Use of complex formation between Congo Red and polysaccharides in detection and assay of polysaccharide hydrolases

Wood, Peter; Erfle, J. D.; Teather, R. M.

doi:10.1016/0076-6879(88)60107-8

Cited by 131 publications

(84 citation statements)

References 17 publications

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“…The genomic library was screened for CMCase-producing clones by using an overlay of 0.7% (wt/vol) top agar containing 0.2% (wt/vol) carboxymethyl cellulose (CMC). Plaques having CMCase activity were recognized by the formation of clear haloes on a red background after staining with 0.1% (wt/vol) Congo red and destaining with 1 M NaCl (27). Positive plaques were reisolated three times to ensure purity.…”

Section: Methodsmentioning

confidence: 99%

Cloning, Sequencing, and Expression of a Eubacterium cellulosolvens 5 Gene Encoding an Endoglucanase (Cel5A) with Novel Carbohydrate-Binding Modules, and Properties of Cel5A

Yoda

Toyoda

Mukoyama

et al. 2005

Appl Environ Microbiol

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A novel Eubacterium cellulosolvens 5 gene encoding an endoglucanase (Cel5A) was cloned and expressed in Escherichia coli, and its enzymatic properties were characterized. The cel5A gene consists of a 3,444-bp open reading frame and encodes a 1,148-amino-acid protein with a molecular mass of 127,047 Da. Cel5A is a modular enzyme consisting of an N-terminal signal peptide, two glycosyl hydrolase family 5 catalytic modules, two novel carbohydrate-binding modules (CBMs), two linker sequences, and a C-terminal sequence with an unknown function. The amino acid sequences of the two catalytic modules and the two CBMs are 94% and 73% identical to each other, respectively. Two regions that consisted of one CBM and one catalytic module were tandemly connected via a linker sequence. The CBMs did not exhibit significant sequence similarity with any other CBMs. Analyses of the hydrolytic activity of the recombinant Cel5A (rCel5A) comprising the CBMs and the catalytic modules showed that the enzyme is an endoglucanase with activities with carboxymethyl cellulose, lichenan, acid-swollen cellulose, and oat spelt xylan. To investigate the functions of the CBMs and the catalytic modules, truncated derivatives of rCel5A were constructed and characterized. There were no differences in the hydrolytic activities with various polysaccharides or in the hydrolytic products obtained from cellooligosaccharides between the two catalytic modules. Both CBMs had the same substrate affinity with intact rCel5A. Removal of the CBMs from rCel5A reduced the catalytic activities with various polysaccharides remarkably. These observations show that CBMs play an important role in the catalytic function of the enzyme.The rumen microbial ecosystem is composed of anaerobic microorganisms, such as bacteria, fungi, and protozoa. Some of these rumen microorganisms, the cellulolytic bacteria, are able to digest cellulosic material of plants and produce energy for the host animals. Many cellulolytic enzymes have been isolated from rumen microorganisms, and the genes encoding these enzymes have been cloned and sequenced (5, 9, 17). However, the precise mechanisms of lignocellulose degradation in the rumen are not yet fully understood. In order to clarify these mechanisms, it is necessary to study the microbial cellulolytic enzymes biochemically and genetically.The anaerobic cellulolytic bacterium Eubacterium cellulosolvens is sporadically dominant in the rumen (18). It is known that E. cellulosolvens 5 adheres tightly to cellulose, so studies of this adhesion have been performed (13,14,(23)(24)(25)(26). Some cellulose-binding proteins (CBPs) have been found in culture supernatant and cell lysate of the organism (14, 26). A gene encoding cellulose-binding protein A (CBPA), which is one of these CBPs, has been cloned and characterized (23-25). Additionally, the presence of some proteins exhibiting carboxymethyl cellulase (CMCase) activity in culture supernatant and cell lysate of E. cellulosolvens 5 was revealed by zymogram analysis (26). In order to advance res...

