Screening Cellulase producers using Congo Red clearing zone assay

Cellulose is a polysaccharide which is made up of 100 to 1000 glucose unit linked together by glycosidic bond. Cellulose is the major component in plant cellwall. Cellulose can be obtained from wood pulp and is mainly used to produce paper. it is a renewable source of energy, Cellulose may be hydrolyzed using enzymes to produce glucose, which can be used for the production of ethanol, organic acids and other chemicals. Cellulose is hydrolyzed using enzyme cellulase, which cleaves cellulose into glucose units.

Cellulase enzymes produced chiefly by microbial sources, starting from prokaryotic organisms like bacteria, and protozoans to eukaryotic organisms, that catalyze the cellulolysis. However, there are also the cellulases produced by animal sources and plant materials. Cellulases are inducible enzymes which are synthesized by microorganisms during their growth on cellulosic materials, the screening of cellulase producing microbes can be done using congo red.

Congo Red Clearing Zone Assay

grow isolated microbes, which is needed to be screened for cellulase production in CMC agar, (carboxy methyl cellulose it is soluble form of cellulose) NaNO3, K2HPO4, KCl; MgSO4, yeast extract, glucose. The medium should be solidified using 1.7 % w/v agar., grow culture 2-3 days at 25-30^oC and the screening can be done using congo red clearing zone assay.

Congo red clearing zone assay is suitable for qualitative display of cellulase activity. After incubation plates which contain the circular batches of isolated microorganisms are flooded with 0.1% congo red solution and left for 15 min with intermittent shaking. Then destained with 1M NaCl solution. The clearing zone of enzymatic activity will be visible around the batch of growth. The NaCl solution elutes the dye in the clearing zone where the cellulose has been degraded into simple sugars by the enzymatic activity.

List of few cellulase producing microbes

Trichoderma koningii, Trichoderma reesei, Trichoderma viride, Aspergillus niger

Thermoactinomyces sp., Thermomonospora curvata

Cellulomonas sp.

References

Qualitative Display and Measurement of Enzyme Activity of Isolated Cellulolytic Bacteria

A.S. Ponnambalam1, R.S. Deepthi2, A.R. Ghosh

Research Article, Biotechnol. Bioinf. Bioeng. 2011, Society for Applied Biotechnology.

Cellulase production Kasing Apun, Sarawak, Malaysia, Practical Biotechnology, NCBE

http://www.fao.org/docrep/w7241e/w7241e08.htm