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Cellulase (endo-1,4-β-D-glucanase)
(Aspergillus niger)

Product code: E-CELAN
€197.00

2,000 Units

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Content: 2,000 Units
Shipping Temperature: Ambient
Storage Temperature: 2-8oC
Formulation: In 3.2 M ammonium sulphate
Physical Form: Suspension
Stability: > 1 year under recommended storage conditions
Enzyme Activity: endo-Cellulase
EC Number: 3.2.1.4
CAZy Family: GH12
CAS Number: 9012-54-8
Synonyms: cellulase; 4-beta-D-glucan 4-glucanohydrolase
Source: Aspergillus niger
Molecular Weight: 27,000
Concentration: Supplied at ~ 1,200 U/mL
Expression: Purified from Aspergillus niger
Specificity: endo-hydrolysis of (1,4)-β-D-glucosidic linkages in cellulose.
Specific Activity: ~ 50 U/mg (40oC, pH 4.5 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 acetate buffer (100 mM), pH 4.5 at 40oC.
Temperature Optima: 60oC
pH Optima: 4.5
Application examples: Applications established in diagnostics and research within the textiles, food and feed, carbohydrate and biofuels industries.

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

Display all Carbohydrate Active enZYme products.

Documents
Certificate of Analysis
Safety Data Sheet
Data Sheet
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

Structural and mechanistic insights into fungal β-1, 3-glucan synthase FKS1.

Hu, X., Yang, P., Chai, C., Liu, J., Sun, H., Wu, Y., Zhang, M., Zhang, M, Liu, X. & Yu, H. (2023). Nature, 38, 1-9.

The membrane-integrated synthase FKS is involved in the biosynthesis of β-1,3-glucan, the core component of the fungal cell wall. FKS is the target of widely prescribed antifungal drugs, including echinocandin and ibrexafungerp. Unfortunately, the mechanism of action of FKS remains enigmatic and this has hampered development of more effective medicines targeting the enzyme. Here we present the cryo-electron microscopy structures of Saccharomyces cerevisiae FKS1 and the echinocandin-resistant mutant FKS1(S643P). These structures reveal the active site of the enzyme at the membrane-cytoplasm interface and a glucan translocation path spanning the membrane bilayer. Multiple bound lipids and notable membrane distortions are observed in the FKS1 structures, suggesting active FKS1-membrane interactions. Echinocandin-resistant mutations are clustered at a region near TM5-6 and TM8 of FKS1. The structure of FKS1(S643P) reveals altered lipid arrangements in this region, suggesting a drug-resistant mechanism of the mutant enzyme. The structures, the catalytic mechanism and the molecular insights into drug-resistant mutations of FKS1 revealed in this study advance the mechanistic understanding of fungal β-1,3-glucan biosynthesis and establish a foundation for developing new antifungal drugs by targeting FKS.

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Publication

Oligosaccharides production by enzymatic hydrolysis of banana pseudostem pulp.

Díaz, S., Ortega, Z., Benítez, A. N., Marrero, M. D., Carvalheiro, F., Duarte, L. C., Leonidas Matsakas, L., Eleni Krikigianni, E., Ulrika Rova, U., Christakopoulos, P. & Fernandes, M. C. (2021). Biomass Conversion and Biorefinery, 136, 1-12.

Banana production generates significant amounts of agricultural wastes, being fiber extraction one of the most relevant alternatives for their valorization. This process produces banana’s pseudostem pulp (BPP) as a byproduct, which shows an interesting composition for the biorefinery’s biochemical platform, with high polysaccharides (68%) and low lignin contents. This work deals with the enzymatic hydrolysis (EH) of raw and hydrothermally pre-treated BPP, focusing on the production of oligosaccharides (OS). Raw BPP hydrolysis with cellulase at different dosages rendered only 3.2% OS yields (OSY). Pectinase addition has not affected EH performance. On the other hand, EH of hydrothermally pre-treated BPP at 150°C and 170°C (P150 and P170) allowed to increase OSY up to 28% (P150, 1 FPU of cellulase/g dry biomass, 12 h), being 72% of the solubilized sugars in the form of cello-oligosaccharides. This last condition was subjected to a multi-stage EH strategy without improvements in OSY. An endo-glucanase was also tested, but obtained OSY were lower than cellulase results. Finally, obtained OS demonstrated to stimulate the growth of two Lactobacilli strains. The results show that BPP pre-treated under mild operational conditions is a good candidate for cello-oligosaccharides production by EH using 1 FPU/g DB of cellulase with a simple strategy.

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Publication

High-throughput screening of environmental polysaccharide-degrading bacteria using biomass containment and complex insoluble substrates.

Monge, E. C., Levi, M., Forbin, J. N., Legesse, M. D., Udo, B. A., deCarvalho, T. N. & Gardner, J. G. (2020). Applied Microbiology and Biotechnology, 1-11.

