemblies are able to reveal the unique features of affinity of CBMs. In particular, the results show that oligomerization of CBMs and the presence of ferulic acid motifs in the assemblies play an important role in the binding affinity of CBMs. Thus we propose that these features should be finely tuned when CBMs are used in plant cell walls to optimise bioprocesses.

Degradation of Granular Starch by the Bacterium Microbacterium aurum Strain B8.A Involves a Modular α-Amylase Enzyme System with FNIII and CBM25 Domains.

PubMed

Valk, Vincent; Eeuwema, Wieger; Sarian, Fean D; van der Kaaij, Rachel M; Dijkhuizen, Lubbert

2015-10-01

The bacterium Microbacterium aurum strain B8.A, originally isolated from a potato plant wastewater facility, is able to degrade different types of starch granules. Here we report the characterization of an unusually large, multidomain M. aurum B8.A α-amylase enzyme (MaAmyA). MaAmyA is a 1,417-amino-acid (aa) protein with a predicted molecular mass of 148 kDa. Sequence analysis of MaAmyA showed that its catalytic core is a family GH13_32 α-amylase with the typical ABC domain structure, followed by a fibronectin (FNIII) domain, two carbohydrate binding modules (CBM25), and another three FNIII domains. Recombinant expression and purification yielded an enzyme with the ability to degrade wheat and potato starch granules by introducing pores. Characterization of various truncated mutants of MaAmyA revealed a direct relationship between the presence of CBM25 domains and the ability of MaAmyA to form pores in starch granules, while the FNIII domains most likely function as stable linkers. At the C terminus, MaAmyA carries a 300-aa domain which is uniquely associated with large multidomain amylases; its function remains to be elucidated. We concluded that M. aurum B8.A employs a multidomain enzyme system to initiate degradation of starch granules via pore formation. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Deletion of uncharacterized domain from α-1,3-glucanase of Bacillus circulans KA-304 enhances heterologous enzyme production in Escherichia coli.

PubMed

Yano, Shigekazu; Suyotha, Wasana; Zanma, Sumika; Konno, Hiroyuki; Cherdvorapong, Vipavee; Wakayama, Mamoru

2018-05-08

α-1,3-Glucanase (Agl-KA) of Bacillus circulans KA-304 consists of an N-terminal discoidin domain (DS1), a carbohydrate binding module family 6 (CBM6), threonine and proline repeats (TP), a second discoidin domain (DS2), an uncharacterized conserved domain (UCD), and a C-terminal catalytic domain. Previously, we reported that DS1, CBM6, and DS2 have α-1,3-glucan-binding activity and contribute to α-1,3-glucan hydrolysis. In this study, UCD deletion mutant (AglΔUCD) was constructed, and its properties were compared with those of Agl-KA. α-1,3-Glucan hydrolyzing, α-1,3-glucan binding, and protoplast-forming activities of AglΔUCD were almost the same as those of Agl-KA. k cat /K m values of AgΔUCD and Agl-KA were 11.4 and 11.1 s -1 mg -1 mL, respectively. AglΔUCD and Agl-KA exhibited similar characteristics, such as optimal pH, pH stability, optimal temperature, and thermostability. These results suggest that UCD is not α-1,3-glucan-binding and flexible linker domain, and that deletion of UCD does not affect the affinity of N-terminal binding domains and the catalytic action of the C-terminal domain. Subsequently, heterologous UCenzyme productivity of AglΔD in Escherichia coli was compared with that of Agl-KA. The productivity of AglΔUCD was about 4-fold larger than that of Agl-KA after an 8-h induction at 30°C. In the case of induction at 20°C, the productivity of AglΔUCD was also larger than that of Agl-KA. These findings indicate that deletion of only UCD enhances the enzyme productivity in E. coli.

Identification and Characterization of a Large Protein Essential for Degradation of the Crystalline Region of Cellulose by Cytophaga hutchinsonii

PubMed Central

Wang, Sen; Zhao, Dong; Bai, Xinfeng; Zhang, Weican

2016-01-01

ABSTRACT Cytophaga hutchinsonii is a Gram-negative bacterium that can efficiently degrade crystalline cellulose by a unique mechanism different from the free cellulase or cellulosome strategy. In this study, chu_3220, encoding the hypothetical protein CHU_3220 (205 kDa), was identified by insertional mutation and gene deletion as the first gene essential for degradation of the crystalline region but not the amorphous region of cellulose by C. hutchinsonii. A chu_3220 deletion mutant was defective in the degradation of crystalline cellulose and increased the degree of crystallinity of Avicel PH101 but could still degrade amorphous cellulose completely. CHU_3220 was found to be located on the outer surface of the outer membrane and could bind to cellulose. It contains 15 PbH1 domains and a C-terminal domain (CHU_C) that was proved to be critical for the localization of CHU_3220 on the cell surface and the function of CHU_3220 in crystalline cellulose degradation. Moreover, the degradation of crystalline cellulose was intact-cell dependent and inhibited by NaN3. Further study showed that chu_3220 was induced by cellulose and that the endoglucanase activity on the cell surface was significantly reduced without chu_3220. Real-time PCR revealed that the transcription of most genes encoding endoglucanases located on the cell surface was decreased in the chu_3220 deletion mutant, indicating that chu_3220 might also play a role in the regulation of the expression of some endoglucanases. IMPORTANCE Cytophaga hutchinsonii could efficiently degrade crystalline cellulose with a unique mechanism without cellulosomes and free cellulases. It lacks proteins that are thought to play important roles in disruption of the crystalline region of cellulose, including exoglucanases, lytic polysaccharide monooxygenases, expansins, expansin-like proteins, or swollenins, and most of its endoglucanases lack carbohydrate binding modules. The mechanism of the degradation of crystalline cellulose is still unknown. In this study, chu_3220 was identified as the first gene essential for the degradation of the crystalline region but not the amorphous region of cellulose. CHU_3220 is a high-molecular-weight protein located on the outer surface of the outer membrane and could bind to cellulose. We proposed that CHU_3220 might be an essential component of a protein complex on the cell surface in charge of the decrystallization of crystalline cellulose. The degradation of crystalline cellulose by C. hutchinsonii was not only dependent on intact cells but also required the energy supplied by the cells. This was obviously different from other known cellulose depolymerization system. Our study has shed more light on the novel strategy of crystalline cellulose degradation by C. hutchinsonii. PMID:27742681

Complete genome of the cellyloytic thermophile Acidothermus cellulolyticus 11B provides insights into its ecophysiological and evloutionary adaptations

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

Barabote, Ravi D.; Xie, Gary; Leu, David H.

We present here the complete 2.4 Mb genome of the cellulolytic actinobacterial thermophile, Acidothermus cellulolyticus 11B. New secreted glycoside hydrolases and carbohydrate esterases were identified in the genome, revealing a diverse biomass-degrading enzyme repertoire far greater than previously characterized, and significantly elevating the industrial value of this organism. A sizable fraction of these hydrolytic enzymes break down plant cell walls and the remaining either degrade components in fungal cell walls or metabolize storage carbohydrates such as glycogen and trehalose, implicating the relative importance of these different carbon sources. A novel feature of the A. cellulolyticus secreted cellulolytic and xylanolytic enzymesmore » is that they are fused to multiple tandemly arranged carbohydrate binding modules (CBM), from families 2 and 3. Interestingly, CBM3 was found to be always N-terminal to CBM2, suggesting a functional constraint driving this organization. While the catalytic domains of these modular enzymes are either diverse or unrelated, the CBMs were found to be highly conserved in sequence and may suggest selective substrate-binding interactions. For the most part, thermophilic patterns in the genome and proteome of A. cellulolyticus were weak, which may be reflective of the recent evolutionary history of A. cellulolyticus since its divergence from its closest phylogenetic neighbor Frankia, a mesophilic plant endosymbiont and soil dweller. However, ribosomal proteins and non-coding RNAs (rRNA and tRNAs) in A. cellulolyticus showed thermophilic traits suggesting the importance of adaptation of cellular translational machinery to environmental temperature. Elevated occurrence of IVYWREL amino acids in A. cellulolyticus orthologs compared to mesophiles, and inverse preferences for G and A at the first and third codon positions also point to its ongoing thermoadaptation. Additional interesting features in the genome of this cellulolytic, hot-springs dwelling prokaryote include a low occurrence of pseudogenes or mobile genetic elements, an unexpected complement of flagellar genes, and presence of three laterally-acquired genomic islands of likely ecophysiological value.« less

Crystal Structure and Computational Characterization of the Lytic Polysaccharide Monooxygenase GH61D from the Basidiomycota Fungus Phanerochaete chrysosporium*

PubMed Central

Wu, Miao; Beckham, Gregg T.; Larsson, Anna M.; Ishida, Takuya; Kim, Seonah; Payne, Christina M.; Himmel, Michael E.; Crowley, Michael F.; Horn, Svein J.; Westereng, Bjørge; Igarashi, Kiyohiko; Samejima, Masahiro; Ståhlberg, Jerry; Eijsink, Vincent G. H.; Sandgren, Mats

2013-01-01

Carbohydrate structures are modified and degraded in the biosphere by a myriad of mostly hydrolytic enzymes. Recently, lytic polysaccharide mono-oxygenases (LPMOs) were discovered as a new class of enzymes for cleavage of recalcitrant polysaccharides that instead employ an oxidative mechanism. LPMOs employ copper as the catalytic metal and are dependent on oxygen and reducing agents for activity. LPMOs are found in many fungi and bacteria, but to date no basidiomycete LPMO has been structurally characterized. Here we present the three-dimensional crystal structure of the basidiomycete Phanerochaete chrysosporium GH61D LPMO, and, for the first time, measure the product distribution of LPMO action on a lignocellulosic substrate. The structure reveals a copper-bound active site common to LPMOs, a collection of aromatic and polar residues near the binding surface that may be responsible for regio-selectivity, and substantial differences in loop structures near the binding face compared with other LPMO structures. The activity assays indicate that this LPMO primarily produces aldonic acids. Last, molecular simulations reveal conformational changes, including the binding of several regions to the cellulose surface, leading to alignment of three tyrosine residues on the binding face of the enzyme with individual cellulose chains, similar to what has been observed for family 1 carbohydrate-binding modules. A calculated potential energy surface for surface translation indicates that P. chrysosporium GH61D exhibits energy wells whose spacing seems adapted to the spacing of cellobiose units along a cellulose chain. PMID:23525113

Responses of cerebral GABA-containing CBM neuron to taste stimulation with seaweed extracts in Aplysia kurodai.

PubMed

Narusuye, Kenji; Kinugawa, Aiko; Nagahama, Tatsumi

2005-11-01

Aplysia kurodai distributed along Japan feeds well on Ulva pertusa but rejects Gelidium amansii with distinctive patterned movements of the jaws and radula. On the ventral side of the cerebral M cluster, four cell bodies of higher order neurons that send axons to the buccal ganglia are distributed (CBM neurons). We have previously shown that the dopaminergic CBM1 modulates basic feeding circuits in the buccal ganglia for rejection by firing at higher frequency after application of the aversive taste of seaweed such as Gelidium amansii. In the present experiments immunohistochemical techniques showed that the CBM3 exhibited gamma-aminobutyric acid (GABA)-like immunoreactivity. The CBM3 may be equivalent to the CBI-3 involved in changing the motor programs from rejection to ingestion in Aplysia californica. The responses of the CBM3 to taste stimulation of the lips with seaweed extracts were investigated by the use of calcium imaging. The calcium-sensitive dye, Calcium Green-1, was iontophoretically introduced into a cell body of the CBM3 using a microelectrode. Application of Ulva pertusa or Gelidium amansii extract induced different changes in fluorescence in the CBM3 cell body, indicating that taste of Ulva pertusa initially induced longer-lasting continuous spike responses at slightly higher frequency compared with that of Gelidium amansii. Considering a role of the CBM3 in the pattern selection, these results suggest that elongation of the initial firing response may be a major factor for the CBM3 to switch the buccal motor programs from rejection to ingestion after application of different tastes of seaweeds in Aplysia kurodai. (c) 2005 Wiley Periodicals, Inc.

Discovery of Microorganisms and Enzymes Involved in High-Solids Decomposition of Rice Straw Using Metagenomic Analyses

PubMed Central

D’haeseleer, Patrik; Khudyakov, Jane; Burd, Helcio; Hadi, Masood; Simmons, Blake A.; Singer, Steven W.; Thelen, Michael P.; VanderGheynst, Jean S.

2013-01-01

High-solids incubations were performed to enrich for microbial communities and enzymes that decompose rice straw under mesophilic (35°C) and thermophilic (55°C) conditions. Thermophilic enrichments yielded a community that was 7.5 times more metabolically active on rice straw than mesophilic enrichments. Extracted xylanase and endoglucanse activities were also 2.6 and 13.4 times greater, respectively, for thermophilic enrichments. Metagenome sequencing was performed on enriched communities to determine community composition and mine for genes encoding lignocellulolytic enzymes. Proteobacteria were found to dominate the mesophilic community while Actinobacteria were most abundant in the thermophilic community. Analysis of protein family representation in each metagenome indicated that cellobiohydrolases containing carbohydrate binding module 2 (CBM2) were significantly overrepresented in the thermophilic community. Micromonospora, a member of Actinobacteria, primarily housed these genes in the thermophilic community. In light of these findings, Micromonospora and other closely related Actinobacteria genera appear to be promising sources of thermophilic lignocellulolytic enzymes for rice straw deconstruction under high-solids conditions. Furthermore, these discoveries warrant future research to determine if exoglucanases with CBM2 represent thermostable enzymes tolerant to the process conditions expected to be encountered during industrial biofuel production. PMID:24205054

Evidence for in vitro binding of pectin side chains to cellulose.

PubMed

Zykwinska, Agata W; Ralet, Marie-Christine J; Garnier, Catherine D; Thibault, Jean-François J

2005-09-01

Pectins of varying structures were tested for their ability to interact with cellulose in comparison to the well-known adsorption of xyloglucan. Our results reveal that sugar beet (Beta vulgaris) and potato (Solanum tuberosum) pectins, which are rich in neutral sugar side chains, can bind in vitro to cellulose. The extent of binding varies with respect to the nature and structure of the side chains. Additionally, branched arabinans (Br-Arabinans) or debranched arabinans (Deb-Arabinans; isolated from sugar beet) and galactans (isolated from potato) were shown bind to cellulose microfibrils. The adsorption of Br-Arabinan and galactan was lower than that of Deb-Arabinan. The maximum adsorption affinity of Deb-Arabinan to cellulose was comparable to that of xyloglucan. The study of sugar beet and potato alkali-treated cell walls supports the hypothesis of pectin-cellulose interaction. Natural composites enriched in arabinans or galactans and cellulose were recovered. The binding of pectins to cellulose microfibrils may be of considerable significance in the modeling of primary cell walls of plants as well as in the process of cell wall assembly.

Functionalization of Recombinant Amelogenin Nanospheres Allows Their Binding to Cellulose Materials.

PubMed

Butler, Samuel J; Bülow, Leif; Bonde, Johan

2016-10-01

Protein engineering to functionalize the self-assembling enamel matrix protein amelogenin with a cellulose binding domain (CBD) is used. The purpose is to examine the binding of the engineered protein, rh174CBD, to cellulose materials, and the possibility to immobilize self-assembled amelogenin nanospheres on cellulose. rh174CBD assembled to nanospheres ≈35 nm in hydrodynamic diameter, very similar in size to wild type amelogenin (rh174). Uniform particles are formed at pH 10 for both rh174 and rh174CBD, but only rh174CBD nanospheres showes significant binding to cellulose (Avicel). Cellulose binding of rh174CBD is promoted when the protein is self-assembled to nanospheres, compared to being in a monomeric form, suggesting a synergistic effect of the multiple CBDs on the nanospheres. The amount of bound rh174CBD nanospheres reached ≈15 mg/g Avicel, which corresponds to 4.2 to 6.3 × 10 -7 mole/m 2 . By mixing rh174 and rh174CBD, and then inducing self-assembly, composite nanospheres with a high degree of cellulose binding can be formed, despite a lower proportion of rh174CBD. This demonstrates that amelogenin variants like rh174 can be incorporated into the nanospheres, and still retain most of the binding to cellulose. Engineered amelogenin nanoparticles can thus be utilized to construct a range of new cellulose based hybrid materials, e.g. for wound treatment. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Increases thermal stability and cellulose-binding capacity of Cryptococcus sp. S-2 lipase by fusion of cellulose binding domain derived from Trichoderma reesei.

PubMed

Thongekkaew, Jantaporn; Ikeda, Hiroko; Iefuji, Haruyuki

2012-03-30

To improve the thermal stability and cellulose-binding capacity of Cryptococcus sp. S-2 lipase (CSLP), the cellulose-binding domain originates from Trichoderma reesei cellobiohydrolase I was engineered into C-terminal region of the CSLP (CSLP-CBD). The CSLP and CSLP-CBD were successfully expressed in the Pichia pastoris using the strong methanol inducible alcohol oxidase 1 (AOX1) promoter and the secretion signal sequence from Saccharomyces cerevisiae (α factor). The recombinant CSLP and CSLP-CBD were secreted into culture medium and estimated by SDS-PAGE to be 22 and 27 kDa, respectively. The fusion enzyme was stable at 80 °C and retained more than 80% of its activity after 120-min incubation at this temperature. Our results also found that the fusion of fungal exoglucanase cellulose-binding domain to CSLP is responsible for cellulose-binding capacity. This attribute should make it an attractive applicant for enzyme immobilization. Copyright © 2012 Elsevier Inc. All rights reserved.

Structural Insights of Glucan Phosphatase Dynamics using Amide Hydrogen/Deuterium Exchange Mass Spectrometry

PubMed Central

Hsu, Simon; Kim, Youngjun; Li, Sheng; Durrant, Eric S.; Pace, Rachel M.; Woods, Virgil L.; Gentry, Matthew S.

2009-01-01

Laforin and Starch Excess 4 (SEX4) are founding members of a class of phosphatases that dephosphorylate phosphoglucans. Each protein contains a carbohydrate binding module (CBM) and a dual specificity phosphatase (DSP) domain. The gene encoding laforin is mutated in a fatal neurodegenerative disease called Lafora disease (LD). In the absence of laforin function, insoluble glucans accumulate that are hyperphosphorylated and exhibit sparse branching. It is hypothesized that these accumulations trigger the neurodegeneration and premature death of LD patients. We recently demonstrated that laforin removes phosphate from phosphoglucans and hypothesized that this function inhibits insoluble glucan accumulation. Loss of SEX4 function in plants yields a similar cellular phenotype; cells accumulate an excess amount of insoluble, hyperphosphorylated glucans. While multiple groups have shown that these phosphatases dephosphorylate phosphoglucans, there is no structure of a glucan phosphatase and little is known about the mechanism whereby they perform this action. We utilized hydrogen-deuterium exchange mass spectrometry (DXMS) and structural modeling to probe the conformational and structural dynamics of the glucan phosphatase SEX4. We found that the enzyme does not undergo a global conformational change upon glucan binding, but instead undergoes minimal rearrangement upon binding. The CBM undergoes increased protection from deuteration when bound to glucans, confirming its role in glucan binding. More interestingly, we identified structural components of the DSP that also undergo increased protection from deuteration upon glucan addition. To determine the position of these regions, we generated a homology model of the SEX4 DSP. The homology model shows that all of these regions are adjacent the DSP active site. Therefore, our results suggest that these regions of the DSP participate in presenting the phosphoglucan to the active site and provide the first structural analysis and mode of action of this unique class of phosphatases. PMID:19754155

Investigation of the binding properties of a multi-modular GH45 cellulase using bioinspired model assemblies.

PubMed

Fong, Monica; Berrin, Jean-Guy; Paës, Gabriel

2016-01-01

Enzymes degrading plant biomass polymers are widely used in biotechnological applications. Their efficiency can be limited by non-specific interactions occurring with some chemical motifs. In particular, the lignin component is known to bind enzymes irreversibly. In order to determine interactions of enzymes with their substrates, experiments are usually performed on isolated simple polymers which are not representative of plant cell wall complexity. But when using natural plant substrates, the role of individual chemical and structural features affecting enzyme-binding properties is also difficult to decipher. We have designed and used lignified model assemblies of plant cell walls as templates to characterize binding properties of multi-modular cellulases. These three-dimensional assemblies are modulated in their composition using the three principal polymers found in secondary plant cell walls (cellulose, hemicellulose, and lignin). Binding properties of enzymes are obtained from the measurement of their mobility that depends on their interactions with the polymers and chemical motifs of the assemblies. The affinity of the multi-modular GH45 cellulase was characterized using a statistical analysis to determine the role played by each assembly polymer. Presence of hemicellulose had much less impact on affinity than cellulose and model lignin. Depending on the number of CBMs appended to the cellulase catalytic core, binding properties toward cellulose and lignin were highly contrasted. Model assemblies bring new insights into the molecular determinants that are responsible for interactions between enzymes and substrate without the need of complex analysis. Consequently, we believe that model bioinspired assemblies will provide relevant information for the design and optimization of enzyme cocktails in the context of biorefineries.

Structure-based Reassessment of the Caveolin Signaling Model: Do Caveolae Regulate Signaling Through Caveolin-Protein Interactions?

PubMed Central

Collins, Brett M.; Davis, Melissa J.; Hancock, John F.; Parton, Robert G.

2012-01-01

Summary Caveolin proteins drive formation of caveolae, specialized cell-surface microdomains that influence cell signaling. Signaling proteins are proposed to use conserved caveolin-binding motifs (CBMs) to associate with caveolae via the caveolin scaffolding domain (CSD). However, structural and bioinformatic analyses argue against such direct physical interactions: In the majority of signaling proteins, the CBM is buried and inaccessible. Putative CBMs do not form a common structure for caveolin recognition, are not enriched amongst caveolin-binding proteins, and are even more common in yeast, which lack caveolae. We propose that CBM/CSD-dependent interactions are unlikely to mediate caveolar signaling, and the basis for signaling effects should therefore be reassessed. PMID:22814599

A small cellulose binding domain protein (CBD1) is highly variable in the nonbinding amino terminus

USDA-ARS?s Scientific Manuscript database

The small cellulose binding domain protein CBD1 is tightly bound to the cellulosic cell wall of the plant pathogenic stramenophile Phytophthora infestans. Transgene expression of the protein in plants has also demonstrated binding to plant cell walls. A study was undertaken using 47 isolates of P. ...

Structure/Function Analysis of Cotton-Based Peptide-Cellulose Conjugates: Spatiotemporal/Kinetic Assessment of Protease Aerogels Compared to Nanocrystalline and Paper Cellulose

PubMed Central

Edwards, J. Vincent; Fontenot, Krystal; Liebner, Falk; Pircher, Nicole Doyle nee; French, Alfred D.; Condon, Brian D.

2018-01-01

Nanocellulose has high specific surface area, hydration properties, and ease of derivatization to prepare protease sensors. A Human Neutrophil Elastase sensor designed with a nanocellulose aerogel transducer surface derived from cotton is compared with cotton filter paper, and nanocrystalline cellulose versions of the sensor. X-ray crystallography was employed along with Michaelis–Menten enzyme kinetics, and circular dichroism to contrast the structure/function relations of the peptide-cellulose conjugate conformation to enzyme/substrate binding and turnover rates. The nanocellulosic aerogel was found to have a cellulose II structure. The spatiotemporal relation of crystallite surface to peptide-cellulose conformation is discussed in light of observed enzyme kinetics. A higher substrate binding affinity (Km) of elastase was observed with the nanocellulose aerogel and nanocrystalline peptide-cellulose conjugates than with the solution-based elastase substrate. An increased Km observed for the nanocellulosic aerogel sensor yields a higher enzyme efficiency (kcat/Km), attributable to binding of the serine protease to the negatively charged cellulose surface. The effect of crystallite size and β-turn peptide conformation are related to the peptide-cellulose kinetics. Models demonstrating the orientation of cellulose to peptide O6-hydroxymethyl rotamers of the conjugates at the surface of the cellulose crystal suggest the relative accessibility of the peptide-cellulose conjugates for enzyme active site binding. PMID:29534033

Structure/Function Analysis of Cotton-Based Peptide-Cellulose Conjugates: Spatiotemporal/Kinetic Assessment of Protease Aerogels Compared to Nanocrystalline and Paper Cellulose.

PubMed

Edwards, J Vincent; Fontenot, Krystal; Liebner, Falk; Pircher, Nicole Doyle Nee; French, Alfred D; Condon, Brian D

2018-03-13

Nanocellulose has high specific surface area, hydration properties, and ease of derivatization to prepare protease sensors. A Human Neutrophil Elastase sensor designed with a nanocellulose aerogel transducer surface derived from cotton is compared with cotton filter paper, and nanocrystalline cellulose versions of the sensor. X-ray crystallography was employed along with Michaelis-Menten enzyme kinetics, and circular dichroism to contrast the structure/function relations of the peptide-cellulose conjugate conformation to enzyme/substrate binding and turnover rates. The nanocellulosic aerogel was found to have a cellulose II structure. The spatiotemporal relation of crystallite surface to peptide-cellulose conformation is discussed in light of observed enzyme kinetics. A higher substrate binding affinity ( K m ) of elastase was observed with the nanocellulose aerogel and nanocrystalline peptide-cellulose conjugates than with the solution-based elastase substrate. An increased K m observed for the nanocellulosic aerogel sensor yields a higher enzyme efficiency ( k cat / K m ), attributable to binding of the serine protease to the negatively charged cellulose surface. The effect of crystallite size and β-turn peptide conformation are related to the peptide-cellulose kinetics. Models demonstrating the orientation of cellulose to peptide O6-hydroxymethyl rotamers of the conjugates at the surface of the cellulose crystal suggest the relative accessibility of the peptide-cellulose conjugates for enzyme active site binding.

Increases thermal stability and cellulose-binding capacity of Cryptococcus sp. S-2 lipase by fusion of cellulose binding domain derived from Trichoderma reesei

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

Thongekkaew, Jantaporn, E-mail: jantaporn_25@yahoo.com; Ikeda, Hiroko; Iefuji, Haruyuki

Highlights: Black-Right-Pointing-Pointer The CSLP and fusion enzyme were successfully expressed in the Pichia pastoris. Black-Right-Pointing-Pointer The fusion enzyme was stable at 80 Degree-Sign C for 120-min. Black-Right-Pointing-Pointer The fusion enzyme was responsible for cellulose-binding capacity. Black-Right-Pointing-Pointer The fusion enzyme has an attractive applicant for enzyme immobilization. -- Abstract: To improve the thermal stability and cellulose-binding capacity of Cryptococcus sp. S-2 lipase (CSLP), the cellulose-binding domain originates from Trichoderma reesei cellobiohydrolase I was engineered into C-terminal region of the CSLP (CSLP-CBD). The CSLP and CSLP-CBD were successfully expressed in the Pichia pastoris using the strong methanol inducible alcohol oxidase 1 (AOX1)more » promoter and the secretion signal sequence from Saccharomyces cerevisiae ({alpha} factor). The recombinant CSLP and CSLP-CBD were secreted into culture medium and estimated by SDS-PAGE to be 22 and 27 kDa, respectively. The fusion enzyme was stable at 80 Degree-Sign C and retained more than 80% of its activity after 120-min incubation at this temperature. Our results also found that the fusion of fungal exoglucanase cellulose-binding domain to CSLP is responsible for cellulose-binding capacity. This attribute should make it an attractive applicant for enzyme immobilization.« less

Polypeptides having endoglucanase activity and polynucleotides encoding same

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

Spodsberg, Nikolaj; Shagasi, Tarana

The present invention relates to isolated polypeptides having endoglucanase activity, catalytic domains, cellulose binding domains and polynucleotides encoding the polypeptides, catalytic domains or cellulose binding domains. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, catalytic domains or cellulose binding domains.

Polypeptides having endoglucanase activity and polynucleotides encoding same

DOEpatents

Spodsberg, Nikolaj; Shagasi, Tarana

2015-06-30

The present invention relates to isolated polypeptides having endoglucanase activity, catalytic domains, cellulose binding domains and polynucleotides encoding the polypeptides, catalytic domains or cellulose binding domains. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, catalytic domains or cellulose binding domains.

Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

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

Stringer, Mary Ann; McBrayer, Brett

2016-11-29

The present invention relates to isolated polypeptides having cellobiohydrolase activity, catalytic domains, and cellulose binding domains and polynucleotides encoding the polypeptides, catalytic domains, and cellulose binding domains. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, catalytic domains, or cellulose binding domains.

The Collagen Binding Proteins of Streptococcus mutans and Related Streptococci

PubMed Central

Avilés-Reyes, Alejandro; Miller, James H.; Lemos, José A.; Abranches, Jacqueline

2016-01-01

Summary The ability of Streptococcus mutans to interact with collagen through the expression of collagen-binding proteins (CBPs) bestows this oral pathogen with an alternative to the sucrose-dependent mechanism of colonization classically attributed to caries development. Based on the abundance and distribution of collagen throughout the human body, stringent adherence to this molecule grants S. mutans with the opportunity to establish infection at different host sites. Surface proteins, such as SpaP, WapA, Cnm and Cbm, have been shown to bind collagen in vitro, and it has been suggested that these molecules play a role in colonization of oral and extra-oral tissues. However, robust collagen binding is not achieved by all strains of S. mutans, particularly those that lack Cnm or Cbm. These observations merit careful dissection of the contribution from these different CBPs towards tissue colonization and virulence. In this review, we will discuss the current understanding of mechanisms utilized by S. mutans and related streptococci to colonize collagenous tissues, and the possible contribution of CBPs to infections in different sites of the host. PMID:26991416

Aromatic amino acids in the cellulose binding domain of Penicillium crustosum endoglucanase EGL1 differentially contribute to the cellulose affinity of the enzyme

PubMed Central

Xiong, Wei; Chen, Fang-Yuan; Xu, Li; Han, Zheng-Gang

2017-01-01

The cellulose binding domain (CBD) of cellulase binding to cellulosic materials is the initiation of a synergistic action on the enzymatic hydrolysis of the most abundant renewable biomass resources in nature. The binding of the CBD domain to cellulosic substrates generally relies on the interaction between the aromatic amino acids structurally located on the flat face of the CBD domain and the glucose rings of cellulose. In this study, we found the CBD domain of a newly cloned Penicillium crustosum endoglucanase EGL1, which was phylogenetically related to Aspergillus, Fusarium and Rhizopus, and divergent from the well-characterized Trichoderma reeseis cellulase CBD domain, contain two conserved aromatic amino acid-rich regions, Y451-Y452 and Y477-Y478-Y479, among which three amino acids Y451, Y477, and Y478 structurally sited on a flat face of this domain. Cellulose binding assays with green fluorescence protein as the marker, adsorption isotherm assays and an isothermal titration calorimetry assays revealed that although these three amino acids participated in this process, the Y451-Y452 appears to contribute more to the cellulose binding than Y477-Y478-Y479. Further glycine scanning mutagenesis and structural modelling revealed that the binding between CBD domain and cellulosic materials might be multi-amino-acids that participated in this process. The flexible poly-glucose molecule could contact Y451, Y477, and Y478 which form the contacting flat face of CBD domain as the typical model, some other amino acids in or outside the flat face might also participate in the interaction. Thus, it is possible that the conserved Y451-Y452 of CBD might have a higher chance of contacting the cellulosic substrates, contributing more to the affinity of CBD than the other amino acids. PMID:28475645

Imaging Cell Wall Architecture in Single Zinnia elegans Tracheary Elements1[OA

PubMed Central

Lacayo, Catherine I.; Malkin, Alexander J.; Holman, Hoi-Ying N.; Chen, Liang; Ding, Shi-You; Hwang, Mona S.; Thelen, Michael P.

2010-01-01

The chemical and structural organization of the plant cell wall was examined in Zinnia elegans tracheary elements (TEs), which specialize by developing prominent secondary wall thickenings underlying the primary wall during xylogenesis in vitro. Three imaging platforms were used in conjunction with chemical extraction of wall components to investigate the composition and structure of single Zinnia TEs. Using fluorescence microscopy with a green fluorescent protein-tagged Clostridium thermocellum family 3 carbohydrate-binding module specific for crystalline cellulose, we found that cellulose accessibility and binding in TEs increased significantly following an acidified chlorite treatment. Examination of chemical composition by synchrotron radiation-based Fourier-transform infrared spectromicroscopy indicated a loss of lignin and a modest loss of other polysaccharides in treated TEs. Atomic force microscopy was used to extensively characterize the topography of cell wall surfaces in TEs, revealing an outer granular matrix covering the underlying meshwork of cellulose fibrils. The internal organization of TEs was determined using secondary wall fragments generated by sonication. Atomic force microscopy revealed that the resulting rings, spirals, and reticulate structures were composed of fibrils arranged in parallel. Based on these combined results, we generated an architectural model of Zinnia TEs composed of three layers: an outermost granular layer, a middle primary wall composed of a meshwork of cellulose fibrils, and inner secondary wall thickenings containing parallel cellulose fibrils. In addition to insights in plant biology, studies using Zinnia TEs could prove especially productive in assessing cell wall responses to enzymatic and microbial degradation, thus aiding current efforts in lignocellulosic biofuel production. PMID:20592039

Microscopic Analysis of Corn Fiber Using Corn Starch- and Cellulose-Specific Molecular Probes

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

Porter, S. E.; Donohoe, B. S.; Beery, K. E.

Ethanol is the primary liquid transportation fuel produced from renewable feedstocks in the United States today. The majority of corn grain, the primary feedstock for ethanol production, has been historically processed in wet mills yielding products such as gluten feed, gluten meal, starch, and germ. Starch extracted from the grain is used to produce ethanol in saccharification and fermentation steps; however the extraction of starch is not 100% efficient. To better understand starch extraction during the wet milling process, we have developed fluorescent probes that can be used to visually localize starch and cellulose in samples using confocal microscopy. Thesemore » probes are based on the binding specificities of two types of carbohydrate binding modules (CBMs), which are small substrate-specific protein domains derived from carbohydrate degrading enzymes. CBMs were fused, using molecular cloning techniques, to a green fluorescent protein (GFP) or to the red fluorescent protein DsRed (RFP). Using these engineered probes, we found that the binding of the starch-specific probe correlates with starch content in corn fiber samples. We also demonstrate that there is starch internally localized in the endosperm that may contribute to the high starch content in corn fiber. We also surprisingly found that the cellulose-specific probe did not bind to most corn fiber samples, but only to corn fiber that had been hydrolyzed using a thermochemical process that removes the residual starch and much of the hemicellulose. Our findings should be of interest to those working to increase the efficiency of the corn grain to ethanol process.« less

Clock and trigger distribution for CBM-TOF quality evaluation of RPC super module detector assemblies

NASA Astrophysics Data System (ADS)

Li, C.; Huang, X.; Cao, P.; Wang, J.; An, Q.

2018-03-01

RPC Super module (SM) detector assemblies are used for charged hadron identification in the Time-of-Flight (TOF) spectrometer at the Compressed Baryonic Matter (CBM) experiment. Each SM contains several multi-gap Resistive Plate Chambers (MRPCs) and provides up to 320 electronic channels in total for high-precision time measurements. Time resolution of the Time-to-Digital Converter (TDC) is required to be better than 20 ps. During mass production, the quality of each SM needs to be evaluated. In order to meet the requirements, the system clock signal as well as the trigger signal should be distributed precisely and synchronously to all electronics modules within the evaluation readout system. In this paper, a hierarchical clock and trigger distribution method is proposed for the quality evaluation of CBM-TOF SM detectors. In a first stage, the master clock and trigger module (CTM) allocated in a 6U PXI chassis distributes the clock and trigger signals to the slave CTM in the same chassis. In a second stage, the slave CTM transmits the clock and trigger signals to the TDC readout module (TRM) through one optical link. In a third stage, the TRM distributes the clock and trigger signals synchronously to 10 individual TDC boards. Laboratory test results show that the clock jitter at the third stage is less than 4 ps (RMS) and the trigger transmission latency from the master CTM to the TDC is about 272 ns with 11 ps (RMS) jitter. The overall performance complies well with the required specifications.

An optimization method for condition based maintenance of aircraft fleet considering prognostics uncertainty.

PubMed

Feng, Qiang; Chen, Yiran; Sun, Bo; Li, Songjie

2014-01-01

An optimization method for condition based maintenance (CBM) of aircraft fleet considering prognostics uncertainty is proposed. The CBM and dispatch process of aircraft fleet is analyzed first, and the alternative strategy sets for single aircraft are given. Then, the optimization problem of fleet CBM with lower maintenance cost and dispatch risk is translated to the combinatorial optimization problem of single aircraft strategy. Remain useful life (RUL) distribution of the key line replaceable Module (LRM) has been transformed into the failure probability of the aircraft and the fleet health status matrix is established. And the calculation method of the costs and risks for mission based on health status matrix and maintenance matrix is given. Further, an optimization method for fleet dispatch and CBM under acceptable risk is proposed based on an improved genetic algorithm. Finally, a fleet of 10 aircrafts is studied to verify the proposed method. The results shows that it could realize optimization and control of the aircraft fleet oriented to mission success.

An Optimization Method for Condition Based Maintenance of Aircraft Fleet Considering Prognostics Uncertainty

PubMed Central

Chen, Yiran; Sun, Bo; Li, Songjie

2014-01-01

An optimization method for condition based maintenance (CBM) of aircraft fleet considering prognostics uncertainty is proposed. The CBM and dispatch process of aircraft fleet is analyzed first, and the alternative strategy sets for single aircraft are given. Then, the optimization problem of fleet CBM with lower maintenance cost and dispatch risk is translated to the combinatorial optimization problem of single aircraft strategy. Remain useful life (RUL) distribution of the key line replaceable Module (LRM) has been transformed into the failure probability of the aircraft and the fleet health status matrix is established. And the calculation method of the costs and risks for mission based on health status matrix and maintenance matrix is given. Further, an optimization method for fleet dispatch and CBM under acceptable risk is proposed based on an improved genetic algorithm. Finally, a fleet of 10 aircrafts is studied to verify the proposed method. The results shows that it could realize optimization and control of the aircraft fleet oriented to mission success. PMID:24892046

The cellulose-binding activity of the PsB multiprotein complex is required for proper assembly of the spore coat and spore viability in Dictyostelium discoideum.

PubMed

Srinivasan, S; Griffiths, K R; McGuire, V; Champion, A; Williams, K L; Alexander, S

2000-08-01

The terminal event of spore differentiation in the cellular slime mould Dictyostelium discoideum is the assembly of the spore coat, which surrounds the dormant amoeba and allows the organism to survive during extended periods of environmental stress. The spore coat is a polarized extracellular matrix composed of glycoproteins and cellulose. The process of spore coat formation begins by the regulated secretion of spore coat proteins from the prespore vesicles (PSVs). Four of the major spore coat proteins (SP96, PsB/SP85, SP70 and SP60) exist as a preassembled multiprotein complex within the PSVs. This complete complex has an endogenous cellulose-binding activity. Mutant strains lacking either the SP96 or SP70 proteins produce partial complexes that do not have cellulose-binding activity, while mutants lacking SP60 produce a partial complex that retains this activity. Using a combination of immunofluorescence microscopy and biochemical methods we now show that the lack of cellulose-binding activity in the SP96 and SP70 mutants results in abnormally assembled spore coats and spores with greatly reduced viability. In contrast, the SP60 mutant, in which the PsB complex retains its cellulose-binding activity, produces spores with apparently unaltered structure and viability. Thus, it is the loss of the cellulose-binding activity of the PsB complex, rather than the mere loss of individual spore coat proteins, that results in compromised spore coat structure. These results support the idea that the cellulose-binding activity associated with the complete PsB complex plays an active role in the assembly of the spore coat.

Caldicellulosiruptor Core and Pangenomes Reveal Determinants for Noncellulosomal Thermophilic Deconstruction of Plant Biomass

PubMed Central

Blumer-Schuette, Sara E.; Giannone, Richard J.; Zurawski, Jeffrey V.; Ozdemir, Inci; Ma, Qin; Yin, Yanbin; Xu, Ying; Kataeva, Irina; Poole, Farris L.; Adams, Michael W. W.; Hamilton-Brehm, Scott D.; Elkins, James G.; Larimer, Frank W.; Land, Miriam L.; Hauser, Loren J.; Cottingham, Robert W.; Hettich, Robert L.

2012-01-01

Extremely thermophilic bacteria of the genus Caldicellulosiruptor utilize carbohydrate components of plant cell walls, including cellulose and hemicellulose, facilitated by a diverse set of glycoside hydrolases (GHs). From a biofuel perspective, this capability is crucial for deconstruction of plant biomass into fermentable sugars. While all species from the genus grow on xylan and acid-pretreated switchgrass, growth on crystalline cellulose is variable. The basis for this variability was examined using microbiological, genomic, and proteomic analyses of eight globally diverse Caldicellulosiruptor species. The open Caldicellulosiruptor pangenome (4,009 open reading frames [ORFs]) encodes 106 GHs, representing 43 GH families, but only 26 GHs from 17 families are included in the core (noncellulosic) genome (1,543 ORFs). Differentiating the strongly cellulolytic Caldicellulosiruptor species from the others is a specific genomic locus that encodes multidomain cellulases from GH families 9 and 48, which are associated with cellulose-binding modules. This locus also encodes a novel adhesin associated with type IV pili, which was identified in the exoproteome bound to crystalline cellulose. Taking into account the core genomes, pangenomes, and individual genomes, the ancestral Caldicellulosiruptor was likely cellulolytic and evolved, in some cases, into species that lost the ability to degrade crystalline cellulose while maintaining the capacity to hydrolyze amorphous cellulose and hemicellulose. PMID:22636774

Accelerating the Rate-Limiting Step in Novel Enzymatic Carbohydrate-to-Hydrogen Technology by Enzyme Engineering

DTIC Science & Technology

2011-10-30

stable phosphoglucose isomerase through immobilization of cellulose-binding module-tagged thermophilic enzyme on low- cost high-capacity celiulosic...NOVEL ENZYMATIC CARBOHYDRATE-TO-HYDROGEN TECHNOLOGY BY ENZYME ENGINEERING Grant/Contract Number: FA9550-08-1-0145 Program Manager: Dr. Walt...bbtransformation (SyPaB) is the implementation of complicated biochemical reactions by in vitro assembly of enzyme and coenzymes. Different from in vivo

Dissecting the functional significance of non-catalytic carbohydrate binding modules in the deconstruction of plant cell walls

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

Hahn, Michael G.

The project seeks to investigate the mechanism by which CBMs potentiate the activity of glycoside hydrolases against complete plant cell walls. The project is based on the hypothesis that the wide range of CBMs present in bacterial enzymes maximize the potential target substrates by directing the cognate enzymes not only to different regions of a specific plant cell wall, but also increases the range of plant cell walls that can be degraded. In addition to maximizing substrate access, it was also proposed that CBMs can target specific subsets of hydrolases with complementary activities to the same region of the plantmore » cell wall, thereby maximizing the synergistic interactions between these enzymes. This synergy is based on the premise that the hydrolysis of a specific polysaccharide will increase the access of closely associated polymers to enzyme attack. In addition, it is unclear whether the catalytic module and appended CBM of modular enzymes have evolved unique complementary activities.« less

The HADES-RICH upgrade using Hamamatsu H12700 MAPMTs with DiRICH FEE + Readout

NASA Astrophysics Data System (ADS)

Patel, V.; Traxler, M.

2018-03-01

The High Acceptance Di-Electron Spectrometer (HADES) is operational since the year 2000 and uses a hadron blind RICH detector for electron identification. The RICH photon detector is currently replaced by Hamamatsu H12700 MAPMTs with a readout system based on the DiRICH front-end module. The electronic readout chain is being developed as a joint effort of the HADES-, CBM- and PANDA collaborations and will also be used in the photon detectors for the upcoming Compressed Baryonic Matter (CBM) and PANDA experiments at FAIR . This article gives a brief overview on the photomultipliers and their quality assurance test measurements, as well as first measurements of the new DiRICH front-end module in final configurations.

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 based on the complexity (mono-/disaccharides vs. polysaccharides) and type of carbon (C5 vs. C6) available to the microorganism. The presence or increased abundance of extracellular proteins as a response to specific substrates helps to further elucidate C. bescii’s utilization and conversion of lignocellulosic biomass to biofuel and other valuable products. This includes improved characterization of extracellular proteins that lack discrete functional roles and are poorly/not annotated.« less

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 based on the complexity (mono-/disaccharides vs. polysaccharides) and type of carbon (C5 vs. C6) available to the microorganism. The presence or increased abundance of extracellular proteins as a response to specific substrates helps to further elucidate C. bescii’s utilization and conversion of lignocellulosic biomass to biofuel and other valuable products. This includes improved characterization of extracellular proteins that lack discrete functional roles and are poorly/not annotated.« less

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-/disaccharides vs. polysaccharides) and type of carbon (C5 vs. C6) available to the microorganism. The presence or increased abundance of extracellular proteins as a response to specific substrates helps to further elucidate C. bescii 's utilization and conversion of lignocellulosic biomass to biofuel and other valuable products. This includes improved characterization of extracellular proteins that lack discrete functional roles and are poorly/not annotated.

Multi-Mode Binding of Cellobiohydrolase Cel7A from Trichoderma reesei to Cellulose

PubMed Central

Jalak, Jürgen; Väljamäe, Priit

2014-01-01

Enzymatic hydrolysis of recalcitrant polysaccharides like cellulose takes place on the solid-liquid interface. Therefore the adsorption of enzymes to the solid surface is a pre-requisite for catalysis. Here we used enzymatic activity measurements with fluorescent model-substrate 4-methyl-umbelliferyl-β-D-lactoside for sensitive monitoring of the binding of cellobiohydrolase TrCel7A from Trichoderma reesei to bacterial cellulose (BC). The binding at low nanomolar free TrCel7A concentrations was exclusively active site mediated and was consistent with Langmuir's one binding site model with K d and A max values of 2.9 nM and 126 nmol/g BC, respectively. This is the strongest binding observed with non-complexed cellulases and apparently represents the productive binding of TrCel7A to cellulose chain ends on the hydrophobic face of BC microfibril. With increasing free TrCel7A concentrations the isotherm gradually deviated from the Langmuir's one binding site model. This was caused by the increasing contribution of lower affinity binding modes that included both active site mediated binding and non-productive binding with active site free from cellulose chain. The binding of TrCel7A to BC was found to be only partially reversible. Furthermore, the isotherm was dependent on the concentration of BC with more efficient binding observed at lower BC concentrations. The phenomenon can be ascribed to the BC concentration dependent aggregation of BC microfibrils with concomitant reduction of specific surface area. PMID:25265511

Evidence for glucocorticoid receptor binding to a site(s) in a remote region of the 5' flanking sequences of the human proopiomelanocortin gene

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

Tully, D.B.; Hillman, D.; Herbert, E.

1986-05-01

Glucocorticoids negatively regulate expression of the human proopiomelanocortin (POMC) gene. It has been postulated that this effect may be modulated by a direct interaction of the glucocorticoid receptor (GR) with DNA in the vicinity of the POMC promoter. In order to investigate interactions of GR with POMC DNA, DNA-cellulose competitive binding assays have been performed using isolated fragments of cloned POMC DNA to compete with calf thymus DNA-cellulose for binding of triamcinolone acetonide affinity-labelled GR prepared from HeLa S/sub 3/ cells. In these assays, two fragments isolated from the 5' flanking sequences of POMC DNA (Fragment 3,-1765 to -677 andmore » Fragment 4, -676 to +125 with respect to the mRNA cap site) have competed favorably, with Fragment 3 consistently competing more strongly than Fragment 4. Additional studies have been conducted utilizing a newly developed South-western Blot procedure in which specific /sup 32/P-labelled DNA fragments are allowed to bind to dexamethasone mesylate labelled GR immobilized on nitrocellulose filters. Results from these studies have also shown preferential binding by POMC DNA fragments 3 and 4. DNA footprinting and gene transfer experiments are now being conducted to further characterize the nature of GR interaction with POMC DNA.« less

Chromatographic and traditional albumin isotherms on cellulose: a model for wound protein adsorption on modified cotton

USDA-ARS?s Scientific Manuscript database

Albumin is the most abundant protein found in healing wounds. Traditional and chromatogrpahic protein isotherms of albumin binding on modified cotton fibers are useful in understanding albumin binding to cellulose wound dressings. An important consideration in the design of cellulosic wound dressin...

Biochemical characterization of a thermophilic β-mannanase from Talaromyces leycettanus JCM12802 with high specific activity.

PubMed

Wang, Caihong; Luo, Huiying; Niu, Canfang; Shi, Pengjun; Huang, Huoqing; Meng, Kun; Bai, Yingguo; Wang, Kun; Hua, Huifang; Yao, Bin

2015-02-01

Thermophilic β-mannanases are of increasing importance for wide industrial applications. In the current study, gene cloning, functional expression in Pichia pastoris, and characterization of a thermophilic β-mannanase (Man5A) from thermophilic Talaromyces leycettanus JCM12802 are reported. Deduced Man5A exhibits the highest identity with a putative β-mannanase from Talaromyces stipitatus ATCC10500 (70.3 %) and is composed of an N-terminal signal peptide, a fungal-type carbohydrate-binding module (CBM) of family 1, and a catalytic domain of glycosyl hydrolase (GH) family 5 at the C-terminus. Two recombinant proteins with different glycosylation levels, termed Man5A1 (72 kDa) and Man5A2 (60 kDa), were identified after purification. Both enzymes were thermophilic, exhibiting optimal activity at 85-90 °C, and were highly stable at 70 °C. Man5A1 and Man5A2 had a pH optimum of 4.5 and 4.0, respectively, and were highly stable over the broad pH range of 3.0-10.0. Most metal ions and sodium dodecyl sulfate (SDS) had no effect on the enzymatic activities. Man5A1 and Man5A2 exhibited high specific activity (2,160 and 1,800 U/mg, respectively) when using locust bean gum as the substrate. The CBM1 and two key residues D191 and R286 were found to affect Man5A thermostability. Man5A displays a classical four-site-binding mode, hydrolyzing mannooligosaccharides into smaller units, galactomannan into mannose and mannobiose, and glucomanman into mannose, mannobiose, and mannopentaose, respectively. All these properties make Man5A a good candidate for extensive applications in the bioconversion, pulp bleaching, textile, food, and feed industries.

Cellulose-hemicellulose interaction in wood secondary cell-wall

NASA Astrophysics Data System (ADS)

Zhang, Ning; Li, Shi; Xiong, Liming; Hong, Yu; Chen, Youping

2015-12-01

The wood cell wall features a tough and relatively rigid fiber reinforced composite structure. It acts as a pressure vessel, offering protection against mechanical stress. Cellulose microfibrils, hemicellulose and amorphous lignin are the three major components of wood. The structure of secondary cell wall could be imagined as the same as reinforced concrete, in which cellulose microfibrils acts as reinforcing steel bar and hemicellulose-lignin matrices act as the concrete. Therefore, the interface between cellulose and hemicellulose/lignin plays a significant role in determine the mechanical behavior of wood secondary cell wall. To this end, we present a molecular dynamics (MD) simulation study attempting to quantify the strength of the interface between cellulose microfibrils and hemicellulose. Since hemicellulose binds with adjacent cellulose microfibrils in various patterns, the atomistic models of hemicellulose-cellulose composites with three typical binding modes, i.e. bridge, loop and random binding modes are constructed. The effect of the shape of hemicellulose chain on the strength of hemicellulose-cellulose composites under shear loadings is investigated. The contact area as well as hydrogen bonds between cellulose and hemicellulose, together with the covalent bonds in backbone of hemicellulose chain are found to be the controlling parameters which determine the strength of the interfaces in the composite system. For the bridge binding model, the effect of shear loading direction on the strength of the cellulose material is also studied. The obtained results suggest that the shear strength of wood-inspired engineering composites can be optimized through maximizing the formations of the contributing hydrogen bonds between cellulose and hemicellulose.

Micelle-Triggered β-Hairpin to α-Helix Transition in a 14-Residue Peptide from a Choline-Binding Repeat of the Pneumococcal Autolysin LytA

PubMed Central

Zamora-Carreras, Héctor; Maestro, Beatriz; Strandberg, Erik; Ulrich, Anne S; Sanz, Jesús M; Jiménez, M Ángeles

2015-01-01

Choline-binding modules (CBMs) have a ββ-solenoid structure composed of choline-binding repeats (CBR), which consist of a β-hairpin followed by a short linker. To find minimal peptides that are able to maintain the CBR native structure and to evaluate their remaining choline-binding ability, we have analysed the third β-hairpin of the CBM from the pneumococcal LytA autolysin. Circular dichroism and NMR data reveal that this peptide forms a highly stable native-like β-hairpin both in aqueous solution and in the presence of trifluoroethanol, but, strikingly, the peptide structure is a stable amphipathic α-helix in both zwitterionic (dodecylphosphocholine) and anionic (sodium dodecylsulfate) detergent micelles, as well as in small unilamellar vesicles. This β-hairpin to α-helix conversion is reversible. Given that the β-hairpin and α-helix differ greatly in the distribution of hydrophobic and hydrophilic side chains, we propose that the amphipathicity is a requirement for a peptide structure to interact and to be stable in micelles or lipid vesicles. To our knowledge, this “chameleonic” behaviour is the only described case of a micelle-induced structural transition between two ordered peptide structures. PMID:25917218

Collagen-binding proteins of Streptococcus mutans and related streptococci.

PubMed

Avilés-Reyes, A; Miller, J H; Lemos, J A; Abranches, J

2017-04-01

The ability of Streptococcus mutans to interact with collagen through the expression of collagen-binding proteins (CBPs) bestows this oral pathogen with an alternative to the sucrose-dependent mechanism of colonization classically attributed to caries development. Based on the abundance and distribution of collagen throughout the human body, stringent adherence to this molecule grants S. mutans with the opportunity to establish infection at different host sites. Surface proteins, such as SpaP, WapA, Cnm and Cbm, have been shown to bind collagen in vitro, and it has been suggested that these molecules play a role in colonization of oral and extra-oral tissues. However, robust collagen binding is not achieved by all strains of S. mutans, particularly those that lack Cnm or Cbm. These observations merit careful dissection of the contribution from these different CBPs towards tissue colonization and virulence. In this review, we will discuss the current understanding of mechanisms used by S. mutans and related streptococci to colonize collagenous tissues, and the possible contribution of CBPs to infections in different sites of the host. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Domain wise docking analyses of the modular chitin binding protein CBP50 from Bacillus thuringiensis serovar konkukian S4.

