A long-wavelength fluorescent substrate for continuous fluorometric determination of cellulase activity: resorufin-β-d-cellobioside

Coleman, Daniel J.; Studler, Missy J.; Naleway, John J.

doi:10.1016/j.ab.2007.08.027

Cited by 38 publications

(52 citation statements)

References 25 publications

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“…The favorable spectroscopic properties of resorufin have been previously harnessed for the production of sensitive in vitro substrates for esterases and glycosidases (Tokutake et al, 1990;Beisson et al, 2000;Coleman et al, 2007), including single-molecule detection (English et al, 2006;Gorris et al, 2007). Indeed, earlier work has highlighted the utility of resorufin b-glycosides for imaging the activity of GHs, including exo-b-galactosidase in yeast (Saccharomyces cerevisiae; Wittrup and Bailey, 1988) and exo-b-glucosidase in animal cells (Hays et al, 1998).…”

Section: Discussionmentioning

confidence: 99%

“…This method was readily extended to the synthesis of the unsubstituted glucanase substrate, resorufin b-cellobioside (GG-b-Res; Fig. 1 [2]; Coleman et al, 2007). In a preliminary study, we were likewise able to perform the glycosylation step using variation of the Königs- , substrates for determination of (xylo)glucanase activity.…”

Section: Synthesis Of the Novel Xeh Substrate Xxxg-b-resmentioning

confidence: 99%

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A Real-Time Fluorogenic Assay for the Visualization of Glycoside Hydrolase Activity in Planta

Ibatullin

Banasiak²,

Baumann³

et al. 2009

Plant Physiol.

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There currently exists a diverse array of molecular probes for the in situ localization of polysaccharides, nucleic acids, and proteins in plant cells, including reporter enzyme strategies (e.g. protein-glucuronidase fusions). In contrast, however, there is a paucity of methods for the direct analysis of endogenous glycoside hydrolases and transglycosidases responsible for cell wall remodeling. To exemplify the potential of fluorogenic resorufin glycosides to address this issue, a resorufin b-glycoside of a xylogluco-oligosaccharide (XXXG-b-Res) was synthesized as a specific substrate for in planta analysis of XEH activity. The resorufin aglycone is particularly distinguished for high sensitivity in muro assays due to a low pK a (5.8) and large extinction coefficient (« 62,000 M 21 cm 21 ), long-wavelength fluorescence (excitation 571 nm/emission 585 nm), and high quantum yield (0.74) of the corresponding anion. In vitro analyses demonstrated that XXXG-b-Res is hydrolyzed by the archetypal plant XEH, nasturtium (Tropaeolum majus) NXG1, with classical Michaelis-Menten substrate saturation kinetics and a linear dependence on both enzyme concentration and incubation time. Further, XEH activity could be visualized in real time by observing the localized increase in fluorescence in germinating nasturtium seeds and Arabidopsis (Arabidopsis thaliana) inflorescent stems by confocal microscopy. Importantly, this new in situ XEH assay provides an essential complement to the in situ xyloglucan endotransglycosylase assay, thus allowing delineation of the disparate activities encoded by xyloglucan endotransglycosylase/ hydrolase genes directly in plant tissues. The observation that XXXG-b-Res is also hydrolyzed by diverse microbial XEHs indicates that this substrate, and resorufin glycosides in general, may find broad applicability for the analysis of wall restructuring by polysaccharide hydrolases during morphogenesis and plant-microbe interactions.

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Section: Discussionmentioning

confidence: 99%

Section: Synthesis Of the Novel Xeh Substrate Xxxg-b-resmentioning

confidence: 99%

A Real-Time Fluorogenic Assay for the Visualization of Glycoside Hydrolase Activity in Planta

Ibatullin

Banasiak²,

Baumann³

et al. 2009

Plant Physiol.

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“…Cellulase activity was analyzed with a MarkerGene fluorescentcellulase assay kit (MarkerGene Technologies, Inc.) with the fluorescent substrate resorufin cellobioside (6). Fluorescence was recorded using a Gen5 microtiter plate reader (BioTeck Instruments, Inc.) with a 530/25-nm excitation filter and a 595/35-nm emission filter.…”

Section: Methodsmentioning

confidence: 99%

Cellulose- and Xylan-Degrading Thermophilic Anaerobic Bacteria from Biocompost

Sizova

Izquierdo

Panikov

et al. 2011

Appl Environ Microbiol

114

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

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“…The absorbance was measured by the spectrophotometeric method at the wavelength of around 540 nm (Fig. 3) (Coleman et al, 2007). Enzyme activity with 0.83 mg/0.5mL CMC concentration was found to be 52.5 µmoL/mg protein and 69.37 µmol/mg protein at 4°C and 50°C respectively (Fig.…”

Section: Resultsmentioning

confidence: 99%

Testing Carboxymethyl Cellulase Activity Secreted by Trichophyton Terrestre in Carboxymethyl Cellulose Solution

Lone¹,

Sahay²,

Ravinder³

2014

American Journal of Microbiology

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The techniques employed for bioconversion of cellulosic biomass to simple sugars by cellulases have a great industrial brunt. Cold-adapted microorganisms are potential resource of cold-active carboxymethyl cellulases secluded from the cold regions. In this study, Trichophyton terrestre-a rare fungal species in Indian soils, isolated from the rhizosphere of Juglans regia L. during winter season was subjugated for the production and commotion of an enzyme carboxymethyl cellulase in carboxymethyl cellulose solution by DNS method at an ample range of temperatures, using Lineweaver-Burk plot which offers a practical graphical method for the analysis of Michealis-Menten equation, to establish the imperative terms in enzyme kinetics as K m and V max . The enzyme kinetic parameters like maximum activity (V max ), K m and turnover number were recorded at varying concentrations of CMC and different temperatures (4°C and 50°C). The enzyme was found to be tolerant and stable at two varying temperatures which enables this fungal species to survive in the extreme environmental conditions of northern India. Such property of carboxymethyl cellulase enzyme has extensive range of applications and the potential to open new application fields in the areas of industrial processes.

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A long-wavelength fluorescent substrate for continuous fluorometric determination of cellulase activity: resorufin-β-d-cellobioside

Cited by 38 publications

References 25 publications

A Real-Time Fluorogenic Assay for the Visualization of Glycoside Hydrolase Activity in Planta

A Real-Time Fluorogenic Assay for the Visualization of Glycoside Hydrolase Activity in Planta

Cellulose- and Xylan-Degrading Thermophilic Anaerobic Bacteria from Biocompost

Testing Carboxymethyl Cellulase Activity Secreted by Trichophyton Terrestre in Carboxymethyl Cellulose Solution

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