scholarly journals A mannanase, ManA, of the polycentric anaerobic fungusOrpinomycessp. strain PC-2 has carbohydrate binding and docking modules

2005 ◽  
Vol 51 (7) ◽  
pp. 559-568 ◽  
Author(s):  
Eduardo A Ximenes ◽  
Huizhong Chen ◽  
Irina A Kataeva ◽  
Michael A Cotta ◽  
Carlos R Felix ◽  
...  
Keyword(s):  
Specific Activity ◽  
High Specific Activity ◽  
Glycoside Hydrolases ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Amino Acid Residues ◽  
Anaerobic Fungus ◽  
Ph Profile ◽  
Catalytic Module ◽  
Cellulose Binding

The anaerobic fungus Orpinomyces sp. strain PC-2 produces a broad spectrum of glycoside hydrolases, most of which are components of a high molecular mass cellulosomal complex. Here we report about a cDNA (manA) having 1924 bp isolated from the fungus and found to encode a polypeptide of 579 amino acid residues. Analysis of the deduced sequence revealed that it had a mannanase catalytic module, a family 1 carbohydrate-binding module, and a noncatalytic docking module. The catalytic module was homologous to aerobic fungal mannanases belonging to family 5 glycoside hydrolases, but unrelated to the previously isolated mannanases (family 26) of the anaerobic fungus Piromyces. No mannanase activity could be detected in Escherichia coli harboring a manA-containing plasmid. The manA was expressed in Saccharomyces cerevisiae and ManA was secreted into the culture medium in multiple forms. The purified extracellular heterologous mannanase hydrolyzed several types of mannan but lacked activity against cellulose, chitin, or β-glucan. The enzyme had high specific activity toward locust bean mannan and an extremely broad pH profile. It was stable for several hours at 50 °C, but was rapidly inactivated at 60 °C. The carbohydrate-binding module of the Man A produced separately in E. coli bound preferably to insoluble lignocellulosic substrates, suggesting that it might play an important role in the complex enzyme system of the fungus for lignocellulose degradation.Key words: Orpinomyces, anaerobic fungi, mannanase, cellulose-binding module, cellulosome.

scholarly journals Modular Glucuronoxylan-Specific Xylanase with a Family CBM35 Carbohydrate-Binding Module

2012 ◽  
Vol 78 (11) ◽  
pp. 3923-3931 ◽  
Author(s):  
Susana Valeria Valenzuela ◽  
Pilar Diaz ◽  
F. I. Javier Pastor
Keyword(s):  
Catalytic Activity ◽  
Kinetic Parameters ◽  
Glucuronic Acid ◽  
Specific Activity ◽  
Dimensional Structure ◽  
Titration Calorimetry ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Content Type ◽  
Catalytic Module

ABSTRACTXyn30D from the xylanolytic strainPaenibacillus barcinonensishas been identified and characterized. The enzyme shows a modular structure comprising a catalytic module family 30 (GH30) and a carbohydrate-binding module family 35 (CBM35). Like GH30 xylanases, recombinant Xyn30D efficiently hydrolyzed glucuronoxylans and methyl-glucuronic acid branched xylooligosaccharides but showed no catalytic activity on arabinose-substituted xylans. Kinetic parameters of Xyn30D were determined on beechwood xylan, showing aKmof 14.72 mg/ml and akcatvalue of 1,510 min−1. The multidomain structure of Xyn30D clearly distinguishes it from the GH30 xylanases characterized to date, which are single-domain enzymes. The modules of the enzyme were individually expressed in a recombinant host and characterized. The isolated GH30 catalytic module showed specific activity, mode of action on xylan, and kinetic parameters that were similar to those of the full-length enzyme. Computer modeling of the three-dimensional structure of Xyn30D showed that the catalytic module is comprised of a common (β/α)8barrel linked to a side-associated β-structure. Several derivatives of the catalytic module with decreasing deletions of this associated structure were constructed. None of them showed catalytic activity, indicating the importance of the side β-structure in the catalysis of Xyn30D. Binding properties of the isolated carbohydrate-binding module were analyzed by affinity gel electrophoresis, which showed that the CBM35 of the enzyme binds to soluble glucuronoxylans and arabinoxylans. Analysis by isothermal titration calorimetry showed that CBM35 binds to glucuronic acid and requires calcium ions for binding. Occurrence of a CBM35 in a glucuronoxylan-specific xylanase is a differential trait of the enzyme characterized.