PubMed

Sehar, Ujala; Mehmood, Muhammad Aamer; Hussain, Khadim; Nawaz, Salman; Nadeem, Shahid; Siddique, Muhammad Hussnain; Nadeem, Habibullah; Gull, Munazza; Ahmad, Niaz; Sohail, Iqra; Gill, Saba Shahid; Majeed, Summera

2013-01-01

This paper presents an in silico characterization of the chitin binding protein CBP50 from B. thuringiensis serovar konkukian S4 through homology modeling and molecular docking. The CBP50 has shown a modular structure containing an N-terminal CBM33 domain, two consecutive fibronectin-III (Fn-III) like domains and a C-terminal CBM5 domain. The protein presented a unique modular structure which could not be modeled using ordinary procedures. So, domain wise modeling using MODELLER and docking analyses using Autodock Vina were performed. The best conformation for each domain was selected using standard procedure. It was revealed that four amino acid residues Glu-71, Ser-74, Glu-76 and Gln-90 from N-terminal domain are involved in protein-substrate interaction. Similarly, amino acid residues Trp-20, Asn-21, Ser-23 and Val-30 of Fn-III like domains and Glu-15, Ala-17, Ser-18 and Leu-35 of C-terminal domain were involved in substrate binding. Site-directed mutagenesis of these proposed amino acid residues in future will elucidate the key amino acids involved in chitin binding activity of CBP50 protein.

The cellulose binding region in Trichoderma reesei cellobiohydrolase I has a higher capacity in improving crystalline cellulose degradation than that of Penicillium oxalicum.

PubMed

Du, Jian; Zhang, Xiu; Li, Xuezhi; Zhao, Jian; Liu, Guodong; Gao, Baoyu; Qu, Yinbo

2018-06-19

Commercial cellulase preparations for lignocellulose bioconversion are mainly produced by the fungus Trichoderma reesei. The maximum cellulose conversion of T. reesei cellulase mixture was 15%-20% higher than that of Penicillium oxalicum in the hydrolysis of corncob residue and Avicel. Nevertheless, both preparations hydrolyzed more than 92% of cellulose in NaOH-mercerized Avicel. When added to Avicel hydrolysis residue that was less reactive to P. oxalicum cellulases, cellobiohydrolase I (CBH I) from T. reesei resulted in a higher cellulose conversion than its homologous proteins from P. oxalicum and Aspergillus niger at the same protein loadings. Further domain exchange experiment attributed the high hydrolytic efficiency of T. reesei CBH I to its inter-domain linker and cellulose-binding domain. The results in part explained the superior performance of T. reesei cellulases on the degradation of native crystalline cellulose, and highlighted the important role of cellulose-binding region in determining the degree of hydrolysis by cellulases. Copyright © 2018 Elsevier Ltd. All rights reserved.

Computational engineering of cellulase Cel9A-68 functional motions through mutations in its linker region.

PubMed

Costa, M G S; Silva, Y F; Batista, P R

2018-03-14

Microbial cellulosic degradation by cellulases has become a complementary approach for biofuel production. However, its efficiency is hindered by the recalcitrance of cellulose fibres. In this context, computational protein design methods may offer an efficient way to obtain variants with improved enzymatic activity. Cel9A-68 is a cellulase from Thermobifida fusca that is still active at high temperatures. In a previous work, we described a collective bending motion, which governs the overall cellulase dynamics. This movement promotes the approximation of its CBM and CD structural domains (that are connected by a flexible linker). We have identified two residues (G460 and P461) located at the linker that act as a hinge point. Herein, we applied a new level of protein design, focusing on the modulation of this collective motion to obtain cellulase variants with enhanced functional dynamics. We probed whether specific linker mutations would affect Cel9A-68 dynamics through computational simulations. We assumed that P461G and G460+ (with an extra glycine) constructs would present enhanced interdomain motions, while the G460P mutant would be rigid. From our results, the P461G mutation resulted in a broader exploration of the conformational space, as confirmed by clustering and free energy analyses. The WT enzyme was the most rigid system. However, G460P and P460+ explored distinct conformational states described by opposite directions of low-frequency normal modes; they sampled preferentially closed and open conformations, respectively. Overall, we highlight two significant findings: (i) all mutants explored larger conformational spaces than the WT; (ii) the selection of distinct conformational populations was intimately associated with the mutation considered. Thus, the engineering of Cel9A-68 motions through linker mutations may constitute an efficient way to improve cellulase activity, facilitating the disruption of cellulose fibres.

Identification of the uridine 5'-diphosphoglucose (UDP-Glc) binding subunit of cellulose synthase in Acetobacter xylinum using the photoaffinity probe 5-azido-UDP-Glc

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

Lin, F.C.; Brown, R.M. Jr.; Drake, R.R. Jr.

1990-03-25

Photoaffinity labeling of purified cellulose synthase with (beta-32P)5-azidouridine 5'-diphosphoglucose (UDP-Glc) has been used to identify the UDP-Glc binding subunit of the cellulose synthase from Acetobacter xylinum strain ATCC 53582. The results showed exclusive labeling of an 83-kDa polypeptide. Photoinsertion of (beta-32P)5-azido-UDP-Glc is stimulated by the cellulose synthase activator, bis-(3'----5') cyclic diguanylic acid. Addition of increasing amounts of UDP-Glc prevents photolabeling of the 83-kDa polypeptide. The reversible and photocatalyzed binding of this photoprobe also showed saturation kinetics. These studies demonstrate that the 83-kDa polypeptide is the catalytic subunit of the cellulose synthase in A. xylinum strain ATCC 53582.

Bratislava Symposium on Saccharides (7th) Programme and Abstracts

DTIC Science & Technology

1994-09-01

that of cellulose (1). Althoug the binding capacity of cellulose microfibrils is dependent on the sace of the binding un of the kmfbrul& xyloglucans...are not only party embedded in but are also parly free between microfibrils . suggesting cross-link to cellulose microfibuils (2). Xyloglucan...desediftcoli Y.-C.-M a 12. KoIlkovd B., Hricovfrni M., Sirmoutti R.: 43C NMR study of solid-stal, reaction of cellulose with lIgnin monomers 13. Joniak D

Cellulose binding domain proteins

DOEpatents

Shoseyov, O.; Shpiegl, I.; Goldstein, M.; Doi, R.

1998-11-17

A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 16 figs.

Cellulose binding domain proteins

DOEpatents

Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc; Doi, Roy

1998-01-01

A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

Cellulose binding domain fusion proteins

DOEpatents

Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc A.; Doi, Roy H.

1998-01-01

A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

Performance enhancement of perovskite solar cells with Mg-doped TiO{sub 2} compact film as the hole-blocking layer

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

Wang, Jing; Qin, Minchao; Tao, Hong

2015-03-23

In this letter, we report perovskite solar cells with thin dense Mg-doped TiO{sub 2} as hole-blocking layers (HBLs), which outperform cells using TiO{sub 2} HBLs in several ways: higher open-circuit voltage (V{sub oc}) (1.08 V), power conversion efficiency (12.28%), short-circuit current, and fill factor. These properties improvements are attributed to the better properties of Mg-modulated TiO{sub 2} as compared to TiO{sub 2} such as better optical transmission properties, upshifted conduction band minimum (CBM) and downshifted valence band maximum (VBM), better hole-blocking effect, and higher electron life time. The higher-lying CBM due to the modulation with wider band gap MgO and themore » formation of magnesium oxide and magnesium hydroxides together resulted in an increment of V{sub oc}. In addition, the Mg-modulated TiO{sub 2} with lower VBM played a better role in the hole-blocking. The HBL with modulated band position provided better electron transport and hole blocking effects within the device.« less

The inhibition of hemicellulosic sugars on cellulose hydrolysis are highly dependant on the cellulase productive binding, processivity, and substrate surface charges.

PubMed

Zhai, Rui; Hu, Jinguang; Saddler, Jack N

2018-06-01

In this study, the influence of major hemicellulosic sugars (mannose and xylose) on cellulose hydrolysis and major enzyme activities were evaluated by using both commercial enzyme cocktail and purified cellulase monocomponents over a "library" of cellulosic substrates. Surprisingly, the results showed that unlike glucose, mannose/xylose did not inhibit individual cellulase activities but significantly decreased their hydrolytic performance on cellulose substrates. When various enzyme-substrate interactions (e.g. adsorption/desorption, productive binding, and processive moving) were evaluated, it appeared that these hemicellulosic sugars significantly reduced the productive binding and processivity of Cel7A, which in turn limited cellulase hydrolytic efficacy. Among a range of major cellulose characteristics (e.g. crystallinity, degree of polymerization, accessibility, and surface charges), the acid group content of the cellulosic substrates seemed to be the main driver that determined the extent of hemicellulosic sugar inhibition. Our results provided new insights for better understanding the sugar inhibition mechanisms of cellulose hydrolysis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cellulose binding domain fusion proteins

DOEpatents

Shoseyov, O.; Yosef, K.; Shpiegl, I.; Goldstein, M.A.; Doi, R.H.

1998-02-17

A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 16 figs.

Methods of use of cellulose binding domain proteins

DOEpatents

Shoseyov, O.; Shpiegl, I.; Goldstein, M.A.; Doi, R.H.

1997-09-23

A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 16 figs.

Nucleic acids encoding a cellulose binding domain

DOEpatents

Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc A.; Doi, Roy H.

1996-01-01

A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

Nucleic acids encoding a cellulose binding domain

DOEpatents

Shoseyov, O.; Shpiegl, I.; Goldstein, M.A.; Doi, R.H.

1996-03-05

A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 15 figs.

Methods of use of cellulose binding domain proteins

DOEpatents

Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc A.; Doi, Roy H.

1997-01-01

A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

A kinetic study of Trichoderma reesei Cel7B catalyzed cellulose hydrolysis.

PubMed

Song, Xiangfei; Zhang, Shujun; Wang, Yefei; Li, Jingwen; He, Chunyan; Yao, Lishan

2016-06-01

One prominent feature of Trichoderma reesei (Tr) endoglucanases catalyzed cellulose hydrolysis is that the reaction slows down quickly after it starts (within minutes). But the mechanism of the slowdown is not well understood. A structural model of Tr- Cel7B catalytic domain bound to cellulose was built computationally and the potentially important binding residues were identified and tested experimentally. The 13 tested mutants show different binding properties in the adsorption to phosphoric acid swollen cellulose and filter paper. Though the partitioning parameter to filter paper is about 10 times smaller than that to phosphoric acid swollen cellulose, a positive correlation is shown for two substrates. The kinetic studies show that the reactions slow down quickly for both substrates. This slowdown is not correlated to the binding constant but anticorrelated to the enzyme initial activity. The amount of reducing sugars released after 24h by Cel7B in phosphoric acid swollen cellulose, Avicel and filter paper cellulose hydrolysis is correlated with the enzyme activity against a soluble substrate p-nitrophenyl lactoside. Six of the 13 tested mutants, including N47A, N52D, S99A, N323D, S324A, and S346A, yield ∼15-35% more reducing sugars than the wild type (WT) Cel7B in phosphoric acid swollen cellulose and filter paper hydrolysis. This study reveals that the slowdown of the reaction is not due to the binding of the enzyme to cellulose. The activity of Tr- Cel7B against the insoluble substrate cellulose is determined by the enzyme's capability in hydrolyzing the soluble substrate. Copyright © 2016 Elsevier Inc. All rights reserved.

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

Cellulolytic potential of probiotic Bacillus Subtilis AMS6 isolated from traditional fermented soybean (Churpi): An in-vitro study with regards to application as an animal feed additive.

PubMed

Manhar, Ajay K; Bashir, Yasir; Saikia, Devabrata; Nath, Dhrubajyoti; Gupta, Kuldeep; Konwar, Bolin K; Kumar, Rahul; Namsa, Nima D; Mandal, Manabendra

2016-01-01

The aim of the present study is to evaluate the probiotic attributes of Bacillus subtilis AMS6 isolated from fermented soybean (Churpi). This isolate exhibited tolerance to low pH (pH 2.0) and bile salt (0.3%), capability to autoaggregate and coaggregate. AMS6 also showed highest antibacterial activity against the pathogenic indicator strain Salmonella enterica typhimurium (MTCC 1252) and susceptibility towards different antibiotics tested. The isolate was effective in inhibiting the adherence of food borne pathogens to Caco-2 epithelial cell lines, and was also found to be non-hemolytic which further strengthen the candidature of the isolate as a potential probiotic. Further studies revealed B. subtilis AMS6 showed cellulolytic activity (0.54±0.05 filter paper units mL(-1)) at 37°C. The isolate was found to hydrolyze carboxymethyl cellulose, filter paper and maize (Zea mays) straw. The maize straw digestion was confirmed by scanning electron microscopy studies. The isolate was able to degrade filter paper within 96h of incubation. A full length cellulase gene of AMS6 was amplified using degenerate primers consisting of 1499 nucleotides. The ORF encoded for a protein of 499 amino acids residues with a predicted molecular mass of 55.04kDa. The amino acids sequence consisted of a glycosyl hydrolase family 5 domain at N-terminal; Glycosyl hydrolase catalytic core and a CBM-3 cellulose binding domain at its C terminal. The study suggests potential probiotic B. subtilis AMS6 as a promising candidate envisaging its application as an animal feed additive for enhanced fiber digestion and gut health of animal. Copyright © 2016 Elsevier GmbH. All rights reserved.

New perspective on glycoside hydrolase binding to lignin from pretreated corn stover

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

Yarbrough, John M.; Mittal, Ashutosh; Mansfield, Elisabeth

Background: Non-specific binding of cellulases to lignin has been implicated as a major factor in the loss of cellulase activity during biomass conversion to sugars. It is believed that this binding may strongly impact process economics through loss of enzyme activities during hydrolysis and enzyme recycling scenarios. The current model suggests glycoside hydrolase activities are lost though non-specific/non-productive binding of carbohydrate-binding domains to lignin, limiting catalytic site access to the carbohydrate components of the cell wall. Results: In this study, we compared component enzyme affinities of a commercial Trichoderma reesei cellulase formulation, Cellic CTec2, towards extracted corn stover lignin usingmore » sodium dodecyl sulfate-polyacrylamide gel electrophoresis and p-nitrophenyl substrate activities to monitor component binding, activity loss, and total protein binding. Protein binding was strongly affected by pH and ionic strength. β-D-glucosidases and xylanases, which do not have carbohydrate-binding modules (CBMs) and are basic proteins, demonstrated the strongest binding at low ionic strength, suggesting that CBMs are not the dominant factor in enzyme adsorption to lignin. Despite strong adsorption to insoluble lignin, β-D-glucosidase and xylanase activities remained high, with process yields decreasing only 4–15 % depending on lignin concentration. Conclusion: We propose that specific enzyme adsorption to lignin from a mixture of biomass-hydrolyzing enzymes is a competitive affinity where β-D-glucosidases and xylanases can displace CBM interactions with lignin. Process parameters, such as temperature, pH, and salt concentration influence the individual enzymes’ affinity for lignin, and both hydrophobic and electrostatic interactions are responsible for this binding phenomenon. Moreover, our results suggest that concern regarding loss of critical cell wall degrading enzymes to lignin adsorption may be unwarranted when complex enzyme mixtures are used to digest biomass.« less

New perspective on glycoside hydrolase binding to lignin from pretreated corn stover

DOE PAGES

Yarbrough, John M.; Mittal, Ashutosh; Mansfield, Elisabeth; ...

2015-12-18

Background: Non-specific binding of cellulases to lignin has been implicated as a major factor in the loss of cellulase activity during biomass conversion to sugars. It is believed that this binding may strongly impact process economics through loss of enzyme activities during hydrolysis and enzyme recycling scenarios. The current model suggests glycoside hydrolase activities are lost though non-specific/non-productive binding of carbohydrate-binding domains to lignin, limiting catalytic site access to the carbohydrate components of the cell wall. Results: In this study, we compared component enzyme affinities of a commercial Trichoderma reesei cellulase formulation, Cellic CTec2, towards extracted corn stover lignin usingmore » sodium dodecyl sulfate-polyacrylamide gel electrophoresis and p-nitrophenyl substrate activities to monitor component binding, activity loss, and total protein binding. Protein binding was strongly affected by pH and ionic strength. β-D-glucosidases and xylanases, which do not have carbohydrate-binding modules (CBMs) and are basic proteins, demonstrated the strongest binding at low ionic strength, suggesting that CBMs are not the dominant factor in enzyme adsorption to lignin. Despite strong adsorption to insoluble lignin, β-D-glucosidase and xylanase activities remained high, with process yields decreasing only 4–15 % depending on lignin concentration. Conclusion: We propose that specific enzyme adsorption to lignin from a mixture of biomass-hydrolyzing enzymes is a competitive affinity where β-D-glucosidases and xylanases can displace CBM interactions with lignin. Process parameters, such as temperature, pH, and salt concentration influence the individual enzymes’ affinity for lignin, and both hydrophobic and electrostatic interactions are responsible for this binding phenomenon. Moreover, our results suggest that concern regarding loss of critical cell wall degrading enzymes to lignin adsorption may be unwarranted when complex enzyme mixtures are used to digest biomass.« less

Role of IκB kinase β in regulating the remodeling of the CARMA1-Bcl10-MALT1 complex.

PubMed

Karim, Zubair A; Hensch, Nicole R; Qasim, Hanan; Alshbool, Fatima Z; Khasawneh, Fadi T

2018-06-02

The current work investigates the notion that inducible clustering of signaling mediators of the IKK pathway is important for platelet activation. Thus, while the CARMA1, Bcl10, and MALT1 (CBM) complex is essential for triggering IKK/NF-κB activation upon platelet stimulation, the signals that elicit its formation and downstream effector activation remain elusive. We demonstrate herein that IKKβ is involved in membrane fusion, and serves as a critical protein kinase required for initial formation and the regulation of the CARMA1/MALT1/Bcl10/CBM complex in platelets. We also show that IKKβ regulates these processes via modulation of phosphorylation of Bcl10 and IKKγ polyubiquitination. Collectively, our data demonstrate that IKKβ regulates membrane fusion and the remodeling of the CBM complex formation. Copyright © 2018 Elsevier Inc. All rights reserved.

Methods of detection using a cellulose binding domain fusion product

DOEpatents

Shoseyov, O.; Shpiegl, I.; Goldstein, M.A.; Doi, R.H.

1999-01-05

A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 34 figs.

Kits and methods of detection using cellulose binding domain fusion proteins

DOEpatents

Shoseyov, O.; Yosef, K.

1998-04-14

A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 16 figs.

Kits and methods of detection using cellulose binding domain fusion proteins

DOEpatents

Shoseyov, Oded

1998-01-01

A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

Methods of detection using a cellulose binding domain fusion product

DOEpatents

Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc A.; Doi, Roy H.

1999-01-01

A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

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 inexpensive plant feedstocks to products at high yields.« less

PROTEIN TARGETING TO STARCH Is Required for Localising GRANULE-BOUND STARCH SYNTHASE to Starch Granules and for Normal Amylose Synthesis in Arabidopsis

PubMed Central

Seung, David; Soyk, Sebastian; Coiro, Mario; Maier, Benjamin A.; Eicke, Simona; Zeeman, Samuel C.

2015-01-01

The domestication of starch crops underpinned the development of human civilisation, yet we still do not fully understand how plants make starch. Starch is composed of glucose polymers that are branched (amylopectin) or linear (amylose). The amount of amylose strongly influences the physico-chemical behaviour of starchy foods during cooking and of starch mixtures in non-food manufacturing processes. The GRANULE-BOUND STARCH SYNTHASE (GBSS) is the glucosyltransferase specifically responsible for elongating amylose polymers and was the only protein known to be required for its biosynthesis. Here, we demonstrate that PROTEIN TARGETING TO STARCH (PTST) is also specifically required for amylose synthesis in Arabidopsis. PTST is a plastidial protein possessing an N-terminal coiled coil domain and a C-terminal carbohydrate binding module (CBM). We discovered that Arabidopsis ptst mutants synthesise amylose-free starch and are phenotypically similar to mutants lacking GBSS. Analysis of granule-bound proteins showed a dramatic reduction of GBSS protein in ptst mutant starch granules. Pull-down assays with recombinant proteins in vitro, as well as immunoprecipitation assays in planta, revealed that GBSS physically interacts with PTST via a coiled coil. Furthermore, we show that the CBM domain of PTST, which mediates its interaction with starch granules, is also required for correct GBSS localisation. Fluorescently tagged Arabidopsis GBSS, expressed either in tobacco or Arabidopsis leaves, required the presence of Arabidopsis PTST to localise to starch granules. Mutation of the CBM of PTST caused GBSS to remain in the plastid stroma. PTST fulfils a previously unknown function in targeting GBSS to starch. This sheds new light on the importance of targeting biosynthetic enzymes to sub-cellular sites where their action is required. Importantly, PTST represents a promising new gene target for the biotechnological modification of starch composition, as it is exclusively involved in amylose synthesis. PMID:25710501

Cellulose production, activated by cyclic di-GMP through BcsA and BcsZ, is a virulence factor and an essential determinant of the three-dimensional architectures of biofilms formed by Erwinia amylovora Ea1189.

PubMed

Castiblanco, Luisa F; Sundin, George W

2018-01-01

Bacterial biofilms are multicellular aggregates encased in an extracellular matrix mainly composed of exopolysaccharides (EPSs), protein and nucleic acids, which determines the architecture of the biofilm. Erwinia amylovora Ea1189 forms a biofilm inside the xylem of its host, which results in vessel plugging and water transport impairment. The production of the EPSs amylovoran and levan is critical for the formation of a mature biofilm. In addition, cyclic dimeric GMP (c-di-GMP) has been reported to positively regulate amylovoran biosynthesis and biofilm formation in E. amylovora Ea1189. In this study, we demonstrate that cellulose is synthesized by E. amylovora Ea1189 and is a major modulator of the three-dimensional characteristics of biofilms formed by this bacterium, and also contributes to virulence during systemic host invasion. In addition, we demonstrate that the activation of cellulose biosynthesis in E. amylovora is a c-di-GMP-dependent process, through allosteric binding to the cellulose catalytic subunit BcsA. We also report that the endoglucanase BcsZ is a key player in c-di-GMP activation of cellulose biosynthesis. Our results provide evidence of the complex composition of the extracellular matrix produced by E. amylovora and the implications of cellulose biosynthesis in shaping the architecture of the biofilm and in the expression of one of the main virulence phenotypes of this pathogen. © 2016 BSPP AND JOHN WILEY & SONS LTD.

Evolution of GHF5 endoglucanase gene structure in plant-parasitic nematodes: no evidence for an early domain shuffling event.

PubMed

Kyndt, Tina; Haegeman, Annelies; Gheysen, Godelieve

2008-11-03

Endo-1,4-beta-glucanases or cellulases from the glycosyl hydrolase family 5 (GHF5) have been found in numerous bacteria and fungi, and recently also in higher eukaryotes, particularly in plant-parasitic nematodes (PPN). The origin of these genes has been attributed to horizontal gene transfer from bacteria, although there still is a lot of uncertainty about the origin and structure of the ancestral GHF5 PPN endoglucanase. It is not clear whether this ancestral endoglucanase consisted of the whole gene cassette, containing a catalytic domain and a carbohydrate-binding module (CBM, type 2 in PPN and bacteria) or only of the catalytic domain while the CBM2 was retrieved by domain shuffling later in evolution. Previous studies on the evolution of these genes have focused primarily on data of sedentary nematodes, while in this study, extra data from migratory nematodes were included. Two new endoglucanases from the migratory nematodes Pratylenchus coffeae and Ditylenchus africanus were included in this study. The latter one is the first gene isolated from a PPN of a different superfamily (Sphaerularioidea); all previously known nematode endoglucanases belong to the superfamily Tylenchoidea (order Rhabditida). Phylogenetic analyses were conducted with the PPN GHF5 endoglucanases and homologous endoglucanases from bacterial and other eukaryotic lineages such as beetles, fungi and plants. No statistical incongruence between the phylogenetic trees deduced from the catalytic domain and the CBM2 was found, which could suggest that both domains have evolved together. Furthermore, based on gene structure data, we inferred a model for the evolution of the GHF5 endoglucanase gene structure in plant-parasitic nematodes. Our data confirm a close relationship between Pratylenchus spp. and the root knot nematodes, while some Radopholus similis endoglucanases are more similar to cyst nematode genes. We conclude that the ancestral PPN GHF5 endoglucanase gene most probably consisted of the whole gene cassette, i.e. the GHF5 catalytic domain and the CBM2, rather than that it evolved by domain shuffling. Our evolutionary model for the gene structure in PPN GHF5 endoglucanases implies the occurrence of an early duplication event, and more recent gene duplications at genus or species level.

Outer membrane proteins related to SusC and SusD are not required for Cytophaga hutchinsonii cellulose utilization.

PubMed

Zhu, Yongtao; Kwiatkowski, Kurt J; Yang, Tengteng; Kharade, Sampada S; Bahr, Constance M; Koropatkin, Nicole M; Liu, Weifeng; McBride, Mark J

2015-08-01

Cytophaga hutchinsonii, a member of the phylum Bacteroidetes, employs a novel collection of cell-associated proteins to digest crystalline cellulose. Other Bacteroidetes rely on cell surface proteins related to the starch utilization system (Sus) proteins SusC and SusD to bind oligosaccharides and import them across the outer membrane for further digestion. These bacteria typically produce dozens of SusC-like porins and SusD-like oligosaccharide-binding proteins to facilitate utilization of diverse polysaccharides. C. hutchinsonii specializes in cellulose digestion and its genome has only two susC-like genes and two susD-like genes. Single and multiple gene deletions were constructed to determine if the susC-like and susD-like genes have roles in cellulose utilization. A mutant lacking all susC-like and all susD-like genes digested cellulose and grew on cellulose as well as wild-type cells. Further, recombinantly expressed SusD-like proteins CHU_0547 and CHU_0554 failed to bind cellulose or β-glucan hemicellulosic polysaccharides. The results suggest that the Bacteroidetes Sus paradigm for polysaccharide utilization may not apply to the cellulolytic bacterium C. hutchinsonii.

Cellulase Linkers Are Optimized Based on Domain Type and Function: Insights from Sequence Analysis, Biophysical Measurements, and Molecular Simulation

PubMed Central

Sammond, Deanne W.; Payne, Christina M.; Brunecky, Roman; Himmel, Michael E.; Crowley, Michael F.; Beckham, Gregg T.

2012-01-01

Cellulase enzymes deconstruct cellulose to glucose, and are often comprised of glycosylated linkers connecting glycoside hydrolases (GHs) to carbohydrate-binding modules (CBMs). Although linker modifications can alter cellulase activity, the functional role of linkers beyond domain connectivity remains unknown. Here we investigate cellulase linkers connecting GH Family 6 or 7 catalytic domains to Family 1 or 2 CBMs, from both bacterial and eukaryotic cellulases to identify conserved characteristics potentially related to function. Sequence analysis suggests that the linker lengths between structured domains are optimized based on the GH domain and CBM type, such that linker length may be important for activity. Longer linkers are observed in eukaryotic GH Family 6 cellulases compared to GH Family 7 cellulases. Bacterial GH Family 6 cellulases are found with structured domains in either N to C terminal order, and similar linker lengths suggest there is no effect of domain order on length. O-glycosylation is uniformly distributed across linkers, suggesting that glycans are required along entire linker lengths for proteolysis protection and, as suggested by simulation, for extension. Sequence comparisons show that proline content for bacterial linkers is more than double that observed in eukaryotic linkers, but with fewer putative O-glycan sites, suggesting alternative methods for extension. Conversely, near linker termini where linkers connect to structured domains, O-glycosylation sites are observed less frequently, whereas glycines are more prevalent, suggesting the need for flexibility to achieve proper domain orientations. Putative N-glycosylation sites are quite rare in cellulase linkers, while an N-P motif, which strongly disfavors the attachment of N-glycans, is commonly observed. These results suggest that linkers exhibit features that are likely tailored for optimal function, despite possessing low sequence identity. This study suggests that cellulase linkers may exhibit function in enzyme action, and highlights the need for additional studies to elucidate cellulase linker functions. PMID:23139804

Polysaccharide structures and interactions in a lithium chloride/urea/water solvent.

PubMed

Winkworth-Smith, Charles G; MacNaughtan, William; Foster, Tim J

2016-09-20

The molten salt hydrate, lithium chloride (LiCl)/urea/water has previously been shown to swell cellulose, but there has so far been no work done to explore its effect on other polysaccharides. In this paper we have investigated the solvent effects of LiCl/urea/water on four natural polysaccharides. Fenugreek gum and xyloglucan, which are both highly branched, were found to increase in viscosity in LiCl/urea/water relative to water, possibly due to the breakage of all intra-molecular associations whereas the viscosity of konjac glucomannan which is predominantly unbranched did not change. Locust bean gum (LBG) had a lower viscosity in LiCl/urea/water compared to water due to the disruption of aggregates. Confocal microscopy showed that fenugreek gum and LBG are able to bind to cellulose in water, however, the conformational change of fenugreek gum in these solvent conditions inhibited it from binding to cellulose in LiCl/urea/water whereas conformational change allowed xyloglucan to bind to cellulose in LiCl/urea/water whilst it was unable to bind in water. Konjac glucomannan did not bind to cellulose in either solvent system. These results provide new insights into the impact of polysaccharide fine structure on conformational change in different solvent environments. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

Enzymatic conversion of pretreated biomass into fermentable sugars for biorefinery operation

NASA Astrophysics Data System (ADS)

Gao, Dahai

2011-12-01

Depleting petroleum reserves and potential climate change caused by fossil fuel consumption have attracted significant attention towards the use of alternative renewable resources for production of fuels and chemicals. Lignocellulosic biomass provides a plentiful resource for the sustainable production of biofuels and biochemicals and could serve as an important contributor to the world energy portfolio in the near future. Successful biological conversion of lignocellulosic biomass requires an efficient and economical pretreatment method, high glucose/xylose yields during enzymatic hydrolysis and fermentation of both hexose and pentose to ethanol. High enzyme loading is a major economic bottleneck for the commercial processing of pretreated lignocellulosic biomass to produce fermentable sugars. Optimizing the enzyme cocktail for specific types of pretreated biomass allows for a significant reduction in enzyme loading without sacrificing hydrolysis yield. Core glycosyl hydrolases were isolated and purified from various sources to help rationally optimize an enzyme cocktail to digest ammonia fiber expansion (AFEX) treated corn stover. The four core cellulases were endoglucanase I (EG I), cellobiohydrolase I (CBH I), cellobiohydrolase II (CBH II) and beta-Glucosidase (betaG). The two core hemicellulases were an endoxylanase (EX) and a beta-xylosidase (betaX). A diverse set of accessory hemicellulases from bacterial sources was found necessary to enhance the synergistic action of cellulases hydrolysing AFEX pretreated corn stover. High glucose (around 80%) and xylose (around 70%) yields were achieved with a moderate enzyme loading (˜20 mg protein/g glucan) using an in-house developed enzyme cocktail and this cocktail was compared to commercial enzyme. Studying the binding properties of cellulases to lignocellulosic substrates is critical to achieving a fundamental understanding of plant cell wall saccharification. Lignin auto-fluorescence and degradation products formed during pretreatment impede accurate quantification of individual glycosyl hydrolases (GH) binding to pretreated cell walls. A high-throughput Fast Protein Liquid Chromatography (HT-FPLC) based method has been developed to quantify CBH I, CBH II and EG I present in hydrolyzates of untreated, AFEX, and dilute-acid pretreated corn stover. This method can accurately quantify individual enzymes present in complex binary and ternary protein mixtures without interference from plant cell wall derived components. The binding characteristics of CBH I, CBH II and EG I during 48 hours hydrolysis were studied on different cellulose allomorphs: microcrystalline cellulose Avicel (cellulose Ibeta), liquid ammonia treated cellulose (cellulose III), sodium hydroxide treated cellulose (cellulose II) and phosphoric acid swollen amorphous cellulose (AC). The digestibility ranking is AC>cellulose III>cellulose II>cellulose I. However, AC has the highest initial enzyme binding capacity while cellulose III had the lowest. CBH II is less stable during hydrolysis. Time course binding studies were also performed for pretreated biomass. Ammonia Fiber Expansion (AFEX) treated corn stover (CS), dilute acid (ACID) treated CS and ionic liquid (IL) pretreated CS were compared. The results indicate that presence of lignin is responsible for significant unproductive cellulase binding. These results are critical for improving our understanding of enzyme synergism, productive/unproductive enzyme binding and the role of pretreatment on enzyme accessibility to lignocellulosic plant cell walls. The results also assist in engineering novel low unproductive binding enzyme systems and developing economic enzyme recycle options.

STM/STS study on electronic superstructures in the superconducting state of high-Tc cuprate Bi2Sr2CaCu2O8+δ

NASA Astrophysics Data System (ADS)

Mizuta, S.; Kurosawa, T.; Takeyama, K.; Momono, N.; Ishii, Y.; Yoshida, H.; Oda, M.; Ido, M.

2018-03-01

We report STM/STS measurements at 8 K in underdoped Bi2Sr2CaCu2O8+δ crystals (T c = 76 K and hole-doping level p ∼ 0.12) whose energy spectra around the Fermi level are characterized by a two-gap structure consisting of spatially inhomogeneous pseudogap (PG) and comparatively homogeneous superconducting gap (SCG). Two electronic superstructures, checkerboard modulation (CBM) and Cu-O-Cu bond-centered modulation (BCM), are observed with mapping spectral weights at low energies within the SCG and the ratio of spectral weights at ±ΔPG (PG energy), respectively. On the basis of the present findings, we suggest that the lower-energy scale CBM is an intrinsic property of Cu-O planes and can coexist with the BCM whose characteristic energy is ∼ΔPG in identical regions in real space.

Beam test results of STS prototype modules for the future accelerator experiments FAIR/CBM and NICA/MPD projects

NASA Astrophysics Data System (ADS)

Kharlamov, Petr; Dementev, Dmitrii; Shitenkov, Mikhail

2017-10-01

High-energy heavy-ion collision experiments provide the unique possibility to create and investigate extreme states of strongly-interacted matter and address the fundamental aspects of QCD. The experimental investigation the QCD phase diagram would be a major breakthrough in our understanding of the properties of nuclear matter. The reconstruction of the charged particles created in the nuclear collisions, including the determination of their momenta, is the central detection task in high-energy heavy-ion experiments. It is taken up by the Silicon Tracking System in CBM@FAIR and by Inner Tracker in MPD@NICA currently under development. These experiments requires very fast and radiation hard detectors, a novel data read-out and analysis concept including free streaming front-end electronics. Thermal and beam tests of prototype detector modules for these tracking systems showed the stability of sensors and readout electronics operation.

Single-molecule Imaging Analysis of Elementary Reaction Steps of Trichoderma reesei Cellobiohydrolase I (Cel7A) Hydrolyzing Crystalline Cellulose Iα and IIII*

PubMed Central

Shibafuji, Yusuke; Nakamura, Akihiko; Uchihashi, Takayuki; Sugimoto, Naohisa; Fukuda, Shingo; Watanabe, Hiroki; Samejima, Masahiro; Ando, Toshio; Noji, Hiroyuki; Koivula, Anu; Igarashi, Kiyohiko; Iino, Ryota

2014-01-01

Trichoderma reesei cellobiohydrolase I (TrCel7A) is a molecular motor that directly hydrolyzes crystalline celluloses into water-soluble cellobioses. It has recently drawn attention as a tool that could be used to convert cellulosic materials into biofuel. However, detailed mechanisms of action, including elementary reaction steps such as binding, processive hydrolysis, and dissociation, have not been thoroughly explored because of the inherent challenges associated with monitoring reactions occurring at the solid/liquid interface. The crystalline cellulose Iα and IIII were previously reported as substrates with different crystalline forms and different susceptibilities to hydrolysis by TrCel7A. In this study, we observed that different susceptibilities of cellulose Iα and IIII are highly dependent on enzyme concentration, and at nanomolar enzyme concentration, TrCel7A shows similar rates of hydrolysis against cellulose Iα and IIII. Using single-molecule fluorescence microscopy and high speed atomic force microscopy, we also determined kinetic constants of the elementary reaction steps for TrCel7A against cellulose Iα and IIII. These measurements were performed at picomolar enzyme concentration in which density of TrCel7A on crystalline cellulose was very low. Under this condition, TrCel7A displayed similar binding and dissociation rate constants for cellulose Iα and IIII and similar fractions of productive binding on cellulose Iα and IIII. Furthermore, once productively bound, TrCel7A processively hydrolyzes and moves along cellulose Iα and IIII with similar translational rates. With structural models of cellulose Iα and IIII, we propose that different susceptibilities at high TrCel7A concentration arise from surface properties of substrate, including ratio of hydrophobic surface and number of available lanes. PMID:24692563

The missing link: do cortical microtubules define plasma membrane nanodomains that modulate cellulose biosynthesis?

PubMed

Fujita, Miki; Lechner, Bettina; Barton, Deborah A; Overall, Robyn L; Wasteneys, Geoffrey O

2012-02-01

Cellulose production is a crucial aspect of plant growth and development. It is functionally linked to cortical microtubules, which self-organize into highly ordered arrays often situated in close proximity to plasma membrane-bound cellulose synthase complexes (CSCs). Although most models put forward to explain the microtubule-cellulose relationship have considered mechanisms by which cortical microtubule arrays influence the orientation of cellulose microfibrils, little attention has been paid to how microtubules affect the physicochemical properties of cellulose. A recent study using the model system Arabidopsis, however, indicates that microtubules can modulate the crystalline and amorphous content of cellulose microfibrils. Microtubules are required during rapid growth for reducing crystalline content, which is predicted to increase the degree to which cellulose is tethered by hemicellulosic polysaccharides. Such tethering is, in turn, critical for maintaining unidirectional cell expansion. In this article, we hypothesize that cortical microtubules influence the crystalline content of cellulose either by controlling plasma membrane fluidity or by modulating the deposition of noncellulosic wall components in the vicinity of the CSCs. We discuss the current limitations of imaging technology to address these hypotheses and identify the image acquisition and processing strategies that will integrate live imaging with super resolution three-dimensional information.

Cellulose promotes extracellular assembly of Clostridium cellulovorans cellulosomes.

PubMed Central

Matano, Y; Park, J S; Goldstein, M A; Doi, R H

1994-01-01

Cellulosome synthesis by Clostridium cellulovorans was investigated by growing the cells in media containing different carbon sources. Supernatant from cells grown with cellobiose contained no cellulosomes and only the free forms of cellulosomal major subunits CbpA, P100, and P70 and the minor subunits with enzymatic activity. Supernatant from cells grown on pebble-milled cellulose and Avicel contained cellulosomes capable of degrading crystalline cellulose. Supernatants from cells grown with cellobiose, pebble-milled cellulose, and Avicel contained about the same amount of carboxymethyl cellulase activity. Although the supernatant from the medium containing cellobiose did not initially contain active cellulosomes, the addition of crystalline cellulose to the cell-free supernatant fraction converted the free major forms to cellulosomes with the ability to degrade crystalline cellulose. The binding of P100 and P70 to crystalline cellulose was dependent on their attachment to the endoglucanase-binding domains of CbpA. These data strongly indicate that crystalline cellulose promotes cellulosome assembly. Images PMID:7961457

International Space Station Powered Bolt Nut Anomaly and Failure Analysis Summary

NASA Technical Reports Server (NTRS)

Sievers, Daniel E.; Warden, Harry K.

2010-01-01

A key mechanism used in the on-orbit assembly of the International Space Station (ISS) pressurized elements is the Common Berthing Mechanism (CBM). The mechanism that effects the structural connection of the CBM halves is the Powered Bolt Assembly. There are sixteen Powered Bolt Assemblies per CBM. The CBM has a bolt which engages a self aligning Powered Bolt Nut (PBN) on the mating interface; see Figure 1. The Powered Bolt Assemblies are preloaded to approximately 19 kilo pounds (KIPs) prior to pressurization of the CBM. The PBNs mentioned below, manufactured in 2009, will be used on ISS future missions. An on orbit functional failure of this hardware would be unacceptable and in some instances catastrophic due to the failure of modules to mate and seal the atmosphere, risking loss of crew and ISS functions. The manufacturing processes which create the PBNs need to be strictly controlled. Functional (torque vs. tension) acceptance test failures will be the result of processes not being strictly followed. Without the proper knowledge of thread tolerances, fabrication techniques, and dry film lubricant application processes, PBNs will be, and have been manufactured improperly. The knowledge gained from acceptance test failures and the resolution of those failures, thread fabrication techniques and thread dry film lubrication processes can be applied to many aerospace mechanisms to enhance their performance. Test data and manufactured PBN thread geometry will be discussed for both failed and successfully accepted PBNs.

A small cellulose binding domain protein in Phytophtora is cell wall localized

USDA-ARS?s Scientific Manuscript database

Cellulose binding domains (CBD) are structurally conserved regions linked to catalytic regions of cellulolytic enzymes. While widespread amongst saprophytic fungi that subsist on plant cell wall polysaccharides, they are not generally present in plant pathogenic fungi. A genome wide survey of CBDs w...

Modular organisation and functional analysis of dissected modular beta-mannanase CsMan26 from Caldicellulosiruptor Rt8B.4.

PubMed

Sunna, Anwar

2010-03-01

CsMan26 from Caldicellulosiruptor strain Rt8.B4 is a modular beta-mannanase consisting of two N-terminal family 27 carbohydrate-binding modules (CBMs), followed by a family 35 CBM and a family 26 glycoside hydrolase catalytic module (mannanase). A functional dissection of the full-length CsMan26 and a comprehensive characterisation of the truncated derivatives were undertaken to evaluate the role of the CBMs. Limited proteolysis was used to define biochemically the boundaries of the different structural modules in CsMan26. The full-length CsMan26 and three truncated derivatives were produced in Escherichia coli, purified and characterised. The systematic removal of the CBMs resulted in a decrease in the optimal temperature for activity and in the overall thermostability of the derivatives. Kinetic experiments indicated that the presence of the mannan-specific family 27 CBMs increased the affinity of the enzyme towards the soluble galactomannan substrate but this was accompanied by lower catalytic efficiency. The full-length CsMan26 and its truncated derivatives were unable to hydrolyse mannooligosaccharides with degree of polymerisation (DP) of three or less. The major difference in the hydrolysis pattern of larger mannooligosaccharides (DP >3) by the derivatives was determined by their abilities to further hydrolyse the intermediate sugar mannotetraose.

The Arabidopsis COBRA Protein Facilitates Cellulose Crystallization at the Plasma Membrane*

PubMed Central

Sorek, Nadav; Sorek, Hagit; Kijac, Aleksandra; Szemenyei, Heidi J.; Bauer, Stefan; Hématy, Kian; Wemmer, David E.; Somerville, Chris R.

2014-01-01

Mutations in the Arabidopsis COBRA gene lead to defects in cellulose synthesis but the function of COBRA is unknown. Here we present evidence that COBRA localizes to discrete particles in the plasma membrane and is sensitive to inhibitors of cellulose synthesis, suggesting that COBRA and the cellulose synthase complex reside in close proximity on the plasma membrane. Live-cell imaging of cellulose synthesis indicated that, once initiated, cellulose synthesis appeared to proceed normally in the cobra mutant. Using isothermal calorimetry, COBRA was found to bind individual β1–4-linked glucan chains with a KD of 3.2 μm. Competition assays suggests that COBRA binds individual β1–4-linked glucan chains with higher affinity than crystalline cellulose. Solid-state nuclear magnetic resonance studies of the cell wall of the cobra mutant also indicated that, in addition to decreases in cellulose amount, the properties of the cellulose fibrils and other cell wall polymers differed from wild type by being less crystalline and having an increased number of reducing ends. We interpret the available evidence as suggesting that COBRA facilitates cellulose crystallization from the emerging β1–4-glucan chains by acting as a “polysaccharide chaperone.” PMID:25331944

Defence Technology Strategy for the Demands of the 21st Century

DTIC Science & Technology

2006-10-01

understanding of human capability in the CBM role. Ownership of the intellectual property behind algorithms may be sovereign10, but implementation will...synchronisation schemes. · coding schemes. · modulation techniques. · access schemes. · smart spectrum usage . · low probability of intercept. · implementation...modulation techniques; access schemes; smart spectrum usage ; low probability of intercept Spectrum and bandwidth management · cross layer technologies to

Binding and Movement of Individual Cel7A Cellobiohydrolases on Crystalline Cellulose Surfaces Revealed by Single-molecule Fluorescence Imaging*

PubMed Central

Jung, Jaemyeong; Sethi, Anurag; Gaiotto, Tiziano; Han, Jason J.; Jeoh, Tina; Gnanakaran, Sandrasegaram; Goodwin, Peter M.

2013-01-01

The efficient catalytic conversion of biomass to bioenergy would meet a large portion of energy requirements in the near future. A crucial step in this process is the enzyme-catalyzed hydrolysis of cellulose to glucose that is then converted into fuel such as ethanol by fermentation. Here we use single-molecule fluorescence imaging to directly monitor the movement of individual Cel7A cellobiohydrolases from Trichoderma reesei (TrCel7A) on the surface of insoluble cellulose fibrils to elucidate molecular level details of cellulase activity. The motion of multiple, individual TrCel7A cellobiohydrolases was simultaneously recorded with ∼15-nm spatial resolution. Time-resolved localization microscopy provides insights on the activity of TrCel7A on cellulose and informs on nonproductive binding and diffusion. We measured single-molecule residency time distributions of TrCel7A bound to cellulose both in the presence of and absence of cellobiose the major product and a potent inhibitor of Cel7A activity. Combining these results with a kinetic model of TrCel7A binding provides microscopic insight into interactions between TrCel7A and the cellulose substrate. PMID:23818525

Influence of the amyloid dye Congo red on curli, cellulose, and the extracellular matrix in E. coli during growth and matrix purification.

PubMed

Reichhardt, Courtney; McCrate, Oscar A; Zhou, Xiaoxue; Lee, Jessica; Thongsomboon, Wiriya; Cegelski, Lynette

2016-11-01

Microbial biofilms are communities of cells characterized by a hallmark extracellular matrix (ECM) that confers functional attributes to the community, including enhanced cohesion, adherence to surfaces, and resistance to external stresses. Understanding the composition and properties of the biofilm ECM is crucial to understanding how it functions and protects cells. New methods to isolate and characterize ECM are emerging for different biofilm systems. Solid-state nuclear magnetic resonance was used to quantitatively track the isolation of the insoluble ECM from the uropathogenic Escherichia coli strain UTI89 and understand the role of Congo red in purification protocols. UTI89 assembles amyloid-integrated biofilms when grown on YESCA nutrient agar. The ECM contains curli amyloid fibers and a modified form of cellulose. Biofilms formed by UTI89 and other E. coli and Salmonella strains are often grown in the presence of Congo red to visually emphasize wrinkled agar morphologies and to score the production of ECM. Congo red is a hallmark amyloid-binding dye and binds to curli, yet also binds to cellulose. We found that growth in Congo red enabled more facile extraction of the ECM from UTI89 biofilms and facilitates isolation of cellulose from the curli mutant, UTI89ΔcsgA. Yet, Congo red has no influence on the isolation of curli from curli-producing cells that do not produce cellulose. Sodium dodecyl sulfate can remove Congo red from curli, but not from cellulose. Thus, Congo red binds strongly to cellulose and possibly weakens cellulose interactions with the cell surface, enabling more complete removal of the ECM. The use of Congo red as an extracellular matrix purification aid may be applied broadly to other organisms that assemble extracellular amyloid or cellulosic materials. Graphical abstract Solid-state NMR was used to quantitatively track the isolation of the insoluble amyloid-associated ECM from uropathogenic E. coli and understand the role of Congo red in purification protocols.

Higher order scaffoldin assembly in Ruminococcus flavefaciens cellulosome is coordinated by a discrete cohesin-dockerin interaction.

PubMed

Bule, Pedro; Pires, Virgínia M R; Alves, Victor D; Carvalho, Ana Luísa; Prates, José A M; Ferreira, Luís M A; Smith, Steven P; Gilbert, Harry J; Noach, Ilit; Bayer, Edward A; Najmudin, Shabir; Fontes, Carlos M G A

2018-05-03

Cellulosomes are highly sophisticated molecular nanomachines that participate in the deconstruction of complex polysaccharides, notably cellulose and hemicellulose. Cellulosomal assembly is orchestrated by the interaction of enzyme-borne dockerin (Doc) modules to tandem cohesin (Coh) modules of a non-catalytic primary scaffoldin. In some cases, as exemplified by the cellulosome of the major cellulolytic ruminal bacterium Ruminococcus flavefaciens, primary scaffoldins bind to adaptor scaffoldins that further interact with the cell surface via anchoring scaffoldins, thereby increasing cellulosome complexity. Here we elucidate the structure of the unique Doc of R. flavefaciens FD-1 primary scaffoldin ScaA, bound to Coh 5 of the adaptor scaffoldin ScaB. The RfCohScaB5-DocScaA complex has an elliptical architecture similar to previously described complexes from a variety of ecological niches. ScaA Doc presents a single-binding mode, analogous to that described for the other two Coh-Doc specificities required for cellulosome assembly in R. flavefaciens. The exclusive reliance on a single-mode of Coh recognition contrasts with the majority of cellulosomes from other bacterial species described to date, where Docs contain two similar Coh-binding interfaces promoting a dual-binding mode. The discrete Coh-Doc interactions observed in ruminal cellulosomes suggest an adaptation to the exquisite properties of the rumen environment.

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.

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.

Observing cellulose biosynthesis and membrane translocation in crystallo

PubMed Central

Morgan, Jacob L.W.; McNamara, Joshua T.; Fischer, Michael; Rich, Jamie; Chen, Hong-Ming; Withers, Stephen G.; Zimmer, Jochen

2016-01-01

Many biopolymers, including polysaccharides, must be translocated across at least one membrane to reach their site of biological function. Cellulose is a linear glucose polymer synthesized and secreted by a membrane-integrated cellulose synthase. In crystallo enzymology with the catalytically-active bacterial cellulose synthase BcsA-B complex reveals structural snapshots of a complete cellulose biosynthesis cycle, from substrate binding to polymer translocation. Substrate and product-bound structures of BcsA provide the basis for substrate recognition and demonstrate the stepwise elongation of cellulose. Furthermore, the structural snapshots show that BcsA translocates cellulose via a ratcheting mechanism involving a “finger helix” that contacts the polymer's terminal glucose. Cooperating with BcsA's gating loop, the finger helix moves ‘up’ and ‘down’ in response to substrate binding and polymer elongation, respectively, thereby pushing the elongated polymer into BcsA’s transmembrane channel. This mechanism is validated experimentally by tethering BcsA's finger helix, which inhibits polymer translocation but not elongation. PMID:26958837

Trichoderma genes

DOEpatents

Foreman, Pamela [Los Altos, CA; Goedegebuur, Frits [Vlaardingen, NL; Van Solingen, Pieter [Naaldwijk, NL; Ward, Michael [San Francisco, CA

2012-06-19

Described herein are novel gene sequences isolated from Trichoderma reesei. Two genes encoding proteins comprising a cellulose binding domain, one encoding an arabionfuranosidase and one encoding an acetylxylanesterase are described. The sequences, CIP1 and CIP2, contain a cellulose binding domain. These proteins are especially useful in the textile and detergent industry and in pulp and paper industry.

Structural Mechanisms of Plant Glucan Phosphatases in Starch Metabolism

PubMed Central

Meekins, David A.; Vander Kooi, Craig W.; Gentry, Matthew S.

2016-01-01

Glucan phosphatases are a recently discovered class of enzymes that dephosphorylate starch and glycogen, thereby regulating energy metabolism. Plant genomes encode for two glucan phosphatases called Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2) that regulate starch metabolism by selectively dephosphorylating glucose moieties within starch glucan chains. Recently, the structures of both SEX4 and LSF2 were determined, with and without phosphoglucan products bound, revealing the mechanism for their unique activities. This review explores the structural and enzymatic features of the plant glucan phosphatases and outlines how they are uniquely adapted for carrying out their cellular functions. We outline the physical mechanisms employed by SEX4 and LSF2 to interact with starch glucans: SEX4 binds glucan chains via a continuous glucan binding platform comprised of its Dual Specificity Phosphatase (DSP) domain and Carbohydrate Binding Module (CBM) while LSF2 utilizes Surface Binding Sites (SBSs). SEX4 and LSF2 both contain a unique network of aromatic residues in their catalytic DSP domains that serve as glucan engagement platforms and are unique to the glucan phosphatases. We also discuss the phosphoglucan substrate specificities inherent to SEX4 and LSF2 and outline structural features within the active site that govern glucan orientation. This review defines the structural mechanism of the plant glucan phosphatases with respect to phosphatases, starch metabolism, and protein-glucan interaction; thereby providing a framework for their applications in both agricultural and industrial settings. PMID:26934589

Bacterial cellulose-kaolin nanocomposites for application as biomedical wound healing materials

NASA Astrophysics Data System (ADS)

Wanna, Dwi; Alam, Catharina; Toivola, Diana M.; Alam, Parvez

2013-12-01

This short communication provides preliminary experimental details on the structure-property relationships of novel biomedical kaolin-bacterial cellulose nanocomposites. Bacterial cellulose is an effective binding agent for kaolin particles forming reticulated structures at kaolin-cellulose interfaces and entanglements when the cellulose fraction is sufficiently high. The mechanical performance of these materials hence improves with an increased fraction of bacterial cellulose, though this also causes the rate of blood clotting to decrease. These composites have combined potential as both short-term (kaolin) and long-term (bacterial cellulose) wound healing materials.

CIP1 polypeptides and their uses

DOEpatents

Foreman, Pamela [Los Altos, CA; Van Solingen, Pieter [Naaldwijk, NL; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA

2011-04-12

Described herein are novel gene sequences isolated from Trichoderma reesei. Two genes encoding proteins comprising a cellulose binding domain, one encoding an arabionfuranosidase and one encoding an acetylxylanesterase are described. The sequences, CIP1 and CIP2, contain a cellulose binding domain. These proteins are especially useful in the textile and detergent industry and in pulp and paper industry.

Evaluation of cellulose-binding domain fused to a lipase for the lipase immobilization.

PubMed

Hwang, Sangpill; Ahn, Jungoh; Lee, Sumin; Lee, Tai Gyu; Haam, Seungjoo; Lee, Kangtaek; Ahn, Ik-Sung; Jung, Joon-Ki

2004-04-01

A cellulose-binding domain (CBD) fragment of a cellulase gene of Trichoderma hazianum was fused to a lipase gene of Bacillus stearothermophilus L1 to make a gene cluster for CBD-BSL lipase. The specific activity of CBD-BSL lipase for oil hydrolysis increased by 33% after being immobilized on Avicel (microcrystalline cellulose), whereas those of CBD-BSL lipase and BSL lipase decreased by 16% and 54%, respectively, after being immobilized on silica gel. Although the loss of activity of an enzyme immobilized by adsorption has been reported previously, the loss of activity of the CBD-BSL lipase immobilized on Avicel was less than 3% after 12 h due to the irreversible binding of CBD to Avicel.

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.

Lignin triggers irreversible cellulase loss during pretreated lignocellulosic biomass saccharification.

