scholarly journals A Noncellulosomal Mannanase26E Contains a CBM59 inClostridium cellulovorans

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Kosuke Yamamoto ◽  
Yutaka Tamaru
Keyword(s):  
Plant Cell Wall ◽  
Glycosyl Hydrolase ◽  
Expression Patterns ◽  
Glycoside Hydrolases ◽  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Sds Page ◽  
Mannanase Activity ◽  
Coomassie Brilliant ◽  
Hydrolase Family

A multicomponent enzyme-complex prevents efficient degradation of the plant cell wall for biorefinery. In this study, the method of identifying glycoside hydrolases (GHs) to degrade hemicelluloses was demonstrated. The competence ofC. cellulovorans, which changes to be suitable for degradation of each carbon source, was used for the method.C. cellulovoranswas cultivated into locust bean gum (LBG) that is composed of galactomannan. The proteins produced byC. cellulovoranswere separated into either fractions binding to crystalline cellulose or not. Proteins obtained from each fraction were further separated by SDS-PAGE and were stained with Coomassie Brilliant Blue and were detected for mannanase activity. The proteins having the enzymatic activity for LBG were cut out and were identified by mass spectrometry. As a result, four protein bands were classified into glycosyl hydrolase family 26 (GH26) mannanases. One of the identified mannanases, Man26E, contains a carbohydrate-binding module (CBM) family 59, which binds to xylan, mannan, and Avicel. Although mannose and galactose are the same as a hexose, the expression patterns of the proteins fromC. cellulovoranswere quite different. More interestingly, zymogram for mannanase activity showed that Man26E was detected in only LBG medium.

scholarly journals The modular architecture of Cellvibrio japonicus mannanases in glycoside hydrolase families 5 and 26 points to differences in their role in mannan degradation

2003 ◽  
Vol 371 (3) ◽  
pp. 1027-1043 ◽  
Author(s):  
Deborah HOGG ◽  
Gavin PELL ◽  
Paul DUPREE ◽  
Florence GOUBET ◽  
Susana M. MARTÍN-ORÚE ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Plant Cell Wall ◽  
Catalytic Domain ◽  
Glycoside Hydrolases ◽  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Molecular Architecture ◽  
Catalytic Domains ◽  
Carbohydrate Binding Modules ◽  
Cellvibrio Japonicus

β-1,4-Mannanases (mannanases), which hydrolyse mannans and glucomannans, are located in glycoside hydrolase families (GHs) 5 and 26. To investigate whether there are fundamental differences in the molecular architecture and biochemical properties of GH5 and GH26 mannanases, four genes encoding these enzymes were isolated from Cellvibrio japonicus and the encoded glycoside hydrolases were characterized. The four genes, man5A, man5B, man5C and man26B, encode the mannanases Man5A, Man5B, Man5C and Man26B, respectively. Man26B consists of an N-terminal signal peptide linked via an extended serine-rich region to a GH26 catalytic domain. Man5A, Man5B and Man5C contain GH5 catalytic domains and non-catalytic carbohydrate-binding modules (CBMs) belonging to families 2a, 5 and 10; Man5C in addition contains a module defined as X4 of unknown function. The family 10 and 2a CBMs bound to crystalline cellulose and ivory nut crystalline mannan, displaying very similar properties to the corresponding family 10 and 2a CBMs from Cellvibrio cellulases and xylanases. CBM5 bound weakly to these crystalline polysaccharides. The catalytic domains of Man5A, Man5B and Man26B hydrolysed galactomannan and glucomannan, but displayed no activity against crystalline mannan or cellulosic substrates. Although Man5C was less active against glucomannan and galactomannan than the other mannanases, it did attack crystalline ivory nut mannan. All the enzymes exhibited classic endo-activity producing a mixture of oligosaccharides during the initial phase of the reaction, although their mode of action against manno-oligosaccharides and glucomannan indicated differences in the topology of the respective substrate-binding sites. This report points to a different role for GH5 and GH26 mannanases from C. japonicus. We propose that as the GH5 enzymes contain CBMs that bind crystalline polysaccharides, these enzymes are likely to target mannans that are integral to the plant cell wall, while GH26 mannanases, which lack CBMs and rapidly release mannose from polysaccharides and oligosaccharides, target the storage polysaccharide galactomannan and manno-oligosaccharides.

scholarly journals A novel α-glucosidase from the acidophilic archaeon Ferroplasma acidiphilum strain Y with high transglycosylation activity and an unusual catalytic nucleophile