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Section: Methodsmentioning

confidence: 99%

Cloning, Sequencing, and Expression of a Eubacterium cellulosolvens 5 Gene Encoding an Endoglucanase (Cel5A) with Novel Carbohydrate-Binding Modules, and Properties of Cel5A

Yoda

Toyoda

Mukoyama

et al. 2005

Appl Environ Microbiol

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“…The kinetic parameters of enzymes produced by deletion mutants were estimated by using centrifuged crude cell lysates and the Congo red agar diffusion assay (47).…”

Section: Methodsmentioning

confidence: 99%

DNA sequence of a Fibrobacter succinogenes mixed-linkage beta-glucanase (1,3-1,4-beta-D-glucan 4-glucanohydrolase) gene

Teather

Erfle

1990

J Bacteriol

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The DNA sequence of a mixed-linkage 0-glucanase (1,3-1,4-0-D-glucan 4-glucanohydrolase [EC 3.2.1.73]) gene from Fibrobacter succinogenes cloned in Escherichia coli was determined. The general features of this gene are very similar to the consensus features for other gram-negative bacterial genes. The gene product was processed for export in E. coli. There is a high level of sequence homology between the structure of this glucanase and the structure of a mixed-linkage ,-glucanase from Bacillus subtilis. The nonhomologous region of the amino acid sequence includes a serine-rich region containing five repeats of the sequence ProXxx-Ser-Ser-Ser-Ser-(Ala or Val) which may be functionally related to the serine-rich region observed in Pseudomonasfluorescens celiulase and the serine-and/or threonine-rich regions observed in Cellulomonasfimi endoglucanase and exoglucanase, in Clostridium thermocellum endoglucanases A and B, and in Trichoderma reesei cellobiohydrolase I, cellobiohydrolase II, and endoglucanase I.

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“…Endo-hydrolyase activity was also assessed by Congo Red staining of polysaccharides embedded into agarose gels (Wood et al, 1988). For this assay, 0.1% (w/v) polysaccharide was incorporated into a layer of 1% (w/v) agarose gel containing 50 mm sodium acetate, pH 4.5.…”

Section: Cel12a Activity Against Different Wall Polysaccharidesmentioning

confidence: 99%

A Fungal Endoglucanase with Plant Cell Wall Extension Activity

2001

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We have identified a wall hydrolytic enzyme from Trichoderma reesei with potent ability to induce extension of heatinactivated type I cell walls. It is a small (23-kD) endo-1,4-␤-glucanase (Cel12A) belonging to glycoside hydrolase family 12. Extension of heat-inactivated walls from cucumber (Cucumis sativus cv Burpee Pickler) hypocotyls was induced by Cel12A after a distinct lag time and was accompanied by a large increase in wall plasticity and elasticity. Cel12A also increased the rate of stress relaxation of isolated walls at very short times (Ͻ200 ms; equivalent to reducing t 0 , a parameter that estimates the minimum relaxation time). Similar changes in wall plasticity and elasticity were observed in wheat (Triticum aestivum cv Pennmore Winter) coleoptile (type II) walls, which showed only a negligible extension in response to Cel12A treatment. Thus, Cel12A modifies both type I and II walls, but substantial extension is found only in type I walls. Cel12A has strong endo-glucanase activity against xyloglucan and (133,134)-␤-glucan, but did not exhibit endo-xylanase, endo-mannase, or endo-galactanase activities. In terms of kinetics of action and effects on wall rheology, wall loosening by Cel12A differs qualitatively from the action by expansins, which induce wall extension by a non-hydrolytic polymer creep mechanism. The action by Cel12A mimics some of the changes in wall rheology found after auxin-induced growth. The strategy used here to identify Cel12A could be used to identify analogous plant enzymes that cause auxin-induced changes in cell wall rheology.

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Use of complex formation between Congo Red and polysaccharides in detection and assay of polysaccharide hydrolases

Cited by 131 publications

References 17 publications

Cloning, Sequencing, and Expression of a Eubacterium cellulosolvens 5 Gene Encoding an Endoglucanase (Cel5A) with Novel Carbohydrate-Binding Modules, and Properties of Cel5A

Cloning, Sequencing, and Expression of a Eubacterium cellulosolvens 5 Gene Encoding an Endoglucanase (Cel5A) with Novel Carbohydrate-Binding Modules, and Properties of Cel5A

DNA sequence of a Fibrobacter succinogenes mixed-linkage beta-glucanase (1,3-1,4-beta-D-glucan 4-glucanohydrolase) gene

A Fungal Endoglucanase with Plant Cell Wall Extension Activity

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