PubMed

Gao, Dahai; Haarmeyer, Carolyn; Balan, Venkatesh; Whitehead, Timothy A; Dale, Bruce E; Chundawat, Shishir Ps

2014-01-01

Non-productive binding of enzymes to lignin is thought to impede the saccharification efficiency of pretreated lignocellulosic biomass to fermentable sugars. Due to a lack of suitable analytical techniques that track binding of individual enzymes within complex protein mixtures and the difficulty in distinguishing the contribution of productive (binding to specific glycans) versus non-productive (binding to lignin) binding of cellulases to lignocellulose, there is currently a poor understanding of individual enzyme adsorption to lignin during the time course of pretreated biomass saccharification. In this study, we have utilized an FPLC (fast protein liquid chromatography)-based methodology to quantify free Trichoderma reesei cellulases (namely CBH I, CBH II, and EG I) concentration within a complex hydrolyzate mixture during the varying time course of biomass saccharification. Three pretreated corn stover (CS) samples were included in this study: Ammonia Fiber Expansion(a) (AFEX™-CS), dilute acid (DA-CS), and ionic liquid (IL-CS) pretreatments. The relative fraction of bound individual cellulases varied depending not only on the pretreated biomass type (and lignin abundance) but also on the type of cellulase. Acid pretreated biomass had the highest levels of non-recoverable cellulases, while ionic liquid pretreated biomass had the highest overall cellulase recovery. CBH II has the lowest thermal stability among the three T. reesei cellulases tested. By preparing recombinant family 1 carbohydrate binding module (CBM) fusion proteins, we have shown that family 1 CBMs are highly implicated in the non-productive binding of full-length T. reesei cellulases to lignin. Our findings aid in further understanding the complex mechanisms of non-productive binding of cellulases to pretreated lignocellulosic biomass. Developing optimized pretreatment processes with reduced or modified lignin content to minimize non-productive enzyme binding or engineering pretreatment-specific, low-lignin binding cellulases will improve enzyme specific activity, facilitate enzyme recycling, and thereby permit production of cheaper biofuels.

Cellulose biogenesis: Polymerization and crystallization are coupled processes in Acetobacter xylinum.

PubMed

Benziman, M; Haigler, C H; Brown, R M; White, A R; Cooper, K M

1980-11-01

Calcofluor White ST, stilbene derivative used commerically as an optical brightener for cellulose, increased the rate of glucose polymerization into cellulose by resting cells of the gram-negative bacterium Acetobacter xylinum. This bacterium normally produces a ribbon of cellulose that is a composite of crystalline microfibrils. In concentrations above 0.1 mM, Calcofluor disrupts the assembly of crystalline cellulose I microfibrils and their integration into a composite ribbon by stoichiometric binding to glucose residues of newly polymerized glucan chains. Under these conditions, the rate of glucose polymerization increases up to 4 times the control rate, whereas oxygen uptake increases only 10-15%. These observed effects are readily reversible. If free Calcofluor is washed away or depleted below the threshold value by binding to cellulose as polymerization continues, ribbon production and the normal rate of polymerization resume. It is concluded that polymerization and crystallization are cell-directed, coupled processes and that the rate of crystallization determines the rate of polymerization. It is suggested that coupling must be maintained for biogenesis of crystalline cellulose I.

Cognitive behaviour modification: a technique for teaching subtraction skills to hearing and deaf/hard-of-hearing elementary students.

PubMed

Al-Hilawani, Y A

2000-09-01

The purpose of this study was to examine the influence of using the Cognitive Behaviour Modification (CBM) technique on the subtraction skills of third grade hearing and deaf/hard-of-hearing students. The results indicated that the CBM deaf/hard-of-hearing students and the CBM and non-CBM hearing students made more progress in solving the subtraction problems than the non-CBM deaf/hard-of-hearing students. The results also showed that there were no significant differences between the CBM deaf/hard-of-hearing and the non-CBM hearing students; and there were no significant differences between the CBM and non-CBM hearing students. The results revealed that the CBM hearing students achieved significantly higher post-test scores than the CBM deaf/hard-of-hearing students. However, the CBM deaf/hard-of-hearing students obtained a significantly higher gain score compared to the CBM and non-CBM hearing students. Implications for teachers and suggestions for future research are discussed in this paper.

Universal Screening of Reading in Late Elementary School: R-CBM versus CBM Maze

ERIC Educational Resources Information Center

Graney, Suzanne Bamonto; Martinez, Rebecca S.; Missall, Kristen N.; Aricak, O. Tolga

2010-01-01

Two curriculum-based measurement tools are commonly used to assess progress in reading in elementary school: R-CBM and CBM maze. R-CBM is used in practice more frequently than CBM maze is, although CBM maze is more time efficient to administer than R-CBM is. The technical adequacy of each of these measures has been reported in the literature;…

Simulation of a cellulose fiber in ionic liquid suggests a synergistic approach to dissolution

DOE PAGES

Mostofian, Barmak; Smith, Jeremy C.; Cheng, Xiaolin

2013-08-11

Ionic liquids dissolve cellulose in a more efficient and environmentally acceptable way than conventional methods in aqueous solution. An understanding of how ionic liquids act on cellulose is essential for improving pretreatment conditions and thus detailed knowledge of the interactions between the cations, anions and cellulose is necessary. Here in this study, to explore ionic liquid effects, we perform all-atom molecular dynamics simulations of a cellulose microfibril in 1-butyl-3-methylimidazolium chloride and analyze site–site interactions and cation orientations at the solute–solvent interface. The results indicate that Cl - anions predominantly interact with cellulose surface hydroxyl groups but with differences between chainsmore » of neighboring cellulose layers, referred to as center and origin chains; Cl- binds to C3-hydroxyls on the origin chains but to C2- and C6-hydroxyls on the center chains, thus resulting in a distinct pattern along glucan chains of the hydrophilic fiber surfaces. In particular, Cl - binding disrupts intrachain O3H–O5 hydrogen bonds on the origin chains but not those on the center chains. In contrast, Bmim + cations stack preferentially on the hydrophobic cellulose surface, governed by non-polar interactions with cellulose. Complementary to the polar interactions between Cl - and cellulose, the stacking interaction between solvent cation rings and cellulose pyranose rings can compensate the interaction between stacked cellulose layers, thus stabilizing detached cellulose chains. Moreover, a frequently occurring intercalation of Bmim + on the hydrophilic surface is observed, which by separating cellulose layers can also potentially facilitate the initiation of fiber disintegration. The results provide a molecular description why ionic liquids are ideal cellulose solvents, the concerted action of anions and cations on the hydrophobic and hydrophilic surfaces being key to the efficient dissolution of the amphiphilic carbohydrate.« less

The hydrophobic repeated domain of the Clostridium cellulovorans cellulose-binding protein (CbpA) has specific interactions with endoglucanases.

PubMed Central

Takagi, M; Hashida, S; Goldstein, M A; Doi, R H

1993-01-01

We overexpressed one of the hydrophobic repeated domains (HBDs) (110 amino acid residues) of the cellulose-binding protein (CbpA) from Clostridium cellulovorans by making a hybrid protein with the Escherichia coli maltose-binding protein (MalE). The HBD was purified to homogeneity, and interactions between the HBD and endoglucanases were analyzed by a novel interaction Western blotting (immunoblotting) method. The HBD had specific interactions with endoglucanases (EngB and EngD) from C. cellulovorans. These results indicated that the HBD was an endoglucanase binding site of CbpA. Images PMID:8226657

A new family of β-helix proteins with similarities to the polysaccharide lyases

DOE PAGES

Close, Devin W.; D'Angelo, Sara; Bradbury, Andrew R. M.

2014-09-27

Microorganisms that degrade biomass produce diverse assortments of carbohydrate-active enzymes and binding modules. Despite tremendous advances in the genomic sequencing of these organisms, many genes do not have an ascribed function owing to low sequence identity to genes that have been annotated. Consequently, biochemical and structural characterization of genes with unknown function is required to complement the rapidly growing pool of genomic sequencing data. A protein with previously unknown function (Cthe_2159) was recently isolated in a genome-wide screen using phage display to identify cellulose-binding protein domains from the biomass-degrading bacterium Clostridium thermocellum. Here, the crystal structure of Cthe_2159 is presentedmore » and it is shown that it is a unique right-handed parallel β-helix protein. Despite very low sequence identity to known β-helix or carbohydrate-active proteins, Cthe_2159 displays structural features that are very similar to those of polysaccharide lyase (PL) families 1, 3, 6 and 9. Cthe_2159 is conserved across bacteria and some archaea and is a member of the domain of unknown function family DUF4353. This suggests that Cthe_2159 is the first representative of a previously unknown family of cellulose and/or acid-sugar binding β-helix proteins that share structural similarities with PLs. More importantly, these results demonstrate how functional annotation by biochemical and structural analysis remains a critical tool in the characterization of new gene products.« less

A new family of β-helix proteins with similarities to the polysaccharide lyases

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

Close, Devin W.; D'Angelo, Sara; Bradbury, Andrew R. M.

Microorganisms that degrade biomass produce diverse assortments of carbohydrate-active enzymes and binding modules. Despite tremendous advances in the genomic sequencing of these organisms, many genes do not have an ascribed function owing to low sequence identity to genes that have been annotated. Consequently, biochemical and structural characterization of genes with unknown function is required to complement the rapidly growing pool of genomic sequencing data. A protein with previously unknown function (Cthe_2159) was recently isolated in a genome-wide screen using phage display to identify cellulose-binding protein domains from the biomass-degrading bacterium Clostridium thermocellum. Here, the crystal structure of Cthe_2159 is presentedmore » and it is shown that it is a unique right-handed parallel β-helix protein. Despite very low sequence identity to known β-helix or carbohydrate-active proteins, Cthe_2159 displays structural features that are very similar to those of polysaccharide lyase (PL) families 1, 3, 6 and 9. Cthe_2159 is conserved across bacteria and some archaea and is a member of the domain of unknown function family DUF4353. This suggests that Cthe_2159 is the first representative of a previously unknown family of cellulose and/or acid-sugar binding β-helix proteins that share structural similarities with PLs. More importantly, these results demonstrate how functional annotation by biochemical and structural analysis remains a critical tool in the characterization of new gene products.« less

Compositions and methods comprising cellulase variants with reduced affinity to non-cellulosic materials

DOEpatents

Cascao-Pereira, Luis; Kaper, Thijs; Kelemen, Bradley R.; Liu, Amy D.

2017-07-04

The present disclosure relates to cellulase variants. In particular the present disclosure relates to cellulase variants having reduced binding to non-cellulosic materials. Also described are nucleic acids encoding the cellulase, compositions comprising said cellulase, methods of identifying cellulose variants and methods of using the compositions.

Compositions and methods comprising cellulase variants with reduced affinity to non-cellulosic materials

DOEpatents

Cascao-Pereira, Luis G; Kaper, Thijs; Kelemen, Bradley R; Liu, Amy D

2015-04-07

The present disclosure relates to cellulase variants. In particular the present disclosure relates to cellulase variants having reduced binding to non-cellulosic materials. Also described are nucleic acids encoding the cellulase, compositions comprising said cellulase, methods of identifying cellulose variants and methods of using the compositions.

Compositions and methods comprising cellulase variants with reduced affinity to non-cellulosic materials

DOEpatents

Cascao-Pereira, Luis G.; Kaper, Thijs; Kelemen, Bradley R; Liu, Amy D.

2012-08-07

The present disclosure relates to cellulase variants. In particular the present disclosure relates to cellulase variants having reduced binding to non-cellulosic materials. Also described are nucleic acids encoding the cellulase, compositions comprising said cellulase, methods of identifying cellulose variants and methods of using the compositions.

CBD binding domain fused γ-lactamase from Sulfolobus solfataricus is an efficient catalyst for (-) γ-lactam production.

PubMed

Wang, Jianjun; Zhu, Junge; Min, Cong; Wu, Sheng

2014-05-13

γ-lactamase is used for the resolution of γ-lactam which is utilized in the synthesizing of abacavir and peramivir. In some cases, enzymatic method is the most utilized method because of its high efficiency and productivity. The cellulose binding domain (CBD) of cellulose is often used as the bio-specific affinity matrix for enzyme immobilization. Cellulose is cheap and it has excellent chemical and physical properties. Meanwhile, binding between cellulose and CBD is tight and the desorption rarely happened. We prepared two fusion constructs of the γ-lactamase gene gla, which was from Sulfolobus solfataricus P2. These two constructs had Cbd (cellulose binding domain from Clostridium thermocellum) fused at amino or carboxyl terminus of the γ-lactamase. These two constructs were heterogeneously expressed in E. coli rosetta (DE3) as two fusion proteins. Both of them were immobilized well on Avicel (microcrystalline cellulose matrix). The apparent kinetic parameters revealed that carboxyl terminus fused protein (Gla-linker-Cbd) was a better catalyst. The V(max) and k(cat) value of Avicel immobilized Gla-linker-Cbd were 381 U mg⁻¹ and 4.7 × 10⁵ s⁻¹ respectively. And the values of the free Gla-linker-Cbd were 151 U mg⁻¹ and 1.8 × 10⁵ s⁻¹ respectively. These data indicated that the catalytic efficiency of the enzyme was upgraded after immobilization. The immobilized Gla-linker-Cbd had a 10-degree temperature optimum dropping from 80°C to 70°C but it was stable when incubated at 60°C for 48 h. It remained stable in catalyzing 20-batch reactions. After optimization, the immobilized enzyme concentration in transformation was set as 200 mg/mL. We found out that there was inhibition that occurred to the immobilized enzyme when substrate concentration exceeded 60 mM. Finally a 10 mL-volume transformation was conducted, in which 0.6 M substrate was hydrolyzed and the resolution was completed within 9 h with a 99.5% ee value. Cellulose is the most abundant and renewable material on the Earth. The absorption between Cbd domain and cellulose is a bio-green process. The cellulose immobilized fusion Gla exhibited good catalytic characters, therefore we think the cellulose immobilized Gla is a promising catalyst for the industrial preparation of (-) - γ-lactam.

Expression of the Acidothermus cellulolyticus E1 endoglucanase in Caldicellulosiruptor bescii enhances its ability to deconstruct crystalline cellulose

DOE PAGES

Chung, Daehwan; Young, Jenna; Cha, Minseok; ...

2015-08-13

The Caldicellulosiruptor bescii genome encodes a potent set of carbohydrate-active enzymes (CAZymes), found primarily as multi-domain enzymes that exhibit high cellulolytic and hemicellulolytic activity on and allow utilization of a broad range of substrates, including plant biomass without conventional pretreatment. CelA, the most abundant cellulase in the C. bescii secretome, uniquely combines a GH9 endoglucanase and a GH48 exoglucanase in one protein. The most effective commercial enzyme cocktails used in vitro to pretreat biomass are derived from fungal cellulases (cellobiohydrolases, endoglucanases and a β-d-glucosidases) that act synergistically to release sugars for microbial conversion. The C. bescii genome contains six GH5more » domains in five different open reading frames. Four exist in multi-domain proteins and two as single catalytic domains. E1 is a GH5 endoglucanase reported to have high specific activity and simple architecture and is active at the growth temperature of C. bescii. E1 is an endo-1,4-β-glucanase linked to a family 2 carbohydrate-binding module shown to bind primarily to cellulosic substrates. As a result, we tested if the addition of this protein to the C. bescii secretome would improve its cellulolytic activity.« less

Biofilm formation, cellulose production, and curli biosynthesis by Salmonella originating from produce, animal, and clinical sources.

PubMed

Solomon, Ethan B; Niemira, Brendan A; Sapers, Gerald M; Annous, Bassam A

2005-05-01

The ability of 71 strains of Salmonella enterica originating from produce, meat, or clinical sources to form biofilms was investigated. A crystal violet binding assay demonstrated no significant differences in biofilm formation by isolates from any source when tested in any of the following three media: Luria-Bertani broth supplemented with 2% glucose, tryptic soy broth (TSB), or 1/20th-strength TSB. Incubation was overnight at 30 degrees C under static conditions. Curli production and cellulose production were monitored by assessing morphotypes on Luria-Bertani agar without salt containing Congo red and by assessing fluorescence on Luria-Bertani agar containing calcofluor, respectively. One hundred percent of the clinical isolates exhibited curli biosynthesis, and 73% demonstrated cellulose production. All meat-related isolates formed curli, and 84% produced cellulose. A total of 80% of produce-related isolates produced curli, but only 52% produced cellulose. Crystal violet binding was not statistically different between isolates representing the three morphotypes when grown in TSB; however, significant differences were observed when strains were cultured in the two other media tested. These data demonstrate that the ability to form biofilms is not dependent on the source of the test isolate and suggest a relationship between crystal violet binding and morphotype, with curli- and cellulose-deficient isolates being least effective in biofilm formation.

High Potential Source for Biomass Degradation Enzyme Discovery and Environmental Aspects Revealed through Metagenomics of Indian Buffalo Rumen

PubMed Central

Singh, K. M.; Reddy, Bhaskar; Patel, Dishita; Patel, A. K.; Patel, J. B.; Joshi, C. G.

2014-01-01

The complex microbiomes of the rumen functions as an effective system for plant cell wall degradation, and biomass utilization provide genetic resource for degrading microbial enzymes that could be used in the production of biofuel. Therefore the buffalo rumen microbiota was surveyed using shot gun sequencing. This metagenomic sequencing generated 3.9 GB of sequences and data were assembled into 137270 contiguous sequences (contigs). We identified potential 2614 contigs encoding biomass degrading enzymes including glycoside hydrolases (GH: 1943 contigs), carbohydrate binding module (CBM: 23 contigs), glycosyl transferase (GT: 373 contigs), carbohydrate esterases (CE: 259 contigs), and polysaccharide lyases (PE: 16 contigs). The hierarchical clustering of buffalo metagenomes demonstrated the similarities and dissimilarity in microbial community structures and functional capacity. This demonstrates that buffalo rumen microbiome was considerably enriched in functional genes involved in polysaccharide degradation with great prospects to obtain new molecules that may be applied in the biofuel industry. PMID:25136572

DNA immobilization and detection on cellulose paper using a surface grown cationic polymer via ATRP.

PubMed

Aied, Ahmed; Zheng, Yu; Pandit, Abhay; Wang, Wenxin

2012-02-01

Cationic polymers with various structures have been widely investigated in the areas of medical diagnostics and molecular biology because of their unique binding properties and capability to interact with biological molecules in complex biological environments. In this work, we report the grafting of a linear cationic polymer from an atom transfer radical polymerization (ATRP) initiator bound to cellulose paper surface. We show successful binding of ATRP initiator onto cellulose paper and grafting of polymer chains from the immobilized initiator with ATRP. The cellulose paper grafted polymer was used in combination with PicoGreen (PG) to demonstrate detection of nucleic acids in the nanogram range in homogeneous solution and in a biological sample (serum). The results showed specific identification of hybridized DNA after addition of PG in both solutions.

The effect of cellulose overproduction on binding and biofilm formation on roots by Agrobacterium tumefaciens.

PubMed

Matthysse, Ann G; Marry, Mazz; Krall, Leonard; Kaye, Mitchell; Ramey, Bronwyn E; Fuqua, Clay; White, Alan R

2005-09-01

Agrobacterium tumefaciens growing in liquid attaches to the surface of tomato and Arabidopsis thaliana roots, forming a biofilm. The bacteria also colonize roots grown in sterile quartz sand. Attachment, root colonization, and biofilm formation all were markedly reduced in celA and chvB mutants, deficient in production of cellulose and cyclic beta-(1,2)-D-glucans, respectively. We have identified two genes (celG and cell) in which mutations result in the overproduction of cellulose as judged by chemical fractionation and methylation analysis. Wild-type and chvB mutant strains carrying a cDNA clone of a cellulose synthase gene from the marine urochordate Ciona savignyi also overproduced cellulose. The overproduction in a wild-type strain resulted in increased biofilm formation on roots, as evaluated by light microscopy, and levels of root colonization intermediate between those of cellulose-minus mutants and the wild type. Overproduction of cellulose by a nonattaching chvB mutant restored biofilm formation and bacterial attachment in microscopic and viable cell count assays and partially restored root colonization. Although attachment to plant surfaces was restored, overproduction of cellulose did not restore virulence in the chvB mutant strain, suggesting that simple bacterial binding to plant surfaces is not sufficient for pathogenesis.

Molecular-Scale Features that Govern the Effects of O-Glycosylation on a Carbohydrate-Binding Module

DOE PAGES

Guan, Xiaoyang; Chaffey, Patrick K.; Zeng, Chen; ...

2015-09-21

The protein glycosylation is a ubiquitous post-translational modification in all kingdoms of life. Despite its importance in molecular and cellular biology, the molecular-level ramifications of O-glycosylation on biomolecular structure and function remain elusive. Here, we took a small model glycoprotein and changed the glycan structure and size, amino acid residues near the glycosylation site, and glycosidic linkage while monitoring any corresponding changes to physical stability and cellulose binding affinity. The results of this study reveal the collective importance of all the studied features in controlling the most pronounced effects of O-glycosylation in this system. This study suggests the possibility ofmore » designing proteins with multiple improved properties by simultaneously varying the structures of O-glycans and amino acids local to the glycosylation site.« less

Enzymatic properties of Thermoanaerobacterium thermosaccharolyticum β-glucosidase fused to Clostridium cellulovorans cellulose binding domain and its application in hydrolysis of microcrystalline cellulose.

PubMed

Zhao, Linguo; Pang, Qian; Xie, Jingcong; Pei, Jianjun; Wang, Fei; Fan, Song

2013-11-14

The complete degradation of the cellulose requires the synergistic action of endo-β-glucanase, exo-β-glucanase, and β-glucosidase. But endo-β-glucanase and exo-β-glucanase can be recovered by solid-liquid separation in cellulose hydrolysis by their cellulose binding domain (CBD), however, the β-glucosidases cannot be recovered because of most β-glucosidases without the CBD, so additional β-glucosidases are necessary for the next cellulose degradation. This will increase the cost of cellulose degradation. The glucose-tolerant β-glucosidase (BGL) from Thermoanaerobacterium thermosaccharolyticum DSM 571 was fused with cellulose binding domain (CBD) of Clostridium cellulovorans cellulosome anchoring protein by a peptide linker. The fusion enzyme (BGL-CBD) gene was overexpressed in Escherichia coli with the maximum β-glucosidase activity of 17 U/mL. Recombinant BGL-CBD was purified by heat treatment and following by Ni-NTA affinity. The enzymatic characteristics of the BGL-CBD showed optimal activities at pH 6.0 and 65°C. The fusion of CBD structure enhanced the hydrolytic efficiency of the BGL-CBD against cellobiose, which displayed a 6-fold increase in Vmax/Km in comparison with the BGL. A gram of cellulose was found to absorb 643 U of the fusion enzyme (BGL-CBD) in pH 6.0 at 50°C for 25 min with a high immobilization efficiency of 90%. Using the BGL-CBD as the catalyst, the yield of glucose reached a maximum of 90% from 100 g/L cellobiose and the BGL-CBD could retain over 85% activity after five batches with the yield of glucose all above 70%. The performance of the BGL-CBD on microcrystalline cellulose was also studied. The yield of the glucose was increased from 47% to 58% by adding the BGL-CBD to the cellulase, instead of adding the Novozyme 188. The hydrolytic activity of BGL-CBD is greater than that of the Novozyme 188 in cellulose degradation. The article provides a prospect to decrease significantly the operational cost of the hydrolysis process.

Enzymatic properties of Thermoanaerobacterium thermosaccharolyticum β-glucosidase fused to Clostridium cellulovorans cellulose binding domain and its application in hydrolysis of microcrystalline cellulose

PubMed Central

2013-01-01

Background The complete degradation of the cellulose requires the synergistic action of endo-β-glucanase, exo-β-glucanase, and β-glucosidase. But endo-β-glucanase and exo-β-glucanase can be recovered by solid–liquid separation in cellulose hydrolysis by their cellulose binding domain (CBD), however, the β-glucosidases cannot be recovered because of most β-glucosidases without the CBD, so additional β-glucosidases are necessary for the next cellulose degradation. This will increase the cost of cellulose degradation. Results The glucose-tolerant β-glucosidase (BGL) from Thermoanaerobacterium thermosaccharolyticum DSM 571 was fused with cellulose binding domain (CBD) of Clostridium cellulovorans cellulosome anchoring protein by a peptide linker. The fusion enzyme (BGL-CBD) gene was overexpressed in Escherichia coli with the maximum β-glucosidase activity of 17 U/mL. Recombinant BGL-CBD was purified by heat treatment and following by Ni-NTA affinity. The enzymatic characteristics of the BGL-CBD showed optimal activities at pH 6.0 and 65°C. The fusion of CBD structure enhanced the hydrolytic efficiency of the BGL-CBD against cellobiose, which displayed a 6-fold increase in V max /K m in comparison with the BGL. A gram of cellulose was found to absorb 643 U of the fusion enzyme (BGL-CBD) in pH 6.0 at 50°C for 25 min with a high immobilization efficiency of 90%. Using the BGL-CBD as the catalyst, the yield of glucose reached a maximum of 90% from 100 g/L cellobiose and the BGL-CBD could retain over 85% activity after five batches with the yield of glucose all above 70%. The performance of the BGL-CBD on microcrystalline cellulose was also studied. The yield of the glucose was increased from 47% to 58% by adding the BGL-CBD to the cellulase, instead of adding the Novozyme 188. Conclusions The hydrolytic activity of BGL-CBD is greater than that of the Novozyme 188 in cellulose degradation. The article provides a prospect to decrease significantly the operational cost of the hydrolysis process. PMID:24228818

The anchorage function of CipA (CelL), a scaffolding protein of the Clostridium thermocellum cellulosome

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

Kruus, K.; Wu, J.H.D.; Lua, A.C.

1995-09-26

Enzymatic cellulose degradation is a heterogeneous reaction requiring binding of soluble cellulase molecules to the solid substrate. Based on our studies of the cellulase complex of Clostridium thermocellum (the cellulosome), we have previously proposed that such binding can be brought about by a special {open_quotes}anchorage subunit.{close_quotes} In this {open_quotes}anchor-enzyme{close_quotes} model, CipA (a major subunit of the cellulosome) enhances the activity of CelS (the most abundant catalytic subunit of the cellulosome) by anchoring it to the cellulose surface. We have subsequently reported that CelS contains a conserved duplicated sequence at its C terminus and the CipA contains nine repeated sequences withmore » a cellulose binding domain (CBD) in between the second and third repeats. In this work, we reexamined the anchor-enzyme mechanism by using recombinant CelS (rCelS) and various CipA domains, CBD, R3 (the repeat next to CBD), and CBD/R3, expressed in Escherichia coli. As analyzed by non-denaturing gel electrophoresis, rCelS, through its conserved duplicated sequence, formed a stable complex with R3 or CBD/R3 but not with CBD. Although R3 or CBD alone did not affect the binding of rCelS to cellulose, such binding was dependent on CBD/R3, indicating the anchorage role of CBD/R3. Such anchorage apparently increased the rCelS activity toward crystalline cellulose. These results substantiate the proposed anchor-enzyme model and the expected roles of individual CipA domains and the conserved duplicated sequence of CelS.« less

Control of cellulose biosynthesis by overexpression of a transcription factor

DOEpatents

Han, Kyung-Hwan; Ko, Jae-Heung; Kim, Won-Chan; Kim; , Joo-Yeol

2017-05-16

The invention relates to the over-expression of a transcription factor selected from the group consisting of MYB46, HAM1, HAM2, MYB112, WRKY11, ERF6, and any combination thereof in a plant, which can modulate and thereby modulating the cellulose content of the plant.

Evaluation of Binding Effects in Wood Flour Board Containing Ligno-Cellulose Nanofibers

PubMed Central

Kojima, Yoichi; Isa, Akiko; Kobori, Hikaru; Suzuki, Shigehiko; Ito, Hirokazu; Makise, Rie; Okamoto, Masaki

2014-01-01

Wood-based materials are used extensively in residual construction worldwide. Most of the adhesives used in wood-based materials are derived from fossil resources, and some are not environmentally friendly. This study explores nanofiber technology as an alternative to such adhesives. Previous studies have shown that the three-dimensional binding effects of cellulose nanofiber (CNF), when mixed with wood flour, can significantly improve the physical and mechanical properties of wood flour board. In this study, ligno-cellulose nanofibers (LCNF) were fabricated by wet disk milling of wood flour. Composite boards of wood flour and LCNF were produced to investigate the binding effect(s) of LCNF. The fabrication of LCNF by disk milling was simple and effective, and its incorporation into wood flour board significantly enhanced the physical and mechanical properties of the board. PMID:28788217

PsB multiprotein complex of Dictyostelium discoideum. Demonstration of cellulose binding activity and order of protein subunit assembly.

PubMed

McGuire, V; Alexander, S

1996-06-14

The differentiated spores of Dictyostelium are surrounded by an extracellular matrix, the spore coat, which protects them from environmental factors allowing them to remain viable for extended periods of time. This presumably is a major evolutionary advantage. This unique extracellular matrix is composed of cellulose and glycoproteins. Previous work has shown that some of these spore coat glycoproteins exist as a preassembled multiprotein complex (the PsB multiprotein complex) which is stored in the prespore vesicles (Watson, N., McGuire, V., and Alexander, S (1994) J. Cell Sci. 107, 2567-2579). Later in development, the complex is synchronously secreted from the prespore vesicles and incorporated into the spore coat. We now have shown that the PsB complex has a specific in vitro cellulose binding activity. The analysis of mutants lacking individual subunits of the PsB complex revealed the relative order of assembly of the subunit proteins and demonstrated that the protein subunits must be assembled for cellulose binding activity. These results provide a biochemical explanation for the localization of this multiprotein complex in the spore coat.

Condition-Based Maintenance (CBM): A Working Partnership between Government, Industry, and Academia

DTIC Science & Technology

2006-06-01

Listing [UH-611 COMPONENT NSN PART NUMNBER FEDLOG NOM \\ENCL.ATURE NOMNENC(LATLURE Oil Cooler Fan Bearing 3110-01-329-8573 1 IOKSZZ-401 BEARING,BALL,ANNULAR...Main Rotor Blade 1615-01-106-1903 70150-09100- 043 BLADE,MAIN ROTOR Pump Module Assembly 4320-01-207-7228 70652-02300-050 MODULE ASSY,PUMP Damper...Drive Shaft 2835-01-123-7648 70361-08004- 043 DRIVE SHAFT ASSEMBLYROTARY WING Intermediate Gear Box 1615-01-074-5152 70357-06300-042 GEAR BOX ASSEMBLY 39

Salmonella biofilm formation on Aspergillus niger involves cellulose--chitin interactions.

PubMed

Brandl, Maria T; Carter, Michelle Q; Parker, Craig T; Chapman, Matthew R; Huynh, Steven; Zhou, Yaguang

2011-01-01

Salmonella cycles between host and nonhost environments, where it can become an active member of complex microbial communities. The role of fungi in the environmental adaptation of enteric pathogens remains relatively unexplored. We have discovered that S. enterica Typhimurium rapidly attaches to and forms biofilms on the hyphae of the common fungus, Aspergillus niger. Several Salmonella enterica serovars displayed a similar interaction, whereas other bacterial species were unable to bind to the fungus. Bacterial attachment to chitin, a major constituent of fungal cell walls, mirrored this specificity. Pre-incubation of S. Typhimurium with N-acetylglucosamine, the monomeric component of chitin, reduced binding to chitin beads by as much as 727-fold and inhibited attachment to A. niger hyphae considerably. A cellulose-deficient mutant of S. Typhimurium failed to attach to chitin beads and to the fungus. Complementation of this mutant with the cellulose operon restored binding to chitin beads to 79% of that of the parental strain and allowed for attachment and biofilm formation on A. niger, indicating that cellulose is involved in bacterial attachment to the fungus via the chitin component of its cell wall. In contrast to cellulose, S. Typhimurium curli fimbriae were not required for attachment and biofilm development on the hyphae but were critical for its stability. Our results suggest that cellulose-chitin interactions are required for the production of mixed Salmonella-A. niger biofilms, and support the hypothesis that encounters with chitinaceous alternate hosts may contribute to the ecological success of human pathogens.

Re-constructing our models of cellulose and primary cell wall assembly

PubMed Central

Cosgrove, Daniel J.

2014-01-01

The cellulose microfibril has more subtlety than is commonly recognized. Details of its structure may influence how matrix polysaccharides interact with its distinctive hydrophobic and hydrophilic surfaces to form a strong yet extensible structure. Recent advances in this field include the first structures of bacterial and plant cellulose synthases and revised estimates of microfibril structure, reduced from 36 to 18 chains. New results also indicate that cellulose interactions with xyloglucan are more limited than commonly believed, whereas pectin-cellulose interactions are more prevalent. Computational results indicate that xyloglucan binds tightest to the hydrophobic surface of cellulose microfibrils. Wall extensibility may be controlled at limited regions (“biomechanical hotspots”) where cellulose-cellulose contacts are made, potentially mediated by trace amounts of xyloglucan. PMID:25460077

Electronic structure in 1T-ZrS2 monolayer by strain

NASA Astrophysics Data System (ADS)

Xin, Qianqian; Zhao, Xu; Ma, Xu; Wu, Ninghua; Liu, Xiaomeng; Wei, Shuyi

2017-09-01

We report electronic structure of 1T-ZrS2 monolayer with biaxial strain from -10% to 15%, basing the first principles calculations. Our calculation results indicate that the band structure of ZrS2 monolayer was changed clearly. The location of conduction band minimum (CBM) and valence band maximum (VBM) changed with the variation of isotropic strain. At compressive strain, the location of CBM and VBM retains at M and Γ point, respectively. The band gap of ZrS2 monolayer decreases from 1.111 eV to 0 eV when compressive strain increases from 0% to -8%, which means that the ZrS2 monolayer turns to metal at -8% compressive strain. Under the tensile strain, the ZrS2 monolayer also retains be an indirect band gap semiconductor. The location of CBM moves from M to Γ point and the location of VBM moves along Γ-A-K-Γ direction. The band gap of ZrS2 monolayer firstly increases and then decreases and the biggest band gap is 1.577 eV at tensile strain 6%. We can see the compression strain is more effective than tensile strain in modulating band gap of 1T-ZrS2 monolayer.

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.

Coalbed methane accumulation and dissipation patterns: A Case study of the Junggar Basin, NW China

NASA Astrophysics Data System (ADS)

Li, Xin; Fu, Xuehai; Yang, Xuesong; Ge, Yanyan; Quan, Fangkai

2018-07-01

The Junggar Basin is a potential replacement area of coalbed methane (CBM) development in China. To improve the efficiency of CBM exploration, we investigated CBM accumulation and dissipation patterns of coal profiles located in the northwestern, southern, eastern, and central Junggar Basin based on the following criteria: burial depth, hydrogeological zone, CBM origin, CBM phase, and CBM migration type. We identified four types of CBM accumulation patterns: (1) a self-sourcing CBM pattern containing adsorbed gas of biogenic origin from shallow-depth coal within a weak runoff zone; (2) an endogenic migration pattern containing adsorbed gas of thermogenic origin from the medium and deep coals within a stagnant zone; (3) an exogenic migration pattern containing adsorbed gas of thermogenic origin from deep coal within a stagnant zone; and (4) an exogenic migration pattern containing adsorbed and free gas of thermogenic origin from ultra-deep coal within a stagnant zone. We also identified two types of CBM dissipation patterns: (1) shallow-depth coal within a runoff zone with mixed origin CBM; and (2) shallow and medium-deep coal seams with mixed origin CBM. CBM migration in low-rank coals was more substantial than that adsorbed in high-rank coal. CBM in shallow coal could easily escape, in the absence of closed structures or hydrogeological seals. CBM reservoirs occurred in deep coal where oversaturated gas may accumulate. Future exploration should focus on gas-water sealing structures in shallow coalbeds. CBM that occurred in adsorbed and free phases and other unconventional natural gas dominated by free gas in the coal stratum should be co-explored and co-developed.

Evaluation of the binding effect of human serum albumin on the properties of granules.

PubMed

Kristó, Katalin; Bajdik, János; Eros, István; Pintye-Hódi, Klára

2008-11-01

The main objective of this study was the application of a solution of human serum albumin as a granulating fluid. The properties of the granules formed were evaluated and compared with those when a conventional binder was applied in the same concentration. The powder mixture contained a soluble (mannitol) and an insoluble component (different types of cellulose). The protein solution applied exerted an appropriate aggregating effect if the system contained microcrystalline celluloses. Powdered cellulose was not suitable for the granulation with human serum albumin solution. As compared with the same concentration of the conventionally applied cellulose ethers as binder, the prepared granules exhibited a larger particle size, a significantly better compressibility, a higher breaking hardness and a favourable deformation process. These findings mainly reflect the good adhesive properties of the protein. The best compressibility and mechanical behaviour were attained on the application of the microcrystalline cellulose Vivapur type 105. This favourable behaviour may be connected with the wettability of cellulose. These results suggest that the formulation of tablets may be easier from an active agent in the serum that binds to albumin (e.g. interferon) since the amount of additives (binder) can be reduced.

Re-constructing our models of cellulose and primary cell wall assembly

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

Cosgrove, Daniel J.

2014-11-16

The cellulose microfibril has more subtlety than is commonly recognized. Details of its structure may influence how matrix polysaccharides interact with its distinctive hydrophobic and hydrophilic surfaces to form a strong yet extensible structure. We report that recent advances in this field include the first structures of bacterial and plant cellulose synthases and revised estimates of microfibril structure, reduced from 36 to 18 chains. New results also indicate that cellulose interactions with xyloglucan are more limited than commonly believed, whereas pectin-cellulose interactions are more prevalent. Computational results indicate that xyloglucan binds tightest to the hydrophobic surface of cellulose microfibrils. Finally,more » wall extensibility may be controlled at limited regions (“biomechanical hotspots”) where cellulose-cellulose contacts are made, potentially mediated by trace amounts of xyloglucan.« less

Elaborate cellulosome architecture of Acetivibrio cellulolyticus revealed by selective screening of cohesin-dockerin interactions.

PubMed

Hamberg, Yuval; Ruimy-Israeli, Vered; Dassa, Bareket; Barak, Yoav; Lamed, Raphael; Cameron, Kate; Fontes, Carlos M G A; Bayer, Edward A; Fried, Daniel B

2014-01-01

Cellulosic waste represents a significant and underutilized carbon source for the biofuel industry. Owing to the recalcitrance of crystalline cellulose to enzymatic degradation, it is necessary to design economical methods of liberating the fermentable sugars required for bioethanol production. One route towards unlocking the potential of cellulosic waste lies in a highly complex class of molecular machines, the cellulosomes. Secreted mainly by anaerobic bacteria, cellulosomes are structurally diverse, cell surface-bound protein assemblies that can contain dozens of catalytic components. The key feature of the cellulosome is its modularity, facilitated by the ultra-high affinity cohesin-dockerin interaction. Due to the enormous number of cohesin and dockerin modules found in a typical cellulolytic organism, a major bottleneck in understanding the biology of cellulosomics is the purification of each cohesin- and dockerin-containing component, prior to analyses of their interaction. As opposed to previous approaches, the present study utilized proteins contained in unpurified whole-cell extracts. This strategy was made possible due to an experimental design that allowed for the relevant proteins to be "purified" via targeted affinity interactions as a function of the binding assay. The approach thus represents a new strategy, appropriate for future medium- to high-throughput screening of whole genomes, to determine the interactions between cohesins and dockerins. We have selected the cellulosome of Acetivibrio cellulolyticus for this work due to its exceptionally complex cellulosome systems and intriguing diversity of its cellulosomal modular components. Containing 41 cohesins and 143 dockerins, A. cellulolyticus has one of the largest number of potential cohesin-dockerin interactions of any organism, and contains unusual and novel cellulosomal features. We have surveyed a representative library of cohesin and dockerin modules spanning the cellulosome's total cohesin and dockerin sequence diversity, emphasizing the testing of unusual and previously-unknown protein modules. The screen revealed several novel cell-bound cellulosome architectures, thus expanding on those previously known, as well as soluble cellulose systems that are not bound to the bacterial cell surface. This study sets the stage for screening the entire complement of cellulosomal components from A. cellulolyticus and other organisms with large cellulosome systems. The knowledge gained by such efforts brings us closer to understanding the exceptional catalytic abilities of cellulosomes and will allow the use of novel cellulosomal components in artificial assemblies and in enzyme cocktails for sustainable energy-related research programs.

Re-constructing our models of cellulose and primary cell wall assembly.

PubMed

Cosgrove, Daniel J

2014-12-01

The cellulose microfibril has more subtlety than is commonly recognized. Details of its structure may influence how matrix polysaccharides interact with its distinctive hydrophobic and hydrophilic surfaces to form a strong yet extensible structure. Recent advances in this field include the first structures of bacterial and plant cellulose synthases and revised estimates of microfibril structure, reduced from 36 to 18 chains. New results also indicate that cellulose interactions with xyloglucan are more limited than commonly believed, whereas pectin–cellulose interactions are more prevalent. Computational results indicate that xyloglucan binds tightest to the hydrophobic surface of cellulose microfibrils. Wall extensibility may be controlled at limited regions (‘biomechanical hotspots’) where cellulose–cellulose contacts are made, potentially mediated by trace amounts of xyloglucan.

The cellodextrinase from Pseudomonas fluorescens subsp. cellulosa consists of multiple functional domains.

PubMed Central

Ferreira, L M; Hazlewood, G P; Barker, P J; Gilbert, H J

1991-01-01

A genomic library of Pseudomonas fluorescens subsp. cellulosa DNA was constructed in pUC18 and Escherichia coli recombinants expressing 4-methylumbelliferyl beta-D-cellobioside-hydrolysing activity (MUCase) were isolated. Enzyme produced by MUCase-positive clones did not hydrolyse either cellobiose or cellotriose but converted cellotetraose into cellobiose and cleaved cellopentaose and cellohexaose, producing a mixture of cellobiose and cellotriose. There was no activity against CM-cellulose, insoluble cellulose or xylan. On this basis, the enzyme is identified as an endo-acting cellodextrinase and is designated cellodextrinase C (CELC). Nucleotide sequencing of the gene (celC) which directs the synthesis of CELC revealed an open reading frame of 2153 bp, encoding a protein of Mr 80,189. The deduced primary sequence of CELC was confirmed by the Mr of purified CELC (77,000) and by the experimentally determined N-terminus of the enzyme which was identical with residues 38-47 of the translated sequence. The N-terminal region of CELC showed strong homology with endoglucanase, xylanases and an arabinofuranosidase of Ps. fluorescens subsp. cellulosa; homologous sequences included highly conserved serine-rich regions. Full-length CELC bound tightly to crystalline cellulose. Truncated forms of celC from which the DNA sequence encoding the conserved domain had been deleted, directed the synthesis of a functional cellodextrinase that did not bind to crystalline cellulose. This is consistent with the N-terminal region of CELC comprising a non-catalytic cellulose-binding domain which is distinct from the catalytic domain. The role of the cellulose-binding region is discussed. Images Fig. 2. Fig. 6. PMID:1953673

Active-site copper reduction promotes substrate binding of fungal lytic polysaccharide monooxygenase and reduces stability.

PubMed

Kracher, Daniel; Andlar, Martina; Furtmüller, Paul G; Ludwig, Roland

2018-02-02

Lytic polysaccharide monooxygenases (LPMOs) are a class of copper-containing enzymes that oxidatively degrade insoluble plant polysaccharides and soluble oligosaccharides. Upon reductive activation, they cleave the substrate and promote biomass degradation by hydrolytic enzymes. In this study, we employed LPMO9C from Neurospora crassa , which is active toward cellulose and soluble β-glucans, to study the enzyme-substrate interaction and thermal stability. Binding studies showed that the reduction of the mononuclear active-site copper by ascorbic acid increased the affinity and the maximum binding capacity of LPMO for cellulose. The reduced redox state of the active-site copper and not the subsequent formation of the activated oxygen species increased the affinity toward cellulose. The lower affinity of oxidized LPMO could support its desorption after catalysis and allow hydrolases to access the cleavage site. It also suggests that the copper reduction is not necessarily performed in the substrate-bound state of LPMO. Differential scanning fluorimetry showed a stabilizing effect of the substrates cellulose and xyloglucan on the apparent transition midpoint temperature of the reduced, catalytically active enzyme. Oxidative auto-inactivation and destabilization were observed in the absence of a suitable substrate. Our data reveal the determinants of LPMO stability under turnover and non-turnover conditions and indicate that the reduction of the active-site copper initiates substrate binding. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Xyloglucan-cellulose interaction depends on the sidechains and molecular weight of xyloglucan.

PubMed

Lima, Denis U; Loh, Watson; Buckeridge, Marcos S

2004-05-01

Recent papers have brought evidence against the hypothesis that the fucosyl branching of primary wall xyloglucans (Xg) are responsible for their higher capacity of binding to cellulose. Reinforcement of this questioning has been obtained in this work where we show that the binding capacity was improved when the molecular weight (MW) of the Xg polymers is decreased by enzymatic hydrolysis. Moreover, the enthalpy changes associated with the adsorption process between Xg and cellulose is similar for Xgs with similar MW (but differing in the fine structure such as the presence/absence of fucose). On the basis of these results, we suggest that the fine structure and MW of Xg determines the energy and amount of binding to cellulose, respectively. Thus, the occurrence of different fine structural domains of Xg (e.g. the presence of fucose and the distribution of galactoses) might have several different functions in the wall. Besides the structural function in primary wall, these results might have impact on the packing features of storage Xg in seed cotyledons, since the MW and absence of fucose could also be associated with the self-association capacity. Copyright 2004 Elsevier SAS

Novel method for detection of glycogen in cells.

PubMed

Skurat, Alexander V; Segvich, Dyann M; DePaoli-Roach, Anna A; Roach, Peter J

2017-05-01

Glycogen, a branched polymer of glucose, functions as an energy reserve in many living organisms. Abnormalities in glycogen metabolism, usually excessive accumulation, can be caused genetically, most often through mutation of the enzymes directly involved in synthesis and degradation of the polymer leading to a variety of glycogen storage diseases (GSDs). Microscopic visualization of glycogen deposits in cells and tissues is important for the study of normal glycogen metabolism as well as diagnosis of GSDs. Here, we describe a method for the detection of glycogen using a renewable, recombinant protein which contains the carbohydrate-binding module (CBM) from starch-binding domain containing protein 1 (Stbd1). We generated a fusion protein containing g lutathione S-transferase, a cM c eptitope and the tbd1 BM (GYSC) for use as a glycogen-binding probe, which can be detected with secondary antibodies against glutathione S-transferase or cMyc. By enzyme-linked immunosorbent assay, we demonstrate that GYSC binds glycogen and two other polymers of glucose, amylopectin and amylose. Immunofluorescence staining of cultured cells indicate a GYSC-specific signal that is co-localized with signals obtained with anti-glycogen or anti-glycogen synthase antibodies. GYSC-positive staining inside of lysosomes is observed in individual muscle fibers isolated from mice deficient in lysosomal enzyme acid alpha-glucosidase, a well-characterized model of GSD II (Pompe disease). Co-localized GYSC and glycogen signals are also found in muscle fibers isolated from mice deficient in malin, a model for Lafora disease. These data indicate that GYSC is a novel probe that can be used to study glycogen metabolism under normal and pathological conditions. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions.

PubMed

Römling, Ute; Galperin, Michael Y

2015-09-01

Recent studies of bacterial cellulose biosynthesis, including structural characterization of a functional cellulose synthase complex, provided the first mechanistic insight into this fascinating process. In most studied bacteria, just two subunits, BcsA and BcsB, are necessary and sufficient for the formation of the polysaccharide chain in vitro. Other subunits - which differ among various taxa - affect the enzymatic activity and product yield in vivo by modulating (i) the expression of the biosynthesis apparatus, (ii) the export of the nascent β-D-glucan polymer to the cell surface, and (iii) the organization of cellulose fibers into a higher-order structure. These auxiliary subunits play key roles in determining the quantity and structure of resulting biofilms, which is particularly important for the interactions of bacteria with higher organisms - leading to rhizosphere colonization and modulating the virulence of cellulose-producing bacterial pathogens inside and outside of host cells. We review the organization of four principal types of cellulose synthase operon found in various bacterial genomes, identify additional bcs genes that encode components of the cellulose biosynthesis and secretion machinery, and propose a unified nomenclature for these genes and subunits. We also discuss the role of cellulose as a key component of biofilms and in the choice between acute infection and persistence in the host. Copyright © 2015 Elsevier Ltd. All rights reserved.

Structural motif screening reveals a novel, conserved carbohydrate-binding surface in the pathogenesis-related protein PR-5d.

PubMed

Doxey, Andrew C; Cheng, Zhenyu; Moffatt, Barbara A; McConkey, Brendan J

2010-08-03

Aromatic amino acids play a critical role in protein-glycan interactions. Clusters of surface aromatic residues and their features may therefore be useful in distinguishing glycan-binding sites as well as predicting novel glycan-binding proteins. In this work, a structural bioinformatics approach was used to screen the Protein Data Bank (PDB) for coplanar aromatic motifs similar to those found in known glycan-binding proteins. The proteins identified in the screen were significantly associated with carbohydrate-related functions according to gene ontology (GO) enrichment analysis, and predicted motifs were found frequently within novel folds and glycan-binding sites not included in the training set. In addition to numerous binding sites predicted in structural genomics proteins of unknown function, one novel prediction was a surface motif (W34/W36/W192) in the tobacco pathogenesis-related protein, PR-5d. Phylogenetic analysis revealed that the surface motif is exclusive to a subfamily of PR-5 proteins from the Solanaceae family of plants, and is absent completely in more distant homologs. To confirm PR-5d's insoluble-polysaccharide binding activity, a cellulose-pulldown assay of tobacco proteins was performed and PR-5d was identified in the cellulose-binding fraction by mass spectrometry. Based on the combined results, we propose that the putative binding site in PR-5d may be an evolutionary adaptation of Solanaceae plants including potato, tomato, and tobacco, towards defense against cellulose-containing pathogens such as species of the deadly oomycete genus, Phytophthora. More generally, the results demonstrate that coplanar aromatic clusters on protein surfaces are a structural signature of glycan-binding proteins, and can be used to computationally predict novel glycan-binding proteins from 3 D structure.

A coarse-grained model for synergistic action of multiple enzymes on cellulose

DOE PAGES

Asztalos, Andrea; Daniels, Marcus; Sethi, Anurag; ...

2012-08-01

In this study, degradation of cellulose to glucose requires the cooperative action of three classes of enzymes, collectively known as cellulases. Endoglucanases randomly bind to cellulose surfaces and generate new chain ends by hydrolyzing -1,4-D-glycosidic bonds. Exoglucanases bind to free chain ends and hydrolyze glycosidic bonds in a processive manner releasing cellobiose units. Then, -glucosidases hydrolyze soluble cellobiose to glucose. Optimal synergistic action of these enzymes is essential for efficient digestion of cellulose. Experiments show that as hydrolysis proceeds and the cellulose substrate becomes more heterogeneous, the overall degradation slows down. As catalysis occurs on the surface of crystalline cellulose,more » several factors affect the overall hydrolysis. Therefore, spatial models of cellulose degradation must capture effects such as enzyme crowding and surface heterogeneity, which have been shown to lead to a reduction in hydrolysis rates. As a result, we present a coarse-grained stochastic model for capturing the key events associated with the enzymatic degradation of cellulose at the mesoscopic level. This functional model accounts for the mobility and action of a single cellulase enzyme as well as the synergy of multiple endo- and exo-cellulases on a cellulose surface. The quantitative description of cellulose degradation is calculated on a spatial model by including free and bound states of both endo- and exo-cellulases with explicit reactive surface terms (e.g., hydrogen bond breaking, covalent bond cleavages) and corresponding reaction rates. The dynamical evolution of the system is simulated by including physical interactions between cellulases and cellulose. In conclusion, our coarse-grained model reproduces the qualitative behavior of endoglucanases and exoglucanases by accounting for the spatial heterogeneity of the cellulose surface as well as other spatial factors such as enzyme crowding. Importantly, it captures the endo-exo synergism of cellulase enzyme cocktails. This model constitutes a critical step towards testing hypotheses and understanding approaches for maximizing synergy and substrate properties with a goal of cost effective enzymatic hydrolysis.« less

Cellulose synthase interactive protein 1 (CSI1) mediates the intimate relationship between cellulose microfibrils and cortical microtubules.

PubMed

Lei, Lei; Li, Shundai; Gu, Ying

2012-07-01

Cellulose is synthesized at the plasma membrane by protein complexes known as cellulose synthase complexes (CSCs). The cellulose-microtubule alignment hypothesis states that there is a causal link between the orientation of cortical microtubules and orientation of nascent cellulose microfibrils. The mechanism behind the alignment hypothesis is largely unknown. CESA interactive protein 1 (CSI1) interacts with CSCs and potentially links CSCs to the cytoskeleton. CSI1 not only co-localizes with CSCs but also travels bi-directionally in a speed indistinguishable from CSCs. The linear trajectories of CSI1-RFP coincide with the underlying microtubules labeled by YFP-TUA5. In the absence of CSI1, both the distribution and the motility of CSCs are defective and the alignment of CSCs and microtubules is disrupted. These observations led to the hypothesis that CSI1 directly mediates the interaction between CSCs and microtubules. In support of this hypothesis, CSI1 binds to microtubules directly by an in vitro microtubule-binding assay. In addition to a role in serving as a messenger from microtubule to CSCs, CSI1 labels SmaCCs/MASCs, a compartment that has been proposed to be involved in CESA trafficking and/or delivery to the plasma membrane.

Cellulose synthase interactive protein 1 (CSI1) mediates the intimate relationship between cellulose microfibrils and cortical microtubules

PubMed Central

Lei, Lei; Li, Shundai; Gu, Ying

2012-01-01

Cellulose is synthesized at the plasma membrane by protein complexes known as cellulose synthase complexes (CSCs). The cellulose-microtubule alignment hypothesis states that there is a causal link between the orientation of cortical microtubules and orientation of nascent cellulose microfibrils. The mechanism behind the alignment hypothesis is largely unknown. CESA interactive protein 1 (CSI1) interacts with CSCs and potentially links CSCs to the cytoskeleton. CSI1 not only co-localizes with CSCs but also travels bi-directionally in a speed indistinguishable from CSCs. The linear trajectories of CSI1-RFP coincide with the underlying microtubules labeled by YFP-TUA5. In the absence of CSI1, both the distribution and the motility of CSCs are defective and the alignment of CSCs and microtubules is disrupted. These observations led to the hypothesis that CSI1 directly mediates the interaction between CSCs and microtubules. In support of this hypothesis, CSI1 binds to microtubules directly by an in vitro microtubule-binding assay. In addition to a role in serving as a messenger from microtubule to CSCs, CSI1 labels SmaCCs/MASCs, a compartment that has been proposed to be involved in CESA trafficking and/or delivery to the plasma membrane. PMID:22751327

Parameter and Process Significance in Mechanistic Modeling of Cellulose Hydrolysis

NASA Astrophysics Data System (ADS)

Rotter, B.; Barry, A.; Gerhard, J.; Small, J.; Tahar, B.

