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Cellulase (endo-1,4-β-D-glucanase)
(Thermotoga maritima)

Cellulase endo-1-4-beta-D-glucanase Thermotoga maritima E-CELTM
Product code: E-CELTM
€173.00

2,000 Units at 80oC

Prices exclude VAT

Available for shipping

Content: 2,000 Units at 80oC
Shipping Temperature: Ambient
Storage Temperature: 2-8oC
Formulation: In 3.2 M ammonium sulphate
Physical Form: Suspension
Stability: Minimum 1 year at 4oC. Check vial for details.
Enzyme Activity: endo-Cellulase
EC Number: 3.2.1.4
CAZy Family: GH5
CAS Number: 9012-54-8
Synonyms: cellulase; 4-beta-D-glucan 4-glucanohydrolase
Source: Thermotoga maritima
Molecular Weight: 38,200
Concentration: Supplied at ~ 500 U/mL
Expression: Recombinant from Thermotoga maritima
Specificity: endo-hydrolysis of (1,4)-β-D-glucosidic linkages in cellulose.
Specific Activity: ~ 245 U/mg (80oC, pH 6.0 on CM-cellulose 4M);
~ 50 U/mg (40oC, pH 6.0 on CM-cellulose 4M)
Unit Definition: One Unit of cellulase activity is defined as the amount of enzyme required to release one μmole of glucose reducing-sugar equivalents per minute from CM-Cellulose 4M (10 mg/mL) in sodium phosphate buffer (100 mM), pH 6.
Temperature Optima: 80oC
pH Optima: 6
Application examples: Applications established in diagnostics and research within the textiles, food and feed, carbohydrate and biofuels industries.

High purity recombinant Cellulase (endo-1,4-β-D-glucanase) (Thermotoga maritima) for use in research, biochemical enzyme assays and in vitro diagnostic analysis.

Documents
Certificate of Analysis
Safety Data Sheet
Booklet
Publications
Megazyme publication
Measurement of endo-1,4-β-glucanase.

McCleary, B. V., McKie, V. & Draga, A. (2012). “Methods in Enzymology”, Volume 510, (H. Gilbert, Ed.), Elsevier Inc., pp. 1-17.

Several procedures are available for the measurement of endo-1,4-β-glucanase (EG). Primary methods employ defined oligosaccharides or highly purified polysaccharides and measure the rate of hydrolysis of glycosidic bonds using a reducing-sugar method. However, these primary methods are not suitable for the measurement of EG in crude fermentation broths due to the presence of reducing sugars and other enzymes active on these substrates. In such cases, dyed soluble or insoluble substrates are preferred as they are specific, sensitive, easy to use, and are not affected by other components, such as reducing sugars, in the enzyme preparation.

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Megazyme publication
New developments in the measurement of α-amylase, endo-protease, β-glucanase and β-xylanase.

McCleary, B. V. & Monaghan, D. (2000). “Proceedings of the Second European Symposium on Enzymes in Grain Processing”, (M. Tenkanen, Ed.), VTT Information Service, pp. 31-38.

Over the past 8 years, we have been actively involved in the development of simple and reliable assay procedures, for the measurement of enzymes of interest to the cereals and related industries. In some instances, different procedures have been developed for the measurement of the same enzyme activity (e.g. α-amylase) in a range of different materials (e.g. malt, cereal grains and fungal preparations). The reasons for different procedures may depend on several factors, such as the need for sensitivity, ease of use, robustness of the substrate mixture, or the possibility for automation. In this presentation, we will present information on our most up-to-date procedures for the measurement of α-amylase, endo-protease, β-glucanase and β-xylanase, with special reference to the use of particular assay formats in particular applications.

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Megazyme publication

Measurement of polysaccharide-degrading enzymes in plants using chromogenic and colorimetric substrates.

McCleary, B. V. (1995). “New Diagnostics in Crop Sciences”, (J. R. Skerritt and R. Appels, Eds.), CAB International, pp. 277-301.

Enzymatic degradation of carbohydrates is of major significance in the industrial processing of cereals and fruits. In the production of beer, barley is germinated under well-defined conditions (malting) to induce maximum enzyme synthesis with minimum respiration of reserve carbohydrates. The grains are dried and then extracted with water under controlled conditions. The amylolytic enzymes synthesized during malting, as well as those present in the original barley, convert the starch reserves to fermentable sugars. Other enzymes act on the cell wall polysaccharides, mixed-linkage β-glucan and arabinoxylan, reducing the viscosity and thus aiding filtration, and reducing the possibility of subsequent precipitation of polymeric material (Bamforth, 1982). In baking, β-amylase and α-amylase give controlled degradation of starch to fermentable sugars so as to sustain yeast growth and gas production. Excess quantities of α-amylase in the flour result in excessive degradation of starch during baking which in turn gives a sticky crumb texture and subsequent problems with bread slicing. Juice yield from fruit pulp is significantly improved if cell-wall-degrading enzymes are used to destroy the three-dimensional structure and water-binding capacity of the pectic polysaccharide components of the cell walls. Problems of routine and reliable assay of carbohydrate-degrading enzymes in the presence of high levels of sugar compounds are experienced with such industrial processes.