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.

Effect of Family 22 Carbohydrate-Binding Module on the Thermostability of Xyn10B Catalytic Module fromClostridium stercorarium

2006 ◽  
Vol 70 (12) ◽  
pp. 3039-3041 ◽  
Author(s):  
Rie ARAKI ◽  
Shuichi KARITA ◽  
Akiyoshi TANAKA ◽  
Tetsuya KIMURA ◽  
Kazuo SAKKA
Keyword(s):  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Catalytic Module

scholarly journals Arabinoxylan Oligosaccharide Hydrolysis by Family 43 and 51 Glycosidases from Lactobacillus brevis DSM 20054

2013 ◽  
Vol 79 (21) ◽  
pp. 6747-6754 ◽  
Author(s):  
Herbert Michlmayr ◽  
Johannes Hell ◽  
Cindy Lorenz ◽  
Stefan Böhmdorfer ◽  
Thomas Rosenau ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Functional Food ◽  
Specific Activity ◽  
High Specific Activity ◽  
Lactobacillus Brevis ◽  
Glycoside Hydrolases ◽  
Content Type ◽  
Food And Feed ◽  
Oligosaccharide Hydrolysis

ABSTRACTDue to their potential prebiotic properties, arabinoxylan-derived oligosaccharides [(A)XOS] are of great interest as functional food and feed ingredients. While the (A)XOS metabolism ofBifidobacteriaceaehas been extensively studied, information regarding lactic acid bacteria (LAB) is still limited in this context. The aim of the present study was to fill this important gap by characterizing candidate (A)XOS hydrolyzing glycoside hydrolases (GHs) identified in the genome ofLactobacillus brevisDSM 20054. Two putative GH family 43 xylosidases (XynB1 and XynB2) and a GH family 43 arabinofuranosidase (Abf3) were heterologously expressed and characterized. While the function of XynB1 remains unclear, XynB2 could efficiently hydrolyze xylooligosaccharides. Abf3 displayed high specific activity for arabinobiose but could not release arabinose from an (A)XOS preparation. However, two previously reported GH 51 arabinofuranosidases fromLb. breviswere able to specifically remove α-1,3-linked arabinofuranosyl residues from arabino-xylooligosaccharides (AXHm3 specificity). These results imply thatLb. brevisis at least genetically equipped with functional enzymes in order to hydrolyze the depolymerization products of (arabino)xylans and arabinans. The distribution of related genes inLactobacillalesgenomes indicates that GH 43 and, especially, GH 51 glycosidase genes are rare among LAB and mainly occur in obligately heterofermentativeLactobacillusspp.,Pediococcusspp., members of theLeuconostoc/Weissellabranch, andEnterococcusspp. Apart from the prebiotic viewpoint, this information also adds new perspectives on the carbohydrate (i.e., pentose-oligomer) metabolism of LAB species involved in the fermentation of hemicellulose-containing substrates.

scholarly journals Oxidized Cellulose Binding to Allergens with a Carbohydrate-Binding Module Attenuates Allergic Reactions

2010 ◽  
Vol 186 (2) ◽  
pp. 1240-1247 ◽  
Author(s):  
Nir Shani ◽  
Ziv Shani ◽  
Oded Shoseyov ◽  
Rufayda Mruwat ◽  
David Shoseyov
Keyword(s):  
Carbohydrate Binding ◽  
Allergic Reactions ◽  
Carbohydrate Binding Module ◽  
Oxidized Cellulose ◽  
Cellulose Binding

scholarly journals Functional Characterization of Carbohydrate-Binding Modules in a New Alginate Lyase, TsAly7B, from Thalassomonas sp. LD5

Marine Drugs ◽  
2019 ◽  
Vol 18 (1) ◽  
pp. 25 ◽  
Author(s):  
Zhelun Zhang ◽  
Luyao Tang ◽  
Mengmeng Bao ◽  
Zhigang Liu ◽  
Wengong Yu ◽  
...  
Keyword(s):  
Specific Activity ◽  
Biological Activities ◽  
Functional Characterization ◽  
Alginate Lyase ◽  
Carbohydrate Binding ◽  
Enzyme Degradation ◽  
Carbohydrate Binding Modules ◽  
Catalytic Module ◽  
Alginate Lyases