2005-12-01

The rate of cellulose hydrolysis, and of associated microbial processes, is important in determining the stability of landfills and their potential impact on the environment, as well as associated time scales. To permit further exploration in this field, a process-based model of cellulose hydrolysis was developed. The model, which is relevant to both landfill and anaerobic digesters, includes a novel approach to biomass transfer between a cellulose-bound biofilm and biomass in the surrounding liquid. Model results highlight the significance of the bacterial colonization of cellulose particles by attachment through contact in solution. Simulations revealed that enhanced colonization, and therefore cellulose degradation, was associated with reduced cellulose particle size, higher biomass populations in solution, and increased cellulose-binding ability of the biomass. A sensitivity analysis of the system parameters revealed different sensitivities to model parameters for a typical landfill scenario versus that for an anaerobic digester. The results indicate that relative surface area of cellulose and proximity of hydrolyzing bacteria are key factors determining the cellulose degradation rate.

Study of response nonuniformity for the LHCb calorimeter module and the prototype of the CBM calorimeter module

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

Korolko, I. E.; Prokudin, M. S.

A spatial nonuniformity of the response to high-energy muons is studied in the modules of the LHCb electromagnetic calorimeter and the prototype of the calorimeter module with lead plates and scintillator tiles 0.5 mm thick. The nonuniformity of the response of the inner LHCb modules to 50-GeV electrons is also measured. Software is developed for a thorough simulation of light collection in scintillator plates of a shashlik calorimeter. A model is elaborated to describe light transmission from the initial scintillation to the wavelength-shifting fiber with a subsequent reradiation and propagation of light over the fiber to the photodetector. The resultsmore » of the simulation are in good agreement with data.« less

Formation of the outer layer of the Dictyostelium spore coat depends on the inner-layer protein SP85/PsB.

PubMed

Metcalf, Talibah; Kelley, Karen; Erdos, Gregory W; Kaplan, Lee; West, Christopher M

2003-02-01

The Dictyostelium spore is surrounded by a 220 microm thick trilaminar coat that consists of inner and outer electron-dense layers surrounding a central region of cellulose microfibrils. In previous studies, a mutant strain (TL56) lacking three proteins associated with the outer layer exhibited increased permeability to macromolecular tracers, suggesting that this layer contributes to the coat permeability barrier. Electron microscopy now shows that the outer layer is incomplete in the coats of this mutant and consists of a residual regular array of punctate electron densities. The outer layer is also incomplete in a mutant lacking a cellulose-binding protein associated with the inner layer, and these coats are deficient in an outer-layer protein and another coat protein. To examine the mechanism by which this inner-layer protein, SP85, contributes to outer-layer formation, various domain fragments were overexpressed in forming spores. Most of these exert dominant negative effects similar to the deletion of outer-layer proteins, but one construct, consisting of a fusion of the N-terminal and Cys-rich C1 domain, induces a dense mat of novel filaments at the surface of the outer layer. Biochemical studies show that the C1 domain binds cellulose, and a combination of site-directed mutations that inhibits its cellulose-binding activity suppresses outer-layer filament induction. The results suggest that, in addition to a previously described early role in regulating cellulose synthesis, SP85 subsequently contributes a cross-bridging function between cellulose and other coat proteins to organize previously unrecognized structural elements in the outer layer of the coat.

Coordinated Cyclic-Di-GMP Repression of Salmonella Motility through YcgR and Cellulose

PubMed Central

Zorraquino, Violeta; García, Begoña; Latasa, Cristina; Echeverz, Maite; Toledo-Arana, Alejandro; Valle, Jaione

2013-01-01

Cyclic di-GMP (c-di-GMP) is a secondary messenger that controls a variety of cellular processes, including the switch between a biofilm and a planktonic bacterial lifestyle. This nucleotide binds to cellular effectors in order to exert its regulatory functions. In Salmonella, two proteins, BcsA and YcgR, both of them containing a c-di-GMP binding PilZ domain, are the only known c-di-GMP receptors. BcsA, upon c-di-GMP binding, synthesizes cellulose, the main exopolysaccharide of the biofilm matrix. YcgR is dedicated to c-di-GMP-dependent inhibition of motility through its interaction with flagellar motor proteins. However, previous evidences indicate that in the absence of YcgR, there is still an additional element that mediates motility impairment under high c-di-GMP levels. Here we have uncovered that cellulose per se is the factor that further promotes inhibition of bacterial motility once high c-di-GMP contents drive the activation of a sessile lifestyle. Inactivation of different genes of the bcsABZC operon, mutation of the conserved residues in the RxxxR motif of the BcsA PilZ domain, or degradation of the cellulose produced by BcsA rescued the motility defect of ΔycgR strains in which high c-di-GMP levels were reached through the overexpression of diguanylate cyclases. High c-di-GMP levels provoked cellulose accumulation around cells that impeded flagellar rotation, probably by means of steric hindrance, without affecting flagellum gene expression, exportation, or assembly. Our results highlight the relevance of cellulose in Salmonella lifestyle switching as an architectural element that is both essential for biofilm development and required, in collaboration with YcgR, for complete motility inhibition. PMID:23161026

Salmonella Biofilm Formation on Aspergillus niger Involves Cellulose – Chitin Interactions

PubMed Central

Brandl, Maria T.; Carter, Michelle Q.; Parker, Craig T.; Chapman, Matthew R.; Huynh, Steven; Zhou, Yaguang

2011-01-01

Salmonella cycles between host and nonhost environments, where it can become an active member of complex microbial communities. The role of fungi in the environmental adaptation of enteric pathogens remains relatively unexplored. We have discovered that S. enterica Typhimurium rapidly attaches to and forms biofilms on the hyphae of the common fungus, Aspergillus niger. Several Salmonella enterica serovars displayed a similar interaction, whereas other bacterial species were unable to bind to the fungus. Bacterial attachment to chitin, a major constituent of fungal cell walls, mirrored this specificity. Pre-incubation of S. Typhimurium with N-acetylglucosamine, the monomeric component of chitin, reduced binding to chitin beads by as much as 727-fold and inhibited attachment to A. niger hyphae considerably. A cellulose-deficient mutant of S. Typhimurium failed to attach to chitin beads and to the fungus. Complementation of this mutant with the cellulose operon restored binding to chitin beads to 79% of that of the parental strain and allowed for attachment and biofilm formation on A. niger, indicating that cellulose is involved in bacterial attachment to the fungus via the chitin component of its cell wall. In contrast to cellulose, S. Typhimurium curli fimbriae were not required for attachment and biofilm development on the hyphae but were critical for its stability. Our results suggest that cellulose–chitin interactions are required for the production of mixed Salmonella-A. niger biofilms, and support the hypothesis that encounters with chitinaceous alternate hosts may contribute to the ecological success of human pathogens. PMID:22003399

Sorption of poly(hexamethylenebiguanide) on cellulose: mechanism of binding and molecular recognition.

PubMed

Blackburn, Richard S; Harvey, Anna; Kettle, Lorna L; Payne, John D; Russell, Stephen J

2006-06-20

Antimicrobial agents such as poly(hexamethylene biguanide) (PHMB) find application in medical, apparel, and household textile sectors; although it is understood that certain concentrations need to be applied to achieve suitable performance, there has been very little work published concerning the interactions of the polymer and its adsorption mechanism on cellulose. In this paper, such physical chemistry parameters are examined and related to computational chemistry studies. Adsorption isotherms were constructed: at low concentrations, these were typical Langmuir isotherms; at higher concentrations, they were more indicative of Freundlich isotherms, attributed to a combination of electrostatic and hydrogen-bonding forces, which endorsed computational chemistry proposals. At lower concentrations, electrostatic interactions between PHMB and carboxylic acid groups in the cellulose dominate with a contribution to binding through hydrogen bonding; as the concentration of PHMB increases, hydrogen bonding with cellulose becomes increasingly dominant. At high PHMB concentrations, observations of increasing PHMB adsorption are attributed to monolayer aggregation and multilayer stacking of PHMB through electrostatic interactions with counterions and hydrogen bonding of biguanide groups.

The O-Glycosylated Linker from the Trichoderma reesei Family 7 Cellulase Is a Flexible, Disordered Protein

PubMed Central

Beckham, Gregg T.; Bomble, Yannick J.; Matthews, James F.; Taylor, Courtney B.; Resch, Michael G.; Yarbrough, John M.; Decker, Steve R.; Bu, Lintao; Zhao, Xiongce; McCabe, Clare; Wohlert, Jakob; Bergenstråhle, Malin; Brady, John W.; Adney, William S.; Himmel, Michael E.; Crowley, Michael F.

2010-01-01

Fungi and bacteria secrete glycoprotein cocktails to deconstruct cellulose. Cellulose-degrading enzymes (cellulases) are often modular, with catalytic domains for cellulose hydrolysis and carbohydrate-binding modules connected by linkers rich in serine and threonine with O-glycosylation. Few studies have probed the role that the linker and O-glycans play in catalysis. Since different expression and growth conditions produce different glycosylation patterns that affect enzyme activity, the structure-function relationships that glycosylation imparts to linkers are relevant for understanding cellulase mechanisms. Here, the linker of the Trichoderma reesei Family 7 cellobiohydrolase (Cel7A) is examined by simulation. Our results suggest that the Cel7A linker is an intrinsically disordered protein with and without glycosylation. Contrary to the predominant view, the O-glycosylation does not change the stiffness of the linker, as measured by the relative fluctuations in the end-to-end distance; rather, it provides a 16 Å extension, thus expanding the operating range of Cel7A. We explain observations from previous biochemical experiments in the light of results obtained here, and compare the Cel7A linker with linkers from other cellulases with sequence-based tools to predict disorder. This preliminary screen indicates that linkers from Family 7 enzymes from other genera and other cellulases within T. reesei may not be as disordered, warranting further study. PMID:21112302

Linking community-based monitoring to water policy: Perceptions of citizen scientists.

PubMed

Carlson, Tyler; Cohen, Alice

2018-05-05

This paper examines the relationships between Community-Based Water Monitoring (CBM) and government-led water initiatives. Drawing on a cross-Canada survey of over one hundred organizations, we explore the reasons why communities undertake CBM, the monitoring protocols they follow, and the extent to which CBM program members feel their findings are incorporated into formal (i.e., government-led) decision-making processes. Our results indicate that despite following standardized and credible monitoring protocols, fewer than half of CBM organizations report that their data is being used to inform water policy at any level of government. Moreover, respondents report higher rates of cooperation and data-sharing between CBM organizations themselves than between CBM organizations and their respective governments. These findings are significant, because many governments continue to express support for CBM. We explore the barriers between CBM data collection and government policy, and suggest that structural barriers include lack of multi-year funding, inconsistent protocols, and poor communication. More broadly, we argue that the distinction between formal and informal programming is unclear, and that addressing known CBM challenges will rely on a change in perception: CBM cannot simply be a less expensive alternative to government-driven data collection. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Cultivation of a native alga for biomass and biofuel accumulation in coal bed methane production water

USGS Publications Warehouse

Hodgskiss, Logan H.; Nagy, Justin; Barnhart, Elliott P.; Cunningham, Alfred B.; Fields, Matthew W.

2016-01-01

Coal bed methane (CBM) production has resulted in thousands of ponds in the Powder River Basin of low-quality water in a water-challenged region. A green alga isolate, PW95, was isolated from a CBM production pond, and analysis of a partial ribosomal gene sequence indicated the isolate belongs to the Chlorococcaceae family. Different combinations of macro- and micronutrients were evaluated for PW95 growth in CBM water compared to a defined medium. A small level of growth was observed in unamended CBM water (0.15 g/l), and biomass increased (2-fold) in amended CBM water or defined growth medium. The highest growth rate was observed in CBM water amended with both N and P, and the unamended CBM water displayed the lowest growth rate. The highest lipid content (27%) was observed in CBM water with nitrate, and a significant level of lipid accumulation was not observed in the defined growth medium. Growth analysis indicated that nitrate deprivation coincided with lipid accumulation in CBM production water, and lipid accumulation did not increase with additional phosphorus limitation. The presented results show that CBM production wastewater can be minimally amended and used for the cultivation of a native, lipid-accumulating alga.

Controlled Aggregation and Increased Stability of β-Glucuronidase by Cellulose Binding Domain Fusion

PubMed Central

Kim, Moonjung; Kwon, Kil Koang; Fu, Yaoyao; Kim, Haseong; Lee, Hyewon; Lee, Dae-Hee; Jung, Heungchae; Lee, Seung-Goo

2017-01-01

Cellulose-binding domains (CBDs) are protein domains with cellulose-binding activity, and some act as leaders in the localization of cellulosomal scaffoldin proteins to the hydrophobic surface of crystalline cellulose. In this study, we found that a CBD fusion enhanced and improved soluble β-glucuronidase (GusA) enzyme properties through the formation of an artificially oligomeric state. First, a soluble CBD fused to the C-terminus of GusA (GusA-CBD) was obtained and characterized. Interestingly, the soluble GusA-CBD showed maximum activity at higher temperatures (65°C) and more acidic pH values (pH 6.0) than free GusA did (60°C and pH 7.5). Moreover, the GusA-CBD enzyme showed higher thermal and pH stabilities than the free GusA enzyme did. Additionally, GusA-CBD showed higher enzymatic activity in the presence of methanol than free GusA did. Evaluation of the protease accessibility of both enzymes revealed that GusA-CBD retained 100% of its activity after 1 h incubation in 0.5 mg/ml protease K, while free GusA completely lost its activity. Simple fusion of CBD as a single domain may be useful for tunable enzyme states to improve enzyme stability in industrial applications. PMID:28099480

Controlled Aggregation and Increased Stability of β-Glucuronidase by Cellulose Binding Domain Fusion.

PubMed

Yeom, Soo-Jin; Han, Gui Hwan; Kim, Moonjung; Kwon, Kil Koang; Fu, Yaoyao; Kim, Haseong; Lee, Hyewon; Lee, Dae-Hee; Jung, Heungchae; Lee, Seung-Goo

2017-01-01

Cellulose-binding domains (CBDs) are protein domains with cellulose-binding activity, and some act as leaders in the localization of cellulosomal scaffoldin proteins to the hydrophobic surface of crystalline cellulose. In this study, we found that a CBD fusion enhanced and improved soluble β-glucuronidase (GusA) enzyme properties through the formation of an artificially oligomeric state. First, a soluble CBD fused to the C-terminus of GusA (GusA-CBD) was obtained and characterized. Interestingly, the soluble GusA-CBD showed maximum activity at higher temperatures (65°C) and more acidic pH values (pH 6.0) than free GusA did (60°C and pH 7.5). Moreover, the GusA-CBD enzyme showed higher thermal and pH stabilities than the free GusA enzyme did. Additionally, GusA-CBD showed higher enzymatic activity in the presence of methanol than free GusA did. Evaluation of the protease accessibility of both enzymes revealed that GusA-CBD retained 100% of its activity after 1 h incubation in 0.5 mg/ml protease K, while free GusA completely lost its activity. Simple fusion of CBD as a single domain may be useful for tunable enzyme states to improve enzyme stability in industrial applications.

The Silicon Tracking System of the CBM experiment at FAIR

NASA Astrophysics Data System (ADS)

Teklishyn, Maksym

2018-03-01

The Silicon Tracking System (STS) is the central detector in the Compressed Baryonic Matter (CBM) experiment at FAIR. Operating in the 1Tm dipole magnetic field, the STS will enable pile-up free detection and momentum measurement of the charged particles originating from beam-target nuclear interactions at rates up to 10 MHz. The STS consists of 8 tracking stations based on double-sided silicon micro-strip sensors equipped with fast, self-triggering read-out electronics. With about two million read-out channels, the STS will deliver a high-rate stream of time-stamped data that is transferred to a computing farm for on-line event determination and analysis. The functional building block is a detector module consisting of a sensor, micro-cables and two front-end electronics boards. In this contribution, the development status of the STS components and the system integration is discussed and an outlook on the detector construction is given.

Construction of a chimeric thermostable pyrophosphatase to facilitate its purification and immobilization by using the choline-binding tag.

PubMed

Moldes, Cristina; García, José L; García, Pedro

2004-08-01

The thermophilic inorganic pyrophosphatase (Pyr) from Thermus thermophilus has been produced in Escherichia coli fused to the C terminus of the choline-binding tag (ChB tag) derived from the choline-binding domain (ChBD) of pneumococcal LytA autolysin. The chimeric ChBD-Pyr protein retains its thermostable activity and can be purified in a single step by DEAE-cellulose affinity chromatography. Pyr can be further released from the ChBD by thrombin, using the specific protease recognition site incorporated in the C terminus of this tag. Remarkably, the ChB tag provides a selective and very strong thermostable noncovalent immobilization of ChBD-Pyr in the DEAE-cellulose matrix. The binding of choline or choline analogues, such as DEAE, confers a high thermal stability to this tag; therefore, the immobilized chimeric enzyme can be assayed at high temperature without protein leakage, demonstrating the usefulness of the ChB tag for noncovalent immobilization of thermophilic proteins. Moreover, ChBD-Pyr can be purified and immobilized in a single step on commercial DEAE-cellulose paper. The affinity of the ChB tag for this versatile solid support can be very helpful in developing many biotechnological applications.

Thermodynamics of Interaction between Some Cellulose Ethers and SDS by Titration Microcalorimetry.

PubMed

Singh; Nilsson

1999-05-01

The interaction between certain nonionic cellulose ethers (ethyl hydroxyethyl cellulose and hydroxypropyl methyl cellulose) and sodium dodecyl sulphate (SDS) has been investigated using isothermal titration microcalorimetry at temperatures between 25-50 degrees C. The observed heat flow curves have been interpreted in terms of a plausible mechanism of the interaction of the substituent groups with SDS monomers and clusters. The data have been related to changes occuring in the system at the macro- and microscopic levels with the addition of surfactants and with temperature. The process consists predominantly of polymer-surfactant interactions initially and surfactant-surfactant interactions at the later stages. A phenomenological model of the cooperative interaction (adsorption) process has been derived, and earlier published equilibrium binding data have been used to recover binding constants and Gibbs energy changes for this process. The adsorption enthalpies and entropies have been recovered along with the heat capacity change. The enthalpic cost of confining the nonpolar regions of the polymers in surfactant clusters is high, but the entropy gain from release of hydration shell water molecules as well as increased freedom of movement of these nonpolar regions in the clusters gives the process a strong entropic driving force. The process is entropy-driven initially and converts to being both enthalpy and entropy-driven at high SDS concentrations. An enthalpy-entropy compensation behavior is seen. Strongly negative heat capacity changes have been obtained resulting from the transfer of nonpolar groups from aqueous into nonpolar environments, as well as a reduction of conformational domains that the chains can populate. Changes in these two components cause the heat capacity change to become less negative at the higher binding levels. The system can be classified as exhibiting nonclassical hydrophobic binding at the later stages of binding. Copyright 1999 Academic Press.

Bacterial cellulose biosynthesis: diversity of operons, subunits, products and functions

PubMed Central

Römling, Ute; Galperin, Michael Y.

2015-01-01

Summary Recent studies of bacterial cellulose biosynthesis, including structural characterization of a functional cellulose synthase complex, provided the first mechanistic insight into this fascinating process. In most studied bacteria, just two subunits, BcsA and BcsB, are necessary and sufficient for the formation of the polysaccharide chain in vitro. Other subunits – which differ among various taxa – affect the enzymatic activity and product yield in vivo by modulating expression of biosynthesis apparatus, export of the nascent β-D-glucan polymer to the cell surface, and the organization of cellulose fibers into a higher-order structure. These auxiliary subunits play key roles in determining the quantity and structure of the resulting biofilm, which is particularly important for interactions of bacteria with higher organisms that lead to rhizosphere colonization and modulate virulence of cellulose-producing bacterial pathogens inside and outside of host cells. Here we review the organization of four principal types of cellulose synthase operons found in various bacterial genomes, identify additional bcs genes that encode likely components of the cellulose biosynthesis and secretion machinery, and propose a unified nomenclature for these genes and subunits. We also discuss the role of cellulose as a key component of biofilms formed by a variety of free-living and pathogenic bacteria and, for the latter, in the choice between acute infection and persistence in the host. PMID:26077867

The Dual Activity Responsible for the Elongation and Branching of β-(1,3)-Glucan in the Fungal Cell Wall.

PubMed

Aimanianda, Vishukumar; Simenel, Catherine; Garnaud, Cecile; Clavaud, Cecile; Tada, Rui; Barbin, Lise; Mouyna, Isabelle; Heddergott, Christoph; Popolo, Laura; Ohya, Yoshikazu; Delepierre, Muriel; Latge, Jean-Paul

2017-06-20

β-(1,3)-Glucan, the major fungal cell wall component, ramifies through β-(1,6)-glycosidic linkages, which facilitates its binding with other cell wall components contributing to proper cell wall assembly. Using Saccharomyces cerevisiae as a model, we developed a protocol to quantify β-(1,6)-branching on β-(1,3)-glucan. Permeabilized S. cerevisiae and radiolabeled substrate UDP-( 14 C)glucose allowed us to determine branching kinetics. A screening aimed at identifying deletion mutants with reduced branching among them revealed only two, the bgl2 Δ and gas1 Δ mutants, showing 15% and 70% reductions in the branching, respectively, compared to the wild-type strain. Interestingly, a recombinant Gas1p introduced β-(1,6)-branching on the β-(1,3)-oligomers following its β-(1,3)-elongase activity. Sequential elongation and branching activity of Gas1p occurred on linear β-(1,3)-oligomers as well as Bgl2p-catalyzed products [short β-(1,3)-oligomers linked by a linear β-(1,6)-linkage]. The double S. cerevisiae gas1 Δ bgl2 Δ mutant showed a drastically sick phenotype. An Sc Gas1p ortholog, Gel4p from Aspergillus fumigatus , also showed dual β-(1,3)-glucan elongating and branching activity. Both Sc Gas1p and A. fumigatus Gel4p sequences are endowed with a carbohydrate binding module (CBM), CBM43, which was required for the dual β-(1,3)-glucan elongating and branching activity. Our report unravels the β-(1,3)-glucan branching mechanism, a phenomenon occurring during construction of the cell wall which is essential for fungal life. IMPORTANCE The fungal cell wall is essential for growth, morphogenesis, protection, and survival. In spite of being essential, cell wall biogenesis, especially the core β-(1,3)-glucan ramification, is poorly understood; the ramified β-(1,3)-glucan interconnects other cell wall components. Once linear β-(1,3)-glucan is synthesized by plasma membrane-bound glucan synthase, the subsequent event is its branching event in the cell wall space. Using Saccharomyces cerevisiae as a model, we identified GH72 and GH17 family glycosyltransferases, Gas1p and Bgl2p, respectively, involved in the β-(1,3)-glucan branching. The sick phenotype of the double Scgas1 Δ bgl2 Δ mutant suggested that β-(1,3)-glucan branching is essential. In addition to Sc Gas1p, GH72 family Sc Gas2p and Aspergillus fumigatus Gel4p, having CBM43 in their sequences, showed dual β-(1,3)-glucan elongating and branching activity. Our report identifies the fungal cell wall β-(1,3)-glucan branching mechanism. The essentiality of β-(1,3)-glucan branching suggests that enzymes involved in the glucan branching could be exploited as antifungal targets. Copyright © 2017 Aimanianda et al.

Online Event Reconstruction in the CBM Experiment at FAIR

NASA Astrophysics Data System (ADS)

Akishina, Valentina; Kisel, Ivan

2018-02-01

Targeting for rare observables, the CBM experiment will operate at high interaction rates of up to 10 MHz, which is unprecedented in heavy-ion experiments so far. It requires a novel free-streaming readout system and a new concept of data processing. The huge data rates of the CBM experiment will be reduced online to the recordable rate before saving the data to the mass storage. Full collision reconstruction and selection will be performed online in a dedicated processor farm. In order to make an efficient event selection online a clean sample of particles has to be provided by the reconstruction package called First Level Event Selection (FLES). The FLES reconstruction and selection package consists of several modules: track finding, track fitting, event building, short-lived particles finding, and event selection. Since detector measurements contain also time information, the event building is done at all stages of the reconstruction process. The input data are distributed within the FLES farm in a form of time-slices. A time-slice is reconstructed in parallel between processor cores. After all tracks of the whole time-slice are found and fitted, they are collected into clusters of tracks originated from common primary vertices, which then are fitted, thus identifying the interaction points. Secondary tracks are associated with primary vertices according to their estimated production time. After that short-lived particles are found and the full event building process is finished. The last stage of the FLES package is a selection of events according to the requested trigger signatures. The event reconstruction procedure and the results of its application to simulated collisions in the CBM detector setup are presented and discussed in detail.

Monitoring In Vivo Changes in Tonic Extracellular Dopamine Level by Charge-Balancing Multiple Waveform Fast-Scan Cyclic Voltammetry.

PubMed

Oh, Yoonbae; Park, Cheonho; Kim, Do Hyoung; Shin, Hojin; Kang, Yu Min; DeWaele, Mark; Lee, Jeyeon; Min, Hoon-Ki; Blaha, Charles D; Bennet, Kevin E; Kim, In Young; Lee, Kendall H; Jang, Dong Pyo

2016-11-15

Dopamine (DA) modulates central neuronal activity through both phasic (second to second) and tonic (minutes to hours) terminal release. Conventional fast-scan cyclic voltammetry (FSCV), in combination with carbon fiber microelectrodes, has been used to measure phasic DA release in vivo by adopting a background subtraction procedure to remove background capacitive currents. However, measuring tonic changes in DA concentrations using conventional FSCV has been difficult because background capacitive currents are inherently unstable over long recording periods. To measure tonic changes in DA concentrations over several hours, we applied a novel charge-balancing multiple waveform FSCV (CBM-FSCV), combined with a dual background subtraction technique, to minimize temporal variations in background capacitive currents. Using this method, in vitro, charge variations from a reference time point were nearly zero for 48 h, whereas with conventional background subtraction, charge variations progressively increased. CBM-FSCV also demonstrated a high selectivity against 3,4-dihydroxyphenylacetic acid and ascorbic acid, two major chemical interferents in the brain, yielding a sensitivity of 85.40 ± 14.30 nA/μM and limit of detection of 5.8 ± 0.9 nM for DA while maintaining selectivity. Recorded in vivo by CBM-FSCV, pharmacological inhibition of DA reuptake (nomifensine) resulted in a 235 ± 60 nM increase in tonic extracellular DA concentrations, while inhibition of DA synthesis (α-methyl-dl-tyrosine) resulted in a 72.5 ± 4.8 nM decrease in DA concentrations over a 2 h period. This study showed that CBM-FSCV may serve as a unique voltammetric technique to monitor relatively slow changes in tonic extracellular DA concentrations in vivo over a prolonged time period.

Molecular cloning, overexpression, and enzymatic characterization of glycosyl hydrolase family 16 β-Agarase from marine bacterium Saccharophagus sp. AG21 in Escherichia coli.

PubMed

Lee, Youngdeuk; Oh, Chulhong; De Zoysa, Mahanama; Kim, Hyowon; Wickramaarachchi, Wickramaarachchige Don Niroshana; Whang, Ilson; Kang, Do-Hyung; Lee, Jehee

2013-01-01

An agar-degrading bacterium was isolated from red seaweed (Gelidium amansii) on a natural seawater agar plate, and identified as Saccharophagus sp. AG21. The β-agarase gene from Saccharophagus sp. AG21 (agy1) was screened by long and accurate (LA)-PCR. The predicted sequence has a 1,908 bp open reading frame encoding 636 amino acids (aa), and includes a glycosyl hydrolase family 16 (GH16) β-agarase module and two carbohydrate binding modules of family 6 (CBM6). The deduced aa sequence showed 93.7% and 84.9% similarity to β-agarase of Saccharophagus degradans and Microbulbifer agarilyticus, respectively. The mature agy1 was cloned and overexpressed as a His-tagged recombinant β-agarase (rAgy1) in Escherichia coli, and had a predicted molecular mass of 69 kDa and an isoelectric point of 4.5. rAgy1 showed optimum activity at 55oC and pH 7.6, and had a specific activity of 85 U/mg. The rAgy1 activity was enhanced by FeSO4 (40%), KCl (34%), and NaCl (34%), compared with the control. The newly identified rAgy1 is a β-agarase, which acts to degrade agarose to neoagarotetraose (NA4) and neoagarohexaose (NA6) and may be useful for applications in the cosmetics, food, bioethanol, and reagent industries.

CBM Reading, Mathematics, and Written Expression at the Secondary Level: Examining Latent Composite Relations Among Indices and Unique Predictions With a State Achievement Test

PubMed Central

Codding, Robin S.; Petscher, Yaacov; Truckenmiller, Adrea

2015-01-01

A paucity of research has examined the utility of curriculum-based measurement (CBM) for data-based decision making at the secondary level. As schools move to multitiered systems of service delivery, it is conceivable that multiple screening measures will be used that address various academic subject areas. The value of including different CBM indices measures is not well understood. The purpose of this study was to (a) examine the relationship among a variety of reading, writing, and mathematics CBM indices administered to 249 seventh-grade students; (b) investigate amount and patterns of growth; and (c) examine predictive validity to a high-stakes state test using latent factor analysis and multiple indicator growth models. Results indicated strong correspondence among CBM types for fall static scores but weak relationships among slopes. Different patterns of growth were yielded for CBM writing than for CBM reading and mathematics. Findings from this study suggested that although reading, mathematics, and writing CBM were independently and moderately related to both English Language Arts and Math test scores, reading was the strongest predictor when all 3 CBM constructs were considered jointly. PMID:26347201

Intelligent and integrated techniques for coalbed methane (CBM) recovery and reduction of greenhouse gas emission.

PubMed

Qianting, Hu; Yunpei, Liang; Han, Wang; Quanle, Zou; Haitao, Sun

2017-07-01

Coalbed methane (CBM) recovery is a crucial approach to realize the exploitation of a clean energy and the reduction of the greenhouse gas emission. In the past 10 years, remarkable achievements on CBM recovery have been obtained in China. However, some key difficulties still exist such as long borehole drilling in complicated geological condition, and poor gas drainage effect due to low permeability. In this study, intelligent and integrated techniques for CBM recovery are introduced. These integrated techniques mainly include underground CBM recovery techniques and ground well CBM recovery techniques. The underground CBM recovery techniques consist of the borehole formation technique, gas concentration improvement technique, and permeability enhancement technique. According to the division of mining-induced disturbance area, the ground well arrangement area and well structure type in mining-induced disturbance developing area and mining-induced disturbance stable area are optimized to significantly improve the ground well CBM recovery. Besides, automatic devices such as drilling pipe installation device are also developed to achieve remote control of data recording, which makes the integrated techniques intelligent. These techniques can provide key solutions to some long-term difficulties in CBM recovery.

CBM Reading, Mathematics, and Written Expression at the Secondary Level: Examining Latent Composite Relations Among Indices and Unique Predictions With a State Achievement Test.

PubMed

Codding, Robin S; Petscher, Yaacov; Truckenmiller, Adrea

2015-05-01

A paucity of research has examined the utility of curriculum-based measurement (CBM) for data-based decision making at the secondary level. As schools move to multitiered systems of service delivery, it is conceivable that multiple screening measures will be used that address various academic subject areas. The value of including different CBM indices measures is not well understood. The purpose of this study was to (a) examine the relationship among a variety of reading, writing, and mathematics CBM indices administered to 249 seventh-grade students; (b) investigate amount and patterns of growth; and (c) examine predictive validity to a high-stakes state test using latent factor analysis and multiple indicator growth models. Results indicated strong correspondence among CBM types for fall static scores but weak relationships among slopes. Different patterns of growth were yielded for CBM writing than for CBM reading and mathematics. Findings from this study suggested that although reading, mathematics, and writing CBM were independently and moderately related to both English Language Arts and Math test scores, reading was the strongest predictor when all 3 CBM constructs were considered jointly.

Adsorption mechanism for xanthene dyes to cellulose granules.

PubMed

Tabara, Aya; Yamane, Chihiro; Seguchi, Masaharu

2012-01-01

The xanthene dyes, erythrosine, phloxine, and rose bengal, were adsorbed to charred cellulose granules. The charred cellulose granules were preliminarily steeped in ionic (NaOH, NaCl, KOH, KCl, and sodium dodecyl sulfate (SDS)), nonionic (glucose, sucrose, and ethanol), and amphipathic sucrose fatty acid ester (SFAE) solutions, and adsorption tests on the dye to the steeped and charred cellulose granules were conducted. Almost none of the dye was adsorbed when the solutions of ionic and amphipathic molecules were used, but were adsorbed in the case of steeping in the nonionic molecule solutions. Thin-layer chromatography (TLC) and the Fourier transform infra-red (FT-IR) profiles of SFAE which was adsorbed to the charred cellulose granules and extracted by ethyl ether suggested the presence of hydrophobic sites on the surface of the charred cellulose granules. We confirmed that the xanthene dyes could bind to the charred cellulose granules by ionic and hydrophobic bonds.

Covalent Binding of Antibodies to Cellulose Paper Discs and Their Applications in Naked-eye Colorimetric Immunoassays.

PubMed

Peng, Yanfen; Gelder, Victor Van; Amaladoss, Anburaj; Patel, Kadamb Haribhai

2016-10-21

This report presents two methods for the covalent immobilization of capture antibodies on cellulose filter paper grade No. 1 (medium-flow filter paper) discs and grade No. 113 (fast-flow filter paper) discs. These cellulose paper discs were grafted with amine functional groups through a silane coupling technique before the antibodies were immobilized on them. Periodate oxidation and glutaraldehyde cross-linking methods were used to graft capture antibodies on the cellulose paper discs. In order to ensure the maximum binding capacity of the capture antibodies to their targets after immobilization, the effects of various concentrations of sodium periodate, glutaraldehyde, and capture antibodies on the surface of the paper discs were investigated. The antibodies that were coated on the amine-functionalized cellulose paper discs through a glutaraldehyde cross-linking agent showed enhanced binding activity to the target when compared to the periodate oxidation method. IgG (in mouse reference serum) was used as a reference target in this study to test the application of covalently immobilized antibodies through glutaraldehyde. A new paper-based, enzyme-linked immunosorbent assay (ELISA) was successfully developed and validated for the detection of IgG. This method does not require equipment, and it can detect 100 ng/ml of IgG. The fast-flow filter paper was more sensitive than the medium-flow filter paper. The incubation period of this assay was short and required small sample volumes. This naked-eye, colorimetric immunoassay can be extended to detect other targets that are identified with conventional ELISA.

A Molecular Description of Cellulose Biosynthesis

PubMed Central

McNamara, Joshua T.; Morgan, Jacob L.W.; Zimmer, Jochen

2016-01-01

Cellulose is the most abundant biopolymer on Earth, and certain organisms from bacteria to plants and animals synthesize cellulose as an extracellular polymer for various biological functions. Humans have used cellulose for millennia as a material and an energy source, and the advent of a lignocellulosic fuel industry will elevate it to the primary carbon source for the burgeoning renewable energy sector. Despite the biological and societal importance of cellulose, the molecular mechanism by which it is synthesized is now only beginning to emerge. On the basis of recent advances in structural and molecular biology on bacterial cellulose synthases, we review emerging concepts of how the enzymes polymerize glucose molecules, how the nascent polymer is transported across the plasma membrane, and how bacterial cellulose biosynthesis is regulated during biofilm formation. Additionally, we review evolutionary commonalities and differences between cellulose synthases that modulate the nature of the cellulose product formed. PMID:26034894

The Predictive Validity of CBM Writing Indices for Eighth-Grade Students

ERIC Educational Resources Information Center

Amato, Janelle M.; Watkins, Marley W.

2011-01-01

Curriculum-based measurement (CBM) is an alternative to traditional assessment techniques. Technical work has begun to identify CBM writing indices that are psychometrically sound for monitoring older students' writing proficiency. This study examined the predictive validity of CBM writing indices in a sample of 447 eighth-grade students.…

Thermodynamic Analysis on of Skid-Mounted Coal-bed Methane Liquefaction Device using Cryogenic Turbo-Expander

NASA Astrophysics Data System (ADS)

Chen, Shuangtao; Niu, Lu; Zeng, Qiang; Li, Xiaojiang; Lou, Fang; Chen, Liang; Hou, Yu

2017-12-01

Coal-bed methane (CBM) reserves are rich in Sinkiang of China, and liquefaction is a critical step for the CBM exploration and utilization. Different from other CBM gas fields in China, CBM distribution in Sinkiang is widespread but scattered, and the pressure, flow-rate and nitrogen content of CBM feed vary significantly. The skid-mounted liquefaction device is suggested as an efficient and economical way to recover methane. Turbo-expander is one of the most important parts which generates the cooling capacity for the cryogenic liquefaction system. Using turbo-expander, more cooling capacity and higher liquefied fraction can be achieved. In this study, skid-mounted CBM liquefaction processes based on Claude cycle are established. Cryogenic turbo-expander with high expansion ratio is employed to improve the efficiency of CBM liquefaction process. The unit power consumption per liquefaction mole flow-rate for CBM feed gas is used as the object function for process optimization, compressor discharge pressure, flow ratio of feed gas to turbo-expander and nitrogen friction are analyzed, and optimum operation range of the liquefaction processes are obtained.

Three-dimensional (3D) structure prediction and function analysis of the chitin-binding domain 3 protein HD73_3189 from Bacillus thuringiensis HD73.

PubMed

Zhan, Yiling; Guo, Shuyuan

2015-01-01

Bacillus thuringiensis (Bt) is capable of producing a chitin-binding protein believed to be functionally important to bacteria during the stationary phase of its growth cycle. In this paper, the chitin-binding domain 3 protein HD73_3189 from B. thuringiensis has been analyzed by computer technology. Primary and secondary structural analyses demonstrated that HD73_3189 is negatively charged and contains several α-helices, aperiodical coils and β-strands. Domain and motif analyses revealed that HD73_3189 contains a signal peptide, an N-terminal chitin binding 3 domains, two copies of a fibronectin-like domain 3 and a C-terminal carbohydrate binding domain classified as CBM_5_12. Moreover, analysis predicted the protein's associated localization site to be the cell wall. Ligand site prediction determined that amino acid residues GLU-312, TRP-334, ILE-341 and VAL-382 exposed on the surface of the target protein exhibit polar interactions with the substrate.

Cellulose-Pectin Spatial Contacts Are Inherent to Never-Dried Arabidopsis Primary Cell Walls: Evidence from Solid-State Nuclear Magnetic Resonance1[OPEN

PubMed Central

Wang, Tuo; Park, Yong Bum; Hong, Mei

2015-01-01

The structural role of pectins in plant primary cell walls is not yet well understood because of the complex and disordered nature of the cell wall polymers. We recently introduced multidimensional solid-state nuclear magnetic resonance spectroscopy to characterize the spatial proximities of wall polysaccharides. The data showed extensive cross peaks between pectins and cellulose in the primary wall of Arabidopsis (Arabidopsis thaliana), indicating subnanometer contacts between the two polysaccharides. This result was unexpected because stable pectin-cellulose interactions are not predicted by in vitro binding assays and prevailing cell wall models. To investigate whether the spatial contacts that give rise to the cross peaks are artifacts of sample preparation, we now compare never-dried Arabidopsis primary walls with dehydrated and rehydrated samples. One-dimensional 13C spectra, two-dimensional 13C-13C correlation spectra, water-polysaccharide correlation spectra, and dynamics data all indicate that the structure, mobility, and intermolecular contacts of the polysaccharides are indistinguishable between never-dried and rehydrated walls. Moreover, a partially depectinated cell wall in which 40% of homogalacturonan is extracted retains cellulose-pectin cross peaks, indicating that the cellulose-pectin contacts are not due to molecular crowding. The cross peaks are observed both at −20°C and at ambient temperature, thus ruling out freezing as a cause of spatial contacts. These results indicate that rhamnogalacturonan I and a portion of homogalacturonan have significant interactions with cellulose microfibrils in the native primary wall. This pectin-cellulose association may be formed during wall biosynthesis and may involve pectin entrapment in or between cellulose microfibrils, which cannot be mimicked by in vitro binding assays. PMID:26036615

Coalbed methane-produced water quality and its management options in Raniganj Basin, West Bengal, India

NASA Astrophysics Data System (ADS)

Mendhe, Vinod Atmaram; Mishra, Subhashree; Varma, Atul Kumar; Singh, Awanindra Pratap

2017-06-01

Coalbed methane (CBM) recovery is associated with production of large quantity of groundwater. The coal seams are depressurized by pumping of water for regular and consistent gas production. Usually, CBM operators need to pump >10 m3 of water per day from one well, which depends on the aquifer characteristics, drainage and recharge pattern. In India, 32 CBM blocks have been awarded for exploration and production, out of which six blocks are commercially producing methane gas at 0.5 million metric standard cubic feet per day. Large amount of water is being produced from CBM producing blocks, but no specific information or data are available for geochemical properties of CBM-produced water and its suitable disposal or utilization options for better management. CBM operators are in infancy and searching for the suitable solutions for optimal management of produced water. CBM- and mine-produced water needs to be handled considering its physical and geochemical assessment, because it may have environmental as well as long-term impact on aquifer. Investigations were carried out to evaluate geochemical and hydrogeological conditions of CBM blocks in Raniganj Basin. Totally, 15 water samples from CBM well head and nine water samples from mine disposal head were collected from Raniganj Basin. The chemical signature of produced water reveals high sodium and bicarbonate concentrations with low calcium and magnesium, and very low sulphate in CBM water. It is comprehend that CBM water is mainly of Na-HCO3 type and coal mine water is of Ca-Mg-SO4 and HCO3-Cl-SO4 type. The comparative studies are also carried out for CBM- and mine-produced water considering the geochemical properties, aquifer type, depth of occurrence and lithological formations. Suitable options like impounding, reverse osmosis, irrigation and industrial use after prerequisite treatments are suggested. However, use of this huge volume of CBM- and mine-produced water for irrigation or other beneficial purposes may require careful management based on water pH, EC, TDS, alkalinity, bicarbonate, sodium, fluoride, metals content and SAR values.

Coalbed methane potential in the Appalachian states of Pennsylvania, West Virginia, Maryland, Ohio, Virginia, Kentucky, and Tennessee; an overview

USGS Publications Warehouse

Lyons, Paul C.

1996-01-01

This report focuses on the coalbed methane (CBM) potential of the central Appalachian basin (Virginia, eastern Kentucky, southern West Virginia, and Tennessee) and the northern Appalachian basin (Pennsylvania, northern West Virginia, Maryland, and Ohio). As of April 1996, there were about 800 wells producing CBM in the central and northern Appalachian basin. For the Appalchian basin as a whole (including the Cahaba coal field, Alabama, and excluding the Black Warrior Basin, Alabama), the total CBM production for 1992, 1993, 1994, and 1995, is here estimated at 7.77, 21.51, 29.99, and 32 billion cubic feet (Bcf), respectively. These production data compare with 91.38, 104.70, 110.70, and 112.11 Bcf, respectively, for the same years for the Black Warrior Basin, which is the second largest CBM producing basin in the United States. For 1992-1995, 92-95% of central and northern Appalachian CBM production came from southwestern Virginia, which has by far the largest CBM production the Appalachian states, exclusive of Alabama. For 1994, the average daily production of CBM wells in Virginia was 119.6 Mcf/day, which is about two to four times the average daily production rates for many of the CBM wells in the northern Appalachian basin. For 1992-1995, there is a clear increase in the percentage of CBM being produced in the central and northern Appalachian basin as compared with the Black Warrior Basin. In 1992, this percentage was 8% of the combined central and northern Appalachian and Black Warrior Basin CBM production as compared with 22% in 1995. These trends imply that the Appalachian states, except for Alabama and Virginia, are in their infancy with respect to CBM production. Total in place CBM resources in the central and northern Appalachian basin have been variously estimated at 66-76 trillion cubic feet (Tcf), of which an estimated 14.55 Tcf (3.07 Tcf for central Appalachian basin and 11.48 Tcf for northern Appalachian basin) is technically recoverable according to Ricei s (1995) report. This compares with 20 Tcf in place and 2.30 Tcf as technically recoverable CBM for the Black Warrior Basin. These estimates should be considered preliminary because of unknown CBM potential in Ohio, Maryland, Tennessee, and eastern Kentucky. The largest potential for CBM development in the central Appalachian basin is in the Pocahontas coal beds, which have total gas values as much as 700 cf/ton, and in the New River coal beds. In the northern Appalachian basin, the greatest CBM potential is in the Middle Pennsylvanian Allegheny coal beds, which have total gas values as much as 252 cf/ton. Rice (1995) estimated a mean estimated ultimate recovery per well of 521 MMcfg for the central Appalachian basin and means of 121 and 216 MMcfg for the anticlinal and synclinal areas, respectively, of the northern Applachian basin. There is potential for CBM development in the Valley coal fields and Richmond basin of Virginia, the bituminous region of southeastern Kentucky, eastern Ohio, northern Tennessee, and the Georges Creek coal field of western Maryland and adjacent parts of Pennsylvania. Moreover, the Anthracite region of eastern Pennsylvania, which has the second highest known total gas content for a single coal bed (687 cf/ton) in the central and northern Appalachian basin, should be considered to have a fair to good potential for CBM development where structure, bed continuity, and permeability are favorable. CBM is mainly an undeveloped unconventional fossil-fuel resource in the central and northern Appalachian basin states, except in Virginia, and will probably contribute an increasing part of total Appalachian gas production into the next century as development in Pennsylvania, West Virginia, Ohio, and other Appalachian states continue. The central and northern Appalachian basins are frontier or emerging regions for CBM exploration and development, which will probably extend well into the next century. On the basis of CBM production

Generalizability Theory Analysis of CBM Maze Reliability in Third- through Fifth-Grade Students

ERIC Educational Resources Information Center

Mercer, Sterett H.; Dufrene, Brad A.; Zoder-Martell, Kimberly; Harpole, Lauren Lestremau; Mitchell, Rachel R.; Blaze, John T.

2012-01-01

Despite growing use of CBM Maze in universal screening and research, little information is available regarding the number of CBM Maze probes needed for reliable decisions. The current study extends existing research on the technical adequacy of CBM Maze by investigating the number of probes and assessment durations (1-3 min) needed for reliable…

Relation between CBM-R and CBM-mR Slopes: An Application of Latent Growth Modeling

ERIC Educational Resources Information Center

Yeo, Seungsoo; Fearrington, Jamie Y.; Christ, Theodore J.

2012-01-01

Oral reading tasks and Maze reading tasks are often used interchangeably to assess the level and rate of reading skill development. This study examined the concurrent validity of growth estimates derived from "Curriculum-Based Measurement of Oral Reading" (CBM-R) and "Maze Reading" (CBM-mR). Participants were 1,528 students…

Loosenin, a novel protein with cellulose-disrupting activity from Bjerkandera adusta.

PubMed

Quiroz-Castañeda, Rosa E; Martínez-Anaya, Claudia; Cuervo-Soto, Laura I; Segovia, Lorenzo; Folch-Mallol, Jorge L

2011-02-11

Expansins and expansin-like proteins loosen cellulose microfibrils, possibly through the rupture of intramolecular hydrogen bonds. Together with the use of lignocellulolytic enzymes, these proteins are potential molecular tools to treat plant biomass to improve saccharification yields. Here we describe a new type of expansin-related fungal protein that we have called loosenin. Its corresponding gene, loos1, from the basidiomycete Bjerkandera adusta, was cloned and heterologously expressed in Saccharomyces cerevisiae. LOOS1 is distantly related to plant expansins through the shared presence of a DPBB domain, however domain II found in plant expansins is absent. LOOS1 binds tightly to cellulose and chitin, and we demonstrate that cotton fibers become susceptible to the action of a commercial cellulase following treatment with LOOS1. Natural fibers of Agave tequilana also become susceptible to hydrolysis by cellulases after loosenin treatment. LOOS1 is a new type of protein with disrupting activity on cellulose. LOOS1 binds polysaccharides, and given its enhancing properties on the action of hydrolytic enzymes, LOOS1 represents a potential additive in the production of fermentable sugars from lignocellulose.

Loosenin, a novel protein with cellulose-disrupting activity from Bjerkandera adusta

PubMed Central

2011-01-01

Background Expansins and expansin-like proteins loosen cellulose microfibrils, possibly through the rupture of intramolecular hydrogen bonds. Together with the use of lignocellulolytic enzymes, these proteins are potential molecular tools to treat plant biomass to improve saccharification yields. Results Here we describe a new type of expansin-related fungal protein that we have called loosenin. Its corresponding gene, loos1, from the basidiomycete Bjerkandera adusta, was cloned and heterologously expressed in Saccharomyces cerevisiae. LOOS1 is distantly related to plant expansins through the shared presence of a DPBB domain, however domain II found in plant expansins is absent. LOOS1 binds tightly to cellulose and chitin, and we demonstrate that cotton fibers become susceptible to the action of a commercial cellulase following treatment with LOOS1. Natural fibers of Agave tequilana also become susceptible to hydrolysis by cellulases after loosenin treatment. Conclusions LOOS1 is a new type of protein with disrupting activity on cellulose. LOOS1 binds polysaccharides, and given its enhancing properties on the action of hydrolytic enzymes, LOOS1 represents a potential additive in the production of fermentable sugars from lignocellulose. PMID:21314954

Composition dependence of band alignments in GaxIn1-xAsySb1-y heterojunctions lattice matched to GaSb and InAs

NASA Astrophysics Data System (ADS)

Shim, Kyurhee

2013-11-01

A theoretical model utilizing a universal tight binding method and a correlated function expansion technique is presented to calculate the valence band maximum (VBM) and the conduction band minimum (CBM) of the binary (GaAs, InAS, GaSb, and InSb) and quaternary alloy GaxIn1-xAsySb1-y systems. By organizing the relative positions of the VBM and CBM between semiconductors, the band alignments and band types in the heterojunctions are determined. A straddling (type-I) band alignment in InAs/GaAs, InSb/GaAs, and GaSb/InSb, staggered (type-II) band alignment in GaSb/GaAs, and broken (type-III) band alignment in InSb/InAs and InAs/GaSb are found respectively. In addition, the compositional variations of VBM, CBM, valence band offset, conduction band offset, and band type for the alloy GaxIn1-xAsySb1-y lattice matched on GaSb and InAs are obtained as increasing the composition x. A pronounced upward bowing for the VBM and a very slight upward bowing (almost linear) for CBM are found, respectively. By controlling the compositions (x, y), band type transitions occur. The GaxIn1-xAsySb1-y heterojunctions lattice matched to GaSb changes their band types from type-III at x ˜0→ to type-II at x = 0.07, and → to type-I at x = 0.38. In contrast, the GaxIn1-xAsySb1-y heterojunctions lattice matched to InAs changes their band types from type-II x ˜0→ to type-III at x = 0.32. Reasonable agreement is obtained between our theoretical results and existing experimental data.

Spider Silk-CBD-Cellulose Nanocrystal Composites: Mechanism of Assembly

PubMed Central

Meirovitch, Sigal; Shtein, Zvi; Ben-Shalom, Tal; Lapidot, Shaul; Tamburu, Carmen; Hu, Xiao; Kluge, Jonathan A.; Raviv, Uri; Kaplan, David L.; Shoseyov, Oded

2016-01-01

The fabrication of cellulose-spider silk bio-nanocomposites comprised of cellulose nanocrystals (CNCs) and recombinant spider silk protein fused to a cellulose binding domain (CBD) is described. Silk-CBD successfully binds cellulose, and unlike recombinant silk alone, silk-CBD self-assembles into microfibrils even in the absence of CNCs. Silk-CBD-CNC composite sponges and films show changes in internal structure and CNC alignment related to the addition of silk-CBD. The silk-CBD sponges exhibit improved thermal and structural characteristics in comparison to control recombinant spider silk sponges. The glass transition temperature (Tg) of the silk-CBD sponge was higher than the control silk sponge and similar to native dragline spider silk fibers. Gel filtration analysis, dynamic light scattering (DLS), small angle X-ray scattering (SAXS) and cryo-transmission electron microscopy (TEM) indicated that silk-CBD, but not the recombinant silk control, formed a nematic liquid crystalline phase similar to that observed in native spider silk during the silk spinning process. Silk-CBD microfibrils spontaneously formed in solution upon ultrasonication. We suggest a model for silk-CBD assembly that implicates CBD in the central role of driving the dimerization of spider silk monomers, a process essential to the molecular assembly of spider-silk nanofibers and silk-CNC composites. PMID:27649169

Spider Silk-CBD-Cellulose Nanocrystal Composites: Mechanism of Assembly.

PubMed

Meirovitch, Sigal; Shtein, Zvi; Ben-Shalom, Tal; Lapidot, Shaul; Tamburu, Carmen; Hu, Xiao; Kluge, Jonathan A; Raviv, Uri; Kaplan, David L; Shoseyov, Oded

2016-09-18

The fabrication of cellulose-spider silk bio-nanocomposites comprised of cellulose nanocrystals (CNCs) and recombinant spider silk protein fused to a cellulose binding domain (CBD) is described. Silk-CBD successfully binds cellulose, and unlike recombinant silk alone, silk-CBD self-assembles into microfibrils even in the absence of CNCs. Silk-CBD-CNC composite sponges and films show changes in internal structure and CNC alignment related to the addition of silk-CBD. The silk-CBD sponges exhibit improved thermal and structural characteristics in comparison to control recombinant spider silk sponges. The glass transition temperature (Tg) of the silk-CBD sponge was higher than the control silk sponge and similar to native dragline spider silk fibers. Gel filtration analysis, dynamic light scattering (DLS), small angle X-ray scattering (SAXS) and cryo-transmission electron microscopy (TEM) indicated that silk-CBD, but not the recombinant silk control, formed a nematic liquid crystalline phase similar to that observed in native spider silk during the silk spinning process. Silk-CBD microfibrils spontaneously formed in solution upon ultrasonication. We suggest a model for silk-CBD assembly that implicates CBD in the central role of driving the dimerization of spider silk monomers, a process essential to the molecular assembly of spider-silk nanofibers and silk-CNC composites.

The influence of social comparison on cognitive bias modification and emotional vulnerability.

PubMed

Standage, Helen; Harris, Jemma; Fox, Elaine

2014-02-01

The Cognitive Bias Modification (CBM) paradigm was devised to test predictions that cognitive biases have a causal influence on emotional status. Increasingly, however, researchers are testing the potential clinical applications of CBM. Although generally successful in reducing emotional vulnerability in clinical populations, the impact of CBM interventions has been somewhat variable. The aim of the current experiment was to investigate whether social comparison processing might be an important moderator of CBM. Healthy participants were presented with 80 valenced scenarios devised to induce a positive or negative interpretative bias. Critically, participants answered a series of questions designed to establish whether they assimilated or contrasted themselves with the valenced descriptions. The induction of an interpretation bias that was congruent with the valence of the training scenarios was successful only for participants who tended to assimilate the valenced scenarios, and not for those participants who tended to evaluate themselves against the scenarios. Furthermore, the predicted influence of CBM on emotional outcomes occurred only for those who had an assimilative rather than evaluative orientation toward CBM training material. Of key importance, results indicated that "evaluators" showed increased emotional vulnerability following positive CBM training. This result has both theoretical and clinical implications in suggesting that the success of CBM is dependent upon the way in which participants socially compare themselves with CBM training material. PsycINFO Database Record (c) 2014 APA, all rights reserved.