Carbohydrate degradation by microbes plays an important role in global nutrient cycling, human nutrition, and biotechnological applications. Studies that focus on the degradation of complex recalcitrant polysaccharides are challenging because of the insolubility of these substrates as found in their natural contexts. Specifically, current methods to examine carbohydrate-based biomass degradation using bacterial strains or purified enzymes are not compatible with high-throughput screening using complex insoluble materials. In this report, we developed a small 3D printed filter device that fits inside a microplate well that allows for the free movement of bacterial cells, media, and enzymes while containing insoluble biomass. These devices do not interfere with standard microplate readers and can be used for both short- (24-48 h) and long-duration (> 100 h) experiments using complex insoluble substrates. These devices were used to quantitatively screen in a high-throughput manner environmental isolates for their ability to grow using lignocellulose or rice grains as a sole nutrient source. Additionally, we determined that the microplate-based containment devices are compatible with existing enzymatic assays to measure activity against insoluble biomass. Overall, these microplate containment devices provide a platform to study the degradation of complex insoluble materials in a high-throughput manner and have the potential to help uncover ecologically important aspects of bacterial metabolism as well as to accelerate biotechnological innovation.

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Publication

An engineered GH1 β-glucosidase displays enhanced glucose tolerance and increased sugar release from lignocellulosic materials.

Santos, C. A., Morais, M. A., Terrett, O. M., Lyczakowski, J. J., Zanphorlin, L. M., Ferreira-Filho, J. A., Tonoli, C. C. C., Murakami, M. T., Dupree, P. & Souza, A. P. (2019). Scientific Reports, 9(1), 1-10.

β-glucosidases play a critical role among the enzymes in enzymatic cocktails designed for plant biomass deconstruction. By catalysing the breakdown of β-1, 4-glycosidic linkages, β-glucosidases produce free fermentable glucose and alleviate the inhibition of other cellulases by cellobiose during saccharification. Despite this benefit, most characterised fungal β-glucosidases show weak activity at high glucose concentrations, limiting enzymatic hydrolysis of plant biomass in industrial settings. In this study, structural analyses combined with site-directed mutagenesis efficiently improved the functional properties of a GH1 β-glucosidase highly expressed by Trichoderma harzianum (ThBgl) under biomass degradation conditions. The tailored enzyme displayed high glucose tolerance levels, confirming that glucose tolerance can be achieved by the substitution of two amino acids that act as gatekeepers, changing active-site accessibility and preventing product inhibition. Furthermore, the enhanced efficiency of the engineered enzyme in terms of the amount of glucose released and ethanol yield was confirmed by saccharification and simultaneous saccharification and fermentation experiments using a wide range of plant biomass feedstocks. Our results not only experimentally confirm the structural basis of glucose tolerance in GH1 β-glucosidases but also demonstrate a strategy to improve technologies for bioethanol production based on enzymatic hydrolysis.

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Publication
Discovery of Novel Cellulases Using Proteomic Strategies.

Zoglowek, M., Brewer, H. & Norbeck, A. (2018). Cellulases, pp. 103-113, Humana Press, New York, NY.

In order to develop cost-effective processes for conversion of lignocellulosic biomass, discovery of novel enzymes for enhanced lignocellulose hydrolysis is one of the main scientific and industrial goals. This could be achieved by applying proteomic strategies for identification of proteins secreted by filamentous fungi that are among the most powerful producers of biomass-degrading enzymes. Here a strategy for a comparative study of proteins differentially secreted on media inducing production of biomass-degrading enzymes (e.g., lignocellulosic biomass) and media repressing secretion of those enzymes (e.g., glucose) are presented. The protocols presented include preparation of samples for mass spectrometry and identification of cellulolytic and other carbohydrate-degrading enzymes using bioinformatics.

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Publication
Bonds broken and formed during the mixed-linkage glucan: xyloglucan endotransglucosylase reaction catalysed by Equisetum hetero-trans-β-glucanase.

Simmons, T. J. & Fry, S. C. (2017). Biochemical Journal, 474(7), 1055-1070.

Mixed-linkage glucan : xyloglucan endotransglucosylase (MXE) is one of the three activities of the recently characterised hetero-trans-β-glucanase (HTG), which among land-plants is known only from Equisetum species. The biochemical details of the MXE reaction were incompletely understood - details that would promote understanding of MXE's role in vivo and enable its full technological exploitation. We investigated HTG's site of attack on one of its donor substrates, mixed-linkage (1→3),(1→4)-β-D-glucan (MLG), with radioactive oligosaccharides of xyloglucan as acceptor substrate. Comparing three different MLG preparations, we showed that the enzyme favours those with a high content of cellotetraose blocks. The reaction products were analysed by enzymic digestion, thin-layer chromatography, HPLC and gel-permeation chromatography. Equisetum HTG consistently cleaved the MLG at the third consecutive β-( 1→4)-bond following (towards the reducing terminus) a β-( 1→3)-bond. It then formed a β-( 1→4)-bond between the MLG and the non-reducing terminal glucose residue of the xyloglucan oligosaccharide, consistent with its XTH subfamily membership. Using size-homogeneous barley MLG as donor substrate, we showed that HTG does not favour any particular region of the MLG chain relative the polysaccharide's reducing and non-reducing termini; rather, it selects its target cellotetraosyl unit stochastically along the MLG molecule. This work improves our understanding of how enzymes can exhibit promiscuous substrate specificities and provides the foundations to explore strategies for engineering novel substrate specificities into transglycanases.

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Safety Information
Symbol : GHS08
Signal Word : Danger
Hazard Statements : H334
Precautionary Statements : P261, P284, P304+P340, P342+P311, P501
Safety Data Sheet
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