MarkerGene™ Fluorescent Cellulase Assay Kit

Product ID: M1245



Unit SizePriceQuantity 
1kit
$281.58
  • Buy 5 for $225.26 each and save 21%

Availability: In stock


Description

Allows fast and easy detection of most cellulases in a microtiter plate based assay format. Cellulase assays are widely used in the biofuels industry.

Cellulases are a family of enzymes that include β-glucosidases, endoglucanases, and exoglucanases. These enzymes cleave the β-1,4-D-glycosidic bonds that link the glucose units comprising cellulose. In addition to being produced by plants, cellulase activity is found in many fungi and bacteria, including some plant pathogens. Most animal cells are not known to produce cellulase; cellulolytic activity is often carried out in animals by symbionts. However, recent evidence does suggest cellulase production in some animals, such as insects and arthropods. The study of cellulase activity has many applications in plant molecular biology, agriculture, and manufacturing. Cellulase is also becoming important in the development of alternative fuel sources, as glucose obtained from cellulose hydrolysis is easily fermented into ethanol. Activity of most cellulases can be monitored using our long wavelength fluorescent substrate, Resorufin Cellobioside M1238, contained in the kit. Upon cleavage, the fluorescent compound, Resorufin M0202, is released and activity measurements are easily obtained in a microtiter plate based assay format. The kit contains enough substrate for 200 assays and control experiments (100 μL reaction volume) and also contains reference standards and a detailed protocol for use. See the references below for more information and applications.

Technical Data
SKU M1245
Unit Size 1kit
Detection Method Fluorescence

References and Citations

Citations:

  • Speda J, Johansson MA, Jonsson B-H, Karlsson M (2016) "Applying theories of microbial metabolism for induction of targeted enzyme activity in a methanogenic microbial community at a metabolic steady state" Applied Microbiology and Biotechnology: 1-14
  • Sharma A, Tewari R, Rana SS, Soni R, Soni SK. (2016) "Cellulases: Classification, Methods of Determination and Industrial Applications." Appl Biochem Biotechnol DOI 10.1007/s12010-016-2070-3.
  • Lin YS, Yang CC, Hsu CC, Hsu JT, Wu SC, Lin CJ, Cheng WT (2015) "Establishment of a novel, eco-friendly transgenic pig model using porcine pancreatic amylase promoter-driven fungal cellulase transgenes." Transgenic Res. 24(1):61-71.
  • Zels S, Dillen S, Crabbé K, Spit J, Nachman RJ, Vanden Broeck J. (2015) "Sulfakinin is an important regulator of digestive processes in the migratory locust, Locusta migratoria." Insect Biochem Mol Biol. 61:8-16.
  • Ito Y, Yamanishi M, Ikeuchi A, Imamura C, Matsuyama T. (2015) " Combinatorial screening for transgenic yeasts with high cellulase activities in combination with a tunable expression system." PloS ONE 10(12): e0144870. doi:10.1371/journal.pone.0144870.
  • Liu Z, Sun Y, Feng T, Ji Q, Cong P, Chen Y, He Z. (2014) "Mammalian expression levels of cellulase and xylanase genes optimised by human codon usage are not necessarily higher than those optimised by the extremely biased approach." Biotechnol Lett. 36(11):2169-76.
  • Paungfoo-Lonhienne C, Rentsch D, Robatzek S, Webb RI, Sagulenko E, Näsholm T, Schmidt S, Lonhienne TG. (2010) "Turning the table: plants consume microbes as a source of nutrients." PLoS ONE. 5(7):e11915. doi:10.1371/journal.pone.0011915.
  • Coleman DJ, Studler MJ, Naleway JJ. (2007) "A long-wavelength fluorescent substrate for continuous fluorometric determination of cellulase activity: resorufin-β-D-cellobioside." Anal Biochem 371: 146-153.
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References:

  • Shani Z, Dekel M, Roiz L, Horowitz M, Kolosovski N, Lapidot L, Alkan S, Koltai H, Tsabary G, Goren R, Shoseyov O. (2006) "Expression of endo-1,4-β-glucanase (cel1) in Arabidopsis thaliana is associated with plant growth, xylem development and cell wall thickening." Plant Cell Rep 25: 1067-1074.
  • Villena GK, Gutierrez-Correa M. (2006) "Production of cellulase by Aspergillus niger biofilms developed on polyester cloth." Letters in Applied Microbiology. 43: 262-268.
  • Zhang Q, Bai G, Yang W, Li H, Xiong H. (2006) "Pathogenic cellulase assay of pine wilt disease and immunological localization." Biosci Biotechnol Biochem 70(11): 2727-32.
  • Nakata T, Miyafuji H, Saka S. (2006) "Bioethanol from cellulose with supercritical water treatment followed by enzymatic hydrolysis." Appl Biochem Biotechnol 129-132: 476-85.
  • Watanabe H, Tokuda G. (2001) "Animal Cellulases." Cell Mol Life Sci 58: 1167-1178.
  • Han SJ, Yoo WJ, Kang, HS.(1995)"Characterization of a Bifunctional Cellulase and Its Structural Gene." J. Biol Chem, 270(43): 26012-26019.
  • Boschker HTS, Cappenberg TE. (1994) "A sensitive method using 4-Methylumbelliferyl-beta-Cellobiose as a Substrate to Measure (1,4)-beta-Glucanase Activity in Sediments." Applied and Environmental Biology 60(10): 3592-3596.
  • Chernoglazov VM, Jafarova AN, Klyosov AA. (1989) "Continuous photometric determination of endo-1,4-beta-D-glucanase (cellulase) activity using 4-methylumbelliferyl-beta-D-cellobioside as a substrate." Anal Biochem 179(1): 186-189.
  • Thayer DW. (1978) "Carboxymethylcellulase produced by facultative bacteria from the hind-gut of the termite Reticulitermes hesperus." Journal of General Microbiology 106(1) 13-8.
  • Ferrari T, Arnison P. (1974) "Extraction and Partial Characterization of Cellulases from Expanding Pea Epicotyls." Plant Physiol 54: 487-493.
  • Sizova MV, Izquierdo JA, Panikov NS, Lynd LR. (2011) "Cellulose- and xylan-degrading thermophilic anaerobic bacteria from biocompost." Appl Environ Microbiol. 77(7):2282-91
  • Ibatullin FM, Banasiak A, Baumann MJ, Greffe L, Takahashi J, Mellerowicz EJ, Brumer H. (2009) "A real-time fluorogenic assay for the visualization of glycoside hydrolase activity in planta." Plant Physiol. 151(4):1741-50
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Technical Support

Question about this product? Ask a Scientist!

We pride ourselves on the high quality of our products and want you to get the best possible results from your assays. If you have any questions about this product or need help optimizing your protocol check out the product FAQs below or ask your own question and one of our expert scientists will get back to you asap:


⚛ Question: Is this kit suitable for live cell staining?
➸ Answer: Unfortunately this kit is not suitable for live cell imaging due to the requisite low pH of the staining buffer.
⚛ Question: What pH is the reaction buffer?
➸ Answer: The pH of the reaction buffer is 6.0. This is the upper range of optimum activity for most cellulases, which generally have an optimum pH in the range of 4.0-6.0. A reaction buffer of this pH allows for the fluorescence increase over time (as substrate is turned over) to be measured continuously, rather than a stopped assay.
⚛ Question: Can I add detergent to solubilize my enzyme prior to running the assay?
➸ Answer: If you wish to add detergent to solubilize your enzyme, make sure to test whether the enzyme remains soluble upon further dilution with reaction buffer. If you need to have detergent there to maintain the solubility, make sure include the same concentration in the blank wells just in case the detergent interferes with the assay.
⚛ Question: What do I use for my blank wells?
➸ Answer: Blank wells are the well that contain no enzyme or reference standard. You may add 50ul reaction buffer to the blank wells.
⚛ Question: How do I calculate enzyme kinetics for this reaction?
➸ Answer: Since most fluorescence plate readers or fluorometers just give you relative fluorescence readings, it is necessary to convert those readings to micromoles of substrate turned over per time. The usual units for enzyme turnover (velocity) are umole/min/mL. In order to do this, you plot the standard (resorufin) at various concentrations (standard curve). Then you can use that curve to convert the relative fluorescence readings from the plate reader (or fluorometer) into umoles of resorufin produced. You calculate the rate by plotting turnover versus time, and then finding the initial velocity (which should be the tangent to these curves) at various substrate concentrations. Then you usually plot 1/v vs. 1/S as a Lineweaver-Burk plot which can give you the various parameters for the enzyme and substrate, like Km, Vmax, etc. Using the Lineweaver-Burk curve, you can also determine inhibition constants for various inhibitors, if they are added to the reaction.
⚛ Question: How do I calculate the respective activity of the enzyme (U/ml) from the raw data generated with the kit?
➸ Answer: In order to calculate the cellulase activity you should run a standard curve of purified enzyme along with your unknown samples from which you can plot concentration vs RFU. You can then use this graph to extrapolate the amount of cellulase activity based on the RFU of your unknown samples.
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