Effects of organosolv and ammonia pretreatments on lignin properties and its inhibition for enzymatic hydrolysis

DOE PAGES

Yoo, Chang Geun; Li, Mi; Meng, Xianzhi; ...

2017-04-05

Lignin offers structural support and protection for plant cell walls; but, it also contributes to biomass recalcitrance and the costs of biofuel production via the biological pathway. Organosolv and ammonia pretreatments have been developed to reduce biomass recalcitrance and improve sugar release performance during enzymatic hydrolysis. It is believed that lignin properties are related to its inhibition on enzymatic hydrolysis; therefore, understanding the characteristics of lignin is a key for effective biomass conversion to biofuels. In this study, an organosolv pretreatment using 60% ethanol with 1.25% H 2SO 4 significantly deconstructed poplar lignin and reduced its molecular weights due tomore » the cleavage of lignin inter-unit linkages. The organosolv pretreatment increased the contents of phenolic OH units and the lignin residue showed a high cellulase maximum adsorption capacity. Ammonia pretreatment with 5% ammonium hydroxide was not as effective as organosolv pretreatment on lignin deconstruction. Organosolv lignin residue had lower lignin S/G ratio than the untreated one. Furthermore, when compared to the organosolv lignin residue and untreated lignin, ammonia lignin residue had a higher cellulase adsorption affinity. In addition, the effects of lignin on cellulose hydrolysis was investigated and the results suggested that the presence of lignin with cellulose substrates reduced cellulose hydrolysis, and its inhibitory effect was primarily determined by the lignin properties after each pretreatment. The organosolv pretreatment resulted in a slightly lower cellulase binding strength (249.7 mL g -1) on poplar lignin than that on untreated samples (261.1 mL g -1), while ammonia lignin residue showed a higher cellulase binding strength (402.8 mL g -1) and had more significant inhibition effect on cellulose hydrolysis. Our results demonstrated that the binding strength significantly affected the lignin-derived inhibition on enzymatic hydrolysis of cellulose in the cellulose-lignin mixtures.« less

Effects of organosolv and ammonia pretreatments on lignin properties and its inhibition for enzymatic hydrolysis

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

Yoo, Chang Geun; Li, Mi; Meng, Xianzhi

Lignin offers structural support and protection for plant cell walls; but, it also contributes to biomass recalcitrance and the costs of biofuel production via the biological pathway. Organosolv and ammonia pretreatments have been developed to reduce biomass recalcitrance and improve sugar release performance during enzymatic hydrolysis. It is believed that lignin properties are related to its inhibition on enzymatic hydrolysis; therefore, understanding the characteristics of lignin is a key for effective biomass conversion to biofuels. In this study, an organosolv pretreatment using 60% ethanol with 1.25% H 2SO 4 significantly deconstructed poplar lignin and reduced its molecular weights due tomore » the cleavage of lignin inter-unit linkages. The organosolv pretreatment increased the contents of phenolic OH units and the lignin residue showed a high cellulase maximum adsorption capacity. Ammonia pretreatment with 5% ammonium hydroxide was not as effective as organosolv pretreatment on lignin deconstruction. Organosolv lignin residue had lower lignin S/G ratio than the untreated one. Furthermore, when compared to the organosolv lignin residue and untreated lignin, ammonia lignin residue had a higher cellulase adsorption affinity. In addition, the effects of lignin on cellulose hydrolysis was investigated and the results suggested that the presence of lignin with cellulose substrates reduced cellulose hydrolysis, and its inhibitory effect was primarily determined by the lignin properties after each pretreatment. The organosolv pretreatment resulted in a slightly lower cellulase binding strength (249.7 mL g -1) on poplar lignin than that on untreated samples (261.1 mL g -1), while ammonia lignin residue showed a higher cellulase binding strength (402.8 mL g -1) and had more significant inhibition effect on cellulose hydrolysis. Our results demonstrated that the binding strength significantly affected the lignin-derived inhibition on enzymatic hydrolysis of cellulose in the cellulose-lignin mixtures.« less

The nucleoid-associated protein Fis directly modulates the synthesis of cellulose, an essential component of pellicle-biofilms in the phytopathogenic bacterium Dickeya dadantii.

PubMed

Prigent-Combaret, Claire; Zghidi-Abouzid, Ouafa; Effantin, Géraldine; Lejeune, Philippe; Reverchon, Sylvie; Nasser, William

2012-10-01

Bacteria use biofilm structures to colonize surfaces and to survive in hostile conditions, and numerous bacteria produce cellulose as a biofilm matrix polymer. Hence, expression of the bcs operon, responsible for cellulose biosynthesis, must be finely regulated in order to allow bacteria to adopt the proper surface-associated behaviours. Here we show that in the phytopathogenic bacterium, Dickeya dadantii, production of cellulose is required for pellicle-biofilm formation and resistance to chlorine treatments. Expression of the bcs operon is growth phase-regulated and is stimulated in biofilms. Furthermore, we unexpectedly found that the nucleoid-associated protein and global regulator of virulence functions, Fis, directly represses bcs operon expression by interacting with an operator that is absent from the bcs operon of animal pathogenic bacteria and the plant pathogenic bacterium Pectobacterium. Moreover, production of cellulose enhances plant surface colonization by D. dadantii. Overall, these data suggest that cellulose production and biofilm formation may be important factors for surface colonization by D. dadantii and its subsequent survival in hostile environments. This report also presents a new example of how bacteria can modulate the action of a global regulator to co-ordinate basic metabolism, virulence and modifications of lifestyle. © 2012 Blackwell Publishing Ltd.

The central and northern Appalachian Basin-a frontier region for coalbed methane development

USGS Publications Warehouse

Lyons, P.C.

1998-01-01

The Appalachian basin is the world's second largest coalbed-methane (CBM) producing basin. It has nearly 4000 wells with 1996 annual production at 147.8 billion cubic feet (Bcf). Cumulative CBM production is close to 0.9 trillion cubic feet (Tcf). The Black Warrior Basin of Alabama in the southern Appalachian basin (including a very minor amount from the Cahaba coal field) accounts for about 75% of this annual production and about 75% of the wells, and the remainder comes from the central and northern Appalachian basin. The Southwest Virginia coal field accounts for about 95% of the production from the central and northern parts of the Appalachian basin. Production data and trends imply that several of the Appalachian basin states, except for Alabama and Virginia, are in their infancy with respect to CBM development. Total in-place CBM resources in the central and northern Appalachian basin have been variously estimated at 66 to 76 trillion cubic feet (Tcf), of which an estimated 14.55 Tcf (~ 20%) is technically recoverable according to a 1995 U.S. Geological Survey assessment. For comparison in the Black Warrior basin of the 20 Tcf in-place CBM resources, 2.30 Tcf (~ 12%) is technically recoverable. Because close to 0.9 Tcf of CBM has already been produced from the Black Warrior basin and the proved reserves are about 0.8 Tcf for 1996 [Energy Information Administration (EIA), 1997]. U.S. Crude Oil, Natural Gas, and Natural Gas Liquids Reserves, 1996 Annual Report. U.S. Department of Energy DOE/EIA-0216(96), 145 pp.], these data imply that the central and northern Appalachian basin could become increasingly important in the Appalachian basin CBM picture as CBM resources are depleted in the southern Appalachian basin (Black Warrior Basin and Cahaba Coal Field). CBM development in the Appalachian states could decrease the eastern U.S.A.'s dependence on coal for electricity. CBM is expected to provide over the next few decades a virtually untapped source of unconventional fossil fuel in the Appalachian states, where the CBM resources are large and the demand for cleaner fossil-fuel energy is high.The central and northern Appalachian basin could become increasingly important in the Appalachian basin coalbed methane (CBM) picture as CBM resources are depleted in the southern Appalachian basin. Total in-place CBM resources in the central and the northern Appalachian basin have been estimated at 66 to 76 Tcf, of which 14.55 Tcf is technically recoverable.

Utilization of ethyl cellulose polymer and waste materials for roofing tile production

NASA Astrophysics Data System (ADS)

Sam, Suubitaa Spencer; Ng, ChoonAun; Chee, Swee Yong; Habib, NoorZainab; Nadeem, Humayon; Teoh, Wei Ping

2017-05-01

The aim of this study was to utilize ethyl cellulose, mixture of waste engine oil and waste vegetable oil as a binder in the environmental friendly roofing tile production. The waste engine-vegetable oil wasmix together with ethyl cellulose, fly ash, coarse aggregates, fine aggregatesand a catalyst. The Fourier Transform Infrared (FTIR) analysis showed that the oil mixture added with ethyl cellulose has the relatively high binding effect due to the presence of strong carbonyl group especially after being heat cured at 1900C for 24 hours. The mixed proportion of materials with different amount of ethyl cellulose used was studied in the production of tile specimen. The results showed that the ethyl cellulose composed roofing tile specimens passed the transverse breaking strength, durability, permeabilityand the ultraviolet accelerated test. The shrinkage on the tile can be overcome by adding temperature resistance polymer on the exterior of the tile.

Extracellular matrix family proteins that are potential targets of Dd-STATa in Dictyostelium discoideum.

PubMed

Shimada, Nao; Nishio, Keiko; Maeda, Mineko; Urushihara, Hideko; Kawata, Takefumi

2004-10-01

Dd-STATa is a functional Dictyostelium homologue of metazoan STAT (signal transducers and activators of transcription) proteins, which is activated by cAMP and is thereby translocated into the nuclei of anterior tip cells of the prestalk region of the slug. By using in situ hybridization analyses, we found that the SLF308 cDNA clone, which contains the ecmF gene that encodes a putative extracellular matrix protein and is expressed in the anterior tip cells, was greatly down-regulated in the Dd-STATa-null mutant. Disruption of the ecmF gene, however, resulted in almost no phenotypic change. The absence of any obvious mutant phenotype in the ecmF-null mutant could be due to a redundancy of similar genes. In fact, a search of the Dictyostelium whole genome database demonstrates the existence of an additional 16 homologues, all of which contain a cellulose-binding module. Among these homologues, four genes show Dd-STATa-dependent expression, while the others are Dd-STATa-independent. We discuss the potential role of Dd-STATa in morphogenesis via its effect on the interaction between cellulose and these extracellular matrix family proteins.

Effects of standard and explicit cognitive bias modification and computer-administered cognitive-behaviour therapy on cognitive biases and social anxiety.

PubMed

Mobini, Sirous; Mackintosh, Bundy; Illingworth, Jo; Gega, Lina; Langdon, Peter; Hoppitt, Laura

2014-06-01

This study examines the effects of a single session of Cognitive Bias Modification to induce positive Interpretative bias (CBM-I) using standard or explicit instructions and an analogue of computer-administered CBT (c-CBT) program on modifying cognitive biases and social anxiety. A sample of 76 volunteers with social anxiety attended a research site. At both pre- and post-test, participants completed two computer-administered tests of interpretative and attentional biases and a self-report measure of social anxiety. Participants in the training conditions completed a single session of either standard or explicit CBM-I positive training and a c-CBT program. Participants in the Control (no training) condition completed a CBM-I neutral task matched the active CBM-I intervention in format and duration but did not encourage positive disambiguation of socially ambiguous or threatening scenarios. Participants in both CBM-I programs (either standard or explicit instructions) and the c-CBT condition exhibited more positive interpretations of ambiguous social scenarios at post-test and one-week follow-up as compared to the Control condition. Moreover, the results showed that CBM-I and c-CBT, to some extent, changed negative attention biases in a positive direction. Furthermore, the results showed that both CBM-I training conditions and c-CBT reduced social anxiety symptoms at one-week follow-up. This study used a single session of CBM-I training, however multi-sessions intervention might result in more endurable positive CBM-I changes. A computerised single session of CBM-I and an analogue of c-CBT program reduced negative interpretative biases and social anxiety. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Effects of standard and explicit cognitive bias modification and computer-administered cognitive-behaviour therapy on cognitive biases and social anxiety☆

PubMed Central

Mobini, Sirous; Mackintosh, Bundy; Illingworth, Jo; Gega, Lina; Langdon, Peter; Hoppitt, Laura

2014-01-01

Background and objectives This study examines the effects of a single session of Cognitive Bias Modification to induce positive Interpretative bias (CBM-I) using standard or explicit instructions and an analogue of computer-administered CBT (c-CBT) program on modifying cognitive biases and social anxiety. Methods A sample of 76 volunteers with social anxiety attended a research site. At both pre- and post-test, participants completed two computer-administered tests of interpretative and attentional biases and a self-report measure of social anxiety. Participants in the training conditions completed a single session of either standard or explicit CBM-I positive training and a c-CBT program. Participants in the Control (no training) condition completed a CBM-I neutral task matched the active CBM-I intervention in format and duration but did not encourage positive disambiguation of socially ambiguous or threatening scenarios. Results Participants in both CBM-I programs (either standard or explicit instructions) and the c-CBT condition exhibited more positive interpretations of ambiguous social scenarios at post-test and one-week follow-up as compared to the Control condition. Moreover, the results showed that CBM-I and c-CBT, to some extent, changed negative attention biases in a positive direction. Furthermore, the results showed that both CBM-I training conditions and c-CBT reduced social anxiety symptoms at one-week follow-up. Limitations This study used a single session of CBM-I training, however multi-sessions intervention might result in more endurable positive CBM-I changes. Conclusions A computerised single session of CBM-I and an analogue of c-CBT program reduced negative interpretative biases and social anxiety. PMID:24412966

In situ and Enriched Microbial Community Composition and Function Associated with Coal Bed Methane from Powder River Basin Coals

NASA Astrophysics Data System (ADS)

Barnhart, Elliott; Davis, Katherine; Varonka, Matthew; Orem, William; Fields, Matthew

2016-04-01

Coal bed methane (CBM) is a relatively clean source of energy but current CBM production techniques have not sustained long-term production or produced enough methane to remain economically practical with lower natural gas prices. Enhancement of the in situ microbial community that actively generates CBM with the addition of specific nutrients could potentially sustain development. CBM production more than doubled from native microbial populations from Powder River Basin (PRB) coal beds, when yeast extract and several individual components of yeast extract (proteins and amino acids) were added to laboratory microcosms. Microbial populations capable of hydrogenotrophic (hydrogen production/utilization) methanogenesis were detected in situ and under non-stimulated conditions. Stimulation with yeast extract caused a shift in the community to microorganisms capable of acetoclastic (acetate production/utilization) methanogenesis. Previous isotope analysis from CBM production wells indicated a similar microbial community shift as observed in stimulation experiments: hydrogenotrophic methanogenesis was found throughout the PRB, but acetoclastic methanogenesis dominated major recharge areas. In conjunction, a high proportion of cyanobacterial and algal SSU rRNA gene sequences were detected in a CBM well within a major recharge area, suggesting that these phototrophic organisms naturally stimulate methane production. In laboratory studies, adding phototrophic (algal) biomass stimulated CBM production by PRB microorganisms similarly to yeast extract (~40μg methane increase per gram of coal). Analysis of the British thermal unit (BTU) content of coal from long-term incubations indicated >99.5% of BTU content remained after CBM stimulation with either algae or yeast extract. Biomimicry of in situ algal CBM stimulation could lead to technologies that utilize coupled biological systems (photosynthesis and methane production) that sustainably enhance CBM production and generate algal biofuels while also sequestering carbon dioxide (CO2).

SPECIFIC ADHESION TO CELLULOSE AND HYDROLYSIS OF ORGANOPHOSPHATE NERVE AGENTS BY A GENETICALLY ENGINEERED E. COLI WITH SURFACE-EXPRESSED CELLULOSE-BINDING DOMAIN AND ORGANOPHOSPHORUS HYDROLASE. (R827227)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Molecular imprinting of caffeine on cellulose/silica composite and its characterization

NASA Astrophysics Data System (ADS)

Gill, Rajinder Singh

This dissertation presents a study to prepare molecularly imprinted inorganic/organic hybrid composite which not only confirm the higher binding capabilities for the target molecule (template) but also discriminate its structural analogs. Molecularly imprinted Cellulose/Silica composite (MIP) was prepared by using caffeine as the template. Silica derived from TEOS by using sol-gel techniques was deposited on cheap, abundant organic matrix such as cellulose, which can provide a filtering medium while coffee brewing. Removal of the template from the precursor was verified by Diffuse Reflectance Infrared Fourier Transform Spectroscopy (DRIFTS). Remarkably reduced intensity of -NH2 scissor like mode of caffeine and the presence of traces of "N" by elemental analysis, confirmed the complete removal of caffeine on washing with ethanol. Cellulose to TEOS mass ratio of 2:1 was found to be close to optimal during our analysis. Energy dispersive spectroscopy results leads to an important fact that the deposition of silica was stable even at 373 K. Focus was on the adsorption affinities of caffeine by MIP and was tested by performing relative adsorption of caffeine by MIP and blank (standard) using demountable path length cell in IR. It was observed that MIP showed almost 3-folds higher adsorption capabilities as compared to blank. The initial rate of adsorption of caffeine by MIP is much higher than blank which is one of the desirable feature according the its intended use. The higher adsorption of caffeine by MIP not only depends on the amount of silica deposited but also the available binding sites present on its surface. Selectivity of MIP was also verified by the competitive adsorption of caffeine and its structure analogs such as theophylline. Clearly, MIP showed greater and more rapid binding capabilities for caffeine than theophylline. At short contact times, the binding capability for caffeine is almost 1.8 times greater than the binding capabilities for theophylline.

Solution structure, backbone dynamics and chitin binding of the anti-fungal protein from Streptomyces tendae TU901.

PubMed

Campos-Olivas, R; Hörr, I; Bormann, C; Jung, G; Gronenborn, A M

2001-05-11

AFP1 is a recently discovered anti-fungal, chitin-binding protein from Streptomyces tendae Tü901. Mature AFP1 comprises 86 residues and exhibits limited sequence similarity to the cellulose-binding domains of bacterial cellulases and xylanases. No similarity to the Cys and Gly-rich domains of plant chitin-binding proteins (e.g. agglutinins, lectins, hevein) is observed. AFP1 is the first chitin-binding protein from a bacterium for which anti-fungal activity was shown. Here, we report the three-dimensional solution structure of AFP1, determined by nuclear magnetic resonance spectroscopy. The protein contains two antiparallel beta-sheets (five and four beta-strands each), that pack against each other in a parallel beta-sandwich. This type of architecture is conserved in the functionally related family II of cellulose-binding domains, albeit with different connectivity. A similar fold is also observed in other unrelated proteins (spore coat protein from Myxococcus xanthus, beta-B2 and gamma-B crystallins from Bos taurus, canavalin from Jack bean). AFP1 is therefore classified as a new member of the betagamma-crystallin superfamily. The dynamics of the protein was characterized by NMR using amide 15N relaxation and solvent exchange data. We demonstrate that the protein exhibits an axially symmetric (oblate-like) rotational diffusion tensor whose principal axis coincides to within 15 degrees with that of the inertial tensor. After completion of the present structure of AFP1, an identical fold was reported for a Streptomyces killer toxin-like protein. Based on sequence comparisons and clustering of conserved residues on the protein surface for different cellulose and chitin-binding proteins, we postulate a putative sugar-binding site for AFP1. The inability of the protein to bind short chitin fragments suggests that certain particular architectural features of the solid chitin surface are crucial for the interaction. Copyright 2001 Academic Press.

The Use of Cellulose Nanocrystals for Potential Application in Topical Delivery of Hydroquinone.

PubMed

Taheri, Azade; Mohammadi, Mina

2015-07-01

Nanotechnology-based drug delivery systems can enhance drug permeation through the skin and improve the drug stability. The biodegradability and biocompatibility of cellulose nanocrystals have made these nanoparticles good candidates to use in biomedical applications. The hyperpigmentation is a common skin disorder that could be caused by number of reasons such as sun exposure and pregnancy. Hydroquinone could inhibit the production of melanin and eliminate the discolorations of skin. This study is aimed at introducing cellulose nanocrystals as suitable carriers for drug delivery to skin. Prepared cellulose nanocrystals were characterized by dynamic light scattering and atomic force microscopy. The size of cellulose nanocrystals determined using dynamic light scattering was 301 ± 10 nm. Hydroquinone-cellulose nanocrystal complex was prepared by incubating of hydroquinone solution in cellulose nanocrystals suspension. The size of hydroquinone-cellulose nanocrystal complex determined using dynamic light scattering was 310 ± 10 nm. The hydroquinone content of the hydroquinone-cellulose complex was determined using UV/vis spectroscopy. Hydroquinone was bound to cellulose nanocrystals representing 79.3 ± 2% maximum binding efficiency when 1.1 mg hydroquinone was added to 1 mL of cellulose nanocrystals suspension (2 mg cellulose nanocrystal). The hydroquinone-cellulose nanocrystal complex showed an approximately sustained release profile of hydroquinone. Approximately, 80% of bound hydroquinone released in 4 h. © 2014 John Wiley & Sons A/S.

Ground Vehicle Condition Based Maintenance

DTIC Science & Technology

2010-10-04

Diagnostic Process Map 32 FMEAs Developed : • Diesel Engine • Transmission • Alternators Analysis : • Identify failure modes • Derive design factors and...S&T Initiatives  TARDEC P&D Process Map  Component Testing  ARL CBM Research  AMSAA SDC & Terrain Modeling UNCLASSIFIED 3 CBM+ Overview...UNCLASSIFIED 4 RCM and CBM are core processes for CBM+ System Development • Army Regulation 750-1, 20 Sep 2007, p. 79 - Reliability Centered Maintenance (RCM

Cellulose biosynthesis by the beta-proteobacterium, Chromobacterium violaceum.

PubMed

Recouvreux, Derce O S; Carminatti, Claudimir A; Pitlovanciv, Ana K; Rambo, Carlos R; Porto, Luismar M; Antônio, Regina V

2008-11-01

The Chromobacterium violaceum ATCC 12472 genome was sequenced by The Brazilian National Genome Project Consortium. Previous annotation reported the presence of cellulose biosynthesis genes in that genome. Analysis of these genes showed that, as observed in other bacteria, they are organized in two operons. In the present work, experimental evidences of the presence of cellulose in the extracellular matrix of the biofilm produced by C. violaceum in static cultures are shown. Biofilm samples were enzymatically digested by cellulase, releasing glucose units, suggesting the presence of cellulose as an extracellular matrix component. Fluorescence microscopy observations showed that C. violaceum produces a cellulase-sensitive extracellular matrix composed of fibers able to bind calcofluor. C. violaceum grows on medium containing Congo red, forming brown-red colonies. Together, these results suggest that cellulase-susceptible matrix material is cellulose. Scanning electronic microscopy analysis showed that the extracellular matrix exhibited a network of microfibrils, typical of bacterial cellulose. Although cellulose production is widely distributed between several bacterial species, including at least the groups of Gram-negative proteobacteria alpha and gamma, we give for the first time experimental evidence for cellulose production in beta-proteobacteria.

In silico analysis of β-mannanases and β-mannosidase from Aspergillus flavus and Trichoderma virens UKM1

NASA Astrophysics Data System (ADS)

Yee, Chai Sin; Murad, Abdul Munir Abdul; Bakar, Farah Diba Abu

2013-11-01

A gene encoding an endo-β-1,4-mannanase from Trichoderma virens UKM1 (manTV) and Aspergillus flavus UKM1 (manAF) was analysed with bioinformatic tools. In addition, A. flavus NRRL 3357 genome database was screened for a β-mannosidase gene and analysed (mndA-AF). These three genes were analysed to understand their gene properties. manTV and manAF both consists of 1,332-bp and 1,386-bp nucleotides encoding 443 and 461 amino acid residues, respectively. Both the endo-β-1,4-mannanases belong to the glycosyl hydrolase family 5 and contain a carbohydrate-binding module family 1 (CBM1). On the other hand, mndA-AF which is a 2,745-bp gene encodes a protein sequence of 914 amino acid residues. This β-mannosidase belongs to the glycosyl hydrolase family 2. Predicted molecular weight of manTV, manAF and mndA-AF are 47.74 kDa, 49.71 kDa and 103 kDa, respectively. All three predicted protein sequences possessed signal peptide sequence and are highly conserved among other fungal β-mannanases and β-mannosidases.

The Development of a Gas–Liquid Two-Phase Flow Sensor Applicable to CBM Wellbore Annulus

PubMed Central

Wu, Chuan; Wen, Guojun; Han, Lei; Wu, Xiaoming

2016-01-01

The measurement of wellbore annulus gas–liquid two-phase flow in CBM (coalbed methane) wells is of great significance for reasonably developing gas drainage and extraction processes, estimating CBM output, judging the operating conditions of CBM wells and analyzing stratum conditions. Hence, a specially designed sensor is urgently needed for real-time measurement of gas–liquid two-phase flow in CBM wellbore annulus. Existing flow sensors fail to meet the requirements of the operating conditions of CBM wellbore annulus due to such factors as an inapplicable measurement principle, larger size, poor sealability, high installation accuracy, and higher requirements for fluid media. Therefore, based on the principle of a target flowmeter, this paper designs a new two-phase flow sensor that can identify and automatically calibrate different flow patterns of two-phase flows. Upon the successful development of the new flow sensor, lab and field tests were carried out, and the results show that the newly designed sensor, with a measurement accuracy of ±2.5%, can adapt to the operating conditions of CBM wells and is reliable for long-term work. PMID:27869708

The Development of a Gas-Liquid Two-Phase Flow Sensor Applicable to CBM Wellbore Annulus.

PubMed

Wu, Chuan; Wen, Guojun; Han, Lei; Wu, Xiaoming

2016-11-18

The measurement of wellbore annulus gas-liquid two-phase flow in CBM (coalbed methane) wells is of great significance for reasonably developing gas drainage and extraction processes, estimating CBM output, judging the operating conditions of CBM wells and analyzing stratum conditions. Hence, a specially designed sensor is urgently needed for real-time measurement of gas-liquid two-phase flow in CBM wellbore annulus. Existing flow sensors fail to meet the requirements of the operating conditions of CBM wellbore annulus due to such factors as an inapplicable measurement principle, larger size, poor sealability, high installation accuracy, and higher requirements for fluid media. Therefore, based on the principle of a target flowmeter, this paper designs a new two-phase flow sensor that can identify and automatically calibrate different flow patterns of two-phase flows. Upon the successful development of the new flow sensor, lab and field tests were carried out, and the results show that the newly designed sensor, with a measurement accuracy of ±2.5%, can adapt to the operating conditions of CBM wells and is reliable for long-term work.

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.

Structural Analysis of Semi-specific Oligosaccharide Recognition by a Cellulose-binding Protein of Thermotoga maritima Reveals Adaptations for Functional Diversification of the Oligopeptide Periplasmic Binding Protein Fold

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

Cuneo, Matthew J.; Beese, Lorena S.; Hellinga, Homme W.

Periplasmic binding proteins (PBPs) constitute a protein superfamily that binds a wide variety of ligands. In prokaryotes, PBPs function as receptors for ATP-binding cassette or tripartite ATP-independent transporters and chemotaxis systems. In many instances, PBPs bind their cognate ligands with exquisite specificity, distinguishing, for example, between sugar epimers or structurally similar anions. By contrast, oligopeptide-binding proteins bind their ligands through interactions with the peptide backbone but do not distinguish between different side chains. The extremophile Thermotoga maritima possesses a remarkable array of carbohydrate-processing metabolic systems, including the hydrolysis of cellulosic polymers. Here, we present the crystal structure of a T.more » maritima cellobiose-binding protein (tm0031) that is homologous to oligopeptide-binding proteins. T. maritima cellobiose-binding protein binds a variety of lengths of {beta}(1 {yields} 4)-linked glucose oligomers, ranging from two rings (cellobiose) to five (cellopentaose). The structure reveals that binding is semi-specific. The disaccharide at the nonreducing end binds specifically; the other rings are located in a large solvent-filled groove, where the reducing end makes several contacts with the protein, thereby imposing an upper limit of the oligosaccharides that are recognized. Semi-specific recognition, in which a molecular class rather than individual species is selected, provides an efficient solution for the uptake of complex mixtures.« less

Early grade curriculum-based reading measures for students with intellectual disability.

PubMed

Hill, David R; Lemons, Christopher J

2015-12-01

The purpose of this study was to extend previous research on the use of curriculum-based measurement (CBM) for students with intellectual disability by having 19 special education teachers monitor weekly reading progress of 38 students with intellectual disability for approximately 15 weeks and examining whether students exhibited gains on the progress monitoring measures. In addition to the weekly CBM, teachers reported the type and duration of daily reading instruction. Data were analyzed to explore relationships between CBM performance and reading instruction. Our results indicate that teachers are capable of administering and scoring CBM on a weekly basis and that CBM does capture reading growth for some students with intellectual disability. Correlations between CBM performance and a teacher report of skills taught during reading instruction indicate that teachers may be differentiating instruction based on students' reading ability. Directions for future research as well as limitations of the study are discussed. © The Author(s) 2015.

Dissolving process of a cellulose bunch in ionic liquids: a molecular dynamics study.

PubMed

Li, Yao; Liu, Xiaomin; Zhang, Suojiang; Yao, Yingying; Yao, Xiaoqian; Xu, Junli; Lu, Xingmei

2015-07-21

In recent years, a variety of ionic liquids (ILs) were found to be capable of dissolving cellulose and mechanistic studies were also reported. However, there is still a lack of detailed information at the molecular level. Here, long time molecular dynamics simulations of cellulose bunch in 1-ethyl-3-methylimidazolium acetate (EmimAc), 1-ethyl-3-methylimidazolium chloride (EmimCl), 1-butyl-3-methylimidazolium chloride (BmimCl) and water were performed to analyze the inherent interaction and dissolving mechanism. Complete dissolution of the cellulose bunch was observed in EmimAc, while little change took place in EmimCl and BmimCl, and nothing significant happened in water. The deconstruction of the hydrogen bond (H-bond) network in cellulose was found and analyzed quantitatively. The synergistic effect of cations and anions was revealed by analyzing the whole dissolving process. Initially, cations bind to the side face of the cellulose bunch and anions insert into the cellulose strands to form H-bonds with hydroxyl groups. Then cations start to intercalate into cellulose chains due to their strong electrostatic interaction with the entered anions. The H-bonds formed by Cl(-) cannot effectively separate the cellulose chain and that is the reason why EmimCl and BmimCl dissolve cellulose more slowly. These findings deepen people's understanding on how ILs dissolve cellulose and would be helpful for designing new efficient ILs to dissolve cellulose.

Chitinase-like1/pom-pom1 and its homolog CTL2 are glucan-interacting proteins important for cellulose biosynthesis in Arabidopsis.

PubMed

Sánchez-Rodríguez, Clara; Bauer, Stefan; Hématy, Kian; Saxe, Friederike; Ibáñez, Ana Belén; Vodermaier, Vera; Konlechner, Cornelia; Sampathkumar, Arun; Rüggeberg, Markus; Aichinger, Ernst; Neumetzler, Lutz; Burgert, Ingo; Somerville, Chris; Hauser, Marie-Theres; Persson, Staffan

2012-02-01

Plant cells are encased by a cellulose-containing wall that is essential for plant morphogenesis. Cellulose consists of β-1,4-linked glucan chains assembled into paracrystalline microfibrils that are synthesized by plasma membrane-located cellulose synthase (CESA) complexes. Associations with hemicelluloses are important for microfibril spacing and for maintaining cell wall tensile strength. Several components associated with cellulose synthesis have been identified; however, the biological functions for many of them remain elusive. We show that the chitinase-like (CTL) proteins, CTL1/POM1 and CTL2, are functionally equivalent, affect cellulose biosynthesis, and are likely to play a key role in establishing interactions between cellulose microfibrils and hemicelluloses. CTL1/POM1 coincided with CESAs in the endomembrane system and was secreted to the apoplast. The movement of CESAs was compromised in ctl1/pom1 mutant seedlings, and the cellulose content and xyloglucan structures were altered. X-ray analysis revealed reduced crystalline cellulose content in ctl1 ctl2 double mutants, suggesting that the CTLs cooperatively affect assembly of the glucan chains, which may affect interactions between hemicelluloses and cellulose. Consistent with this hypothesis, both CTLs bound glucan-based polymers in vitro. We propose that the apoplastic CTLs regulate cellulose assembly and interaction with hemicelluloses via binding to emerging cellulose microfibrils.

CHITINASE-LIKE1/POM-POM1 and Its Homolog CTL2 Are Glucan-Interacting Proteins Important for Cellulose Biosynthesis in Arabidopsis[W][OA

PubMed Central

Sánchez-Rodríguez, Clara; Bauer, Stefan; Hématy, Kian; Saxe, Friederike; Ibáñez, Ana Belén; Vodermaier, Vera; Konlechner, Cornelia; Sampathkumar, Arun; Rüggeberg, Markus; Aichinger, Ernst; Neumetzler, Lutz; Burgert, Ingo; Somerville, Chris; Hauser, Marie-Theres; Persson, Staffan

2012-01-01

Plant cells are encased by a cellulose-containing wall that is essential for plant morphogenesis. Cellulose consists of β-1,4-linked glucan chains assembled into paracrystalline microfibrils that are synthesized by plasma membrane–located cellulose synthase (CESA) complexes. Associations with hemicelluloses are important for microfibril spacing and for maintaining cell wall tensile strength. Several components associated with cellulose synthesis have been identified; however, the biological functions for many of them remain elusive. We show that the chitinase-like (CTL) proteins, CTL1/POM1 and CTL2, are functionally equivalent, affect cellulose biosynthesis, and are likely to play a key role in establishing interactions between cellulose microfibrils and hemicelluloses. CTL1/POM1 coincided with CESAs in the endomembrane system and was secreted to the apoplast. The movement of CESAs was compromised in ctl1/pom1 mutant seedlings, and the cellulose content and xyloglucan structures were altered. X-ray analysis revealed reduced crystalline cellulose content in ctl1 ctl2 double mutants, suggesting that the CTLs cooperatively affect assembly of the glucan chains, which may affect interactions between hemicelluloses and cellulose. Consistent with this hypothesis, both CTLs bound glucan-based polymers in vitro. We propose that the apoplastic CTLs regulate cellulose assembly and interaction with hemicelluloses via binding to emerging cellulose microfibrils. PMID:22327741

Contribution of the Collagen-Binding Proteins of Streptococcus mutans to Bacterial Colonization of Inflamed Dental Pulp.

PubMed

Nomura, Ryota; Ogaya, Yuko; Nakano, Kazuhiko

2016-01-01

Streptococcus mutans is a major pathogen of dental caries. Collagen-binding proteins (CBPs) (approximately 120 kDa), termed Cnm and Cbm, are regarded as important cell surface antigens related to the adherence of S. mutans to collagenous tissue. Furthermore, CBP-positive S. mutans strains are associated with various systemic diseases involving bacteremia, such as infective endocarditis. Endodontic infection is considered to be an important cause of bacteremia, but little is known regarding the presence of S. mutans in dental pulp tissue. In the present study, the distribution and virulence of S. mutans in dental pulp tissues were investigated by focusing on CBPs. Adhesion and invasion properties of various S. mutans strains were analyzed using human dental pulp fibroblasts (HDPFs). CBP-positive strains had a significantly higher rate of adhesion to HDPFs compared with CBP-defective isogenic mutant strains (P<0.001). In addition, CBP-positive strains induced HDPF proliferation, which is a possible mechanism related to development of hyperplastic pulpitis. The distribution of S. mutans strains isolated from infected root canal specimens was then analyzed by PCR. We found that approximately 50% of the root canal specimens were positive for S. mutans. Approximately 20% of these strains were Cnm-positive, while no Cbm-positive strains were isolated. The Cnm-positive strains isolated from the specimens showed adhesion to HDPFs. Our results suggest that CBP-positive S. mutans strains exhibit high colonization in dental pulp. This could be a possible virulence factor for various systemic diseases.

The structure of the catalytic domain of a plant cellulose synthase and its assembly into dimers

DOE PAGES

Olek, Anna T.; Rayon, Catherine; Makowski, Lee; ...

2014-07-10

Cellulose microfibrils are para-crystalline arrays of several dozen linear (1→4)-β-d-glucan chains synthesized at the surface of the cell membrane by large, multimeric complexes of synthase proteins. Recombinant catalytic domains of rice ( Oryza sativa) CesA8 cellulose synthase form dimers reversibly as the fundamental scaffold units of architecture in the synthase complex. Specificity of binding to UDP and UDP-Glc indicates a properly folded protein, and binding kinetics indicate that each monomer independently synthesizes single glucan chains of cellulose, i.e., two chains per dimer pair. In contrast to structure modeling predictions, solution x-ray scattering studies demonstrate that the monomer is a two-domain,more » elongated structure, with the smaller domain coupling two monomers into a dimer. The catalytic core of the monomer is accommodated only near its center, with the plant-specific sequences occupying the small domain and an extension distal to the catalytic domain. This configuration is in stark contrast to the domain organization obtained in predicted structures of plant CesA. As a result, the arrangement of the catalytic domain within the CesA monomer and dimer provides a foundation for constructing structural models of the synthase complex and defining the relationship between the rosette structure and the cellulose microfibrils they synthesize.« less

The structure of the catalytic domain of a plant cellulose synthase and its assembly into dimers.

PubMed

Olek, Anna T; Rayon, Catherine; Makowski, Lee; Kim, Hyung Rae; Ciesielski, Peter; Badger, John; Paul, Lake N; Ghosh, Subhangi; Kihara, Daisuke; Crowley, Michael; Himmel, Michael E; Bolin, Jeffrey T; Carpita, Nicholas C

2014-07-01

Cellulose microfibrils are para-crystalline arrays of several dozen linear (1→4)-β-d-glucan chains synthesized at the surface of the cell membrane by large, multimeric complexes of synthase proteins. Recombinant catalytic domains of rice (Oryza sativa) CesA8 cellulose synthase form dimers reversibly as the fundamental scaffold units of architecture in the synthase complex. Specificity of binding to UDP and UDP-Glc indicates a properly folded protein, and binding kinetics indicate that each monomer independently synthesizes single glucan chains of cellulose, i.e., two chains per dimer pair. In contrast to structure modeling predictions, solution x-ray scattering studies demonstrate that the monomer is a two-domain, elongated structure, with the smaller domain coupling two monomers into a dimer. The catalytic core of the monomer is accommodated only near its center, with the plant-specific sequences occupying the small domain and an extension distal to the catalytic domain. This configuration is in stark contrast to the domain organization obtained in predicted structures of plant CesA. The arrangement of the catalytic domain within the CesA monomer and dimer provides a foundation for constructing structural models of the synthase complex and defining the relationship between the rosette structure and the cellulose microfibrils they synthesize. © 2014 American Society of Plant Biologists. All rights reserved.

Superconducting dipole magnet for the CBM experiment at FAIR

NASA Astrophysics Data System (ADS)

Kurilkin, P.; Akishin, P.; Bychkov, A.; Floch, E.; Gusakov, Yu.; Ladygin, V.; Malakhov, A.; Moritz, G.; Ramakers, H.; Senger, P.; Shabunov, A.; Szwangruber, P.; Toral, F.

2017-03-01

The scientific goal of the CBM (Compressed Baryonic Matter) experiment at FAIR (Darmstadt) is to explore the phase diagram of strongly interacting matter at highest baryon densities. The physics program of the CBM experiment is complimentary to the programs to be realized at MPD and BMN facilities at NICA and will start with beam derived by the SIS100 synchrotron. The 5.15 MJ superconducting dipole magnet will be used in the silicon tracking system of the CBM detector. The magnet will provide a magnetic field integral of 1 Tm which is required to obtain a momentum resolution of 1% for the track reconstruction. The results of the development of dipole magnet of the CBM experiment are presented.

The unusual cellulose utilization system of the aerobic soil bacterium Cytophaga hutchinsonii.

PubMed

Zhu, Yongtao; McBride, Mark J

2017-10-01

Cellulolytic microorganisms play important roles in global carbon cycling and have evolved diverse strategies to digest cellulose. Some are 'generous,' releasing soluble sugars from cellulose extracellularly to feed both themselves and their neighbors. The gliding soil bacterium Cytophaga hutchinsonii exhibits a more 'selfish' strategy. It digests crystalline cellulose using cell-associated cellulases and releases little soluble sugar outside of the cell. The mechanism of C. hutchinsonii cellulose utilization is still poorly understood. In this review, we discuss novel aspects of the C. hutchinsonii cellulolytic system. Recently developed genetic manipulation tools allowed the identification of proteins involved in C. hutchinsonii cellulose utilization. These include periplasmic and cell-surface endoglucanases and novel cellulose-binding proteins. The recently discovered type IX secretion system is needed for cellulose utilization and appears to deliver some of the cellulolytic enzymes and other proteins to the cell surface. The requirement for periplasmic endoglucanases for cellulose utilization is unusual and suggests that cello-oligomers must be imported across the outer membrane before being further digested. Cellobiohydrolases or other predicted processive cellulases that play important roles in many other cellulolytic bacteria appear to be absent in C. hutchinsonii. Cells of C. hutchinsonii attach to and glide along cellulose fibers, which may allow them to find sites most amenable to attack. A model of C. hutchinsonii cellulose utilization summarizing recent progress is proposed.

Cellulose biosynthesis in Acetobacter xylinum

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

Lin, F.C.

1988-01-01

Time-lapse video microscopy has shown periodic reversals during the synthesis of cellulose. In the presence of Congo Red, Acetobacter produces a band of fine fibrils. The direction of cell movement is perpendicular to the longitudinal axis of cell, and the rate of movement was decreased. A linear row of particles, presumably the cellulose synthesizing complexes, was found on the outer membrane by freeze-fracture technique. During the cell cycle, the increase of particles in linear row, the differentiation to four linear rows and the separation of the linear rows have been observed. A digitonin-solubilized cellulose synthase was prepared from A. xylinum,more » and incubated under conditions known to lead to active in vitro synthesis of 1,4-{beta}-D-glucan polymer. Electron microscopy revealed that clusters of fibrils were assembled within minutes. Individual fibrils are 17 {plus minus} 2 angstroms in diameter. Evidence for the cellulosic composition of newly synthesized fibrils was based on incorporation of tritium from UDP-({sup 3}H) glucose binding of gold-labeled cellobiohydrolase, and an electron diffraction pattern identified as cellulose II polymorph instead of cellulose I.« less

A field conference on Impacts of coalbed methane development in the Powder River basin, Wyoming

USGS Publications Warehouse

Flores, Romeo M.; Stricker, Gary D.; Meyer, Joseph F.; Doll, Thomas E.; Norton, Pierce H.; Livingston, Robert J.; Jennings, M. Craig; Kinney, Scott; Mitchell, Heather; Dunn, Steve

2001-01-01

Coalbed methane (CBM) development from the Paleocene Fort Union Formation coal beds in the Powder River Basin in Wyoming has been rapidly expanding since 1993.  During the past ten years the number of CBM producing wells rose to about 4,000 wells as of October, 2000.  About 3,500 of these wells were completed since 1998.  About 13-14 percent of these CBM wells are on Federal lands while the majority are on State and private lands.  More than 50 percent of the lands in the Powder River Basin contains mineral rights owned by the Federal government.  CBM development on Federal lands creates impacts in the basin resulting from associated drilling, facilities, gas gathering systems (e.g., pipeline networks), access roads, and withdrawal and disposal of co-produced water from CBM wells.  The Bureau of Land Management (BLM) assesses the land-use management and impacts of drilling CBM wells on lands where mineral rights are controlled by the Federal government.

Chemical genetic screening identifies a novel inhibitor of parallel alignment of cortical microtubules and cellulose microfibrils.

PubMed

Yoneda, Arata; Higaki, Takumi; Kutsuna, Natsumaro; Kondo, Yoichi; Osada, Hiroyuki; Hasezawa, Seiichiro; Matsui, Minami

2007-10-01

It is a well-known hypothesis that cortical microtubules control the direction of cellulose microfibril deposition, and that the parallel cellulose microfibrils determine anisotropic cell expansion and plant cell morphogenesis. However, the molecular mechanism by which cortical microtubules regulate the orientation of cellulose microfibrils is still unclear. To investigate this mechanism, chemical genetic screening was performed. From this screening, 'SS compounds' were identified that induced a spherical swelling phenotype in tobacco BY-2 cells. The SS compounds could be categorized into three classes: those that disrupted the cortical microtubules; those that reduced cellulose microfibril content; and thirdly those that had neither of these effects. In the last class, a chemical designated 'cobtorin' was found to induce the spherical swelling phenotype at the lowest concentration, suggesting strong binding activity to the putative target. Examining cellulose microfibril regeneration using taxol-treated protoplasts revealed that the cobtorin compound perturbed the parallel alignment of pre-existing cortical microtubules and nascent cellulose microfibrils. Thus, cobtorin could be a novel inhibitor and an attractive tool for further investigation of the mechanism that enables cortical microtubules to guide the parallel deposition of cellulose microfibrils.

Conformations of low-molecular-weight lignin polymers in water

DOE PAGES

Petridis, Loukas; Smith, Jeremy C.

2016-01-13

Low-molecular-weight lignin binds to cellulose during the thermochemical pretreatment of biomass for biofuel production, which prevents the efficient hydrolysis of the cellulose to sugars. The binding properties of lignin are influenced strongly by the conformations it adopts. Here, we use molecular dynamics simulations in aqueous solution to investigate the dependence of the shape of lignin polymers on chain length and temperature. Lignin is found to adopt collapsed conformations in water at 300 and 500 K. However, at 300 K, a discontinuous transition is found in the shape of the polymer as a function of the chain length. Below a criticalmore » degree of polymerization, N c=15, the polymer adopts less spherical conformations than above N c. The transition disappears at high temperatures (500 K) at which only spherical shapes are adopted. As a result, an implication relevant to cellulosic biofuel production is that lignin will self-aggregate even at high pretreatment temperatures.« less

Conformations of Low-Molecular-Weight Lignin Polymers in Water.

PubMed

Petridis, Loukas; Smith, Jeremy C

2016-02-08

Low-molecular-weight lignin binds to cellulose during the thermochemical pretreatment of biomass for biofuel production, which prevents the efficient hydrolysis of the cellulose to sugars. The binding properties of lignin are influenced strongly by the conformations it adopts. Here, we use molecular dynamics simulations in aqueous solution to investigate the dependence of the shape of lignin polymers on chain length and temperature. Lignin is found to adopt collapsed conformations in water at 300 and 500 K. However, at 300 K, a discontinuous transition is found in the shape of the polymer as a function of the chain length. Below a critical degree of polymerization, Nc =15, the polymer adopts less spherical conformations than above Nc. The transition disappears at high temperatures (500 K) at which only spherical shapes are adopted. An implication relevant to cellulosic biofuel production is that lignin will self-aggregate even at high pretreatment temperatures. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Alkali Metal-Glucose Interaction Probed with Infrared Pre-Dissociation Spectroscopy

NASA Astrophysics Data System (ADS)

Kregel, Steven J.; Marsh, Brett; Zhou, Jia; Garand, Etienne

2015-06-01

The efficient extraction of cellulose from biomass and its subsequent conversion to glucose derivatives is an attractive goal in the field of energy science. However, current industrial methods require high ionic strength and harsh conditions. Ionic liquids (IL's) are a class of "green" compounds that have been shown to dissolve cellulose in concentrations of up to 25 wt%. In order to understand IL's extraordinary cellulose dissolving power, a molecular level understanding of the IL-cellulose interaction is needed. Toward that end, we have acquired infrared pre-dissociation spectra of M+-glucose, where M+=Li+, Na+, or K+. Through comparisons with density functional theory calculations, we have determined the relative abundances of various M+-glucose binding motifs in both the thermodynamic and kinetic limits. These results provide insight on the hydrogen bonding dynamics of glucose and are a step towards a fuller understanding of cellulose interactions with ionic liquids.

Positively and Negatively Charged Ionic Modifications to Cellulose Assessed as Cotton-Based Protease-Lowering and Haemostatic Wound Agents

USDA-ARS?s Scientific Manuscript database

Recent developments in cellulose wound dressings targeted to different stages of wound healing have been based on structural and charge modifications that function to modulate events in the complex inflammatory and hemostatic phases of wound healing. Hemostasis and inflammation comprise two overlapp...

A cellulose binding domain protein restores female fertility when expressed in transgenic Bintje potato.

PubMed

Jones, Richard W; Perez, Frances G

2016-03-18

Expression of a gene encoding the family 1 cellulose binding domain protein CBD1, identified in the cellulosic cell wall of the potato late blight pathogen Phytophthora infestans, was tested in transgenic potato to determine if it had an influence on plant cell walls and resistance to late blight. Multiple regenerants of potato (cv. Bintje) were developed and selected for high expression of CBD 1 transcripts. Tests with detached leaflets showed no evidence of increased or decreased resistance to P. infestans, in comparison with the blight susceptible Bintje controls, however, changes in plant morphology were evident in CBD 1 transgenics. Plant height increases were evident, and most importantly, the ability to produce seed berries from a previously sterile cultivar. Immunolocalization of CBD 1 in seed berries revealed the presence throughout the tissue. While Bintje control plants are male and female sterile, CBD 1 transgenics were female fertile. Crosses made using pollen from the late blight resistant Sarpo Mira and transgenic CBD1 Bintje as the female parent demonstrated the ability to introgress P. infestans targeted resistance genes, as well as genes responsible for color and tuber shape, into Bintje germplasm. A family 1 cellulose-binding domain (CBD 1) encoding gene from the potato late blight pathogen P. infestans was used to develop transgenic Bintje potato plants. Transgenic plants became female fertile, allowing for a previously sterile cultivar to be used in breeding improvement. Selection for the absence of the CBD transgene in progeny should allow for immediate use of a genetically enhanced material. Potential for use in other Solanaceous crops is proposed.

Multiscale Modulation of Nanocrystalline Cellulose Hydrogel via Nanocarbon Hybridization for 3D Neuronal Bilayer Formation.

PubMed

Kim, Dongyoon; Park, Subeom; Jo, Insu; Kim, Seong-Min; Kang, Dong Hee; Cho, Sung-Pyo; Park, Jong Bo; Hong, Byung Hee; Yoon, Myung-Han

2017-07-01

Bacterial biopolymers have drawn much attention owing to their unconventional three-dimensional structures and interesting functions, which are closely integrated with bacterial physiology. The nongenetic modulation of bacterial (Acetobacter xylinum) cellulose synthesis via nanocarbon hybridization, and its application to the emulation of layered neuronal tissue, is reported. The controlled dispersion of graphene oxide (GO) nanoflakes into bacterial cellulose (BC) culture media not only induces structural changes within a crystalline cellulose nanofibril, but also modulates their 3D collective association, leading to substantial reduction in Young's modulus (≈50%) and clear definition of water-hydrogel interfaces. Furthermore, real-time investigation of 3D neuronal networks constructed in this GO-incorporated BC hydrogel with broken chiral nematic ordering revealed the vertical locomotion of growth cones, the accelerated neurite outgrowth (≈100 µm per day) with reduced backward travel length, and the efficient formation of synaptic connectivity with distinct axonal bifurcation abundancy at the ≈750 µm outgrowth from a cell body. In comparison with the pristine BC, GO-BC supports the formation of well-defined neuronal bilayer networks with flattened interfacial profiles and vertical axonal outgrowth, apparently emulating the neuronal development in vivo. We envisioned that our findings may contribute to various applications of engineered BC hydrogel to fundamental neurobiology studies and neural engineering. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Combinatorial Libraries of Arrayable Single-Chain Antibodies

NASA Astrophysics Data System (ADS)

Benhar, Itai

Antibodies that bind their respective targets with high affinity and specificity have proven to be essential reagents for biological research. Antibody phage display has become the leading tool for the rapid isolation of single-chain variable fragment (scFv) antibodies in vitro for research applications, but there is usually a gap between scFv isolation and its application in an array format suitable for high-throughput proteomics. In this chapter, we present our antibody phage display system where antibody isolation and scFv immobilization are facilitated by the design of the phagemid vector used as platform. In our system, the scFvs are fused at their C-termini to a cellulose-binding domain (CBD) and can be immobilized onto cellulose-based filters. This made it possible to develop a unique filter lift screen that allowed the efficient screen for multiple binding specificities, and to directly apply library-derived scFvs in an antibody spotted microarray.

Phosphoethanolamine cellulose: A naturally produced chemically modified cellulose.

PubMed

Thongsomboon, Wiriya; Serra, Diego O; Possling, Alexandra; Hadjineophytou, Chris; Hengge, Regine; Cegelski, Lynette

2018-01-19

Cellulose is a major contributor to the chemical and mechanical properties of plants and assumes structural roles in bacterial communities termed biofilms. We find that Escherichia coli produces chemically modified cellulose that is required for extracellular matrix assembly and biofilm architecture. Solid-state nuclear magnetic resonance spectroscopy of the intact and insoluble material elucidates the zwitterionic phosphoethanolamine modification that had evaded detection by conventional methods. Installation of the phosphoethanolamine group requires BcsG, a proposed phosphoethanolamine transferase, with biofilm-promoting cyclic diguanylate monophosphate input through a BcsE-BcsF-BcsG transmembrane signaling pathway. The bcsEFG operon is present in many bacteria, including Salmonella species, that also produce the modified cellulose. The discovery of phosphoethanolamine cellulose and the genetic and molecular basis for its production offers opportunities to modulate its production in bacteria and inspires efforts to biosynthetically engineer alternatively modified cellulosic materials. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Developing and Evaluating a Kindergarten to Third Grade CBM Mathematics Assessment

ERIC Educational Resources Information Center

Lee, Young-Sun; Lembke, Erica

2016-01-01

The present study examined the technical adequacy of curriculum-based measurement (CBM) measure of early numeracy for kindergarten through third grade students. Our CBM measures were developed to reflect broad and theoretically derived categories of mathematical thinking: quick retrieval, written computation, and number sense. The mastery of these…

Decision-Making Accuracy of CBM Progress-Monitoring Data

ERIC Educational Resources Information Center

Hintze, John M.; Wells, Craig S.; Marcotte, Amanda M.; Solomon, Benjamin G.

2018-01-01

This study examined the diagnostic accuracy associated with decision making as is typically conducted with curriculum-based measurement (CBM) approaches to progress monitoring. Using previously published estimates of the standard errors of estimate associated with CBM, 20,000 progress-monitoring data sets were simulated to model student reading…

Deletion of Caldicellulosiruptor bescii CelA reveals its crucial role in the deconstruction of lignocellulosic biomass

DOE PAGES

Young, Jenna; Chung, Daehwan; Bomble, Yannick J.; ...