2005 ◽  
Vol 391 (2) ◽  
pp. 269-276 ◽  
Author(s):  
Manuel Ferrer ◽  
Olga V. Golyshina ◽  
Francisco J. Plou ◽  
Kenneth N. Timmis ◽  
Peter N. Golyshin
Keyword(s):  
Glycosyl Hydrolase ◽  
Soluble Starch ◽  
Glycoside Hydrolases ◽  
Site Directed Mutagenesis ◽  
Preference Order ◽  
Glycosyl Hydrolase Family ◽  
Significant Similarity ◽  
Transglycosylation Activity ◽  
Membrane Bound ◽  
Hydrolase Family

Ferroplasma acidiphilum strain Y (DSM 12658), a ferrous iron-oxidizing, acidophilic and mesophilic archaeon, was found to produce a membrane-bound α-glucosidase (αGluFa) showing no significant similarity to any of the known glycoside hydrolases classified in different families and having an unusual catalytic site consisting of a threonine and a histidine residue. The highest α-glucosidase activity was found at low pH, 2.4–3.5, and the substrate preference order was: sucrose>maltose>maltotriose ≫maltotetraose≫malto-oligosaccharides from maltopentaose to maltoheptaose⋙soluble starch (kcat/Km was 293.0, 197.0, 18.8, 0.3 and 0.02 s−1·mM−1 respectively). The enzyme was able to transfer glucosyl groups from maltose as donor, to produce exclusively maltotriose (up to 300 g/l). Chemical modification and electrospray ionization MS analysis of 5-fluoro-α-D-glucopyranosyl-enzyme derivatives, coupled with site-directed mutagenesis, strongly suggested that the putative catalytic nucleophile in this enzyme is Thr212. Iron was found to be essential for enzyme activity and integrity, and His390 was shown to be essential for iron binding. These results suggest that the metalloenzyme αGluFa is a new member of the glycosyl hydrolase family that uses a novel mechanism for sugar glycosylation and/or transglycosylation.

Plant vascular system-feeding Psyllidae (Hemiptera) and Nematoda genomes encode family 12 glycosyl hydrolases

2019 ◽  
Vol 151 (3) ◽  
pp. 291-297
Author(s):  
Richard W. Jones
Keyword(s):  
Plant Cell Wall ◽  
Vascular Tissue ◽  
Vascular System ◽  
Glycosyl Hydrolase ◽  
Expression System ◽  
Root Tip ◽  
Glycosyl Hydrolases ◽  
Endoglucanase Activity ◽  
Intron Structure ◽  
Hydrolase Family

AbstractInsect-encoded cellulolytic plant cell wall hydrolases have thus far been found mostly from glycosyl hydrolase family 5, 9, 10, and 45. We now report the first evidence for genomic encoding of family 12 glycosyl hydrolases in vascular feeding Psyllidae (Hemiptera) and Nematoda. The genes were identified in three psyllids (Acanthocasuarina muellerianae Taylor, Pachypsylla venusta (Osten-Sacken), and Diaphorina citri Kuwayama) and a root tip feeding dagger nematode (Xiphinema index Thorne and Allen; Dorylaimida: Longidoridae). While the final gene products were highly similar, the genomic intron structure varied, having a 2 kB intron in P. venusta, a 283 base-pair intron in D. citri, and no intron in X. index. Endoglucanase activity was demonstrated using the D. citri genes in an Agrobacterium Conn (Rhizobiaceae) infiltration-based plant expression system. The presence of family 12 endoglucanases in this set of insects suggests a specific role in facilitating feeding on vascular tissue.

scholarly journals Domain Analysis of a Modular α-l-Arabinofuranosidase with a Unique Carbohydrate Binding Strategy from the Fiber-Degrading Bacterium Fibrobacter succinogenes S85

2010 ◽  
Vol 192 (20) ◽  
pp. 5424-5436 ◽  
Author(s):  
Shosuke Yoshida ◽  
Charles W. Hespen ◽  
Robert L. Beverly ◽  
Roderick I. Mackie ◽  
Isaac K. O. Cann
Keyword(s):  
Plant Cell Wall ◽  
Catalytic Efficiency ◽  
Glycoside Hydrolases ◽  
Site Directed Mutagenesis ◽  
Biofuel Production ◽  
Carbohydrate Binding ◽  
Fibrobacter Succinogenes ◽  
Carbohydrate Binding Module ◽  
Degrading Bacterium ◽  
Catalytic Module