Alginate lyases degrade alginate into oligosaccharides, of which the biological activities have vital roles in various fields. Some alginate lyases contain one or more carbohydrate-binding modules (CBMs), which assist the function of the catalytic modules. However, the precise function of CBMs in alginate lyases has yet to be fully elucidated. We have identified a new multi-domain alginate lyase, TsAly7B, in the marine bacterium Thalassomonas sp. LD5. This novel lyase contains an N-terminal CBM9, an internal CBM32, and a C-terminal polysaccharide lyase family 7 (PL7) catalytic module. To investigate the specific function of each of these CBMs, we expressed and characterized the full-length TsAly7B and three truncated mutants: TM1 (CBM32-PL7), TM2 (CBM9-PL7), and TM3 (PL7 catalytic module). CBM9 and CBM32 could enhance the degradation of alginate. Notably, the specific activity of TM2 was 7.6-fold higher than that of TM3. CBM32 enhanced the resistance of the catalytic module to high temperatures. In addition, a combination of CBM9 and CBM32 showed enhanced thermostability when incubated at 80 °C for 1 h. This is the first report that finds CBM9 can significantly improve the ability of enzyme degradation. Our findings provide new insight into the interrelationships of tandem CBMs and alginate lyases and other polysaccharide-degrading enzymes, which may inspire CBM fusion strategies.

scholarly journals Novel Carbohydrate-Binding Module of β-1,3-Xylanase from a Marine Bacterium, Alcaligenes sp. Strain XY-234

2002 ◽  
Vol 184 (9) ◽  
pp. 2399-2403 ◽  
Author(s):  
Fumiyoshi Okazaki ◽  
Yutaka Tamaru ◽  
Shinnosuke Hashikawa ◽  
Yu-Teh Li ◽  
Toshiyoshi Araki
Keyword(s):  
Amino Acid ◽  
Marine Bacterium ◽  
Catalytic Domain ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Amino Acid Residues ◽  
Glycosyl Hydrolases ◽  
Binding Studies ◽  
Calculated Molecular Mass ◽  
Reading Frame

ABSTRACT A β-1,3-xylanase gene (txyA) from a marine bacterium, Alcaligenes sp. strain XY-234, has been cloned and sequenced. txyA consists of a 1,410-bp open reading frame that encodes 469 amino acid residues with a calculated molecular mass of 52,256 Da. The domain structure of the β-1,3-xylanase (TxyA) consists of a signal peptide of 22 amino acid residues, followed by a catalytic domain which belongs to family 26 of the glycosyl hydrolases, a linker region with one array of DGG and six repeats of DNGG, and a novel carbohydrate-binding module (CBM) at the C terminus. The recombinant TxyA hydrolyzed β-1,3-xylan but not other polysaccharides such as β-1,4-xylan, carboxymethylcellulose, curdlan, glucomannan, or β-1,4-mannan. TxyA was capable of binding specifically to β-1,3-xylan. The analysis using truncated TxyA lacking either the N- or C-terminal region indicated that the region encoding the CBM was located between residues 376 and 469. Binding studies on the CBM revealed that the Kd and the maximum amount of protein bound to β-1,3-xylan were 4.2 μM and 18.2 μmol/g of β-1,3-xylan, respectively. Furthermore, comparison of the enzymatic properties between proteins with and without the CBM strongly indicated that the CBM of TxyA plays an important role in the hydrolysis of β-1,3-xylan.

scholarly journals Extra tyrosine in the carbohydrate-binding module ofIrpex lacteusXyn10B enhances its cellulose-binding ability

2015 ◽  
Vol 79 (5) ◽  
pp. 738-746 ◽  
Author(s):  
Hiroto Nishijima ◽  
Kouichi Nozaki ◽  
Masahiro Mizuno ◽  
Tsutomu Arai ◽  
Yoshihiko Amano
Keyword(s):  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Binding Ability ◽  
Cellulose Binding
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