2014-10-09

Background: Members of the bacterial genus Caldicellulosiruptor are the most thermophilic cellulolytic organisms described to date, and have the ability to grow on lignocellulosic biomass without conventional pretreatment. Different species vary in their abilities to degrade cellulose, and the presence of CelA, a bifunctional glycoside hydrolase that contains a Family 48 and a Family 9 catalytic domain, correlates well with cellulolytic ability in members of this genus. For example, C. hydrothermalis, which does not contain a CelA homolog, or a GH48 Family or GH9 Family glycoside hydrolase, is the least cellulolytic of the Caldicellulosiruptor species so far described. C. bescii,more » which contains CelA and expresses it constitutively, is among the most cellulolytic. In fact, CelA is the most abundant extracellular protein produced in C. bescii. The enzyme contains two catalytic units, a Family 9A-CBM3c processive endoglucanase and a Family 48 exoglucanase, joined by two Family 3b carbohydrate-binding domains. Although there are two non-reducing end-specific Family 9 and three reducing end-specific Family 48 glycoside hydrolases (producing primarily glucose and cellobiose; and cellobiose and cellotriose, respectively) in C. bescii, CelA is the only protein that combines both enzymatic activities. Results: A deletion of the celA gene resulted in a dramatic reduction in the microorganism’s ability to grow on crystalline cellulose (Avicel) and diminished growth on lignocellulosic biomass. A comparison of the overall endoglucanase and exoglucanase activities of the mutant compared with the wild-type suggests that the loss of the endoglucanase activity provided by the GH9 family domain is perhaps compensated for by other enzymes produced by the cell. In contrast, it appears that no other enzymes in the C. bescii secretome can compensate for the loss of exoglucanase activity. The change in enzymatic activity in the celA mutant resulted in a 15-fold decrease in sugar release on Avicel compared with the parent and wild-type strains. In conclusion: The exoglucanase activity of the GH48 domain of CelA plays a major role in biomass degradation within the suite of C. bescii biomass-degrading enzymes.« less

Analysis of the cellulose synthase operon genes, bcsA, bcsB, and bcsC in Cronobacter species: Prevalence among species and their roles in biofilm formation and cell-cell aggregation.

PubMed

Hu, Lan; Grim, Christopher J; Franco, Augusto A; Jarvis, Karen G; Sathyamoorthy, Vengopal; Kothary, Mahendra H; McCardell, Barbara A; Tall, Ben D

2015-12-01

Cronobacter species are emerging food-borne pathogens that cause severe sepsis, meningitis, and necrotizing entercolitis in neonates and infants. Bacterial pathogens such as Escherichia coli and Salmonella species produce extracellular cellulose which has been shown to be involved in rugosity, biofilm formation, and host colonization. In this study the distribution and prevalence of cellulose synthase operon genes (bcsABZC) were determined by polymerase chain reaction (PCR) analysis in 231 Cronobacter strains isolated from clinical, food, environmental, and unknown sources. Furthermore, bcsA and bcsB isogenic mutants were constructed in Cronobacter sakazakii BAA894 to determine their roles. In calcofluor binding assays bcsA and bcsB mutants did not produce cellulose, and their colonial morphotypes were different to that of the parent strain. Biofilm formation and bacterial cell-cell aggregation were significantly reduced in bcsA and bcsB mutants compared to the parental strain. bcsA or bcsAB PCR-negative strains of C. sakazakii did not bind calcofluor, and produced less biofilm and cell-cell aggregation compared to strains possessing bcsAB genes. These data indicated that Cronobacter bcsABZC were present in all clinical isolates and most of food and environmental isolates. bcsA and bcsB genes of Cronobacter were necessary to produce cellulose, and were involved in biofilm formation and cell-cell aggregation. Published by Elsevier Ltd.

German version of the Community Balance and Mobility Scale : Translation and evaluation of measurement properties.

PubMed

Gordt, Katharina; Mikolaizak, A Stefanie; Nerz, Corinna; Barz, Carolin; Gerhardy, Thomas; Weber, Michaela; Becker, Clemens; Schwenk, Michael

2018-02-12

Tools to detect subtle balance deficits in high-functioning community-dwelling older adults are lacking. The Community Balance and Mobility Scale (CBM) is a valuable tool to measure balance deficits in this group; however, it is not yet available in the German language. The aim was 1) to translate and cross-culturally adapt the CBM into the German language and 2) to investigate the measurement properties of the German CBM (G-CBM). The original CBM was translated into the German language according to established guidelines. A total of 51 older adults (mean age 69.9 ± 7.1 years) were recruited to measure construct validity by comparing the G‑CBM against standardized balance and/or mobility assessments including the Fullerton Advanced Balance Scale (FAB), Berg Balance Scale (BBS), 3 m Tandem Walk (3MTW), 8 Level Balance Scale (8LBS), 30 s Chair Stand Test (30CST), Timed Up and Go (TUG) test, gait speed, and the Falls Efficacy Scale International (FES-I). Intrarater and interrater reliability and internal consistency reliability were estimated using intraclass correlations (ICC) and Cronbach's alpha, respectively. Ceiling effects were calculated as the percentage of the sample scoring the maximum score. The G‑CBM correlated excellently with FAB and BBS (ρ = 0.78-0.85; P < 0.001), good with 3MTW, TUG, and FES-I (ρ = -0.55 to -0.61; P < 0.001), and moderately with 8LBS, 30CST, and habitual gait speed (ρ = 0.32-0.46; P < 0.001). Intrarater (ICC 3,k  = 0.998; P < 0.001) and interrater (ICC 2,k  = 0.996; P < 0.001) reliability, and internal consistency reliability (α = 0.998) were also high. The G‑CBM did not show ceiling effects. The G‑CBM is a valid and reliable tool for measuring subtle balance deficits in older high-functioning adults. The absence of ceiling effects emphasizes the use of this scale in this cohort. The G‑CBM can now be utilized in clinical practice.

Cellulose modulates biofilm formation by counteracting curli-mediated colonization of solid surfaces in Escherichia coli.

PubMed

Gualdi, Luciana; Tagliabue, Letizia; Bertagnoli, Stefano; Ieranò, Teresa; De Castro, Cristina; Landini, Paolo

2008-07-01

In enterobacteria, the CsgD protein activates production of two extracellular structures: thin aggregative fimbriae (curli) and cellulose. While curli fibres promote biofilm formation and cell aggregation, the evidence for a direct role of cellulose as an additional determinant for biofilm formation is not as straightforward. The MG1655 laboratory strain of Escherichia coli only produces limited amounts of curli and cellulose; however, ectopic csgD expression results in strong stimulation of curli and cellulose production. We show that, in a csgD-overexpressing derivative of MG1655, cellulose production negatively affects curli-mediated surface adhesion and cell aggregation, thus acting as a negative determinant for biofilm formation. Consistent with this observation, deletion of the bcsA gene, necessary for cellulose production, resulted in a significant increase in curli-dependent adhesion. We found that cellulose production increased tolerance to desiccation, suggesting that the function of cellulose might be related to resistance to environmental stresses rather than to biofilm formation. Production of the curli/cellulose network in enterobacteria typically takes place at low growth temperature (<32 degrees C), but not at 37 degrees C. We show that CsgD overexpression can overcome temperature-dependent control of the curli-encoding csgBA operon, but not of the cellulose-related adrA gene, suggesting very tight temperature control of cellulose production in E. coli MG1655.

A new model for the determination of limb segment mass in children.

PubMed

Kuemmerle-Deschner, J B; Hansmann, S; Rapp, H; Dannecker, G E

2007-04-01

The knowledge of limb segment masses is critical for the calculation of joint torques. Several methods for segment mass estimation have been described in the literature. They are either inaccurate or not applicable to the limb segments of children. Therefore, we developed a new cylinder brick model (CBM) to estimate segment mass in children. The aim of this study was to compare CBM and a model based on a polynomial regression equation (PRE) to volume measurement obtained by the water displacement method (WDM). We examined forearms, hands, lower legs, and feet of 121 children using CBM, PRE, and WDM. The differences between CBM and WDM or PRE and WDM were calculated and compared using a Bland-Altman plot of differences. Absolute limb segment mass measured by WDM ranged from 0.16+/-0.04 kg for hands in girls 5-6 years old, up to 2.72+/-1.03 kg for legs in girls 11-12 years old. The differences of normalised segment masses ranged from 0.0002+/-0.0021 to 0.0011+/-0.0036 for CBM-WDM and from 0.0023+/-0.0041 to 0.0127+/-0.036 for PRE-WDM (values are mean+/-2 S.D.). The CBM showed better agreement with WDM than PRE for all limb segments in girls and boys. CBM is accurate and superior to PRE for the estimation of individual limb segment mass of children. Therefore, CBM is a practical and useful tool for the analysis of kinetic parameters and the calculation of resulting forces to assess joint functionality in children.

Cloning, expression and characterization of a novel GH5 exo/endoglucanase of Thermobifida halotolerans YIM 90462(T) by genome mining.

PubMed

Zhang, Feng; Zhang, Xiao-Mei; Yin, Yi-Rui; Li, Wen-Jun

2015-12-01

The 1389-bp thcel5A gene, which encodes a family 5 of glycoside hydrolases (GH5), was screened from the draft genome of Thermobifida halotolerans YIM 90462(T). ThCel5A was most similar (77% identity) to a GH5 endoglucanase from Thermobifida fusca YX, followed by cellulases from Nocardiopsis dassonvillei subsp. dassonvillei DSM 43111, Nocardiopsis alba ATCC BAA-2165, and Kribbella flavida DSM 17836. The deduced amino acid sequence of ThCel5A, which consisted of 462 amino acid residues, encompassed a family 2 cellulose-binding module and a GH5 catalytic domain. Notably, ThCel5A hydrolysed soluble as well as insoluble cellulose substrates. The enzymatic hydrolysis assay showed that the activity of recombinant ThCel5A was optimized at pH 8.0 and 50°C. Moreover, it retained hydrolytic activity in the presence of various metal ions and >90% activity within the range of pH 8.0-9.0 after 30 min at 50°C. These results suggested that this enzyme has considerable potential in industrial applications. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Xyloglucan endotransglucosylase/hydrolases (XTHs) are inactivated by binding to glass and cellulosic surfaces, and released in active form by a heat-stable polymer from cauliflower florets.

PubMed

Sharples, Sandra C; Nguyen-Phan, Tu C; Fry, Stephen C

2017-11-01

Xyloglucan endotransglucosylase (XET) activity, which cuts and re-joins hemicellulose chains in the plant cell wall, contributing to wall assembly and growth regulation, is the major activity of XTH proteins. During purification, XTHs often lose XET activity which, however, is restored by treatment with certain cold-water-extractable, heat-stable polymers (CHPs), e.g. from cauliflower florets. It was not known whether the XTH-activating factor (XAF) present in CHPs works by promoting (e.g. allosterically) XET activity or by re-solubilising sequestered XTH proteins. We now show that XTHs in dilute solution bind to diverse surfaces (e.g. glass and cellulose), and that CHPs can re-solubilise the bound enzyme, re-activating it. Cell walls prepared from cauliflower florets, mung bean shoots and Arabidopsis cell-suspension cultures each contained endogenous, tightly bound, inactive XTHs, which were likewise rapidly solubilised (within 0.5h) and thus activated by cauliflower XAF. We present a convenient quantitative assay for XAF acting on the native sequestered XTHs of Arabidopsis cell walls; using this assay, we show that CHPs from all plants tested possess XAF activity. The XAF activity of diverse CHPs does not correlate with their conductivity, showing that this activity is not a simple ionic effect. The XAF action of cauliflower CHPs was augmented by NaCl, although NaCl alone was much less effective than a CHP solution of similar conductivity, confirming that the cauliflower polymers did not simply exert a salt effect. We suggest that XAF is an endogenous regulator of XET action, modulating cell-wall loosening and/or assembly in vivo. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

The community balance and mobility scale alleviates the ceiling effects observed in the currently used gait and balance assessments for the community-dwelling older adults.

PubMed

Balasubramanian, Chitralakshmi K

2015-01-01

Currently used balance assessments show a ceiling effect and lack activities essential for community mobility in higher-functioning older adults. The aim of this study was to investigate the reliability and validity of the Community Balance and Mobility (CB&M) Scale in a high-functioning community-dwelling older adult population since the CB&M Scale includes assessment of several challenging tasks and may alleviate the ceiling effects observed in commonly used gait and balance assessments for this cohort. A convenience sample of 40 older adults (73.4 ± 6.9 years) participated in this cross-sectional study. Previously standardized balance and mobility assessments measuring similar constructs as the CB&M were used for validation. Outcomes included Timed Up and Go Test, Berg Balance Scale (BBS), Dynamic Gait Index (DGI), Functional Reach Test (FRT), Short Physical Performance Battery (SPPB), 6-Minute Walk Test (6MWT), Activities Specific Balance Confidence scale (ABC), gait speed, and intraindividual gait variability. A falls questionnaire documented the history of falls. Rater reliability (ICC > 0.95) and internal consistency (α= .97) of the CB&M scale were high. CB&M scores demonstrated strong correlations with DGI, BBS, SPPB, and 6MWT (ρ= 0.70-0.87; P < .01); moderate correlations with falls history, TUG, ABC, and gait speed (ρ= 0.44-0.65; P < .01); and low correlations with FRT, swing and stance time variability (ρ= 0.34-0.37; P < .05). Dynamic Gait Index, BBS, SPPB, and ABC assessments demonstrated ceiling effects (7.5%-32.5%), while no floor or ceiling effects were noted on the CB&M. Logistic regression model showed that the CB&M scores significantly predicted falls history (χ(2) = 6.66, odds ratio = 0.92; P < .01). Area under the curve for the CB&M scale was 0.80 (95% CI: 0.65-0.95). A score of CB&M ≤ 39 was the optimal trade-off between sensitivity and specificity (sensitivity = 79%, specificity = 76%) and a score of CB&M ≤ 45 maximized sensitivity (sensitivity = 93%, specificity = 60%) to discriminate persons with 2 or more falls from those with fewer than 2 falls in the past year. CB&M scale is reliable and valid to evaluate gait, balance, and mobility in community-dwelling older adults. Unlike some currently used balance and mobility assessments for the community-dwelling older adults, the CB&M scale did not show a ceiling in detection of balance and mobility deficits. In addition, cutoff scores have been proposed that might serve as criteria to discriminate older adults with balance and mobility deficits. The CB&M scale might enable assessment of balance and mobility limitations masked by other assessments and help design interventions to improve community mobility and sustain independence in the higher-functioning community-dwelling older adult.

Perspective on Certainty-Based Marking: An Interview with Tony Gardner-Medwin

ERIC Educational Resources Information Center

Cornwell, Reid; Gardner-Medwin, Tony

2008-01-01

In this edition of Perspectives, Reid Cornwell discusses certainty-based marking (CBM) with Tony Gardner-Medwin, professor emeritus of physiology at University College London (UCL), which adopted a simple, theoretically sound version of CBM in its medical education program. CBM has been shown to encourage thinking, reflection, improved analysis,…

Diagnostic Efficiency of easyCBM[R] Math: Oregon. Technical Report #1009

ERIC Educational Resources Information Center

Anderson, Daniel; Alonzo, Julie; Tindal, Gerald

2010-01-01

The easyCBM[R] assessment system is an online benchmark and progress monitoring assessment system designed for use within a response to intervention framework. Educators using easyCBM[R] are often interested in using the results to predict students' state test performance. In the following technical document, we report diagnostic efficiency…

Curriculum-Based Measurement of Oral Reading: Passage Equivalence and Probe-Set Development

ERIC Educational Resources Information Center

Christ, Theodore J.; Ardoin, Scott P.

2009-01-01

Curriculum-based measurement of reading (CBM-R) is used to estimate oral reading fluency. Unlike many traditional published tests, CBM-R materials are often comprised of 20 to 30 alternate forms/passages. Historically, CBM-R assessment materials were sampled from curricular materials. Recent research has documented the potentially deleterious…

Curriculum-based Measurement in Assessing Bilingual Students: A Promising New Direction.

ERIC Educational Resources Information Center

Bentz, Johnell; Pavri, Shireen

2000-01-01

This article discusses the problems with traditional methods of assessing bilingual students and describes curriculum-based measurement (CBM) for use with bilingual Hispanic students. Additional information about the features of CBM is presented along with issues related to the use of CBM with bilingual Hispanic students. (Contains references.)…

Development of method to remove weld scallop and ceramic backing material of wedge type and its application

NASA Astrophysics Data System (ADS)

Kang, Sung-Koo; Yang, Jong-Soo; Kim, Ho-Kyung

2015-06-01

The weld scallop has been used for joining T-bars. There are a lot of weld scallops in shipbuilding. It is difficult to perform scallop welding due to the inconvenient welding position. This results in many problems such as porosity, slag inclusion, etc. In this study, a new method is devised to remove weld scallops by incorporating a Ceramic Backing Material (CBM). The weld scallop is removed by an elongation of the v groove. In order to insert a CBM into the groove without a weld scallop, a wedge-shaped CBM is developed. The top side of the developed CBM is similar to the shape of a general back bead. The bottom surface has a saw-toothed shape for cutting at a suitable length. This can be attached to the root side of a face plate using adhesive tape, just like a general CBM. Welding experiments in normal and abnormal conditions are carried out and the possibility of burn-through is examined. This CBM's applicability to shipbuilding is verified.

Structure/function analysis of cotton-based peptide-cellulose conjugates: spatiotemporal/kinetic assessment of protease aerogels compared to nanocrystalline and paper cellulose

USDA-ARS?s Scientific Manuscript database

The growing incidence of chronic wounds in the world population has prompted increased interest in chronic wound dressings with protease-modulating activity and protease point of care sensors to treat and enable monitoring of elevated protease-based wound pathology. However, the overall design featu...

Compressed baryonic matter at FAIR: JINR participation

NASA Astrophysics Data System (ADS)

Kurilkin, P.; Ladygin, V.; Malakhov, A.; Senger, P.

2015-11-01

The scientific mission of the Compressed Baryonic Matter(CBM) experiment is the study of the nuclear matter properties at the high baryon densities in heavy ion collisions at the Facility of Antiproton and Ion Research (FAIR) in Darmstadt. We present the results on JINR participation in the CBM experiment. JINR teams are responsible on the design, the coordination of superconducting(SC) magnet manufacture, its testing and installation in CBM cave. Together with Silicon Tracker System it will provide the momentum resolution better 1% for different configuration of CBM setup. The characteristics and technical aspects of the magnet are discussed. JINR plays also a significant role in the manufacture of two straw tracker station for the muon detection system. JINR team takes part in the development of new method for simulation, processing and analysis experimental data for different basic detectors of CBM.

Starch Binding Domain-containing Protein 1 Plays a Dominant Role in Glycogen Transport to Lysosomes in Liver*

PubMed Central

Sun, Tao; Yi, Haiqing; Yang, Chunyu; Kishnani, Priya S.; Sun, Baodong

2016-01-01

A small portion of cellular glycogen is transported to and degraded in lysosomes by acid α-glucosidase (GAA) in mammals, but it is unclear why and how glycogen is transported to the lysosomes. Stbd1 has recently been proposed to participate in glycogen trafficking to lysosomes. However, our previous study demonstrated that knockdown of Stbd1 in GAA knock-out mice did not alter lysosomal glycogen storage in skeletal muscles. To further determine whether Stbd1 participates in glycogen transport to lysosomes, we generated GAA/Stbd1 double knock-out mice. In fasted double knock-out mice, glycogen accumulation in skeletal and cardiac muscles was not affected, but glycogen content in liver was reduced by nearly 73% at 3 months of age and by 60% at 13 months as compared with GAA knock-out mice, indicating that the transport of glycogen to lysosomes was suppressed in liver by the loss of Stbd1. Exogenous expression of human Stbd1 in double knock-out mice restored the liver lysosomal glycogen content to the level of GAA knock-out mice, as did a mutant lacking the Atg8 family interacting motif (AIM) and another mutant that contains only the N-terminal 24 hydrophobic segment and the C-terminal starch binding domain (CBM20) interlinked by an HA tag. Our results demonstrate that Stbd1 plays a dominant role in glycogen transport to lysosomes in liver and that the N-terminal transmembrane region and the C-terminal CBM20 domain are critical for this function. PMID:27358407

Evaluating models of cellulose degradation by Fibrobacter succinogenes S85

DOE PAGES

Burnet, Meagan C.; Dohnalkova, Alice C.; Neumann, Anthony P.; ...

2015-12-02

Fibrobacter succinogenes S85 is an anaerobic non-cellulosome utilizing cellulolytic bacterium originally isolated from the cow rumen microbial community. Efforts to elucidate its cellulolytic machinery have resulted in the proposal of numerous models which involve a combination of cell-surface attachment via a combination of cellulose-binding fibro-slime proteins and pili, the production of cellulolytic vesicles, and the entry of cellulose fibers into the periplasmic space. Here, we used a combination of RNA-sequencing, proteomics, and transmission electron microscopy (TEM) to further elucidate the cellulolytic mechanism of F. succinogenes. Our RNA-sequence analysis shows that genes encoding Type II and III secretion systems, fibro-slime proteins,more » and pili are differentially expressed on cellulose, relative to glucose. A subcellular fractionation of cells grown on cellulose revealed that carbohydrate active enzymes associated with cellulose deconstruction and fibro-slime proteins were greater in the extracellular media, as compared to the periplasm and outer membrane fractions. TEMs of samples harvested at mid-exponential and stationary phases of growth on cellulose and glucose showed the presence of grooves in the cellulose between the bacterial cells and substrate, suggesting enzymes work extracellularly for cellulose degradation. Membrane vesicles were only observed in stationary phase cultures grown on cellulose. Furthermore, these results provide evidence that F. succinogenes attaches to cellulose fibers using fibro-slime and pili, produces cellulases, such as endoglucanases, that are secreted extracellularly using type II and III secretion systems, and degrades the cellulose into cellodextrins that are then imported back into the periplasm for further digestion by β-glucanases and other cellulases.« less

Evaluating Models of Cellulose Degradation by Fibrobacter succinogenes S85

PubMed Central

Burnet, Meagan C.; Dohnalkova, Alice C.; Neumann, Anthony P.; Lipton, Mary S.; Smith, Richard D.; Suen, Garret; Callister, Stephen J.

2015-01-01

Fibrobacter succinogenes S85 is an anaerobic non-cellulosome utilizing cellulolytic bacterium originally isolated from the cow rumen microbial community. Efforts to elucidate its cellulolytic machinery have resulted in the proposal of numerous models which involve cell-surface attachment via a combination of cellulose-binding fibro-slime proteins and pili, the production of cellulolytic vesicles, and the entry of cellulose fibers into the periplasmic space. Here, we used a combination of RNA-sequencing, proteomics, and transmission electron microscopy (TEM) to further clarify the cellulolytic mechanism of F. succinogenes. Our RNA-sequence analysis shows that genes encoding type II and III secretion systems, fibro-slime proteins, and pili are differentially expressed on cellulose, relative to glucose. A subcellular fractionation of cells grown on cellulose revealed that carbohydrate active enzymes associated with cellulose deconstruction and fibro-slime proteins were greater in the extracellular medium, as compared to the periplasm and outer membrane fractions. TEMs of samples harvested at mid-exponential and stationary phases of growth on cellulose and glucose showed the presence of grooves in the cellulose between the bacterial cells and substrate, suggesting enzymes work extracellularly for cellulose degradation. Membrane vesicles were only observed in stationary phase cultures grown on cellulose. These results provide evidence that F. succinogenes attaches to cellulose fibers using fibro-slime and pili, produces cellulases, such as endoglucanases, that are secreted extracellularly using type II and III secretion systems, and degrades the cellulose into cellodextrins that are then imported back into the periplasm for further digestion by β-glucanases and other cellulases. PMID:26629814

A rapid procedure for measurement of the free testosterone fraction in human plasma using the centria radioimmunoassay centrifugal analyzer.

PubMed

Schwarz, S; Boyd, J

1981-01-01

Following the incubation of plasma with a tracer amount of tritiated testosterone, the reaction mixture is separated into a sex hormone-binding globulin bound and an unbound fraction of radioligand using DEAE-cellulose columns placed in the incubator-separator module of the Centria radioimmunoassay centrifugal analyzer. Neural Tris-buffer elutes unbound steroid from the matrix, while acidic Tris-buffer can remove the protein-bound fraction in a subsequent step. Complementary and thus qualitatively equal results are obtained when counting either eluate. Comparison of this technique with an ammonium sulfate precipitation method showed high correlation. Free testosterone indices as determined by the Centria modification in a number of prepuberal children, normal men and women, as well as pregnant and hirsute women similar to those previously reported.

Cellulase digestibility of pretreated biomass is limited by cellulose accessibility.

PubMed

Jeoh, Tina; Ishizawa, Claudia I; Davis, Mark F; Himmel, Michael E; Adney, William S; Johnson, David K

2007-09-01

Attempts to correlate the physical and chemical properties of biomass to its susceptibility to enzyme digestion are often inconclusive or contradictory depending on variables such as the type of substrate, the pretreatment conditions and measurement techniques. In this study, we present a direct method for measuring the key factors governing cellulose digestibility in a biomass sample by directly probing cellulase binding and activity using a purified cellobiohydrolase (Cel7A) from Trichoderma reesei. Fluorescence-labeled T. reesei Cel7A was used to assay pretreated corn stover samples and pure cellulosic substrates to identify barriers to accessibility by this important component of cellulase preparations. The results showed cellulose conversion improved when T. reesei Cel7A bound in higher concentrations, indicating that the enzyme had greater access to the substrate. Factors such as the pretreatment severity, drying after pretreatment, and cellulose crystallinity were found to directly impact enzyme accessibility. This study provides direct evidence to support the notion that the best pretreatment schemes for rendering biomass more digestible to cellobiohydrolase enzymes are those that improve access to the cellulose in biomass cell walls, as well as those able to reduce the crystallinity of cell wall cellulose.

Extraction of palm tree cellulose and its functionalization via graft copolymerization.

PubMed

Al-Hoqbani, Abdulmajeed A; Abdel-Halim, E S; Al-Deyab, Salem S

2014-09-01

The work in this paper was planned with the aim of extracting the cellulosic component of palm tree waste and functionalizing this cellulose through graft copolymerization with acrylic acid. The cellulose extraction included hot alkali treatment with aqueous sodium hydroxide to remove the non-cellulosic binding materials. The alkali treatment was followed by an oxidative bleaching using peracid/hydrogen peroxide mixture with the aim of removing the rest of non-cellulosic materials to improve the fiber hydrophilicity and accessibility towards further grafting reaction. Optimum conditions for cellulose extraction are boiling in 5% (W/V) NaOH in a material to liquor ratio of 1:20 for 1 h then bleaching with 60 ml/l bleaching mixture at initial pH value of 6.5 for 30 min. The pH of the bleaching medium is turned to the alkaline range 11 and bleaching continues for extra 30 min. Graft copolymerization reaction was initiated by potassium bromate/thiourea dioxide redox system. Optimum conditions for grafting are 30 mmol of potassium bromate, 30 mmol of thiourea dioxide and 150 g of acrylic acid (each per 100 g of cellulose). The polymerization reaction was carried out for 120 min at 50°C using a material to liquor ratio of 1:20. Copyright © 2014 Elsevier B.V. All rights reserved.

Diagnostic Efficiency of easyCBM[R] Math: Washington State. Technical Report #1008

ERIC Educational Resources Information Center

Anderson, Daniel; Alonzo, Julie; Tindal, Gerald

2010-01-01

easyCBM[R] is an online benchmark and progress monitoring assessment system designed for use within a response to intervention framework. Educators using easyCBM[R] are often interested in using the results to predict students' state test performance. In the following technical document, we report diagnostic efficiency statistics using a sample…

Advanced (Measurement) Applications of Curriculum-Based Measurement in Reading

ERIC Educational Resources Information Center

Petscher, Yaacov; Cummings, Kelli Dawn; Biancarosa, Gina; Fien, Hank

2013-01-01

The purpose of this article is to provide a commentary on the current state of several measurement issues pertaining to curriculum-based measures of reading (R-CBM). We begin by providing an overview of the utility of R-CBM, followed by a presentation of five specific measurements considerations: (a) the reliability of R-CBM oral reading fluency…

Curriculum-Based Measurement of Oral Reading: A Preliminary Investigation of Confidence Interval Overlap to Detect Reliable Growth

ERIC Educational Resources Information Center

Van Norman, Ethan R.

2016-01-01

Curriculum-based measurement of oral reading (CBM-R) progress monitoring data is used to measure student response to instruction. Federal legislation permits educators to use CBM-R progress monitoring data as a basis for determining the presence of specific learning disabilities. However, decision making frameworks originally developed for CBM-R…

The xyloglucan-cellulose assembly at the atomic scale.

PubMed

Hanus, Jaroslav; Mazeau, Karim

2006-05-01

The assembly of cell wall components, cellulose and xyloglucan (XG), was investigated at the atomistic scale using molecular dynamics simulations. A molecular model of a cellulose crystal corresponding to the allomorph Ibeta and exhibiting a flexible complex external morphology was employed to mimic the cellulose microfibril. The xyloglucan molecules considered were the three typical basic repeat units, differing only in the size of one of the lateral chain. All the investigated XG fragments adsorb nonspecifically onto cellulose fiber; multiple arrangements are equally probable, and every cellulose surface was capable of binding the short XG molecules. The following structural effects emerged: XG molecules that do not have any long side chains tended to adapt themselves nicely to the topology of the microfibril, forming a flat, outstretched conformation with all the sugar residues interacting with the surface. In contrast, the XG molecules, which have long side chains, were not able to adopt a flat conformation that would enable the interaction of all the XG residues with the surface. In addition to revealing the fundamental atomistic details of the XG adsorption on cellulose, the present calculations give a comprehensive understanding of the way the XG molecules can unsorb from cellulose to create a network that forms the cell wall. Our revisited view of the adsorption features of XG on cellulose microfibrils is consistent with experimental data, and a model of the network is proposed. Copyright (c) 2006 Wiley Periodicals, Inc.

Role of the Filters in the Formation and Stabilization of Semiquinone Radicals Collected from Cigarette Smoke

PubMed Central

Maskos, Zofia; Dellinger, Barry

2013-01-01

The fractional pyrolysis of Bright tobacco was performed in nitrogen atmosphere over the temperature range of 240 – 510 °C in a specially constructed, high temperature flow reactor system. Electron paramagnetic resonance (EPR) spectroscopy was used to analyze the free radicals in the initially produced total particular matter (TPM) and in TPM after exposure to ambient air (aging). Different filters have been used to collect TPM from tobacco smoke: cellulosic, cellulose nitrate, cellulose acetate, nylon, Teflon and Cambridge. The collection of the primary radicals (measured immediately after collection of TPM on filters), the formation and stabilization of the secondary radicals (defined as radicals formed during aging of TPM samples on the filters) depend significantly on the material of the filter. A mechanistic explanation about different binding capability of the filters decreasing in the order: cellulosic < cellulose nitrate < cellulose acetate < nylon ~ teflon is presented. Different properties were observed for the Cambridge filter. Specific care must be taken using the filters for identification of radicals from tobacco smoke to avoid artifacts in each case. PMID:24265513

Trends in major-ion constituents and properties for selected sampling sites in the Tongue and Powder River watersheds, Montana and Wyoming, based on data collected during water years 1980-2010

USGS Publications Warehouse

Sando, Steven K.; Vecchia, Aldo V.; Barnhart, Elliott P.; Sando, Thomas R.; Clark, Melanie L.; Lorenz, David L.

2014-01-01

The primary purpose of this report is to present information relating to flow-adjusted temporal trends in major-ion constituents and properties for 16 sampling sites in the Tongue and Powder River watersheds based on data collected during 1980–2010. In association with this primary purpose, the report presents background information on major-ion characteristics (including specific conductance, calcium, magnesium, potassium, sodium adsorption ratio, sodium, alkalinity, chloride, fluoride, dissolved sulfate, and dissolved solids) of the sampling sites and coal-bed methane (CBM) produced water (groundwater pumped from coal seams) in the site watersheds, trend analysis methods, streamflow conditions, and factors that affect trend results. The Tongue and Powder River watersheds overlie the Powder River structural basin (PRB) in northeastern Wyoming and southeastern Montana. Limited extraction of coal-bed methane (CBM) from the PRB began in the early 1990’s, and increased dramatically during the late 1990’s and early 2000’s. CBM-extraction activities produce discharges of water with high concentrations of dissolved solids (particularly sodium and bicarbonate ions) relative to most stream water in the Tongue and Powder River watersheds. Water-quality of CBM produced water is of concern because of potential effects of sodium on agricultural soils and potential effects of bicarbonate on aquatic biota. Two parametric trend-analysis methods were used in this study: the time-series model (TSM) and ordinary least squares regression (OLS) on time, streamflow, and season. The TSM was used to analyze trends for 11 of the 16 study sites. For five sites, data requirements of the TSM were not met and OLS was used to analyze trends. Two primary 10-year trend-analysis periods were selected. Trend-analysis period 1 (water years 1986–95; hereinafter referred to as period 1) was selected to represent variability in major-ion concentrations in the Tongue and Powder River watersheds before potential effects of CBM-extraction activities. Trend analysis period 2 (water years 2001–10; hereinafter referred to as period 2) was selected because it encompassed substantial CBM-extraction activities and therefore might indicate potential effects of CBM-extraction activities on water quality of receiving streams in the Tongue and Powder River watersheds. For sites that did not satisfy data requirements for the TSM, OLS was used to analyze trends for period 2 (if complete data were available) or a 6-year period (2005–10). Flow-rate characteristics of CBM-produced water were estimated to allow general comparisons with streamflow characteristics of the sampling sites. The information on flow-rate characteristics of CBM-produced water in relation to streamflow does not account for effects of disposal, treatment, or other remediation activities on the potential quantitative effects of CBM-produced water on receiving streams. In many places, CBM-produced water is discharged into impoundments or channels in upper reaches of tributary watersheds where water infiltrates and does not directly contribute to streamflow. For Tongue River at State line (site 4) mean annual pumping rate of CBM-produced water during water years 2001–10 (hereinafter referred to as mean CBM pumping rate) was 6 percent of the mean of annual median streamflows during water years 2001–10 (hereinafter referred to as 2001–10 median streamflow). For main-stem Tongue River sites 5, 7, and 10, mean CBM pumping rate was 8–12 percent of 2001–10 median streamflow. For main-stem Powder River sites (sites 12, 13, and 16), mean CBM pumping rates were 26, 28, and 34 percent of 2001–10 median streamflows, respectively. For main-stem Tongue River sites analyzed by using the TSM and downstream from substantial CBM-extraction activities [Tongue River at State line (site 4), Tongue River at Tongue River Dam (site 5), Tongue River at Birney Day School (site 7), and Tongue River at Miles City (site 10)], generally small significant or nonsignificant decreases in most constituents are indicated for period 1. For period 2 for these sites, the TSM trend results do not allow confident conclusions concerning detection of effects of CBM-extraction activities on stream water quality. Detection of significant trends in major-ion constituents and properties for period 2 generally was infrequent, and direction, magnitudes, and significance of fitted trends were not strongly consistent with relative differences in water quality between stream water and CBM-produced water. The TSM indicated significant or generally large magnitude increases in median values of sodium adsorption ratio (SAR), sodium, and alkalinity for period 2 for sites 5 and 7, which might indicate potential effects of CBM-extraction activities on stream water. However, other factors, including operations of Tongue River Reservoir, irrigation activities, contributions of saline groundwater, and operations of the Decker coal mine, confound confident determination of causes of detected significant trends for sites 5 and 7. For all mainstem Tongue River sites, trends for period 2 generally are within ranges of those for period 1 before substantial CBM-extraction activities. For main-stem Powder River sites analyzed by using the TSM [Powder River at Sussex (site 11), Powder River at Arvada (site 12), Powder River at Moorhead (site 13), and Powder River near Locate (site 16)], significant or generally large magnitude decreases in median values of SAR, sodium, estimated alkalinity, chloride, fluoride, specific conductance, and dissolved solids are indicated for period 1. Patterns in trend results for period 1 for main-stem Powder River sites are consistent with effects of Salt Creek oil-brine reinjection that started in 1990. Trend results for all main-stem Powder River sites downstream from substantial CBM-extraction activities (sites 12, 13, and 16) indicate evidence of potential effects of CBM-extraction activities on stream water quality, although evidence is stronger for sites 12 and 13 than for site 16. Evidence in support of potential CBM effects includes significant increases in median values of SAR, sodium, and estimated alkalinity for period 2 for sites 12, 13, and 16 that are consistent with relative differences between stream water and CBM-produced water. Significant increases in median values of these constituents for period 2 are not indicated for Powder River at Sussex (site 11) upstream from substantial CBM-extraction activities. In interpreting the trend results, it is notable that the fitted trends evaluate changes in median concentrations and also notable that changes in median concentrations that might be attributed to CBM-extraction activities probably are more strongly evident during low to median streamflow conditions than during mean to high streamflow conditions. This observation is relevant in assessing trend results in relation to specific water-quality concerns, including effects of water-quality changes on irrigators and effects on stream biota and ecology.

Antithyroid drug-related hepatotoxicity in hyperthyroidism patients: a population-based cohort study

PubMed Central

Wang, Meng-Ting; Lee, Wan-Ju; Huang, Tien-Yu; Chu, Che-Li; Hsieh, Chang-Hsun

2014-01-01

Aims The evidence of hepatotoxicity of antithyroid drugs (ATDs) is limited to case reports or spontaneous reporting. This study aimed to quantify the incidence and comparative risks of hepatotoxicity for methimazole (MMI)/carbimazole (CBM) vs. propylthiouracil (PTU) in a population-based manner. Methods We conducted a cohort study of hyperthyroidism patients initially receiving MMI/CBM or PTU between 1 January 2004 and 31 December 2008 using the Taiwan National Health Insurance Research Database. The examined hepatotoxicity consisted of cholestasis, non-infectious hepatitis, acute liver failure and liver transplant, with the incidences and relative risks being quantified by Poisson exact methods and Cox proportional hazard models, respectively. Results The study cohort comprised 71 379 ATD initiators, with a median follow-up of 196 days. MMI/CBM vs. PTU users had a higher hepatitis incidence rate (3.17/1000 vs. 1.19/1000 person-years) but a lower incidence of acute liver failure (0.32/1000 vs. 0.68/1000 person-years). The relative risk analysis indicated that any use of MMI/CBM was associated with a 2.89-fold (95% CI 1.81, 4.60) increased hepatitis risk compared with PTU, with the risk increasing to 5.08-fold for high dose MMI/CBM (95% CI 3.15, 8.18). However, any MMI/CBM use vs. PTU was not related to an increased risk of cholestasis (adjusted hazard ratio [HR] 1.14, 95% CI 0.40, 3.72) or acute liver failure (adjusted HR 0.54, 95% CI 0.24, 1.22). Conclusions MMI/CBM and PTU exert dissimilar incidence rates of hepatotoxicity. Compared to PTU, MMI/CBM are associated in a dose-dependent manner with an increased risk for hepatitis while the risks are similar for acute liver failure and cholestasis. PMID:25279406

Attention and interpretation bias modification treatment for social anxiety disorder: A randomized clinical trial of efficacy and synergy.

PubMed

Naim, Reut; Kivity, Yogev; Bar-Haim, Yair; Huppert, Jonathan D

2018-06-01

Attention bias modification treatment (ABMT) and cognitive bias modification of interpretation (CBM-I) both have demonstrated efficacy in alleviating social anxiety, but how they compare with each other, their combination, and with a combined control condition has not been studied. We examined their relative and combined efficacy compared to control conditions in a randomized controlled trial (RCT). Ninety-five adults diagnosed with social anxiety disorder (SAD), were randomly allocated to 4 groups: ABMT + CBM-I control (hereafter ABMT; n = 23), CBM-I + ABMT control (hereafter CBM-I; n = 24), combined ABMT + CBM-I (n = 23), and combined control (n = 25). Treatment included eight sessions over four weeks. Clinician-rated and self-reported measures of social anxiety symptoms, functional impairment, and threat-related attention and interpretive biases were evaluated at baseline, post-treatment, and 3-month follow-up. ABMT yielded greater symptom reduction as measured by both clinician-ratings (Cohen's ds = 0.57-0.70) and self-reports (ds = 0.70-0.85) compared with the CBM-I, the combined ABMT + CBM-I, and the combined control conditions. Neither of the other conditions demonstrated superior symptom change compared to the control condition. No group differences were found for functioning or cognitive biases measures. Limitations mainly include the mix of active and control treatments applied across the different groups. Therefore, the net effect of each of the treatments by itself could not be clearly tested. Results suggest superiority of ABMT compared to CBM-I and their combination in terms of symptom reduction. Possible interpretations and methodological issues underlying the observed findings are discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

Successful engagement: CBM's holistic approach to the work in the area of ear care, deafness, hard of hearing and deafblindness.

PubMed

Tesni, Sian; Santana-Hernández, Diego

2014-09-01

CBM is an international Christian development organisation, committed to improving the quality of life of persons with disabilities in disadvantaged societies. CBM aims at achieving this in partnership with local organisations and in cooperation with stakeholders at regional and international levels. This article aims at sharing CBM's holistic approach for successful engagement in the work area of ear care, deafness, hard of hearing and deafblindness. Review of CBM supporting documents, including strategies, position papers, technical guidelines, criteria of success and information related to partners, centres of excellence and model projects. Description of how partner programmes use participatory methods of planning and implementation, involving persons with disabilities and the community. Recommendations on how to develop national or sub-national strategies to impact the lives, and advocate with governments for the rights and inclusion, of persons with disabilities. CBM's holistic approach to work in the area of ear care; deafness; hard of hearing and deafblindness includes intervention at all levels of health care provision, Education, CBR and Audiological, Speech Therapy and other interdisciplinary services. This article presents CBM's core principles and shares specific strategic planning and results, together with lessons learnt while searching a sustainable engagement for field work. Rehabilitation, in its widest conception, should be approached as a multidisciplinary strategy, and developed in close cooperation with other local, national and international agencies. CBM is positioned in a strategic point from where it can play a key role in the facilitation of an international forum for agencies and stakeholders to reach a consensus to decrease the burden of ear disease and hearing loss, through early intervention and re/habilitation.

Biogas production from various coal types using beef cattle rumen's liquid as a source of microorganisms consortium

NASA Astrophysics Data System (ADS)

Kurnani, Tubagus Benito Achmad; Harlia, Ellin; Hidayati, Yuli Astuti; Marlina, Eulis Tanti; Sugiarto, A. N.; Rahmah, K. N.; Joni, I. M.

2018-02-01

Nowadays, Indonesia is developing Coal-Bed Methane (CBM) production, but its production is not sufficient yet. Basically, CBM is produced naturally along with coal formation, i.e. through the activity of indigenous microorganisms. In this regard, to increase the production of CBM, adding a consortium of microorganisms into the coal deposit can be an option. One source of a consortium of bacteria available in nature is the rumen contents of ruminant livestock such as beef cattle. The purpose of this research was to know the capability of bacteria in rumen contents of beef cattle to produce CBM from various types of coal. In addition, to get a better concentration of bacteria than previous research so that it can be used as a reference for CBM production in the future. This explorative research used an experimental method with descriptive explanation. CBM production was performed using Hungate tube as a digester with three coal substrates, namely lignite, sub-bituminous and bituminous. This experiment also used 10-7 diluted rumen content of beef cattle as a source. The parameters measured were bacterial density, the amount of CBM, carbon dioxide and nitrous oxide on day 2, 5, 10 and 14. The treatment and parameters measurement were carried out in triplicates. This study finding showed that the highest bacterial density in all three types of coal was obtained on day 10 as well as the amount of CBM, carbon dioxide and nitrous oxide. These results are higher than the results from previous research therefore, this treatment can be used as an inoculant in a solid form for easy distribution.

Design, fabrication, and testing of energy-harvesting thermoelectric generator

NASA Astrophysics Data System (ADS)

Jovanovic, Velimir; Ghamaty, Saeid

2006-03-01

An energy-harvesting thermoelectric generator (TEG) is being developed to provide power for wireless sensors used in health monitoring of Navy machinery. TEGs are solid-state devices that convert heat directly into electricity without any moving parts. In this application, the TEGs utilize the heat transfer between shipboard waste heat sources and the ambient air to generate electricity. In order to satisfy the required small design volume of less than one cubic inch, Hi-Z is using its innovative thin-film Quantum Well (QW) thermoelectric technology that will provide a factor of four increase in efficiency and a large reduction in the device volume over the currently used bulk Bi IITe 3 based thermoelectics. QWs are nanostructured multi-layer films. These wireless sensors can be used to detect cracks, corrosion, impact damage, and temperature and vibration excursions as part of the Condition Based Maintenance (CBM) of the Navy ship machinery. The CBM of the ship machinery can be significantly improved by automating the process with the use of self-powered wireless sensors. These power-harvesting TEGs can be used to replace batteries as electrical power sources and to eliminate power cables and data lines. The first QW TEG module was fabricated and initial tests were successful. It is planned to conduct performance tests the entire prototype QW TEG device (consisting of the TEG module, housing, thermal insulation and the heat sink) in a simulated thermal environment of a Navy ship.

Effect of Chicken Bone Meal as Phosphorus Supplement on Blood Metabolites in Fattening Lambs

NASA Astrophysics Data System (ADS)

Pujiastuti, A.; Muktiani, A.

2018-02-01

The aim of this study was to evaluate the effect of chicken bone meal (CBM) as phosphorus supplementon blood metabolites in fattening lambs. The experiment used 16 of 12 months old local male lambs with initial body weight 27.01 ± 1.51 kg. The experiment used a complete randomized design with 4 treatments and 4 replications. The treatments were T0 (basal ration = native grass + soybean curd waste), T1 (basal ration + 0.49% P Dicalcium phosphate), T2 (basal ration + 0.70% P CBM), T3 (basal ration + 1.39 % P CBM). The results indicated that CBM as phosphorus supplement was significantly different (P<0,05) on P intake, phosphorus and glucose serum and did not different significantly on dry matter intake and alkaline phosphatase activity. In conclusion, CBM is one of requirement organic phosphorus supplement which can be applied on ruminants.

An end-to-end communications architecture for condition-based maintenance applications

NASA Astrophysics Data System (ADS)

Kroculick, Joseph

2014-06-01

This paper explores challenges in implementing an end-to-end communications architecture for Condition-Based Maintenance Plus (CBM+) data transmission which aligns with the Army's Network Modernization Strategy. The Army's Network Modernization strategy is based on rolling out network capabilities which connect the smallest unit and Soldier level to enterprise systems. CBM+ is a continuous improvement initiative over the life cycle of a weapon system or equipment to improve the reliability and maintenance effectiveness of Department of Defense (DoD) systems. CBM+ depends on the collection, processing and transport of large volumes of data. An important capability that enables CBM+ is an end-to-end network architecture that enables data to be uploaded from the platform at the tactical level to enterprise data analysis tools. To connect end-to-end maintenance processes in the Army's supply chain, a CBM+ network capability can be developed from available network capabilities.

The CBM RICH detector

NASA Astrophysics Data System (ADS)

Adamczewski-Musch, J.; Akishin, P.; Becker, K.-H.; Belogurov, S.; Bendarouach, J.; Boldyreva, N.; Chernogorov, A.; Deveaux, C.; Dobyrn, V.; Dürr, M.; Eschke, J.; Förtsch, J.; Heep, J.; Höohne, C.; Kampert, K.-H.; Kochenda, L.; Kopfer, J.; Kravtsov, P.; Kres, I.; Lebedev, S.; Lebedeva, E.; Leonova, E.; Linev, S.; Mahmoud, T.; Michel, J.; Miftakhov, N.; Niebur, W.; Ovcharenko, E.; Pauly, C.; Pfeifer, D.; Querchfeld, S.; Rautenberg, J.; Reinecke, S.; Riabov, Y.; Roshchin, E.; Samsonov, V.; Tarasenkova, O.; Traxler, M.; Ugur, C.; Vznuzdaev, E.; Vznuzdaev, M.

2016-05-01

The CBM RICH detector will use CO2 as radiator gas, focussing glass mirrors with Al+MgF2 reflective and protective coating and Hamamatsu H12700 MAPMTs as photon detectors. The detector will serve for electron to pion separation up to momenta of 8 GeV/c and thus enable in CBM the measurement of electromagnetic radiation from the early and dense fireball in A+A collisions at SIS 100. In this article, the current status of the CBM RICH development will be presented including new measurements of the radiation hardness of the H12700 MAPMT and WLS coatings with p-terphenyl, the new concept for the readout electronics, and optimizations ongoing with respect to the mirror mount structure and overall geometry. Prior to the usage in CBM, part of the already ordered MAPMTs will be used to upgrade the HADES RICH detector for a new measurement campaign at SIS 18 from 2018-2020.

Effects of Lytic Polysaccharide Monooxygenase Oxidation on Cellulose Structure and Binding of Oxidized Cellulose Oligomers to Cellulases

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

Vermaas, Josh V.; Crowley, Michael F.; Beckham, Gregg T.

In nature, polysaccharide glycosidic bonds are cleaved by hydrolytic enzymes for a vast array of biological functions. Recently, a new class of enzymes that utilize an oxidative mechanism to cleave glycosidic linkages was discovered; these enzymes are called lytic polysaccharide monooxygenases (LPMO). These oxidative enzymes are synergistic with cocktails of hydrolytic enzymes and are thought to act primarily on crystalline regions, in turn providing new sites of productive attachment and detachment for processive hydrolytic enzymes. In the case of cellulose, the homopolymer of ..beta..-1,4-d-glucose, enzymatic oxidation occurs at either the reducing end or the nonreducing end of glucose, depending onmore » enzymatic specificity, and results in the generation of oxidized chemical substituents at polymer chain ends. LPMO oxidation of cellulose is thought to produce either a lactone at the reducing end of glucose that can spontaneously or enzymatically convert to aldonic acid or 4-keto-aldose at the nonreducing end that may further oxidize to a geminal diol. Here, we use molecular simulation to examine the effect of oxidation on the structure of crystalline cellulose. The simulations highlight variations in behaviors depending on the chemical identity of the oxidized species and its location within the cellulose fibril, as different oxidized species introduce steric effects that disrupt local crystallinity and in some cases reduce the work needed for polymer decrystallization. Reducing-end oxidations are easiest to decrystallize when located at the end of the fibril, whereas nonreducing end oxidations readily decrystallize from internal cleavage sites despite their lower solvent accessibility. The differential in decrystallization free energy suggests a molecular mechanism consistent with experimentally observed LPMO/cellobiohydrolase synergy. Additionally, the soluble oxidized cellobiose products released by hydrolytic cellulases may bind to the active sites of cellulases with different affinities relative to cellobiose itself, which potentially affects hydrolytic turnover through product inhibition. To examine the effect of oxidation on cello-oligomer binding, we use thermodynamic integration to compute the relative change in binding free energy between the hydrolyzed and oxidized products in the active site of Family 7 and Family 6 processive glycoside hydrolases, Trichoderma reesei Cel7A and Cel6A, which are key industrial cellulases and commonly used model systems for fungal cellulases. Our results suggest that the equilibrium between the two reducing end oxidized products, favoring the linear aldonic acid, may increase product inhibition, which would in turn reduce processive substrate turnover. In the case of LMPO action at the nonreducing end, oxidation appears to lower affinity with the nonreducing end specific cellulase, reducing product inhibition and potentially promoting processive cellulose turnover. Overall, this suggests that oxidation of recalcitrant polysaccharides by LPMOs accelerates degradation not only by increasing the concentration of chain termini but also by reducing decrystallization work, and that product inhibition may be somewhat reduced as a result.« less

β-subunit myristoylation functions as an energy sensor by modulating the dynamics of AMP-activated Protein Kinase.

PubMed

Ali, Nada; Ling, Naomi; Krishnamurthy, Srinath; Oakhill, Jonathan S; Scott, John W; Stapleton, David I; Kemp, Bruce E; Anand, Ganesh Srinivasan; Gooley, Paul R

2016-12-21

The heterotrimeric AMP-activated protein kinase (AMPK), consisting of α, β and γ subunits, is a stress-sensing enzyme that is activated by phosphorylation of its activation loop in response to increases in cellular AMP. N-terminal myristoylation of the β-subunit has been shown to suppress Thr172 phosphorylation, keeping AMPK in an inactive state. Here we use amide hydrogen-deuterium exchange mass spectrometry (HDX-MS) to investigate the structural and dynamic properties of the mammalian myristoylated and non-myristoylated inactivated AMPK (D139A) in the presence and absence of nucleotides. HDX MS data suggests that the myristoyl group binds near the first helix of the C-terminal lobe of the kinase domain similar to other kinases. Our data, however, also shows that ATP.Mg 2+ results in a global stabilization of myristoylated, but not non-myristoylated AMPK, and most notably for peptides of the activation loop of the α-kinase domain, the autoinhibitory sequence (AIS) and the βCBM. AMP does not have that effect and HDX measurements for myristoylated and non-myristoylated AMPK in the presence of AMP are similar. These differences in dynamics may account for a reduced basal rate of phosphorylation of Thr172 in myristoylated AMPK in skeletal muscle where endogenous ATP concentrations are very high.

easyCBM[R] Mathematics Criterion Related Validity Evidence: Oregon State Test. Technical Report #1011

ERIC Educational Resources Information Center

Anderson, Daniel; Alonzo, Julie; Tindal, Gerald

2010-01-01

In this technical report, we present the results of a study examining the relation between the math measures available on the easyCBM[R] online benchmark and progress monitoring assessment system and the Oregon statewide assessment of mathematics. Designed for use within a response to intervention (RTI) framework, easyCBM[R] is intended to help…

Utilizing Curriculum-Based Measurements of Writing in Third and Fifth Grade

ERIC Educational Resources Information Center

Dombek, Jennifer Lucas

2013-01-01

The purpose of this study was to address the utility of curriculum-based measurements of written expression (CBM-W) for predicting writing growth longitudinally and address the extent to which CBM-W relate to other academic and behavior measures. CBM-W has been shown to be an appropriate and useful method for assessing and monitoring writing skill…

Making the Good Even Better: Feedback from easyCBM Focus Groups, School Year 2009/2010. Technical Report # 1001

ERIC Educational Resources Information Center

Alonzo, Julie; Tindal, Gerald; Lai, Cheng-Fei

2010-01-01

This technical report provides a summary of feedback from teachers, administrators, and support personnel who used the easyCBM progress monitoring and benchmark assessment system during school year 2009/2010. Data were gathered from semi-structured focus groups conducted during the 2010 easyCBM August Institute at the University of Oregon. Results…

Predicting Performance on the Tennessee Comprehensive Assessment for Third Grade Reading Students Using Reading Curriculum Based Measures

ERIC Educational Resources Information Center

Kirkham, Scott; Lampley, James H.