ABSTRACT Family 43 glycoside hydrolases (GH43s) are known to exhibit various activities involved in hemicellulose hydrolysis. Thus, these enzymes contribute to efficient plant cell wall degradation, a topic of much interest for biofuel production. In this study, we characterized a unique GH43 protein from Fibrobacter succinogenes S85. The recombinant protein showed α-l-arabinofuranosidase activity, specifically with arabinoxylan. The enzyme is, therefore, an arabinoxylan arabinofuranohydrolase (AXH). The F. succinogenes AXH (FSUAXH1) is a modular protein that is composed of a signal peptide, a GH43 catalytic module, a unique β-sandwich module (XX domain), a family 6 carbohydrate-binding module (CBM6), and F. succinogenes-specific paralogous module 1 (FPm-1). Truncational analysis and site-directed mutagenesis of the protein revealed that the GH43 domain/XX domain constitute a new form of carbohydrate-binding module and that residue Y484 in the XX domain is essential for binding to arabinoxylan, although protein structural analyses may be required to confirm some of the observations. Kinetic studies demonstrated that the Y484A mutation leads to a higher k cat for a truncated derivative of FSUAXH1 composed of only the GH43 catalytic module and the XX domain. However, an increase in the Km for arabinoxylan led to a 3-fold decrease in catalytic efficiency. Based on the knowledge that most XX domains are found only in GH43 proteins, the evolutionary relationships within the GH43 family were investigated. These analyses showed that in GH43 members with a XX domain, the two modules have coevolved and that the length of a loop within the XX domain may serve as an important determinant of substrate specificity.

scholarly journals Comparative Community Proteomics Demonstrates the Unexpected Importance of Actinobacterial Glycoside Hydrolase Family 12 Protein for Crystalline Cellulose Hydrolysis

mBio ◽  
2016 ◽  
Vol 7 (4) ◽  
Author(s):  
Jennifer Hiras ◽  
Yu-Wei Wu ◽  
Kai Deng ◽  
Carrie D. Nicora ◽  
Joshua T. Aldrich ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Glycoside Hydrolase ◽  
Plant Biomass ◽  
Cellulose Hydrolysis ◽  
Global Carbon Cycle ◽  
Glycoside Hydrolases ◽  
Crystalline Cellulose ◽  
Glycoside Hydrolase Family ◽  
Global Carbon ◽  
Hydrolase Family

ABSTRACT Glycoside hydrolases (GHs) are key enzymes in the depolymerization of plant-derived cellulose, a process central to the global carbon cycle and the conversion of plant biomass to fuels and chemicals. A limited number of GH families hydrolyze crystalline cellulose, often by a processive mechanism along the cellulose chain. During cultivation of thermophilic cellulolytic microbial communities, substantial differences were observed in the crystalline cellulose saccharification activities of supernatants recovered from divergent lineages. Comparative community proteomics identified a set of cellulases from a population closely related to actinobacterium Thermobispora bispora that were highly abundant in the most active consortium. Among the cellulases from T. bispora , the abundance of a GH family 12 (GH12) protein correlated most closely with the changes in crystalline cellulose hydrolysis activity. This result was surprising since GH12 proteins have been predominantly characterized as enzymes active on soluble polysaccharide substrates. Heterologous expression and biochemical characterization of the suite of T. bispora hydrolytic cellulases confirmed that the GH12 protein possessed the highest activity on multiple crystalline cellulose substrates and demonstrated that it hydrolyzes cellulose chains by a predominantly random mechanism. This work suggests that the role of GH12 proteins in crystalline cellulose hydrolysis by cellulolytic microbes should be reconsidered. IMPORTANCE Cellulose is the most abundant organic polymer on earth, and its enzymatic hydrolysis is a key reaction in the global carbon cycle and the conversion of plant biomass to biofuels. The glycoside hydrolases that depolymerize crystalline cellulose have been primarily characterized from isolates. In this study, we demonstrate that adapting microbial consortia from compost to grow on crystalline cellulose generated communities whose soluble enzymes exhibit differential abilities to hydrolyze crystalline cellulose. Comparative proteomics of these communities identified a protein of glycoside hydrolase family 12 (GH12), a family of proteins previously observed to primarily hydrolyze soluble substrates, as a candidate that accounted for some of the differences in hydrolytic activities. Heterologous expression confirmed that the GH12 protein identified by proteomics was active on crystalline cellulose and hydrolyzed cellulose by a random mechanism, in contrast to most cellulases that act on the crystalline polymer in a processive mechanism.

scholarly journals Insights into Plant Cell Wall Degradation from the Genome Sequence of the Soil Bacterium Cellvibrio japonicus