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Megazyme publication

Measurement of polysaccharide degrading enzymes using chromogenic and colorimetric substrates.

McCleary, B. V. (1991). Chemistry in Australia, September, 398-401.

Enzymic degradation of carbohydrates is of major significance in the industrial processing of cereals and fruits. In the production of beer, barley is germinated under well defined conditions (malting) to induce maximum enzyme synthesis with minimum respiration of reserve carbohydrates. The grains are dried and then extracted with water under controlled conditions. The amylolytic enzymes synthesized during malting, as well as those present in the original barley, convert the starch reserves to fermentable sugars. Other enzymes act on the cell wall polysaccharides, mixed-linkage β-glucan and arabinoxylan, reducing the viscosity and thus aiding filtration, and reducing the possibility of subsequent precipitation of polymeric material. In baking, β-amylase and α-amylase give controlled degradation of starch to fermentable sugars so as to sustain yeast growth and gas production. Excess quantities of α-amylase in the flour result in excessive degradation of starch during baking which in turn gives a sticky crumb texture and subsequent problems with bread slicing. Juice yield from fruit pulp is significantly improved if cell-wall degrading enzymes are used to destroy the three-dimensional structure and water binding capacity of the pectic polysaccharide components of the cell walls. Problems of routine and reliable assay of carbohydrate degrading enzymes in the presence of high levels of sugar compounds are experienced with such industrial process.

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Publication
Engineering Geobacillus thermodenitrificans to introduce cellulolytic activity; expression of native and heterologous cellulase genes.

Daas, M. J., Nijsse, B., van de Weijer, A. H., Groenendaal, B. W., Janssen, F., van der Oost, J. & van Kranenburg, R. (2018). BMC biotechnology, 18(1), 42.

Background: Consolidated bioprocessing (CBP) is a cost-effective approach for the conversion of lignocellulosic biomass to biofuels and biochemicals. The enzymatic conversion of cellulose to glucose requires the synergistic action of three types of enzymes: exoglucanases, endoglucanases and β-glucosidases. The thermophilic, hemicellulolytic Geobacillus thermodenitrificans T12 was shown to harbor desired features for CBP, although it lacks the desired endo and exoglucanases required for the conversion of cellulose. Here, we report the expression of both endoglucanase and exoglucanase encoding genes by G. thermodenitrificans T12, in an initial attempt to express cellulolytic enzymes that complement the enzymatic machinery of this strain. Results: A metagenome screen was performed on 73 G. thermodenitrificans strains using HMM profiles of all known CAZy families that contain endo and/or exoglucanases. Two putative endoglucanases, GE39 and GE40, belonging to glucoside hydrolase family 5 (GH5) were isolated and expressed in both E. coli and G. thermodenitrificans T12. Structure modeling of GE39 revealed a folding similar to a GH5 exo-1,3-β-glucanase from S. cerevisiae. However, we determined GE39 to be a β-xylosidase having pronounced activity towards p-nitrophenyl-β-D-xylopyranoside. Structure modelling of GE40 revealed its protein architecture to be similar to a GH5 endoglucanase from B. halodurans, and its endoglucanase activity was confirmed by enzymatic activity against 2-hydroxyethylcellulose, carboxymethylcellulose and barley β-glucan. Additionally, we introduced expression constructs into T12 containing Geobacillus sp. 70PC53 endoglucanase gene celA and both endoglucanase genes (M1 and M2) from Geobacillus sp. WSUCF1. Finally, we introduced expression constructs into T12 containing the C. thermocellum exoglucanases celK and celS genes and the endoglucanase celC gene. Conclusions: We identified a novel G. thermodenitrificans β-xylosidase (GE39) and a novel endoglucanase (GE40) using a metagenome screen based on multiple HMM profiles. We successfully expressed both genes in E. coli and functionally expressed the GE40 endoglucanase in G. thermodenitrificans T12. Additionally, the heterologous production of active CelK, a C. thermocellum derived exoglucanase, and CelA, a Geobacillus derived endoglucanase, was demonstrated with strain T12. The native hemicellulolytic activity and the heterologous cellulolytic activity described in this research provide a good basis for the further development of G. thermodenitrificans T12 as a host for consolidated bioprocessing.

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Safety Information
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Precautionary Statements : Not Applicable
Safety Data Sheet
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