2014-01-01

The purpose of this study was to investigate the relationship between three predictor variables (Fall R-CBM, Winter R-CBM, and Spring R-CBM) and the Tennessee Comprehensive Assessment Program third grade reading and language arts assessment. The population selected for this study included all third grade students from an East Tennessee school…

Cross-Validation of easyCBM Reading Cut Scores in Washington: 2009-2010. Technical Report #1109

ERIC Educational Resources Information Center

Irvin, P. Shawn; Park, Bitnara Jasmine; Anderson, Daniel; Alonzo, Julie; Tindal, Gerald

2011-01-01

This technical report presents results from a cross-validation study designed to identify optimal cut scores when using easyCBM[R] reading tests in Washington state. The cross-validation study analyzes data from the 2009-2010 academic year for easyCBM[R] reading measures. A sample of approximately 900 students per grade, randomly split into two…

Performance of Children on the Community Balance and Mobility Scale

ERIC Educational Resources Information Center

Wright, Marilyn J.; Bos, Cecily

2012-01-01

This study describes the performance of children 8-11 years of age on the Community Balance and Mobility Scale (CB&M) and associations between performance and age, body mass index (BMI), and sex. A convenience sample of 84 was recruited. The CB&M was administered using instructions we developed for children. Mean CB&M total scores (95%…

Nondestructive, real-time determination and visualization of cellulose, hemicellulose and lignin by luminescent oligothiophenes

NASA Astrophysics Data System (ADS)

Choong, Ferdinand X.; Bäck, Marcus; Steiner, Svava E.; Melican, Keira; Nilsson, K. Peter R.; Edlund, Ulrica; Richter-Dahlfors, Agneta

2016-10-01

Enabling technologies for efficient use of the bio-based feedstock are crucial to the replacement of oil-based products. We investigated the feasibility of luminescent conjugated oligothiophenes (LCOs) for non-destructive, rapid detection and quality assessment of lignocellulosic components in complex biomass matrices. A cationic pentameric oligothiophene denoted p-HTEA (pentamer hydrogen thiophene ethyl amine) showed unique binding affinities to cellulose, lignin, hemicelluloses, and cellulose nanofibrils in crystal, liquid and paper form. We exploited this finding using spectrofluorometric methods and fluorescence confocal laser scanning microscopy, for sensitive, simultaneous determination of the structural and compositional complexities of native lignocellulosic biomass. With exceptional photostability, p-HTEA is also demonstrated as a dynamic sensor for real-time monitoring of enzymatic cellulose degradation in cellulolysis. These results demonstrate the use of p-HTEA as a non-destructive tool for the determination of cellulose, hemicellulose and lignin in complex biomass matrices, thereby aiding in the optimization of biomass-converting technologies.

Cellulose acetate layer effect toward aluminium corrosion rate in hydrochloric acid media

NASA Astrophysics Data System (ADS)

Andarany, K. S.; Sagir, A.; Ahmad, A.; Deni, S. K.; Gunawan, W.

2017-09-01

Corrosion occurs due to the oxidation and reduction reactions between the material and its environment. The oxidation reaction defined as reactions that produce electrons and reduction is between two elements that bind the electrons. Corrosion cannot be inevitable in life both within the industry and household. Corrosion cannot eliminate but can be control. According to the voltaic table, Aluminum is a metal that easily corroded. This study attempts to characterize the type of corrosion by using a strong acid media (HCl). Experiment using a strong acid (HCl), at a low concentration that occurs is pitting corrosion, whereas at high concentrations that occurs is corrosion erosion. One of prevention method is by using a coating method. An efforts are made to slow the rate of corrosion is by coating the metal with “cellulose acetate” (CA). cellulose acetate consisted of cellulose powder dissolved in 99% acetic acid, and then applied to the aluminum metal. Soaking experiments using hydrochloric acid, cellulose acetate is able to slow down the corrosion rate of 47 479%.

Nondestructive, real-time determination and visualization of cellulose, hemicellulose and lignin by luminescent oligothiophenes

PubMed Central

Choong, Ferdinand X.; Bäck, Marcus; Steiner, Svava E.; Melican, Keira; Nilsson, K. Peter R.; Edlund, Ulrica; Richter-Dahlfors, Agneta

2016-01-01

Enabling technologies for efficient use of the bio-based feedstock are crucial to the replacement of oil-based products. We investigated the feasibility of luminescent conjugated oligothiophenes (LCOs) for non-destructive, rapid detection and quality assessment of lignocellulosic components in complex biomass matrices. A cationic pentameric oligothiophene denoted p-HTEA (pentamer hydrogen thiophene ethyl amine) showed unique binding affinities to cellulose, lignin, hemicelluloses, and cellulose nanofibrils in crystal, liquid and paper form. We exploited this finding using spectrofluorometric methods and fluorescence confocal laser scanning microscopy, for sensitive, simultaneous determination of the structural and compositional complexities of native lignocellulosic biomass. With exceptional photostability, p-HTEA is also demonstrated as a dynamic sensor for real-time monitoring of enzymatic cellulose degradation in cellulolysis. These results demonstrate the use of p-HTEA as a non-destructive tool for the determination of cellulose, hemicellulose and lignin in complex biomass matrices, thereby aiding in the optimization of biomass-converting technologies. PMID:27759105

Drug release from nanoparticles embedded in four different nanofibrillar cellulose aerogels.

PubMed

Valo, Hanna; Arola, Suvi; Laaksonen, Päivi; Torkkeli, Mika; Peltonen, Leena; Linder, Markus B; Serimaa, Ritva; Kuga, Shigenori; Hirvonen, Jouni; Laaksonen, Timo

2013-09-27

Highly porous nanocellulose aerogels prepared by freeze-drying from various nanofibrillar cellulose (NFC) hydrogels are introduced as nanoparticle reservoirs for oral drug delivery systems. Here we show that beclomethasone dipropionate (BDP) nanoparticles coated with amphiphilic hydrophobin proteins can be well integrated into the NFC aerogels. NFCs from four different origins are introduced and compared to microcrystalline cellulose (MCC). The nanocellulose aerogel scaffolds made from red pepper (RC) and MCC release the drug immediately, while bacterial cellulose (BC), quince seed (QC) and TEMPO-oxidized birch cellulose-based (TC) aerogels show sustained drug release. Since the release of the drug is controlled by the structure and interactions between the nanoparticles and the cellulose matrix, modulation of the matrix formers enable a control of the drug release rate. These nanocomposite structures can be very useful in many pharmaceutical nanoparticle applications and open up new possibilities as carriers for controlled drug delivery. Copyright © 2013 Elsevier B.V. All rights reserved.

Combining cognitive bias modification training with motivational support in alcohol dependent outpatients: study protocol for a randomised controlled trial.

PubMed

Boffo, Marilisa; Pronk, Thomas; Wiers, Reinout W; Mannarini, Stefania

2015-02-26

Addiction research has hypothesised that automatic and reflective cognitive processes play an important role in the onset and maintenance of alcohol (ab)use, wherein automatic reactions to drug-related cues steer the drug user towards consuming before reflective processes can get over and steer towards a different behavioural response. These automatic processes include the tendency to attend and approach alcohol cues. These biases may be trained away from alcohol via computerised cognitive bias modification (CBM). The present protocol describes the design of a double-blind randomised controlled trial (RCT) testing the effectiveness of attentional bias and approach bias re-training with a 2×2 factorial design, alongside a brief motivational support (MS) program. Participants (n = 120) are adult alcohol dependent outpatients, recruited from a public health service for addiction in Italy, who have been abstinent for at least two months, and with a main diagnosis of alcohol dependence disorder. Participants are randomly assigned to one of four experimental conditions and complete 11 sessions of training after a baseline assessment. The MS takes place before each training session. Post-intervention and three-month follow-up assessments examine the change in clinical outcome variables and attentional and approach biases (measured with the Visual Probe Task and the Approach-Avoidance Task, respectively). Alcohol approach-avoidance implicit memory associations (measured with the Brief Implicit Association Test) are also evaluated at pre- and post-intervention to explore generalisation effects. Primary outcome measure is relapse rate at follow-up. Secondary outcome measures include change in cognitive biases, in alcohol-related implicit memory associations, and in the clinical variables assessed. An exploratory analysis is also planned to detect interaction effects between the CBM modules and possible moderators (interference control capacity, gender, age, number of previous detoxifications) and mediators (change in cognitive bias) of the primary outcome measure. This RCT is the first to test the effectiveness of a combined CBM intervention alongside motivational support in alcohol-dependent outpatients. The results of this study can be extremely valuable for future research in the optimisation of CBM treatment for alcohol addiction. Current Controlled Trials ISRCTN01005959 (registration date: 24 October 2013).

MPLM On-Orbit Interface Dynamic Flexibility Modal Test

NASA Technical Reports Server (NTRS)

Bookout, Paul S.; Rodriguez, Pedro I.; Tinson, Ian; Fleming, Paolo

2001-01-01

Now that the International Space Station (ISS) is being constructed, payload developers have to not only verify the Shuttle-to-payload interface, but also the interfaces their payload will have with the ISS. The Multi Purpose Logistic Module (MPLM) being designed and built by Alenia Spazio in Torino, Italy is one such payload. The MPLM is the primary carrier for the ISS Payload Racks, Re-supply Stowage Racks, and the Resupply Stowage Platforms to re-supply the ISS with food, water, experiments, maintenance equipment and etc. During the development of the MPLM there was no requirement for verification of the on-orbit interfaces with the ISS. When this oversight was discovered, all the dynamic test stands had already been disassembled. A method was needed that would not require an extensive testing stand and could be completed in a short amount of time. The residual flexibility testing technique was chosen. The residual flexibility modal testing method consists of measuring the free-free natural frequencies and mode shapes along with the interface frequency response functions (FRF's). Analytically, the residual flexibility method has been investigated in detail by, MacNeal, Martinez, Carne, and Miller, and Rubin, but has not been implemented extensively for model correlation due to difficulties in data acquisition. In recent years improvement of data acquisition equipment has made possible the implementation of the residual flexibility method as in Admire, Tinker, and Ivey, and Klosterman and Lemon. The residual flexibility modal testing technique is applicable to a structure with distinct points (DOF) of contact with its environment, such as the MPLM-to-Station interface through the Common Berthing Mechanism (CBM). The CBM is bolted to a flange on the forward cone of the MPLM. During the fixed base test (to verify Shuttle interfaces) some data was gathered on the forward cone panels. Even though there was some data on the forward cones, an additional modal test was performed to better characterize its behavior. The CBM mounting flange is the only remaining structure of the MPLM that no test data was available. This paper discusses the implementation of the residual flexibility modal testing technique on the CBM flange and the modal test of the forward cone panels.

A primer on the occurrence of coalbed methane in low-rank coals, with special reference to its potential occurrence in Pakistan

USGS Publications Warehouse

SanFilipo, John R.

2000-01-01

Introduction: This report compiles and updates a series of correspondence that took place between 1998 and early 2000 among the author and representatives of various consulting groups operating in the coal sector of Pakistan. The purpose of the original correspondence was to introduce basic concepts of coalbed methane (CBM) in low-rank coals to planners and other parties interested in the development of Pakistan's coal, particularly the large deposits of the Thar desert area of Sindh Province that were recently discovered (SanFilipo and Khan, 1994) by the Geological Survey of Pakistan (GSP) and the U.S. Geological Survey (USGS). The author tested two shallow boreholes in Sindh Province for CBM in 1992, including one in Thar, with very marginal results. Additional targets with better CBM prospects were recommended shortly thereafter (SanFilipo and others, 1994), but these were not followed up during subsequent drilling, nor were any other sites tested. Recent events, notably the rapid pace of CBM development in low-rank coals of the Powder River Basin of the U.S., and a show of CBM in commercial quantities in the Cambay Basin of India - both of which are similar in age and rank to most of Pakistan's coal - have indicated a need for reevaluating the initial CBM investigations made in Pakistan in 1992 and for a reassessment of the CBM prospects for the country at large.

Instrument performance enhancement and modification through an extended instrument paradigm

NASA Astrophysics Data System (ADS)

Mahan, Stephen Lee

An extended instrument paradigm is proposed, developed and shown in various applications. The CBM (Chin, Blass, Mahan) method is an extension to the linear systems model of observing systems. In the most obvious and practical application of image enhancement of an instrument characterized by a time-invariant instrumental response function, CBM can be used to enhance images or spectra through a simple convolution application of the CBM filter for a resolution improvement of as much as a factor of two. The CBM method can be used in many applications. We discuss several within this work including imaging through turbulent atmospheres, or what we've called Adaptive Imaging. Adaptive Imaging provides an alternative approach for the investigator desiring results similar to those obtainable with adaptive optics, however on a minimal budget. The CBM method is also used in a backprojected filtered image reconstruction method for Positron Emission Tomography. In addition, we can use information theoretic methods to aid in the determination of model instrumental response function parameters for images having an unknown origin. Another application presented herein involves the use of the CBM method for the determination of the continuum level of a Fourier transform spectrometer observation of ethylene, which provides a means for obtaining reliable intensity measurements in an automated manner. We also present the application of CBM to hyperspectral image data of the comet Shoemaker-Levy 9 impact with Jupiter taken with an acousto-optical tunable filter equipped CCD camera to an adaptive optics telescope.

Effects of the butyric acid-producing strain Clostridium butyricum MIYAIRI 588 on broiler and piglet zootechnical performance and prevention of necrotic enteritis.

PubMed

Takahashi, Motomichi; McCartney, Elinor; Knox, Anne; Francesch, Maria; Oka, Kentaro; Wada, Kaoruko; Ideno, Marie; Uno, Koji; Kozłowski, Krzysztof; Jankowski, Jan; Gracia, Marta I; Morales, Joaquin; Kritas, Spyridon K; Esteve-Garcia, Enric; Kamiya, Shigeru

2018-06-01

The objective of this study was to assess the effects of a probiotic strain Clostridium butyricumMIYAIRI 588 (CBM588) on broiler and weaned piglet health and zootechnical performance. Five field studies were carried out in broilers and five in weaned piglets under European feed additive guidelines. Each study followed a randomized blocked design with two treatments: Control (basal diet) and CBM588 supplemented groups. The zootechnical performance parameters selected were body weight, daily gain, feed intake and feed efficiency (feed:gain). Broilers fed diets with CBM588 gained significantly more weight (+2%, p < .001) and exhibited significantly better feed efficiency (-1.6%, p < .001) in comparison with Controls. Similarly, analysis of pooled data of weaned piglet trials showed that CBM588-fed piglets were significantly heavier than Controls (+2.6%, p = .014), exhibited significantly higher mean daily gain (+4.7%; p = .004), and significantly improved feed efficiency (-4.2%, p = .001). In addition to the zootechnical efficacy studies, the preventive effect of CBM588 on necrotic enteritis (NE) was assessed in a natural challenge model in broilers where CBM588 reduced the incidence and severity of NE lesions. These data indicate the potential of CBM588 to improve broiler and weaned piglet zootechnical performance, and to make a positive contribution to animal health. © 2018 Japanese Society of Animal Science.

The relative contribution of methanotrophs to microbial communities and carbon cycling in soil overlying a coal-bed methane seep

USGS Publications Warehouse

Mills, Christopher T.; Slater, Gregory F.; Dias, Robert F.; Carr, Stephanie A.; Reddy, Christopher M.; Schmidt, Raleigh; Mandernack, Kevin W.

2013-01-01

Seepage of coal-bed methane (CBM) through soils is a potential source of atmospheric CH4 and also a likely source of ancient (i.e. 14C-dead) carbon to soil microbial communities. Natural abundance 13C and 14C compositions of bacterial membrane phospholipid fatty acids (PLFAs) and soil gas CO2 and CH4 were used to assess the incorporation of CBM-derived carbon into methanotrophs and other members of the soil microbial community. Concentrations of type I and type II methanotroph PLFA biomarkers (16:1ω8c and 18:1ω8c, respectively) were elevated in CBM-impacted soils compared with a control site. Comparison of PLFA and 16s rDNA data suggested type I and II methanotroph populations were well estimated and overestimated by their PLFA biomarkers, respectively. The δ13C values of PLFAs common in type I and II methanotrophs were as negative as −67‰ and consistent with the assimilation of CBM. PLFAs more indicative of nonmethanotrophic bacteria had δ13C values that were intermediate indicating assimilation of both plant- and CBM-derived carbon. Δ14C values of select PLFAs (−351 to −936‰) indicated similar patterns of CBM assimilation by methanotrophs and nonmethanotrophs and were used to estimate that 35–91% of carbon assimilated by nonmethanotrophs was derived from CBM depending on time of sampling and soil depth.

Effects of coal-bed methane discharge waters on the vegetation and soil ecosystem in Powder River Basin, Wyoming

USGS Publications Warehouse

Stearns, M.; Tindall, J.A.; Cronin, G.; Friedel, M.J.; Bergquist, E.

2005-01-01

Coal-bed methane (CBM) co-produced discharge waters in the Powder River Basin of Wyoming, resulting from extraction of methane from coal seams, have become a priority for chemical, hydrological and biological research during the last few years. Soil and vegetation samples were taken from affected and reference sites (upland elevations and wetted gully) in Juniper Draw to investigate the effects of CBM discharge waters on soil physical and chemical properties and on native and introduced vegetation density and diversity. Results indicate an increase of salinity and sodicity within local soil ecosystems at sites directly exposed to CBM discharge waters. Elevated concentrations of sodium in the soil are correlated with consistent exposure to CBM waters. Clay-loam soils in the study area have a much larger specific surface area than the sandy soils and facilitate a greater sodium adsorption. However, there was no significant relation between increasing water sodium adsorption ratio (SAR) values and increasing sediment SAR values downstream; however, soils exposed to the CBM water ranged from the moderate to severe SAR hazard index. Native vegetation species density was highest at the reference (upland and gully) and CBM affected upland sites. The affected gully had the greatest percent composition of introduced vegetation species. Salt-tolerant species had the greatest richness at the affected gully, implying a potential threat of invasion and competition to established native vegetation. These findings suggest that CBM waters could affect agricultural production operations and long-term water quality. ?? Springer 2005.

Cross-Validation of easyCBM Reading Cut Scores in Oregon: 2009-2010. Technical Report #1108

ERIC Educational Resources Information Center

Park, Bitnara Jasmine; Irvin, P. Shawn; Anderson, Daniel; Alonzo, Julie; Tindal, Gerald

2011-01-01

This technical report presents results from a cross-validation study designed to identify optimal cut scores when using easyCBM[R] reading tests in Oregon. The cross-validation study analyzes data from the 2009-2010 academic year for easyCBM[R] reading measures. A sample of approximately 2,000 students per grade, randomly split into two groups of…

Molecular engineering of fracture energy dissipating sacrificial bonds into cellulose nanocrystal nanocomposites.

PubMed

McKee, Jason R; Huokuna, Johannes; Martikainen, Lahja; Karesoja, Mikko; Nykänen, Antti; Kontturi, Eero; Tenhu, Heikki; Ruokolainen, Janne; Ikkala, Olli

2014-05-12

Even though nanocomposites have provided a plethora of routes to increase stiffness and strength, achieving increased toughness with suppressed catastrophic crack growth has remained more challenging. Inspired by the concepts of mechanically excellent natural nanomaterials, one-component nanocomposites were fabricated involving reinforcing colloidal nanorod cores with polymeric grafts containing supramolecular binding units. The concept is based on mechanically strong native cellulose nanocrystals (CNC) grafted with glassy polymethacrylate polymers, with side chains that contain 2-ureido-4[1H]-pyrimidone (UPy) pendant groups. The interdigitation of the grafts and the ensuing UPy hydrogen bonds bind the nanocomposite network together. Under stress, UPy groups act as sacrificial bonds: simultaneously providing adhesion between the CNCs while allowing them to first orient and then gradually slide past each other, thus dissipating fracture energy. We propose that this architecture involving supramolecular binding units within side chains of polymer grafts attached to colloidal reinforcements opens generic approaches for tough nanocomposites. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cognitive neuroscience of cognitive retraining for addiction medicine: From mediating mechanisms to questions of efficacy.

PubMed

Gladwin, Thomas E; Wiers, Corinde E; Wiers, Reinout W

2016-01-01

Cognitive retraining or cognitive bias modification (CBM) involves having subjects repeatedly perform a computerized task designed to reduce the impact of automatic processes that lead to harmful behavior. We first discuss the theory underlying CBM and provide a brief overview of important research progress in its application to addiction. We then focus on cognitive- and neural-mediating mechanisms. We consider recent criticism of both CBM and its theoretical foundations. Evaluations of CBM could benefit from considering theory-driven factors that may determine variations in efficacy, such as motivation. Concerning theory, while there is certainly room for fundamental advances in current models, we argue that the basic view of impulsive behavior and its control remains a useful and productive heuristic. Finally, we briefly discuss some interesting new directions for CBM research: enhancement of training via transcranial direct current stimulation, online training, and gamification, i.e., the use of gameplay elements to increase motivation. © 2016 Elsevier B.V. All rights reserved.

Food-motivated behavior in rats with cortico-basomedial amygdala damage.

PubMed

Lukaszewska, I; Korczynski, R; Kostarczyk, E; Fonberg, E

1984-06-01

Twelve rats with amygdala damage (CBM) and 20 sham-operated controls were tested in several food-related situations. The CBM rats showed a longer latency to eat than controls in a novel environment due to more pronounced exploration. In the competition for food, CBM rats lost 85% of encounters with controls. Immediately after the contest, when allowed to eat singly, CBM rats displayed a higher persistence of alimentary responses to an emptied cup than did controls, presumably because they experienced more losses in the food competition. Both groups were equally able to overcome obstacles on the way to food, which suggests similar alimentary motivation. The only direct indication of a lowered responsiveness to hunger in CBM rats was 24-hr-fasting-induced hypophagia. The results of this study indicate the involvement of the cortico-basomedial amygdaloid region in the control of relations between alimentary and other motivations. The contribution of eventual changes of food motivation in the postoperative alteration of this balance is discussed.

Investigating the technical adequacy of curriculum-based measurement in written expression for students who are deaf or hard of hearing.

PubMed

Cheng, Shu-Fen; Rose, Susan

2009-01-01

This study investigated the technical adequacy of curriculum-based measures of written expression (CBM-W) in terms of writing prompts and scoring methods for deaf and hard-of-hearing students. Twenty-two students at the secondary school-level completed 3-min essays within two weeks, which were scored for nine existing and alternative curriculum-based measurement (CBM) scoring methods. The technical features of the nine scoring methods were examined for interrater reliability, alternate-form reliability, and criterion-related validity. The existing CBM scoring method--number of correct minus incorrect word sequences--yielded the highest reliability and validity coefficients. The findings from this study support the use of the CBM-W as a reliable and valid tool for assessing general writing proficiency with secondary students who are deaf or hard of hearing. The CBM alternative scoring methods that may serve as additional indicators of written expression include correct subject-verb agreements, correct clauses, and correct morphemes.

Synthesis of surface bound silver nanoparticles on cellulose fibers using lignin as multi-functional agent.

PubMed

Hu, Sixiao; Hsieh, You-Lo

2015-10-20

Lignin has proven to be highly effective "green" multi-functional binding, complexing and reducing agents for silver cations as well as capping agents for the synthesis of silver nanoparticles on ultra-fine cellulose fibrous membranes. Silver nanoparticles could be synthesized in 10min to be densely distributed and stably bound on the cellulose fiber surfaces at up to 2.9% in mass. Silver nanoparticle increased in sizes from 5 to 100nm and became more polydispersed in size distribution on larger fibers and with longer synthesis time. These cellulose fiber bound silver nanoparticles did not agglomerate under elevated temperatures and showed improved thermal stability. The presence of alkali lignin conferred moderate UV absorbing ability in both UV-B and UV-C regions whereas the bound silver nanoparticles exhibited excellent antibacterial activities toward Escherichia coli. Copyright © 2015 Elsevier Ltd. All rights reserved.

Technical Adequacy of the easyCBM Primary-Level Reading Measures (Grades K-1), 2009-2010 Version. Technical Report #1003

ERIC Educational Resources Information Center

Lai, Cheng-Fei; Nese, Joseph F. T.; Jamgochian, Elisa M.; Alonzo, Julie; Tindal, Gerald

2010-01-01

In this technical report, we provide the results of a series of studies on the technical adequacy of the early reading measures available on the easyCBM[R] assessment system. The results from the two-level hierarchical linear growth model analyses suggest that the reliability of the slope estimates for the easyCBM[R] reading measures are strong,…

easyCBM CCSS Math Item Scaling and Test Form Revision (2012-2013): Grades 6-8. Technical Report #1313

ERIC Educational Resources Information Center

Anderson, Daniel; Alonzo, Julie; Tindal, Gerald

2012-01-01

The purpose of this technical report is to document the piloting and scaling of new easyCBM mathematics test items aligned with the Common Core State Standards (CCSS) and to describe the process used to revise and supplement the 2012 research version easyCBM CCSS math tests in Grades 6-8. For all operational 2012 research version test forms (10…

Curriculum-Based Measurement of Oral Reading: An Evaluation of Growth Rates and Seasonal Effects among Students Served in General and Special Education

ERIC Educational Resources Information Center

Christ, Theodore J.; Silberglitt, Benjamin; Yeo, Seungsoo; Cormier, Damien

2010-01-01

Curriculum-based measurement of oral reading (CBM-R) is often used to benchmark growth in the fall, winter, and spring. CBM-R is also used to set goals and monitor student progress between benchmarking occasions. The results of previous research establish an expectation that weekly growth on CBM-R tasks is consistently linear throughout the…

Coalbed-methane production in the Appalachian basin: Chapter G.2 in Coal and petroleum resources in the Appalachian basin: distribution, geologic framework, and geochemical character

USGS Publications Warehouse

Milici, Robert C.; Polyak, Désirée E.; Ruppert, Leslie F.; Ryder, Robert T.

2014-01-01

Coalbed methane (CBM) occurs in coal beds of Mississippian and Pennsylvanian (Carboniferous) age in the northern, central, and southern Appalachian basin coal regions, which extend almost continuously from Pennsylvania southward to Alabama. Most commercial CBM production in the Appalachian basin is from three structural subbasins: (1) the Dunkard basin in Pennsylvania, Ohio, and northern West Virginia; (2) the Pocahontas basin in southern West Virginia, eastern Kentucky, and southwestern Virginia; and (3) part of the Black Warrior basin in Alabama. The cumulative CBM production in the Dunkard basin through 2005 was 17 billion cubic feet (BCF), the production in the Pocahontas basin through 2006 was 754 BCF, and the production in the part of the Black Warrior basin in Alabama through 2007 was 2.008 TCF. CBM development may be regarded as mature in Alabama, where annual production from 1998 through 2007 was relatively constant and ranged from 112 to 121 BCF. An opportunity still exists for additional growth in the Pocahontas basin. In 2005, annual CBM production in the Pocahontas basin in Virginia and West Virginia was 85 BCF. In addition, opportunities are emerging for producing the large, diffuse CBM resources in the Dunkard basin as additional wells are drilled and technology improves.

Modification of cognitive biases related to posttraumatic stress: A systematic review and research agenda.

PubMed

Woud, Marcella L; Verwoerd, Johan; Krans, Julie

2017-06-01

Cognitive models of Posttraumatic Stress Disorder (PTSD) postulate that cognitive biases in attention, interpretation, and memory represent key factors involved in the onset and maintenance of PTSD. Developments in experimental research demonstrate that it may be possible to manipulate such biases by means of Cognitive Bias Modification (CBM). In the present paper, we summarize studies assessing cognitive biases in posttraumatic stress to serve as a theoretical and methodological background. However, our main aim was to provide an overview of the scientific literature on CBM in (analogue) posttraumatic stress. Results of our systematic literature review showed that most CBM studies targeted attentional and interpretation biases (attention: five studies; interpretation: three studies), and one study modified memory biases. Overall, results showed that CBM can indeed modify cognitive biases and affect (analog) trauma symptoms in a training congruent manner. Interpretation bias procedures seemed effective in analog samples, and memory bias training proved preliminary success in a clinical PTSD sample. Studies of attention bias modification provided more mixed results. This heterogeneous picture may be explained by differences in the type of population or variations in the CBM procedure. Therefore, we sketched a detailed research agenda targeting the challenges for CBM in posttraumatic stress. Copyright © 2017 Elsevier Ltd. All rights reserved.

Does napping enhance the effects of Cognitive Bias Modification-Appraisal training? An experimental study.

PubMed

Woud, Marcella L; Cwik, Jan C; Blackwell, Simon E; Kleim, Birgit; Holmes, Emily A; Adolph, Dirk; Zhang, Hui; Margraf, Jürgen

2018-01-01

Posttraumatic Stress Disorder (PTSD) is characterised by dysfunctional appraisals of the trauma and its consequences including one's own symptoms. Experimental studies have shown that Cognitive Bias Modification-Appraisal (CBM-App) training can reduce dysfunctional interpretations and analog trauma symptoms. One important question is how to enhance the effects of CBM-App. Following work suggesting that sleep has beneficial effects on consolidation processes and can thus improve learning, the present study investigated whether a brief period of sleep (i.e., a nap) enhances the effects of CBM-App. All participants watched a stressful movie as an analogue trauma induction. After that, participants received either positive or negative CBM-App training. Within each training, half of the participants then had a 90-minute nap or watched a neutral movie. Results showed that the CBM training induced training-congruent appraisals. Sleep did not enhance this effect. Participants who slept, however, experienced fewer intrusive memories of the analogue trauma, but this effect was independent of the CBM condition. These results provide valuable information about the effects of sleep during a 90-minute nap period on encoding of analogue trauma and emotional learning in the context of appraisal, and highlight the importance of sleep as a focus for continued research.

Does napping enhance the effects of Cognitive Bias Modification-Appraisal training? An experimental study

PubMed Central

Cwik, Jan C.; Blackwell, Simon E.; Kleim, Birgit; Holmes, Emily A.; Adolph, Dirk; Zhang, Hui; Margraf, Jürgen

2018-01-01

Posttraumatic Stress Disorder (PTSD) is characterised by dysfunctional appraisals of the trauma and its consequences including one’s own symptoms. Experimental studies have shown that Cognitive Bias Modification—Appraisal (CBM-App) training can reduce dysfunctional interpretations and analog trauma symptoms. One important question is how to enhance the effects of CBM-App. Following work suggesting that sleep has beneficial effects on consolidation processes and can thus improve learning, the present study investigated whether a brief period of sleep (i.e., a nap) enhances the effects of CBM-App. All participants watched a stressful movie as an analogue trauma induction. After that, participants received either positive or negative CBM-App training. Within each training, half of the participants then had a 90-minute nap or watched a neutral movie. Results showed that the CBM training induced training-congruent appraisals. Sleep did not enhance this effect. Participants who slept, however, experienced fewer intrusive memories of the analogue trauma, but this effect was independent of the CBM condition. These results provide valuable information about the effects of sleep during a 90-minute nap period on encoding of analogue trauma and emotional learning in the context of appraisal, and highlight the importance of sleep as a focus for continued research. PMID:29447217

Characterization of the interaction of yeast enolase with polynucleotides.

PubMed

al-Giery, A G; Brewer, J M

1992-09-23

Yeast enolase is inhibited under certain conditions by DNA. The enzyme binds to single-stranded DNA-cellulose. Inhibition was used for routine characterization of the interaction. The presence of the substrate 2-phospho-D-glycerate reduces inhibition and binding. Both yeast enolase isozymes behave similarly. Impure yeast enolase was purified by adsorption onto a single-stranded DNA-cellulose column followed by elution with substrate. Interaction with RNA, double-stranded DNA, or degraded DNA results in less inhibition, suggesting that yeast enolase preferentially binds single-stranded DNA. However, yeast enolase is not a DNA-unwinding protein. The enzyme is inhibited by the short synthetic oligodeoxynucleotides G6, G8 and G10 but not T8 or T6, suggesting some base specificity in the interaction. The interaction is stronger at more acid pH values, with an apparent pK of 5.6. The interaction is prevented by 0.3 M KCl, suggesting that electrostatic factors are important. Histidine or lysine reverse the inhibition at lower concentrations, while phosphate is still more effective. Binding of single-stranded DNA to enolase reduces the reaction of protein histidyl residues with diethylpyrocarbonate. The inhibition of yeast enolase by single-stranded DNA is not total, and suggests the active site is not directly involved in the interaction. Binding of substrate may induce a conformational change in the enzyme that interferes with DNA binding and vice versa.

Microbially-Enhanced Coal Bed Methane: Strategies for Increased Biogenic Production

NASA Astrophysics Data System (ADS)

Davis, K.; Barhart, E. P.; Schweitzer, H. D.; Cunningham, A. B.; Gerlach, R.; Hiebert, R.; Fields, M. W.

2014-12-01

Coal is the largest fossil fuel resource in the United States. Most of this coal is deep in the subsurface making it costly and potentially dangerous to extract. However, in many of these deep coal seams, methane, the main component of natural gas, has been discovered and successfully harvested. Coal bed methane (CBM) currently accounts for approximately 7.5% of the natural gas produced in the U.S. Combustion of natural gas produces substantially less CO2 and toxic emissions (e.g. heavy metals) than combustion of coal or oil thereby making it a cleaner energy source. In the large coal seams of the Powder River Basin (PRB) in southeast Montana and northeast Wyoming, CBM is produced almost entirely by biogenic processes. The in situ conversion of coal to CBM by the native microbial community is of particular interest for present and future natural gas sources as it provides the potential to harvest energy from coal seams with lesser environmental impacts than mining and burning coal. Research at Montana State University has shown the potential for enhancing the subsurface microbial processes that produce CBM. Long-term batch enrichments have investigated the methane enhancement potential of yeast extract as well as algal and cyanobacterial biomass additions with increased methane production observed with all three additions when compared to no addition. Future work includes quantification of CBM enhancement and normalization of additions. This presentation addresses the options thus far investigated for increasing CBM production and the next steps for developing the enhanced in situ conversion of coal to CBM.

Cognitive bias modification for interpretation with and without prior repetitive negative thinking to reduce worry and rumination in generalised anxiety disorder and depression: protocol for a multisession experimental study with an active control condition

PubMed Central

Mathews, Andrew; Whyte, Jessica; Hirsch, Colette R

2016-01-01

Introduction Worry and rumination are two forms of repetitive thinking characterised by their negative content and apparently uncontrollable nature. Although worry and rumination share common features and have been conceptualised as part of a transdiagnostic repetitive negative thinking (RNT) process, it remains unclear whether they share the same underlying cognitive mechanisms. This multisession experimental study investigates the tendency to make negative interpretations regarding ambiguous information as a cognitive mechanism underlying RNT. We compare multisession cognitive bias modification for interpretations (CBM-I) with an active control condition to examine whether repeatedly training positive interpretations reduces worry and rumination in individuals with generalised anxiety disorder or depression, respectively. Further, we examine the potential modulatory effects of engaging in RNT immediately prior to CBM-I. Design, methods and analysis A community sample of individuals meeting diagnostic criteria for either generalised anxiety disorder (n=60) or current major depressive episode (n=60) will be randomly allocated to CBM-I with prior RNT, CBM-I without prior RNT (ie, standard CBM-I), or an active control (no resolution of ambiguity) condition. All conditions receive a 3-week internet-based intervention consisting of one initial session at the first study visit and nine home-based sessions of CBM-I training (or active control). We will assess and compare the effects of CBM-I with and without prior RNT on ‘near-transfer’ measures of interpretation bias closely related to the training as well as ‘far-transfer’ outcomes related to RNT and emotional distress. Impact on questionnaire measures will additionally be assessed at 1-month follow-up. Multigroup analyses will be conducted to assess the impact of CBM-I on near-transfer and far-transfer outcome measures. PMID:27986741

Factor Analysis of the Community Balance and Mobility Scale in Individuals with Knee Osteoarthritis.

PubMed

Takacs, Judit; Krowchuk, Natasha M; Goldsmith, Charles H; Hunt, Michael A

2017-10-01

The clinical assessment of balance is an important first step in characterizing the risk of falls. The Community Balance and Mobility Scale (CB&M) is a test of balance and mobility that was designed to assess performance on advanced tasks necessary for independence in the community. However, other factors that can affect balancing ability may also be present during performance of the real-world tasks on the CB&M. It is important for clinicians to understand fully what other modifiable factors the CB&M may encompass. The purpose of this study was to evaluate the underlying constructs in the CB&M in individuals with knee osteoarthritis (OA). This was an observational study, with a single testing session. Participants with knee OA aged 50 years and older completed the CB&M, a clinical test of balance and mobility. Confirmatory factor analysis was then used to examine whether the tasks on the CB&M measure distinct factors. Three a priori theory-driven models with three (strength, balance, mobility), four (range of motion added) and six (pain and fear added) constructs were evaluated using multiple fit indices. A total of 131 participants (mean [SD] age 66.3 [8.5] years, BMI 27.3 [5.2] kg m -2 ) participated. A three-factor model in which all tasks loaded on these three factors explained 65% of the variance and yielded the most optimal model, as determined using scree plots, chi-squared values and explained variance. The first factor accounted for 49% of the variance and was interpreted as lower limb muscle strength. The second and third factors were interpreted as mobility and balance, respectively. The CB&M demonstrated the measurement of three distinct factors, interpreted as lower limb strength, balance and mobility, supporting the use of the CB&M with people with knee OA for evaluation of these important factors in falls risk and functional mobility. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Stimulating learning-by-doing in advanced biofuels: effectiveness of alternative policies

NASA Astrophysics Data System (ADS)

Chen, Xiaoguang; Khanna, Madhu; Yeh, Sonia

2012-12-01

This letter examines the effectiveness of various biofuel and climate policies in reducing future processing costs of cellulosic biofuels due to learning-by-doing. These policies include a biofuel production mandate alone and supplementing the biofuel mandate with other policies, namely a national low carbon fuel standard, a cellulosic biofuel production tax credit or a carbon price policy. We find that the binding biofuel targets considered here can reduce the unit processing cost of cellulosic ethanol by about 30% to 70% between 2015 and 2035 depending on the assumptions about learning rates and initial costs of biofuel production. The cost in 2035 is more sensitive to the speed with which learning occurs and less sensitive to uncertainty in the initial production cost. With learning rates of 5-10%, cellulosic biofuels will still be at least 40% more expensive than liquid fossil fuels in 2035. The addition of supplementary low carbon/tax credit policies to the mandate that enhance incentives for cellulosic biofuels can achieve similar reductions in these costs several years earlier than the mandate alone; the extent of these incentives differs across policies and different kinds of cellulosic biofuels.

Starch Binding Domain-containing Protein 1 Plays a Dominant Role in Glycogen Transport to Lysosomes in Liver.

PubMed

Sun, Tao; Yi, Haiqing; Yang, Chunyu; Kishnani, Priya S; Sun, Baodong

2016-08-05

A small portion of cellular glycogen is transported to and degraded in lysosomes by acid α-glucosidase (GAA) in mammals, but it is unclear why and how glycogen is transported to the lysosomes. Stbd1 has recently been proposed to participate in glycogen trafficking to lysosomes. However, our previous study demonstrated that knockdown of Stbd1 in GAA knock-out mice did not alter lysosomal glycogen storage in skeletal muscles. To further determine whether Stbd1 participates in glycogen transport to lysosomes, we generated GAA/Stbd1 double knock-out mice. In fasted double knock-out mice, glycogen accumulation in skeletal and cardiac muscles was not affected, but glycogen content in liver was reduced by nearly 73% at 3 months of age and by 60% at 13 months as compared with GAA knock-out mice, indicating that the transport of glycogen to lysosomes was suppressed in liver by the loss of Stbd1. Exogenous expression of human Stbd1 in double knock-out mice restored the liver lysosomal glycogen content to the level of GAA knock-out mice, as did a mutant lacking the Atg8 family interacting motif (AIM) and another mutant that contains only the N-terminal 24 hydrophobic segment and the C-terminal starch binding domain (CBM20) interlinked by an HA tag. Our results demonstrate that Stbd1 plays a dominant role in glycogen transport to lysosomes in liver and that the N-terminal transmembrane region and the C-terminal CBM20 domain are critical for this function. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Advanced Cloning Tools for Construction of Designer Cellulosomes.

PubMed

Kahn, Amaranta; Bayer, Edward A; Moraïs, Sarah

2018-01-01

Cellulose deconstruction is achieved in nature through two main enzymatic paradigms, i.e., free enzymes and enzymatic complexes (called cellulosomes). Gaining insights into the mechanism of action and synergy among the different cellulases is of high interest, notably in the field of renewable energy, and specifically, for the conversion of cellulosic biomass to soluble sugars, en route to biofuels. In this context, designer cellulosomes are artificially assembled, chimaeric protein complexes that are used as a tool to comparatively study cellulose degradation by different enzymatic paradigms, and could also serve to improve cellulose deconstruction. Various molecular biology techniques are employed in order to design and engineer the various components of designer cellulosomes. In this chapter, we describe the cloning processes through which the appropriate modules are selected and assembled at the molecular level.

Survival potential of wild type cellulose deficient Salmonella from the feed industry.

PubMed

Vestby, Lene K; Møretrø, Trond; Ballance, Simon; Langsrud, Solveig; Nesse, Live L

2009-11-23

Biofilm has been shown to be one way for Salmonella to persist in the feed factory environment. Matrix components, such as fimbriae and cellulose, have been suggested to play an important role in the survival of Salmonella in the environment. Multicellular behaviour by Salmonella is often categorized according to colony morphology into rdar (red, dry and rough) expressing curli fimbriae and cellulose, bdar (brown, dry and rough) expressing curli fimbriae and pdar (pink, dry and rough) expressing cellulose. The aim of the study was to look into the distribution of morphotypes among feed and fish meal factory strains of Salmonella, with emphasis on potential differences between morphotypes with regards to survival in the feed factory environment. When screening a total of 148 Salmonella ser. Agona, Salmonella ser. Montevideo, Salmonella ser. Senftenberg and Salmonella ser. Typhimurium strains of feed factory, human clinical and reference collection origin, as many as 99% were able to express rough morphology (rdar or bdar). The dominant morphotype was rdar (74%), however as many as 55% of Salmonella ser. Agona and 19% of Salmonella ser. Senftenberg displayed the bdar morphology. Inconsistency in Calcofluor binding, indicating expression of cellulose, was found among 25% of all the strains tested, however Salmonella ser. Agona showed to be highly consistent in Calcofluor binding (98%). In biofilm, Salmonella ser. Agona strains with bdar mophology was found to be equally tolerant to disinfection treatment as strains with rdar morphotype. However, rdar morphology appeared to be favourable in long term survival in biofilm in a very dry environment. Chemical analysis showed no major differences in polysaccharide content between bdar and rdar strains. Our results indicate that cellulose is not a major component of the Salmonella biofilm matrix. The bdar morphotype is common among Salmonella ser. Agona strains isolated from the factory environment. The rdar and the bdar strains were found to be equally tolerant to disinfectants, while the rdar strain was found to be more tolerant to long-term desiccation and nutrient depletion in biofilm than the bdar strain. Cellulose does not appear to be a major component of the Salmonella biofilm matrix.

A Gibberellin-Mediated DELLA-NAC Signaling Cascade Regulates Cellulose Synthesis in Rice.

PubMed

Huang, Debao; Wang, Shaogan; Zhang, Baocai; Shang-Guan, Keke; Shi, Yanyun; Zhang, Dongmei; Liu, Xiangling; Wu, Kun; Xu, Zuopeng; Fu, Xiangdong; Zhou, Yihua

2015-06-01

Cellulose, which can be converted into numerous industrial products, has important impacts on the global economy. It has long been known that cellulose synthesis in plants is tightly regulated by various phytohormones. However, the underlying mechanism of cellulose synthesis regulation remains elusive. Here, we show that in rice (Oryza sativa), gibberellin (GA) signals promote cellulose synthesis by relieving the interaction between SLENDER RICE1 (SLR1), a DELLA repressor of GA signaling, and NACs, the top-layer transcription factors for secondary wall formation. Mutations in GA-related genes and physiological treatments altered the transcription of CELLULOSE SYNTHASE genes (CESAs) and the cellulose level. Multiple experiments demonstrated that transcription factors NAC29/31 and MYB61 are CESA regulators in rice; NAC29/31 directly regulates MYB61, which in turn activates CESA expression. This hierarchical regulation pathway is blocked by SLR1-NAC29/31 interactions. Based on the results of anatomical analysis and GA content examination in developing rice internodes, this signaling cascade was found to be modulated by varied endogenous GA levels and to be required for internode development. Genetic and gene expression analyses were further performed in Arabidopsis thaliana GA-related mutants. Altogether, our findings reveal a conserved mechanism by which GA regulates secondary wall cellulose synthesis in land plants and provide a strategy for manipulating cellulose production and plant growth. © 2015 American Society of Plant Biologists. All rights reserved.

A Gibberellin-Mediated DELLA-NAC Signaling Cascade Regulates Cellulose Synthesis in Rice[OPEN

PubMed Central

Huang, Debao; Wang, Shaogan; Zhang, Baocai; Shang-Guan, Keke; Shi, Yanyun; Zhang, Dongmei; Liu, Xiangling; Wu, Kun; Xu, Zuopeng; Fu, Xiangdong; Zhou, Yihua

2015-01-01

Cellulose, which can be converted into numerous industrial products, has important impacts on the global economy. It has long been known that cellulose synthesis in plants is tightly regulated by various phytohormones. However, the underlying mechanism of cellulose synthesis regulation remains elusive. Here, we show that in rice (Oryza sativa), gibberellin (GA) signals promote cellulose synthesis by relieving the interaction between SLENDER RICE1 (SLR1), a DELLA repressor of GA signaling, and NACs, the top-layer transcription factors for secondary wall formation. Mutations in GA-related genes and physiological treatments altered the transcription of CELLULOSE SYNTHASE genes (CESAs) and the cellulose level. Multiple experiments demonstrated that transcription factors NAC29/31 and MYB61 are CESA regulators in rice; NAC29/31 directly regulates MYB61, which in turn activates CESA expression. This hierarchical regulation pathway is blocked by SLR1-NAC29/31 interactions. Based on the results of anatomical analysis and GA content examination in developing rice internodes, this signaling cascade was found to be modulated by varied endogenous GA levels and to be required for internode development. Genetic and gene expression analyses were further performed in Arabidopsis thaliana GA-related mutants. Altogether, our findings reveal a conserved mechanism by which GA regulates secondary wall cellulose synthesis in land plants and provide a strategy for manipulating cellulose production and plant growth. PMID:26002868

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

Presidential Green Chemistry Challenge: 2012 Designing Greener Chemicals Award

EPA Pesticide Factsheets

Presidential Green Chemistry Challenge 2012 award winner, Buckman International, developed Maximyze enzymes that modify the cellulose in wood fibers to increase binding between fibers in paper and improve paper strength.

Enhanced plastic deformations of nanofibrillated cellulose film by adsorbed moisture and protein-mediated interactions.

PubMed

Malho, Jani-Markus; Ouellet-Plamondon, Claudiane; Rüggeberg, Markus; Laaksonen, Päivi; Ikkala, Olli; Burgert, Ingo; Linder, Markus B

2015-01-12

Biological composites are typically based on an adhesive matrix that interlocks rigid reinforcing elements in fiber composite or brick-and-mortar assemblies. In nature, the adhesive matrix is often made up of proteins, which are also interesting model systems, as they are unique among polymers in that we know how to engineer their structures with atomic detail and to select protein elements for specific interactions with other components. Here we studied how fusion proteins that consist of cellulose binding proteins linked to proteins that show a natural tendency to form multimer complexes act as an adhesive matrix in combination with nanofibrillated cellulose. We found that the fusion proteins are retained with the cellulose and that the proteins mainly affect the plastic yield behavior of the cellulose material as a function of water content. Interestingly, the proteins increased the moisture absorption of the composite, but the well-known plastifying effect of water was clearly decreased. The work helps to understand the functional basis of nanocellulose composites as materials and aims toward building model systems for molecular biomimetic materials.

Tetrad pollen formation in Annona (Annonaceae): proexine formation andbinding mechanism.

PubMed

Tsou, Chih-Hua; Fu, Yu-Lan

2002-05-01

Meiotic tetrads of Annona glabra and A. montana build up a well-developed proexine (protectum, probaculum, and pronexine) at the proximal side but only a thin pronexine at the distal side during the tetrad stage. The callosic envelope is only partially digested by the end of tetrad stage. The remaining, undigested part is composed of the intersporal mass and thin peripheral layers, and the latter is conjunct with the distal pronexine of the microspore. In this remaining callosic structure celluloses are also present. Later on, due to the continuous slow decomposition of this callose-cellulose structure and microspore expansion, microspores break up the callose-cellulose envelope. Because all the four microspores are bound together by the callose-cellulose structure, they move out of the chamber in rotation. Eventually the thin pronexine is pulled toward the center of the tetrad and the well-developed proexine becomes the distal wall. These descriptions of the partial digestion of callosic envelope, the transformation from a callose-cellulose structure to the binding system of tetrad pollen, and microspore rotation in Annona are unusual in the angiosperms.

Improvement of the enzymatic hydrolysis of furfural residues by pretreatment with combined green liquor and ethanol organosolv.

PubMed

Yu, Hailong; Xing, Yang; Lei, Fuhou; Liu, Zhiping; Liu, Zuguang; Jiang, Jianxin

2014-09-01

Furfural residues (FRs) were pretreated with ethanol and a green liquor (GL) catalyst to produce fermentable sugar. Anthraquinone (AQ) was used as an auxiliary reagent to improve delignification and reduce cellulose decomposition. The results showed that 42.7% of lignin was removed and 96.5% of cellulose was recovered from substrates pretreated with 1.0 mL GL/g of dry substrate and 0.4% (w/w) AQ at 140°C for 1h. Compared with raw material, ethanol-GL pretreatment of FRs increased the glucose yield from 69.0% to 85.9% after 96 h hydrolysis with 18 FPU/g-cellulose for cellulase, 27 CBU/g-cellulose for β-glucosidase. The Brauner-Emmett-Teller surface area was reduced during pretreatment, which did not inhibit the enzymatic hydrolysis. Owing to the reduced surface area, the unproductive binding of cellulase to lignin was decreased, thus improving the enzymatic hydrolysis. The degree of polymerization of cellulose from FRs was too low to be a key factor for improving enzymatic hydrolysis. Copyright © 2014 Elsevier Ltd. All rights reserved.

Three-dimensional audio-magnetotelluric sounding in monitoring coalbed methane reservoirs

NASA Astrophysics Data System (ADS)

Wang, Nan; Zhao, Shanshan; Hui, Jian; Qin, Qiming

2017-03-01

Audio-magnetotelluric (AMT) sounding is widely employed in rapid resistivity delineation of objective geometry in near surface exploration. According to reservoir patterns and electrical parameters obtained in Qinshui Basin, China, two-dimensional and three-dimensional synthetic "objective anomaly" models were designed and inverted with the availability of a modular system for electromagnetic inversion (ModEM). The results revealed that 3-D full impedance inversion yielded the subsurface models closest to synthetic models. One or more conductive targets were correctly recovered. Therefore, conductive aquifers in the study area, including hydrous coalbed methane (CBM) reservoirs, were suggested to be the interpretation signs for reservoir characterization. With the aim of dynamic monitoring of CBM reservoirs, the AMT surveys in continuous years (June 2013-May 2015) were carried out. 3-D inversion results demonstrated that conductive anomalies accumulated around the producing reservoirs at the corresponding depths if CBM reservoirs were in high water production rates. In contrast, smaller conductive anomalies were generally identical with rapid gas production or stopping production of reservoirs. These analyses were in accordance with actual production history of CBM wells. The dynamic traces of conductive anomalies revealed that reservoir water migrated deep or converged in axial parts and wings of folds, which contributed significantly to formations of CBM traps. Then the well spacing scenario was also evaluated based on the dynamic production analysis. Wells distributed near closed faults or flat folds, rather than open faults, had CBM production potential to ascertain stable gas production. Therefore, three-dimensional AMT sounding becomes an attractive option with the ability of dynamic monitoring of CBM reservoirs, and lays a solid foundation of quantitative evaluation of reservoir parameters.

Preliminary efficacy and safety of an oromucosal standardized cannabis extract in chemotherapy-induced nausea and vomiting.

PubMed

Duran, Marta; Pérez, Eulàlia; Abanades, Sergio; Vidal, Xavier; Saura, Cristina; Majem, Margarita; Arriola, Edurne; Rabanal, Manel; Pastor, Antoni; Farré, Magí; Rams, Neus; Laporte, Joan-Ramon; Capellà, Dolors

2010-11-01

Despite progress in anti-emetic treatment, many patients still suffer from chemotherapy-induced nausea and vomiting (CINV). This is a pilot, randomized, double-blind, placebo-controlled phase II clinical trial designed to evaluate the tolerability, preliminary efficacy, and pharmacokinetics of an acute dose titration of a whole-plant cannabis-based medicine (CBM) containing delta-9-tetrahydrocannabinol and cannabidiol, taken in conjunction with standard therapies in the control of CINV. Patients suffering from CINV despite prophylaxis with standard anti-emetic treatment were randomized to CBM or placebo, during the 120 h post-chemotherapy period, added to standard anti-emetic treatment. Tolerability was measured as the number of withdrawals from the study during the titration period because of adverse events (AEs). The endpoint for the preliminary efficacy analysis was the proportion of patients showing complete or partial response. Seven patients were randomized to CBM and nine to placebo. Only one patient in the CBM arm was withdrawn due to AEs. A higher proportion of patients in the CBM group experienced a complete response during the overall observation period [5/7 (71.4%) with CMB vs. 2/9 (22.2%) with placebo, the difference being 49.2% (95% CI 1%, 75%)], due to the delayed period. The incidence of AEs was higher in the CBM group (86% vs. 67%). No serious AEs were reported. The mean daily dose was 4.8 sprays in both groups. Compared with placebo, CBM added to standard antiemetic therapy was well tolerated and provided better protection against delayed CINV. These results should be confirmed in a phase III clinical trial. © 2010 Department of Health, Generalitat of Catalonia. British Journal of Clinical Pharmacology © 2010 The British Pharmacological Society.

Invasive species and coal bed methane development in the Powder River Basin, Wyoming

USGS Publications Warehouse

Bergquist, E.; Evangelista, P.; Stohlgren, T.J.; Alley, N.

2007-01-01

One of the fastest growing areas of natural gas production is coal bed methane (CBM) due to the large monetary returns and increased demand for energy from consumers. The Powder River Basin, Wyoming is one of the most rapidly expanding areas of CBM development with projections of the establishment of up to 50,000 wells. CBM disturbances may make the native ecosystem more susceptible to invasion by non-native species, but there are few studies that have been conducted on the environmental impacts of this type of resource extraction. To evaluate the potential effects of CBM development on native plant species distribution and patterns of non-native plant invasion, 36 modified Forest Inventory and Analysis plots (each comprised of four 168-m2 subplots) were established in the Powder River Basin, Wyoming. There were 73 168-m2 subplots on control sites; 42 subplots on secondary disturbances; 14 on major surface disturbances; eight on well pads; and seven on sites downslope of CBM wells water discharge points. Native plant species cover ranged from 39.5 ?? 2.7% (mean ?? 1 SE) in the secondary disturbance subplots to 17.7 ?? 7.5% in the pad subplots. Non-native plant species cover ranged from 31.0 ?? 8.4% in the discharge areas to 14.7 ?? 8.9% in the pad subplots. The control subplots had significantly less non-native species richness than the combined disturbance types. The combined disturbance subplots had significantly greater soil salinity than the control sites. These results suggest that CBM development and associated disturbances may facilitate the establishment of non-native plants. Future research and management decisions should consider the accumulative landscape-scale effects of CBM development on preserving native plant diversity. ?? Springer Science+Business Media B.V. 2006.