2008 ◽  
Vol 190 (15) ◽  
pp. 5455-5463 ◽  
Author(s):  
Robert T. DeBoy ◽  
Emmanuel F. Mongodin ◽  
Derrick E. Fouts ◽  
Louise E. Tailford ◽  
Hoda Khouri ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Hydrolytic Enzymes ◽  
Soil Bacterium ◽  
Glycoside Hydrolases ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Modules ◽  
Cellvibrio Japonicus ◽  
And Storage

ABSTRACT The plant cell wall, which consists of a highly complex array of interconnecting polysaccharides, is the most abundant source of organic carbon in the biosphere. Microorganisms that degrade the plant cell wall synthesize an extensive portfolio of hydrolytic enzymes that display highly complex molecular architectures. To unravel the intricate repertoire of plant cell wall-degrading enzymes synthesized by the saprophytic soil bacterium Cellvibrio japonicus, we sequenced and analyzed its genome, which predicts that the bacterium contains the complete repertoire of enzymes required to degrade plant cell wall and storage polysaccharides. Approximately one-third of these putative proteins (57) are predicted to contain carbohydrate binding modules derived from 13 of the 49 known families. Sequence analysis reveals approximately 130 predicted glycoside hydrolases that target the major structural and storage plant polysaccharides. In common with that of the colonic prokaryote Bacteroides thetaiotaomicron, the genome of C. japonicus is predicted to encode a large number of GH43 enzymes, suggesting that the extensive arabinose decorations appended to pectins and xylans may represent a major nutrient source, not just for intestinal bacteria but also for microorganisms that occupy terrestrial ecosystems. The results presented here predict that C. japonicus possesses an extensive range of glycoside hydrolases, lyases, and esterases. Most importantly, the genome of C. japonicus is remarkably similar to that of the gram-negative marine bacterium, Saccharophagus degradans 2-40T. Approximately 50% of the predicted C. japonicus plant-degradative apparatus appears to be shared with S. degradans, consistent with the utilization of plant-derived complex carbohydrates as a major substrate by both organisms.

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

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
K. M. Singh ◽  
Bhaskar Reddy ◽  
Dishita Patel ◽  
A. K. Patel ◽  
Nidhi Parmar ◽  
...  
Keyword(s):  
Plant Cell Wall ◽  
Glycoside Hydrolases ◽  
Carbohydrate Binding ◽  
Metagenomic Sequencing ◽  
Environmental Aspects ◽  
Rumen Microbiome ◽  
Polysaccharide Lyases ◽  
Effective System ◽  
Plant Cell Wall Degradation ◽  
Buffalo Rumen

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.

scholarly journals Proteomic Characterization of Lignocellulolytic Enzymes Secreted by the Insect-Associated Fungus Daldinia decipiens oita, Isolated from a Forest in Northern Japan

2020 ◽  
Vol 86 (8) ◽  
Author(s):  
Chiaki Hori ◽  
Ruopu Song ◽  
Kazuki Matsumoto ◽  
Ruy Matsumoto ◽  
Benjamin B. Minkoff ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Glycoside Hydrolases ◽  
Wall Material ◽  
Carbohydrate Binding ◽  
Symbiotic Fungus ◽  
Proteomic Approach ◽  
Carbohydrate Binding Modules ◽  
Activity Measurements

ABSTRACT Wood-devastating insects utilize their symbiotic microbes with lignocellulose-degrading abilities to extract energy from recalcitrant woods. It is well known that free-living lignocellulose-degrading fungi secrete various carbohydrate-active enzymes (CAZymes) to degrade plant cell wall components, mainly cellulose, hemicellulose, and lignin. However, CAZymes from insect-symbiotic fungi have not been well documented except for a few examples. In this study, an insect-associated fungus, Daldinia decipiens oita, was isolated as a potential symbiotic fungus of female Xiphydria albopicta captured from Hokkaido forest. This fungus was grown in seven different media containing a single carbon source, glucose, cellulose, xylan, mannan, pectin, poplar, or larch, and the secreted proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 128 CAZymes, including domains of 92 glycoside hydrolases, 15 carbohydrate esterases, 5 polysaccharide lyases, 17 auxiliary activities, and 11 carbohydrate-binding modules, were identified, and these are involved in degradation of cellulose and hemicellulose but not lignin. Together with the results of polysaccharide-degrading activity measurements, we concluded that D. decipiens oita tightly regulates the expression of these CAZymes in response to the tested plant cell wall materials. Overall, this study described the detailed proteomic approach of a woodwasp-associated fungus and revealed that the new isolate, D. decipiens oita, secretes diverse CAZymes to efficiently degrade lignocellulose in the symbiotic environment. IMPORTANCE Recent studies show the potential impacts of insect symbiont microbes on biofuel application with regard to their degradation capability of a recalcitrant plant cell wall. In this study, we describe a novel fungal isolate, D. decipiens oita, as a single symbiotic fungus from the Xiphydria woodwasp found in the northern forests of Japan. Our detailed secretome analyses of D. decipiens oita, together with activity measurements, reveal that this insect-associated fungus exhibits high and broad activities for plant cell wall material degradation, suggesting potential applications within the biomass conversion industry for plant mass degradation.