Study of compressed baryonic matter at FAIR: JINR participation

NASA Astrophysics Data System (ADS)

Derenovskaya, O.; Kurilkin, P.; Gusakov, Yu.; Ivanov, V.; Ladygin, V.; Ladygina, N.; Malakhov, A.; Peshekhonov, V.; Zinchenko, A.

2017-11-01

The scientific goal of the CBM (Compressed Baryonic Matter) experiment at FAIR (Darmstadt) is to explore the phase diagram of strongly interacting matter at highest baryon densities. The physics program of the CBM experiment is complimentary to the programs to be realized at MPD and BMN facilities at NICA and will start with beam derived by the SIS100 synchrotron. The results of JINR participation in the development of different sub-projects of the CBM experiment are presented.

Reducing children's social anxiety symptoms: exploring a novel parent-administered cognitive bias modification training intervention.

PubMed

Lau, Jennifer Y F; Pettit, Eleanor; Creswell, Cathy

2013-07-01

Social fears and worries in children are common and impairing. Yet, questions have been raised over the efficacy, suitability and accessibility of current frontline treatments. Here, we present data on the effectiveness of a novel parent-administered Cognitive Bias Modification of Interpretations (CBM-I) training tool. CBM-I capitalises on findings demonstrating an association between anxiety symptoms and biased interpretations, the tendency to interpret ambiguous situations negatively. Through CBM-I training, participants are exposed to benign resolutions, and reinforced for selecting these. In adults and adolescents, CBM-I training is effective at reducing symptoms and mood reactivity. In the present study, we developed a novel, child-appropriate form of CBM-I training, by presenting training materials within bedtime stories, read by a parent to the child across three consecutive evenings. Compared to a test-retest control group (n = 17), children receiving CBM-I (n = 19) reported greater endorsement of benign interpretations of ambiguous situations post-training (compared to pre-training). These participants (but not the test-retest control group) also showed a significant reduction in social anxiety symptoms. Pending replication and extensions to a clinical sample, these data may implicate a cost-effective, mechanism-driven and developmentally-appropriate resource for targeting social anxiety problems in children. Copyright © 2013 Elsevier Ltd. All rights reserved.

Constraint-based modeling in microbial food biotechnology

PubMed Central

Rau, Martin H.

2018-01-01

Genome-scale metabolic network reconstruction offers a means to leverage the value of the exponentially growing genomics data and integrate it with other biological knowledge in a structured format. Constraint-based modeling (CBM) enables both the qualitative and quantitative analyses of the reconstructed networks. The rapid advancements in these areas can benefit both the industrial production of microbial food cultures and their application in food processing. CBM provides several avenues for improving our mechanistic understanding of physiology and genotype–phenotype relationships. This is essential for the rational improvement of industrial strains, which can further be facilitated through various model-guided strain design approaches. CBM of microbial communities offers a valuable tool for the rational design of defined food cultures, where it can catalyze hypothesis generation and provide unintuitive rationales for the development of enhanced community phenotypes and, consequently, novel or improved food products. In the industrial-scale production of microorganisms for food cultures, CBM may enable a knowledge-driven bioprocess optimization by rationally identifying strategies for growth and stability improvement. Through these applications, we believe that CBM can become a powerful tool for guiding the areas of strain development, culture development and process optimization in the production of food cultures. Nevertheless, in order to make the correct choice of the modeling framework for a particular application and to interpret model predictions in a biologically meaningful manner, one should be aware of the current limitations of CBM. PMID:29588387

Polypeptide composition of bacterial cyclic diguanylic acid-dependent cellulose synthase and the occurrence of immunologically crossreacting proteins in higher plants

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

Mayer, R.; Ross, P.; Weinhouse, H.

1991-06-15

To comprehend the catalytic and regulatory mechanism of the cyclic diguanylic acid (c-di-GMP)-dependent cellulose synthase of Acetobacter xylinum and its relatedness to similar enzymes in other organisms, the structure of this enzyme was analyzed at the polypeptide level. The enzyme, purified 350-fold by enzyme-product entrapment, contains three major peptides (90, 67, and 54 kDa), which, based on direct photoaffinity and immunochemical labeling and amino acid sequence analysis, are constituents of the native cellulose synthase. Labeling of purified synthase with either ({sup 32}P)c-di-GMP or ({alpha}-{sup 32}P)UDP-glucose indicates that activator- and substrate-specific binding sites are most closely associated with the 67- andmore » 54-kDa peptides, respectively, whereas marginal photolabeling is detected in the 90-k-Da peptide. However, antibodies raised against a protein derived from the cellulose synthase structural gene (bcsB) specifically label all three peptides. The authors suggest that the structurally related 67- and 54-kDa peptides are fragments proteolytically derived from the 90-kDa peptide encoded by bcsB. The anti-cellulose synthase antibodies crossreact with a similar set of peptides derived from other cellulose-producing microorganisms and plants such as Agrobacterium tumefaciens, Rhizobium leguminosarum, mung bean, peas, barley, and cotton. The occurrence of such cellulose synthase-like structures in plant species suggests that a common enzymatic mechanism for cellulose biogenesis is employed throughout nature.« less

Event reconstruction for the CBM-RICH prototype beamtest data in 2014

NASA Astrophysics Data System (ADS)

Adamczewski-Musch, J.; Akishin, P.; Becker, K.-H.; Belogurov, S.; Bendarouach, J.; Boldyreva, N.; Deveaux, C.; Dobyrn, V.; Dürr, M.; Eschke, J.; Förtsch, J.; Heep, J.; Höhne, C.; Kampert, K.-H.; Kochenda, L.; Kopfer, J.; Kravtsov, P.; Kres, I.; Lebedev, S.; Lebedeva, E.; Leonova, E.; Linev, S.; Mahmoud, T.; Michel, J.; Miftakhov, N.; Niebur, W.; Ovcharenko, E.; Patel, V.; Pauly, C.; Pfeifer, D.; Querchfeld, S.; Rautenberg, J.; Reinecke, S.; Riabov, Y.; Roshchin, E.; Samsonov, V.; Schetinin, V.; Tarasenkova, O.; Traxler, M.; Ugur, C.; Vznuzdaev, E.; Vznuzdaev, M.

2017-12-01

The Compressed Baryonic Matter (CBM) experiment at the future FAIR facility will investigate the QCD phase diagram at high net baryon densities and moderate temperatures in A+A collisions from 2 to 11 AGeV (SIS100). Electron identification in CBM will be performed by a Ring Imaging Cherenkov (RICH) detector and Transition Radiation Detectors (TRD). A real size prototype of the RICH detector was tested together with other CBM groups at the CERN PS/T9 beam line in 2014. For the first time the data format used the FLESnet protocol from CBM delivering free streaming data. The analysis was fully performed within the CBMROOT framework. In this contribution the data analysis and the event reconstruction methods which were used for obtained data are discussed. Rings were reconstructed using an algorithm based on the Hough Transform method and their parameters were derived with high accuracy by circle and ellipse fitting procedures. We present results of the application of the presented algorithms. In particular we compare results with and without Wavelength shifting (WLS) coating.

Clonality and serotypes of Streptococcus mutans among children by multilocus sequence typing

PubMed Central

Momeni, Stephanie S.; Whiddon, Jennifer; Cheon, Kyounga; Moser, Stephen A.; Childers, Noel K.

2015-01-01

Studies using multilocus sequence typing (MLST) have demonstrated that Streptococcus mutans isolates are genetically diverse. Our laboratory previously demonstrated clonality of S. mutans using MLST but could not discount the possibility of sampling bias. In this study, the clonality of randomly selected S. mutans plaque isolates from African American children was examined using MLST. Serotype and presence of collagen-binding proteins (CBP) cnm/cbm were also assessed. One hundred S. mutans isolates were randomly selected for MLST analysis. Sequence analysis was performed and phylogenetic trees were generated using START2 and MEGA. Thirty-four sequence types (ST) were identified of which 27 were unique to this population. Seventy-five percent of the isolates clustered into 16 clonal groups. Serotypes observed were c (n=84), e (n=3), and k (n=11). The prevalence of S. mutans isolates serotype k was notably high at 17.5%. All isolates were cnm/cbm negative. The clonality of S. mutans demonstrated in this study illustrates the importance of localized populations studies and are consistent with transmission. The prevalence of serotype k, a recently proposed systemic pathogen, observed in this study is higher than reported in most populations and is the first report of S. mutans serotype k in a US population. PMID:26443288

Molecular Dynamics Simulations of Family 7 Cellobiohydrolase Mutants Aimed at Reducing Product Inhibition.

PubMed

Silveira, Rodrigo L; Skaf, Munir S

2015-07-23

Enzymatic conversion of lignocellulosic biomass into biofuels and chemicals constitutes a potential route for sustainable development. Cellobiohydrolases are key enzymes used in industrial cocktails for depolymerization of crystalline cellulose, and their mechanism of action has been intensely studied in the past several years. Provided with a tunnel-like substrate-binding cavity, cellobiohydrolases possess the ability to processively hydrolyze glycosidic bonds of crystalline cellulose, yielding one molecule of cellobiose per catalytic cycle. As such, cellobiose expulsion from the product binding site is a necessary step in order to allow for the processive hydrolysis mechanism. However, the high-affinity binding of cellobiose to the enzyme impairs the process and causes activity inhibition due to reaction products. Here, we use molecular dynamics simulations to study the binding of cellobiose to the Trichoderma reesei Cel7A (TrCel7A) cellobiohydrolase and the effects of mutations that reduce cellobiose binding, without affecting the structural and dynamical integrities of the enzyme. We observe that the product binding site exhibits an intrinsic flexibility that can sterically hinder cellobiose release. Several point mutations in the product binding site reduce cellobiose-enzyme interactions, but not all modifications are able to maintain the structural integrity of the enzyme. In particular, mutation of charged residues in the TrCel7A product binding site causes perturbations that affect the structure of the loops that form the substrate-binding tunnel of the enzyme and, hence, may affect TrCel7A function in other steps of the hydrolysis mechanism. Our results suggest there is a trade-off between product inhibition and catalytic efficiency, and they provide directions for cellulases engineering.

Hyaluronate-binding proteins of murine brain.

PubMed

Marks, M S; Chi-Rosso, G; Toole, B P

1990-01-01

The distribution of hyaluronate-binding activity was determined in the soluble and membrane fractions derived from adult mouse brain by sonication in low-ionic-strength buffer. Approximately 60% of the total activity was recovered in the soluble fraction and 33% in membrane fractions. In both cases, the hyaluronate-binding activities were found to be of high affinity (KD = 10(-9) M), specific for hyaluronate, and glycoprotein in nature. Most of the hyaluronate-binding activity from the soluble fraction chromatographed in the void volume of Sepharose CL-4B and CL-6B. Approximately 50% of this activity was highly negatively charged, eluting from diethylaminoethyl (DEAE)-cellulose in 0.5 M NaCl, and contained chondroitin sulfate chains. This latter material also reacted with antibodies raised against cartilage link protein and the core protein of cartilage proteoglycan. Thus, the binding and physical characteristics of this hyaluronate-binding activity are consistent with those of a chondroitin sulfate proteoglycan aggregate similar to that found in cartilage. A 500-fold purification of this proteoglycan-like, hyaluronate-binding material was achieved by wheat germ agglutinin affinity chromatography, molecular sieve chromatography on Sepharose CL-6B, and ion exchange chromatography on DEAE-cellulose. Another class of hyaluronate-binding material (25-50% of that recovered) eluted from DEAE with 0.24 M NaCl; this material had the properties of a complex glycoprotein, did not contain chondroitin sulfate, and did not react with the antibodies against cartilage link protein and proteoglycan. Thus, adult mouse brain contains at least three different forms of hyaluronate-binding macromolecules. Two of these have properties similar to the link protein and proteoglycan of cartilage proteoglycan aggregates; the third is distinguishable from these entities.

Preliminary Gulf Coast Coalbed Methane Exploration Maps: Depth to Wilcox, Apparent Wilcox Thickness and Vitrinite Reflectance

USGS Publications Warehouse

Barker, Charles E.; Biewick, Laura R.; Warwick, Peter D.; SanFilipo, John R.

2000-01-01

Strong economic controls on the viability of coalbed methane (CBM) prospects make coal geometry and coal property maps key elements in identifying sweet spots and production fairways. Therefore, this study seeks to identify the apparent prospective areas for CBM exploration in the Wilcox Group (Paleocene-Eocene) lignite and coalbeds by mapping net coal thickness, depth to coal, and coal rank (vitrinite reflectance). Economic factors are not considered in this CBM prospects study. Given the comparatively extensive gas pipeline and other production infrastructure development in the Gulf Coast Region, these factors seem less a control compared to other areas. However, open leasable public lands are minimal or nonexistent in the Gulf Coast region and access to the CBM prospects could be a problem.

The RICH detector of the CBM experiment

NASA Astrophysics Data System (ADS)

Adamczewski-Musch, J.; Akishin, P.; Becker, K.-H.; Belogurov, S.; Bendarouach, J.; Boldyreva, N.; Deveaux, C.; Dobyrn, V.; Dürr, M.; Eschke, J.; Förtsch, J.; Heep, J.; Höhne, C.; Kampert, K.-H.; Kochenda, L.; Kopfer, J.; Kravtsov, P.; Kres, I.; Lebedev, S.; Lebedeva, E.; Leonova, E.; Linev, S.; Mahmoud, T.; Michel, J.; Miftakhov, N.; Niebur, W.; Ovcharenko, E.; Patel, V.; Pauly, C.; Pfeifer, D.; Querchfeld, S.; Rautenberg, J.; Reinecke, S.; Riabov, Y.; Roshchin, E.; Samsonov, V.; Schetinin, V.; Tarasenkova, O.; Traxler, M.; Ugur, C.; Vznuzdaev, E.; Vznuzdaev, M.

2017-12-01

The CBM-RICH detector is designed to identify electrons with momenta up to 8 GeV/c and high purity as this is essential for the CBM physics program. The detector consist of a CO2-gaseous radiator, a spherical mirror system, and Multi-Anode PhotoMultiplier Tubes (MAPMT) of type H12700 from Hamamatsu as photon detectors. The detector concept was verified through R&D studies and a laterally scaled prototype. The results were summarized in a TDR, in which open issues were defined concerning the readout electronics, the shielding of the magnetic stray field in the MAPMT region, the radiation hardness of the MAPMT sensors, and the mechanical holding structure of the mirror system. In this article an overview is given on the CBM RICH development with focus on those open issues.

Multidimensional Self-Assembled Structures of Alkylated Cellulose Oligomers Synthesized via in Vitro Enzymatic Reactions.

PubMed

Yataka, Yusuke; Sawada, Toshiki; Serizawa, Takeshi

2016-10-04

The self-assembly of biomolecules into highly ordered nano-to-macroscale structures is essential in the construction of biological tissues and organs. A variety of biomolecular assemblies composed of nucleic acids, peptides, and lipids have been used as molecular building units for self-assembled materials. However, crystalline polysaccharides have rarely been utilized in self-assembled materials. In this study, we describe multidimensional self-assembled structures of alkylated cellulose oligomers synthesized via in vitro enzymatic reactions. We found that the alkyl chain length drastically affected the assembled morphologies and allomorphs of cellulose moieties. The modulation of the intermolecular interactions of cellulose oligomers by alkyl substituents was highly effective at controlling their assembly into multidimensional structures. This study proposes a new potential of crystalline oligosaccharides for structural components of molecular assemblies with controlled morphologies and crystal structures.

Property rights regimes to optimize natural resource use - future CBM development and sustainability

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

Eaton, C.; Ingelson, A.; Knopff, R.

2007-04-01

Property rights regimes that promote sustainable development in the context of coalbed methane (CBM) exploration and production recognize and optimize the value of multiple natural resources including minerals, water, flora, and fauna. Institutional mechanisms that account for and mitigate both the short- and long-term external impacts from CBM development promote sustainability. The long-term potential for a vibrant recreational and tourist economy on a particular landscape may be compromised by overly shortsighted mineral resource extraction.

Potential water-quality effects of coal-bed methane production water discharged along the upper Tongue River, Wyoming and Montana

USGS Publications Warehouse

Kinsey, Stacy M.; Nimick, David A.

2011-01-01

Water quality in the upper Tongue River from Monarch, Wyoming, downstream to just upstream from the Tongue River Reservoir in Montana potentially could be affected by discharge of coal-bed methane (CBM) production water (hereinafter referred to as CBM discharge). CBM discharge typically contains high concentrations of sodium and other ions that could increase dissolved-solids (salt) concentrations, specific conductance (SC), and sodium-adsorption ratio (SAR) in the river. Increased inputs of sodium and other ions have the potential to alter the river's suitability for agricultural irrigation and aquatic ecosystems. Data from two large tributaries, Goose Creek and Prairie Dog Creek, indicate that these tributaries were large contributors to the increase in SC and SAR in the Tongue River. However, water-quality data were not available for most of the smaller inflows, such as small tributaries, irrigation-return flows, and CBM discharges. Thus, effects of these inflows on the water quality of the Tongue River were not well documented. Effects of these small inflows might be subtle and difficult to determine without more extensive data collection to describe spatial patterns. Therefore, synoptic water-quality sampling trips were conducted in September 2005 and April 2006 to provide a spatially detailed profile of the downstream changes in water quality in this reach of the Tongue River. The purpose of this report is to describe these downstream changes in water quality and to estimate the potential water-quality effects of CBM discharge in the upper Tongue River. Specific conductance of the Tongue River through the study reach increased from 420 to 625 microsiemens per centimeter (.μS/cm; or 49 percent) in the downstream direction in September 2005 and from 373 to 543 .μS/cm (46 percent) in April 2006. Large increases (12 to 24 percent) were measured immediately downstream from Goose Creek and Prairie Dog Creek during both sampling trips. Increases attributed to direct CBM discharges were smaller. In September 2005, the SC of 12 measured CBM discharges ranged from 1,750 to 2,440 .μS/cm, and the combined discharges increased SC in the river by an estimated 4.5 percent. In April 2006, the SC of eight measured CBM discharges ranged from 1,720 to 2,070 μS/cm; the largest of these discharges likely increased SC in the river by 5.8 percent. Estimates of potential effects of the CBM discharges on the SC of the Tongue River near the Tongue River Reservoir were calculated using a two-step process involving linear regression and mass-balance calculations for a range of streamflow and CBM-discharge conditions. Potential effects from CBM discharges are larger increases of SC and SAR at lower flows than at higher flows and relative increases that are substantially smaller for SC than for SAR. For example, if the streamflow was 100 cubic feet per second (ft3/s) in the Tongue River near the Tongue River Reservoir and CBM discharge ranged from 1,250 to 5,000 gallons per minute, the projected increases would range from 4.4 to 16 percent for SC and from 39 to 151 percent for SAR. In comparison, if the streamflow was 600 ft3/s, the projected increases would range from 2.2 to 8.4 percent for SC and from 21 to 79 percent for SAR. This analysis of potential water-quality effects on the SC and SAR of the Tongue River in the study area assumes that the quantity and quality of water flowing into the study reach at the time of this study was the same as during the period before CBM development (data from water years 1985-99).

3D-QSPR Method of Computational Technique Applied on Red Reactive Dyes by Using CoMFA Strategy

PubMed Central

Mahmood, Uzma; Rashid, Sitara; Ali, S. Ishrat; Parveen, Rasheeda; Zaheer-ul-Haq; Ambreen, Nida; Khan, Khalid Mohammed; Perveen, Shahnaz; Voelter, Wolfgang

2011-01-01

Cellulose fiber is a tremendous natural resource that has broad application in various productions including the textile industry. The dyes, which are commonly used for cellulose printing, are “reactive dyes” because of their high wet fastness and brilliant colors. The interaction of various dyes with the cellulose fiber depends upon the physiochemical properties that are governed by specific features of the dye molecule. The binding pattern of the reactive dye with cellulose fiber is called the ligand-receptor concept. In the current study, the three dimensional quantitative structure property relationship (3D-QSPR) technique was applied to understand the red reactive dyes interactions with the cellulose by the Comparative Molecular Field Analysis (CoMFA) method. This method was successfully utilized to predict a reliable model. The predicted model gives satisfactory statistical results and in the light of these, it was further analyzed. Additionally, the graphical outcomes (contour maps) help us to understand the modification pattern and to correlate the structural changes with respect to the absorptivity. Furthermore, the final selected model has potential to assist in understanding the charachteristics of the external test set. The study could be helpful to design new reactive dyes with better affinity and selectivity for the cellulose fiber. PMID:22272108

Lignin blockers and uses thereof

DOEpatents

Yang, Bin [West Lebanon, NH; Wyman, Charles E [Norwich, VT

2011-01-25

Disclosed is a method for converting cellulose in a lignocellulosic biomass. The method provides for a lignin-blocking polypeptide and/or protein treatment of high lignin solids. The treatment enhances cellulase availability in cellulose conversion and allows for the determination of optimized pretreatment conditions. Additionally, ethanol yields from a Simultaneous Saccharification and Fermentation process are improved 5-25% by treatment with a lignin-blocking polypeptide and/or protein. Thus, a more efficient and economical method of processing lignin containing biomass materials utilizes a polypeptide/protein treatment step that effectively blocks lignin binding of cellulase.

A Novel GH7 Endo-β-1,4-Glucanase from Neosartorya fischeri P1 with Good Thermostability, Broad Substrate Specificity and Potential Application in the Brewing Industry.

PubMed

Liu, Yun; Dun, Baoqing; Shi, Pengjun; Ma, Rui; Luo, Huiying; Bai, Yingguo; Xie, Xiangming; Yao, Bin

2015-01-01

An endo-β-1,4-glucanase gene, cel7A, was cloned from the thermophilic cellulase-producing fungus Neosartorya fischeri P1 and expressed in Pichia pastoris. The 1,410-bp full-length gene encodes a polypeptide of 469 amino acids consisting of a putative signal peptide at residues 1-20, a catalytic domain of glycoside hydrolase family 7 (GH7), a short Thr/Ser-rich linker and a family 1 carbohydrate-binding module (CBM 1). The purified recombinant Cel7A had pH and temperature optima of pH 5.0 and 60°C, respectively, and showed broad pH adaptability (pH 3.0-6.0) and excellent stability at pH3.0-8.0 and 60°C. Belonging to the group of nonspecific endoglucanases, Cel7A exhibited the highest activity on barley β-glucan (2020 ± 9 U mg-1), moderate on lichenan and CMC-Na, and weak on laminarin, locust bean galactomannan, Avicel, and filter paper. Under simulated mashing conditions, addition of Cel7A (99 μg) reduced the mash viscosity by 9.1% and filtration time by 24.6%. These favorable enzymatic properties make Cel7A as a good candidate for applications in the brewing industry.

Re-training Automatic Action Tendencies to Approach Cigarettes among Adolescent Smokers: A Pilot Study

PubMed Central

Kong, Grace; Larsen, Helle; Cavallo, Dana; Becker, Daniela; Cousijn, Janna; Salemink, Elske; D'Escury-Koenigs, Annemat L. Collot; Morean, Meghan; Wiers, Reinout; Krishnan-Sarin, Suchitra

2015-01-01

Background This pilot study conducted a preliminary examination of whether Cognitive Bias Modification (CBM), a computerized task to retrain cognitive-approach biases towards smoking stimuli, (1) changed approach bias for cigarettes, and (2) improved smoking cessation outcomes in adolescent smokers. Methods Sixty adolescent smokers received four weeks of Cognitive Behavioral Therapy (CBT) for smoking cessation, with CBM (90% avoidance/10% approach for smoking stimuli and 10% avoidance/90% approach for neutral stimuli) or sham (50% avoidance/50% approach for smoking and neutral stimuli) training in the Netherlands (n = 42) and the United States (n = 18). Results While we did not observe changes in action tendencies related to CBM, adolescents with higher smoking approach biases at baseline had greater decreases in approach biases at follow up, compared to adolescents with smoking avoidance biases, regardless of treatment condition (p = 0.01). Intent-to-treat (ITT) analyses showed that CBM, when compared with sham trended toward higher end-of-treatment, biochemically-confirmed, seven-day point prevalence abstinence, (17.2% vs. 3.2%, p = 0.071). ITT analysis also showed that regardless of treatment condition, cotinine level (p = 0.045) and average number of cigarette smoked (p ≤ 0.001) significantly decreased over the course of treatment. Conclusions The findings from this pilot study suggests that re-training approach biases toward cigarettes shows promise for smoking cessation among adolescent smokers. Future research should utilize larger samples and increased distinction between CBM and sham conditions, and examine mechanisms underlying the CBM approach. PMID:26186485

A Question of Control? Examining the Role of Control Conditions in Experimental Psychopathology using the Example of Cognitive Bias Modification Research.

PubMed

Blackwell, Simon E; Woud, Marcella L; MacLeod, Colin

2017-10-26

While control conditions are vitally important in research, selecting the optimal control condition can be challenging. Problems are likely to arise when the choice of control condition is not tightly guided by the specific question that a given study aims to address. Such problems have become increasingly apparent in experimental psychopathology research investigating the experimental modification of cognitive biases, particularly as the focus of this research has shifted from theoretical questions concerning mechanistic aspects of the association between cognitive bias and emotional vulnerability, to questions that instead concern the clinical efficacy of 'cognitive bias modification' (CBM) procedures. We discuss the kinds of control conditions that have typically been employed in CBM research, illustrating how difficulties can arise when changes in the types of research questions asked are not accompanied by changes in the control conditions employed. Crucially, claims made on the basis of comparing active and control conditions within CBM studies should be restricted to those conclusions allowed by the specific control condition employed. CBM studies aiming to establish clinical utility are likely to require quite different control conditions from CBM studies aiming to illuminate mechanisms. Further, conclusions concerning the clinical utility of CBM interventions cannot necessarily be drawn from studies in which the control condition has been chosen to answer questions concerning mechanisms. Appreciating the need to appropriately alter control conditions in the transition from basic mechanisms-focussed investigations to applied clinical research could greatly facilitate the translational process.

Structure and characteristics of an endo-beta-1,4-glucanase, isolated from Trametes hirsuta, with high degradation to crystalline cellulose.

PubMed

Nozaki, Kouichi; Seki, Takahiro; Matsui, Keiko; Mizuno, Masahiro; Kanda, Takahisa; Amano, Yoshihiko

2007-10-01

Trametes hirsuta produced cellulose-degrading enzymes when it was grown in a cellulosic medium such as Avicel or wheat bran. An endo-beta-1,4-glucanase (ThEG) was purified from the culture filtrate, and the gene and the cDNA were isolated. The gene consisted of an open reading frame encoding 384 amino acids, interrupted by 11 introns. The whole sequence showed high homology with that of family 5 glycoside hydrolase. The properties of the recombinant enzyme (rEG) in Aspergillus oryzae were compared with those of the En-1 from Irpex lacteus, which showed the highest homology among all the endoglucanases reported. The rEG activity against Avicel was about 8 times higher than that of En-1 when based on CMC degradation. A remarkable structural difference between the two enzymes was the length of the linker connecting the cellulose-binding domain to the catalytic domain.

Production of nano bacterial cellulose from waste water of candied jujube-processing industry using Acetobacter xylinum.

PubMed

Li, Zheng; Wang, Lifen; Hua, Jiachuan; Jia, Shiru; Zhang, Jianfei; Liu, Hao

2015-04-20

The work is aimed to investigate the suitability of waste water of candied jujube-processing industry for the production of bacterial cellulose (BC) by Gluconacetobacter xylinum CGMCC No.2955 and to study the structure properties of bacterial cellulose membranes. After acid pretreatment, the glucose of hydrolysate was higher than that of waste water of candied jujube. The volumetric yield of bacterial cellulose in hydrolysate was 2.25 g/L, which was 1.5-folds of that in waste water of candied jujube. The structures indicated that the fiber size distribution was 3-14 nm in those media with an average diameter being around 5.9 nm. The crystallinity index of BC from pretreatment medium was lower than that of without pretreatment medium and BCs from various media had similar chemical binding. Ammonium citrate was a key factor for improving production yield and the crystallinity index of BC. Copyright © 2014 Elsevier Ltd. All rights reserved.

Nanomechanical Sensing of Biological Interfacial Interactions

NASA Astrophysics Data System (ADS)

Du, Wenjian

Cellulose is the most abundant biopolymer on earth. Cellulase is an enzyme capable of converting insoluble cellulose into soluble sugars. Cellulosic biofuel produced from such fermentable simple sugars is a promising substitute as an energy source. However, its economic feasibility is limited by the low efficiency of the enzymatic hydrolysis of cellulose by cellulase. Cellulose is insoluble and resistant to enzymatic degradation, not only because the beta-1,4-glycosidic bonds are strong covalent bonds, but also because cellulose microfibrils are packed into tightly bound, crystalline lattices. Enzymatic hydrolysis of cellulose by cellulase involves three steps--initial binding, decrystallization, and hydrolytic cleavage. Currently, the mechanism for the decrystallization has not yet been elucidated, though it is speculated to be the rate-limiting step of the overall enzymatic activity. The major technical challenge limiting the understanding of the decrystallization is the lack of an effective experimental approach capable of examining the decrystallization, an interfacial enzymatic activity on solid substrates. The work presented develops a nanomechanical sensing approach to investigate both the decrystallization and enzymatic hydrolytic cleavage of cellulose. The first experimental evidence of the decrystallization is obtained by comparing the results from native cellulase and non-hydrolytic cellulase. Surface topography has been applied to examine the activities of native cellulase and non-hydrolytic cellulase on cellulose substrate. The study demonstrates additional experimental evidence of the decrystallization in the hydrolysis of cellulose. By combining simulation and monitoring technology, the current study also investigates the structural changes of cellulose at a molecular level. In particular, the study employs cellulose nanoparticles with a bilayer structure on mica sheets. By comparing results from a molecular dynamic simulation and the distance between cellulose layers monitored by means of the atomic force microscopy (AFM), the current study shows that water molecules can efficiently reduce the energy required for separating two layers of cellulose bilayers during hydration of cellulose bilayer nanoparticles. The findings of the study contribute to explicating the mechanism of cellulose the decrystallization, a free-energetically unfavorable process, through enzymatic hydrolysis of cellulase. The study also investigates the application of a cell-based microcantilever sensor to monitor the real-time ligand-induced response of living cells. These nanomechanical approaches offer unique perspectives on the interfacial activities of biological molecules.

Imparting albumin-binding affinity to a human protein by mimicking the contact surface of a bacterial binding protein.

PubMed

Oshiro, Satoshi; Honda, Shinya

2014-04-18

Attachment of a bacterial albumin-binding protein module is an attractive strategy for extending the plasma residence time of protein therapeutics. However, a protein fused with such a bacterial module could induce unfavorable immune reactions. To address this, we designed an alternative binding protein by imparting albumin-binding affinity to a human protein using molecular surface grafting. The result was a series of human-derived 6 helix-bundle proteins, one of which specifically binds to human serum albumin (HSA) with adequate affinity (KD = 100 nM). The proteins were designed by transferring key binding residues of a bacterial albumin-binding module, Finegoldia magna protein G-related albumin-binding domain (GA) module, onto the human protein scaffold. Despite 13-15 mutations, the designed proteins maintain the original secondary structure by virtue of careful grafting based on structural informatics. Competitive binding assays and thermodynamic analyses of the best binders show that the binding mode resembles that of the GA module, suggesting that the contacting surface of the GA module is mimicked well on the designed protein. These results indicate that the designed protein may act as an alternative low-risk binding module to HSA. Furthermore, molecular surface grafting in combination with structural informatics is an effective approach for avoiding deleterious mutations on a target protein and for imparting the binding function of one protein onto another.

Impact of carboxymethyl cellulose (CMC) and microcrystalline cellulose (MCC) on functional characteristics of emulsified sausages.

PubMed

Schuh, Valerie; Allard, Karin; Herrmann, Kurt; Gibis, Monika; Kohlus, Reinhard; Weiss, Jochen

2013-02-01

Inclusion of fibers, such as carboxymethyl cellulose (CMC) and microcrystalline cellulose (MCC), at the expense of fat or protein in meat batters could be used to produce healthier sausages while lowering production costs. To study the impact of CMC/MCC on structural/functional characteristics of emulsified sausages, standard-fat Lyoner-style sausages were formulated with CMC/MCC at concentrations of 0.3-2.0%. Methods of analysis included rheology, water binding capacity (WBC), texture measurements, and Confocal Laser Scanning Microscopy (CLSM). WBC, texture measurements, and rheology all indicated that addition of CMC (>0.7%) led to destabilization of the batter, which upon heating could no longer be converted into a coherent protein network, a fact that was also revealed in CLSM images. In contrast, MCC was highly compatible with the matrix and improved firmness (1405-1651N/100g) with increasing concentration compared to control (1381N/100g) while keeping WBC (4.6-5.9%) with <2% MCC at the level of the control (4.8%). Results were discussed in terms of molecular interactions of meat proteins with celluloses. Copyright © 2012 Elsevier Ltd. All rights reserved.

Mechanistic kinetic models of enzymatic cellulose hydrolysis-A review.

PubMed

Jeoh, Tina; Cardona, Maria J; Karuna, Nardrapee; Mudinoor, Akshata R; Nill, Jennifer

2017-07-01

Bioconversion of lignocellulose forms the basis for renewable, advanced biofuels, and bioproducts. Mechanisms of hydrolysis of cellulose by cellulases have been actively studied for nearly 70 years with significant gains in understanding of the cellulolytic enzymes. Yet, a full mechanistic understanding of the hydrolysis reaction has been elusive. We present a review to highlight new insights gained since the most recent comprehensive review of cellulose hydrolysis kinetic models by Bansal et al. (2009) Biotechnol Adv 27:833-848. Recent models have taken a two-pronged approach to tackle the challenge of modeling the complex heterogeneous reaction-an enzyme-centric modeling approach centered on the molecularity of the cellulase-cellulose interactions to examine rate limiting elementary steps and a substrate-centric modeling approach aimed at capturing the limiting property of the insoluble cellulose substrate. Collectively, modeling results suggest that at the molecular-scale, how rapidly cellulases can bind productively (complexation) and release from cellulose (decomplexation) is limiting, while the overall hydrolysis rate is largely insensitive to the catalytic rate constant. The surface area of the insoluble substrate and the degrees of polymerization of the cellulose molecules in the reaction both limit initial hydrolysis rates only. Neither enzyme-centric models nor substrate-centric models can consistently capture hydrolysis time course at extended reaction times. Thus, questions of the true reaction limiting factors at extended reaction times and the role of complexation and decomplexation in rate limitation remain unresolved. Biotechnol. Bioeng. 2017;114: 1369-1385. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

The high-rate data challenge: computing for the CBM experiment

NASA Astrophysics Data System (ADS)

Friese, V.; CBM Collaboration

2017-10-01

The Compressed Baryonic Matter experiment (CBM) is a next-generation heavy-ion experiment to be operated at the FAIR facility, currently under construction in Darmstadt, Germany. A key feature of CBM is very high interaction rate, exceeding those of contemporary nuclear collision experiments by several orders of magnitude. Such interaction rates forbid a conventional, hardware-triggered readout; instead, experiment data will be freely streaming from self-triggered front-end electronics. In order to reduce the huge raw data volume to a recordable rate, data will be selected exclusively on CPU, which necessitates partial event reconstruction in real-time. Consequently, the traditional segregation of online and offline software vanishes; an integrated on- and offline data processing concept is called for. In this paper, we will report on concepts and developments for computing for CBM as well as on the status of preparations for its first physics run.

Positive Imagery-Based Cognitive Bias Modification as a Web-Based Treatment Tool for Depressed Adults

PubMed Central

Blackwell, Simon E.; Browning, Michael; Mathews, Andrew; Pictet, Arnaud; Welch, James; Davies, Jim; Watson, Peter; Geddes, John R.

2015-01-01

Depression is a global health problem requiring treatment innovation. Targeting neglected cognitive aspects may provide a useful route. We tested a cognitive-training paradigm using positive mental imagery (imagery cognitive bias modification, imagery CBM), developed via experimental psychopathology studies, in a randomized controlled trial. Training was delivered via the Internet to 150 individuals with current major depression. Unexpectedly, there was no significant advantage for imagery CBM compared with a closely matched control for depression symptoms as a whole in the full sample. In exploratory analyses, compared with the control, imagery CBM significantly improved anhedonia over the intervention and improved depression symptoms as a whole for those participants with fewer than five episodes of depression and those who engaged to a threshold level of imagery. Results suggest avenues for improving imagery CBM to inform low-intensity treatment tools for depression. Anhedonia may be a useful treatment target for future work. PMID:25984421

Fall may be imminent for Kansas Cherokee basin coalbed gas output

USGS Publications Warehouse

Newell, K. David

2010-01-01

Natural gas production in the Kansas portion of the Cherokee basin, Southeastern Kansas, for 2008 was 49.1 bcf. The great majority of Cherokee basin gas production is now coal-bed methane (CBM). The major producers are Quest Energy LLC, Dart Cherokee Basin Operating Co. LLC, and Layne Energy Operating LLC. Most CBM in Southeastern Kansas is from Middle and Upper Pennsylvanian high-volatile B and A rank bituminous coals at 800 to 1,200 ft depth. Rates of decline for the CBM wells generally decrease the longer a well produces. A gentler collective decline of 13.8% is calculated by averaging the number of new producing wells in a given year with that of the previous year. By the calculations using the gentler overall 13.8% decline rate, if more than 918 successful CBM wells are drilled in 2009, then gas production will increase from 2008 to 2009.

Time-based Reconstruction of Free-streaming Data in CBM

NASA Astrophysics Data System (ADS)

Akishina, Valentina; Kisel, Ivan; Vassiliev, Iouri; Zyzak, Maksym

2018-02-01

Traditional latency-limited trigger architectures typical for conventional experiments are inapplicable for the CBM experiment. Instead, CBM will ship and collect time-stamped data into a readout buffer in a form of a time-slice of a certain length and deliver it to a large computer farm, where online event reconstruction and selection will be performed. Grouping measurements into physical collisions must be performed in software and requires reconstruction not only in space, but also in time, the so-called 4-dimensional track reconstruction and event building. The tracks, reconstructed with 4D Cellular Automaton track finder, are combined into event-corresponding clusters according to the estimated time in the target position and the errors, obtained with the Kalman Filter method. The reconstructed events are given as inputs to the KF Particle Finder package for short-lived particle reconstruction. The results of time-based reconstruction of simulated collisions in CBM are presented and discussed in details.

A precision device needs precise simulation: Software description of the CBM Silicon Tracking System

NASA Astrophysics Data System (ADS)

Malygina, Hanna; Friese, Volker; CBM Collaboration

2017-10-01

Precise modelling of detectors in simulations is the key to the understanding of their performance, which, in turn, is a prerequisite for the proper design choice and, later, for the achievement of valid physics results. In this report, we describe the implementation of the Silicon Tracking System (STS), the main tracking device of the CBM experiment, in the CBM software environment. The STS makes uses of double-sided silicon micro-strip sensors with double metal layers. We present a description of transport and detector response simulation, including all relevant physical effects like charge creation and drift, charge collection, cross-talk and digitization. Of particular importance and novelty is the description of the time behaviour of the detector, since its readout will not be externally triggered but continuous. We also cover some aspects of local reconstruction, which in the CBM case has to be performed in real-time and thus requires high-speed algorithms.

Observed mechanism for the breakup of small bundles of cellulose Iα and Iβ in ionic liquids from molecular dynamics simulations.

PubMed

Rabideau, Brooks D; Agarwal, Animesh; Ismail, Ahmed E

2013-04-04

Explicit, all-atom molecular dynamics simulations are used to study the breakup of small bundles of cellulose Iα and Iβ in the ionic liquids [BMIM]Cl, [EMIM]Ac, and [DMIM]DMP. In all cases, significant breakup of the bundles is observed with the initial breakup following a common underlying mechanism. Anions bind strongly to the hydroxyl groups of the exterior strands of the bundle, forming negatively charged complexes. Binding also weakens the intrastrand hydrogen bonds present in the cellulose strands, providing greater strand flexibility. Cations then intercalate between the individual strands, likely due to charge imbalances, providing the bulk to push the individual moieties apart and initiating the separation. The peeling of an individual strand from the main bundle is observed in [EMIM]Ac with an analysis of its hydrogen bonds with other strands showing that the chain detaches glucan by glucan from the main bundle in discrete, rapid events. Further analysis shows that the intrastrand hydrogen bonds of each glucan tend to break for a sustained period of time before the interstrand hydrogen bonds break and strand detachment occurs. Examination of similar nonpeeling strands shows that, without this intrastrand hydrogen bond breakage, the structural rigidity of the individual unit can hinder its peeling despite interstrand hydrogen bond breakage.

A Comparison of Cognitive Bias Modification for Interpretation and Computerized Cognitive Behavior Therapy

PubMed Central

Bowler, Jennifer O.; Mackintosh, Bundy; Dunn, Barnaby D.; Mathews, Andrew; Dalgleish, Tim; Hoppitt, Laura

2012-01-01

Objective: Computerized cognitive behavioral therapy (cCBT) and cognitive bias modification for interpretation (CBM-I) both have demonstrated efficacy in alleviating social anxiety, but how they compare with each other has not been investigated. The present study tested the prediction that both interventions would reduce anxiety relative to a no-intervention comparison condition, but CBM-I would be particularly effective at modifying threat-related cognitive bias under high mental load. Method: Sixty-three primarily Caucasian adults (mean age = 22.7, SD = 5.87; 68.3% female) with high social anxiety, randomly allocated to 3 groups: CBM-I (n = 21), cCBT (n = 21), and a no-intervention control group (n = 21) provided complete data for analysis. Pre- and postintervention (4 sessions lasting 2 weeks, control participants only attended the pre–post sessions) self-report measures of anxiety, depression, attentional control, and threat-related interpretive bias were completed. In addition, interpretive bias under high versus low cognitive load was measured using the Scrambled Sentences Test. Results: Both CBM-I and cCBT groups reported significantly reduced levels of social anxiety, trait anxiety, and depression and improved attentional control, relative to the control group, with no clear superiority of either active intervention. Although both active conditions reduced negative bias on the Scrambled Sentences Test completed under mental load, CBM-I was significantly more effective at doing so. Conclusions: The results suggest that although not differing in therapeutic efficacy, CBM-I and cCBT might differ in the resilience of their effects when under mental load. PMID:22963595

What's in a Trial? On the Importance of Distinguishing Between Experimental Lab Studies and Randomized Controlled Trials: The Case of Cognitive Bias Modification and Alcohol Use Disorders.

PubMed

Wiers, Reinout W; Boffo, Marilisa; Field, Matt

2018-05-01

Recently, the National Institutes of Health (NIH) redefined clinical trials to include any study involving behavioral or biomedical interventions. In line with a general framework from experimental medicine, we argue that it is crucial to distinguish between experimental laboratory studies aimed at revealing psychological mechanisms underlying behavior and randomized controlled trials (RCTs) in clinical samples aimed at testing the efficacy of an intervention. As an illustration, we reviewed the current state of the evidence on the efficacy of cognitive bias modification (CBM) interventions in alcohol use disorders. A recent meta-analysis "cast serious doubts on the clinical utility of CBM interventions for addiction." That analysis combined experimental laboratory studies and RCTs. We demonstrated that, when studies are differentiated regarding study type (experimental laboratory study or RCT), mode of delivery (controlled experiment or Internet), and population (healthy volunteers or patients), the following effects are found: (a) short-lived effects of CBM on drinking behavior in experimental laboratory studies in students, but only when the bias is successfully manipulated; (b) small but robust effects of CBM on treatment outcome when administered as an adjunct to established treatments in clinical settings in RCTs with alcohol-dependent patients; and (c) nonspecific effects (reduced drinking irrespective of condition) in RCTs of CBM administered online to problem drinkers. We discuss how CBM might be improved when it is better integrated into regular treatment, especially cognitive behavioral therapy, and we conclude that disregarding the difference between experimental laboratory studies and RCTs can lead to invalid conclusions.

A systems approach to modeling Community-Based Environmental Monitoring: a case of participatory water quality monitoring in rural Mexico.

PubMed

Burgos, Ana; Páez, Rosaura; Carmona, Estela; Rivas, Hilda

2013-12-01

Community-Based Environmental Monitoring (CBM) is a social practice that makes a valuable contribution to environmental management and construction of active societies for sustainable future. However, its documentation and analysis show deficiencies that hinder contrast and comparison of processes and effects. Based on systems approach, this article presents a model of CBM to orient assessment of programs, with heuristic or practical goals. In a focal level, the model comprises three components, the social subject, the object of monitoring, and the means of action, and five processes, data management, social learning, assimilation/decision making, direct action, and linking. Emergent properties were also identified in the focal and suprafocal levels considering community self-organization, response capacity, and autonomy for environmental management. The model was applied to the assessment of a CBM program of water quality implemented in rural areas in Mexico. Attributes and variables (indicators) for components, processes, and emergent properties were selected to measure changes that emerged since the program implementation. The assessment of the first 3 years (2010-2012) detected changes that indicated movement towards the expected results, but it revealed also the need to adjust the intervention strategy and procedures. Components and processes of the model reflected relevant aspects of the CBM in real world. The component called means of action as a key element to transit "from the data to the action." The CBM model offered a conceptual framework with advantages to understand CBM as a socioecological event and to strengthen its implementation under different conditions and contexts.

Cognitive Bias Modification for adolescents with substance use problems--Can serious games help?

PubMed

Boendermaker, Wouter J; Prins, Pier J M; Wiers, Reinout W

2015-12-01

Excessive use of psychoactive substances and resulting disorders are a major societal problem, and the most prevalent mental disorder in young men. Recent reviews have concluded that Cognitive Bias Modification (CBM) shows promise as an intervention method in this field. As adolescence is a critical formative period, successful early intervention may be key in preventing later substance use disorders that are difficult to treat. One issue with adolescents, however, is that they often lack the motivation to change their behavior, and to engage in multisession cognitive training programs. The upcoming use of serious games for health may provide a solution to this motivational challenge. As the use of game-elements in CBM is fairly new, there are very few published studies in this field. This review therefore focuses on currently available evidence from similar fields, such as cognitive training, as well as several ongoing CBM gamification projects, to illustrate the general principles. A number of steps in the gamification process are identified, starting with the original, evidence-based CBM task, towards full integration in a game. While more data is needed, some steps seem better suited for CBM gamification than others. Based on the current evidence, several recommendations are made. As the field is still in its infancy, further research is needed before firm conclusions can be drawn. Gamified CBM may be a promising way to reach at risk youth, but the term "game" should be used with caution. Suggestions are made for future research. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Cognitive bias modification as an add-on treatment in clinical depression: Results from a placebo-controlled, single-blinded randomized control trial.

PubMed

Vrijsen, Janna N; Fischer, Verena S; Müller, Bernhard W; Scherbaum, Norbert; Becker, Eni S; Rinck, Mike; Tendolkar, Indira

2018-06-06

Only 60% of depressed patients respond sufficiently to treatment, so there is a dire need for novel approaches to improve treatment effects. Cognitive Bias Modification (CBM) may be an effective and easily implemented computerized add-on to treatment-as-usual. Therefore, we investigated the effects of a positivity-attention training and a positivity-approach training compared to control trainings. In a blinded randomized-controlled design, 139 depressed inpatients received either the CBM Attention Dot-Probe Training (DPT) or the CBM Approach-Avoidance Training (AAT), next to treatment as usual. N = 121 finished all four training sessions. Both trainings had an active and a control condition. In both active conditions, patients were trained to preferentially process generally positive pictures over neutral pictures. Depressive symptom severity was assessed before and after CBM, and positivity bias was measured at the start and end of each session. Clinician-rated depressive symptom severity decreased more in patients who received the active condition of the DPT or the AAT compared to patients in the control conditions. Significant change in positivity bias was found for the DPT (not the AAT), but did not mediate the effect of the training on depressive symptoms. The results suggest that both types of CBM (i.e., DPT and AAT) may provide a fitting add-on treatment option for clinical depression. The working mechanisms and optimal dose of CBM trainings, plus their possible combination, should be examined in more detail. Copyright © 2018. Published by Elsevier B.V.

Role of Rhizobium endoglucanase CelC2 in cellulose biosynthesis and biofilm formation on plant roots and abiotic surfaces.

PubMed

Robledo, M; Rivera, L; Jiménez-Zurdo, Jose I; Rivas, R; Dazzo, F; Velázquez, E; Martínez-Molina, E; Hirsch, Ann M; Mateos, Pedro F

2012-09-12

The synthesis of cellulose is among the most important but poorly understood biochemical processes, especially in bacteria, due to its complexity and high degree of regulation. In this study, we analyzed both the production of cellulose by all known members of the Rhizobiaceae and the diversity of Rhizobium celABC operon predicted to be involved in cellulose biosynthesis. We also investigated the involvement in cellulose production and biofilm formation of celC gene encoding an endoglucanase (CelC2) that is required for canonical symbiotic root hair infection by Rhizobium leguminosarum bv. trifolii. ANU843 celC mutants lacking (ANU843ΔC2) or overproducing cellulase (ANU843C2+) produced greatly increased or reduced amounts of external cellulose micro fibrils, respectively. Calcofluor-stained cellulose micro fibrils were considerably longer when formed by ANU843ΔC2 bacteria rather than by the wild-type strain, in correlation with a significant increase in their flocculation in batch culture. In contrast, neither calcofluor-stained extracellular micro fibrils nor flocculation was detectable in ANU843C2+ cells. To clarify the role of cellulose synthesis in Rhizobium cell aggregation and attachment, we analyzed the ability of these mutants to produce biofilms on different surfaces. Alteration of wild-type CelC2 levels resulted in a reduced ability of bacteria to form biofilms both in abiotic surfaces and in planta. Our results support a key role of the CelC2 cellulase in cellulose biosynthesis by modulating the length of the cellulose fibrils that mediate firm adhesion among Rhizobium bacteria leading to biofilm formation. Rhizobium cellulose is an essential component of the biofilm polysaccharidic matrix architecture and either an excess or a defect of this "building material" seem to collapse the biofilm structure. These results position cellulose hydrolytic enzymes as excellent anti-biofilm candidates.

Role of Rhizobium endoglucanase CelC2 in cellulose biosynthesis and biofilm formation on plant roots and abiotic surfaces

PubMed Central

2012-01-01

Background The synthesis of cellulose is among the most important but poorly understood biochemical processes, especially in bacteria, due to its complexity and high degree of regulation. In this study, we analyzed both the production of cellulose by all known members of the Rhizobiaceae and the diversity of Rhizobium celABC operon predicted to be involved in cellulose biosynthesis. We also investigated the involvement in cellulose production and biofilm formation of celC gene encoding an endoglucanase (CelC2) that is required for canonical symbiotic root hair infection by Rhizobium leguminosarum bv. trifolii. Results ANU843 celC mutants lacking (ANU843ΔC2) or overproducing cellulase (ANU843C2+) produced greatly increased or reduced amounts of external cellulose micro fibrils, respectively. Calcofluor-stained cellulose micro fibrils were considerably longer when formed by ANU843ΔC2 bacteria rather than by the wild-type strain, in correlation with a significant increase in their flocculation in batch culture. In contrast, neither calcofluor-stained extracellular micro fibrils nor flocculation was detectable in ANU843C2+ cells. To clarify the role of cellulose synthesis in Rhizobium cell aggregation and attachment, we analyzed the ability of these mutants to produce biofilms on different surfaces. Alteration of wild-type CelC2 levels resulted in a reduced ability of bacteria to form biofilms both in abiotic surfaces and in planta. Conclusions Our results support a key role of the CelC2 cellulase in cellulose biosynthesis by modulating the length of the cellulose fibrils that mediate firm adhesion among Rhizobium bacteria leading to biofilm formation. Rhizobium cellulose is an essential component of the biofilm polysaccharidic matrix architecture and either an excess or a defect of this “building material” seem to collapse the biofilm structure. These results position cellulose hydrolytic enzymes as excellent anti-biofilm candidates. PMID:22970813

Diversity and strain specificity of plant cell wall degrading enzymes revealed by the draft genome of Ruminococcus flavefaciens FD-1.

PubMed

Berg Miller, Margret E; Antonopoulos, Dionysios A; Rincon, Marco T; Band, Mark; Bari, Albert; Akraiko, Tatsiana; Hernandez, Alvaro; Thimmapuram, Jyothi; Henrissat, Bernard; Coutinho, Pedro M; Borovok, Ilya; Jindou, Sadanari; Lamed, Raphael; Flint, Harry J; Bayer, Edward A; White, Bryan A

2009-08-14

Ruminococcus flavefaciens is a predominant cellulolytic rumen bacterium, which forms a multi-enzyme cellulosome complex that could play an integral role in the ability of this bacterium to degrade plant cell wall polysaccharides. Identifying the major enzyme types involved in plant cell wall degradation is essential for gaining a better understanding of the cellulolytic capabilities of this organism as well as highlighting potential enzymes for application in improvement of livestock nutrition and for conversion of cellulosic biomass to liquid fuels. The R. flavefaciens FD-1 genome was sequenced to 29x-coverage, based on pulsed-field gel electrophoresis estimates (4.4 Mb), and assembled into 119 contigs providing 4,576,399 bp of unique sequence. As much as 87.1% of the genome encodes ORFs, tRNA, rRNAs, or repeats. The GC content was calculated at 45%. A total of 4,339 ORFs was detected with an average gene length of 918 bp. The cellulosome model for R. flavefaciens was further refined by sequence analysis, with at least 225 dockerin-containing ORFs, including previously characterized cohesin-containing scaffoldin molecules. These dockerin-containing ORFs encode a variety of catalytic modules including glycoside hydrolases (GHs), polysaccharide lyases, and carbohydrate esterases. Additionally, 56 ORFs encode proteins that contain carbohydrate-binding modules (CBMs). Functional microarray analysis of the genome revealed that 56 of the cellulosome-associated ORFs were up-regulated, 14 were down-regulated, 135 were unaffected, when R. flavefaciens FD-1 was grown on cellulose versus cellobiose. Three multi-modular xylanases (ORF01222, ORF03896, and ORF01315) exhibited the highest levels of up-regulation. The genomic evidence indicates that R. flavefaciens FD-1 has the largest known number of fiber-degrading enzymes likely to be arranged in a cellulosome architecture. Functional analysis of the genome has revealed that the growth substrate drives expression of enzymes predicted to be involved in carbohydrate metabolism as well as expression and assembly of key cellulosomal enzyme components.