scholarly journals Crystallographic analysis shows substrate binding at the −3 to +1 active-site subsites and at the surface of glycoside hydrolase family 11 endo-1,4-β-xylanases

2008 ◽  
Vol 410 (1) ◽  
pp. 71-79 ◽  
Author(s):  
Elien Vandermarliere ◽  
Tine M. Bourgois ◽  
Sigrid Rombouts ◽  
Steven van Campenhout ◽  
Guido Volckaert ◽  
...  
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Glycoside Hydrolase ◽  
Plant Cell Wall ◽  
Substrate Binding ◽  
Crystallographic Analysis ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Modules ◽  
Hydrolase Family

GH 11 (glycoside hydrolase family 11) xylanases are predominant enzymes in the hydrolysis of heteroxylan, an abundant structural polysaccharide in the plant cell wall. To gain more insight into the protein–ligand interactions of the glycone as well as the aglycone subsites of these enzymes, catalytically incompetent mutants of the Bacillus subtilis and Aspergillus niger xylanases were crystallized, soaked with xylo-oligosaccharides and subjected to X-ray analysis. For both xylanases, there was clear density for xylose residues in the −1 and −2 subsites. In addition, for the B. subtilis xylanase, there was also density for xylose residues in the −3 and +1 subsite showing the spanning of the −1/+1 subsites. These results, together with the observation that some residues in the aglycone subsites clearly adopt a different conformation upon substrate binding, allowed us to identify the residues important for substrate binding in the aglycone subsites. In addition to substrate binding in the active site of the enzymes, the existence of an unproductive second ligand-binding site located on the surface of both the B. subtilis and A. niger xylanases was observed. This extra binding site may have a function similar to the separate carbohydrate-binding modules of other glycoside hydrolase families.

scholarly journals Complexity of the Ruminococcus flavefaciens cellulosome reflects an expansion in glycan recognition

2016 ◽  
Vol 113 (26) ◽  
pp. 7136-7141 ◽  
Author(s):  
Immacolata Venditto ◽  
Ana S. Luis ◽  
Maja Rydahl ◽  
Julia Schückel ◽  
Vânia O. Fernandes ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Plant Cell Wall ◽  
Industrial Process ◽  
Site Directed Mutagenesis ◽  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Ruminococcus Flavefaciens ◽  
Pectic Polysaccharide ◽  
Degrading Bacterium ◽  
Carbohydrate Binding Modules

The breakdown of plant cell wall (PCW) glycans is an important biological and industrial process. Noncatalytic carbohydrate binding modules (CBMs) fulfill a critical targeting function in PCW depolymerization. Defining the portfolio of CBMs, the CBMome, of a PCW degrading system is central to understanding the mechanisms by which microbes depolymerize their target substrates. Ruminococcus flavefaciens, a major PCW degrading bacterium, assembles its catalytic apparatus into a large multienzyme complex, the cellulosome. Significantly, bioinformatic analyses of the R. flavefaciens cellulosome failed to identify a CBM predicted to bind to crystalline cellulose, a key feature of the CBMome of other PCW degrading systems. Here, high throughput screening of 177 protein modules of unknown function was used to determine the complete CBMome of R. flavefaciens. The data identified six previously unidentified CBM families that targeted β-glucans, β-mannans, and the pectic polysaccharide homogalacturonan. The crystal structures of four CBMs, in conjunction with site-directed mutagenesis, provide insight into the mechanism of ligand recognition. In the CBMs that recognize β-glucans and β-mannans, differences in the conformation of conserved aromatic residues had a significant impact on the topology of the ligand binding cleft and thus ligand specificity. A cluster of basic residues in CBM77 confers calcium-independent recognition of homogalacturonan, indicating that the carboxylates of galacturonic acid are key specificity determinants. This report shows that the extended repertoire of proteins in the cellulosome of R. flavefaciens contributes to an extended CBMome that supports efficient PCW degradation in the absence of CBMs that specifically target crystalline cellulose.

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