Qualitative Display and Measurement of Enzyme Activity of Isolated Cellulolytic Bacteria A.S. Ponnambalam 1 , R.S. Deepthi 2 , A.R. Ghosh 2 1 Department of Biotechnology, Manipal Institute of Technology, Manipal University, Manipal, Karnataka, 576104, India; Email: setupathy78@gmail.com 2 School of Biosciences and Technology, VIT University, Vellore, 632014, Tamil Nadu, India ABSTRACT Cellulase is the key enzyme in the bio refinery process of producing green chemicals. As cellulase production cost is the major factor in these processes, there is lot of need to isolate the effective producers of cellulase from the natural environment which can degrade the abundantly available lignocellulose materials. Congo red clearing zone assay, gel diffusion assay and dyed congo red filter paper clearing zone assay are used for qualitative display of cellulolytic activity. These techniques are effective in isolation and identification of cellulolytic microorganisms. Six bacterial isolates are isolated and comparatively analysed for effective pro- ducer of cellulase enzyme. Among the six bacterial isolates, a bacterium F is found to be effective producer both qualitatively and quantitatively. It has the cellulolytic activity of 23.9 mg of glucose/ml/min and clearing zone of 1.9 cm compared to next effective producer having activity of 22.9 glucose/ml/min and clearing zone of 1.7 cm. This isolate may be useful in industrial application in near future. Keywords: cellulolytic bacteria, congo red assay, gel diffusion assay, cellulase INTRODUCTION Cellulose is considered as one of the most important sources of carbon on this planet and its annual biosynthesis by both plants and marine algae occurs at a rate of 0.85×10 11 tonnes per annum [1,2]. Cellulase degradation and its subsequent utilizations are important for global carbon sources. The value of cellulose as a renewable source of energy has made cellulose hydrolysis the subject of intense research and industrial interest. Cellulose may be hydrolyzed using enzymes to produce glucose, which can be used for the production of ethanol, organic acids and other chemicals [3]. An important impedi- ment in the exploitation of cellulose is the fact that the production of cellulase is expensive, contributing as much as 50 % to the overall cost of hydrolysis [1]. 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 [4]. The complete enzymatic hydrolysis of cellulosic materials needs different types of cellulose as endoglucanase (1,4-β-D-glucan-4-glucanohydrolase; EC 3.2.1.4), exocellobiohydrolase (1,4-β-D-glucan glucohydrolase; EC 3.2.1.74) and β-glucosidase (β-D-gluco- side glucohydrolase; EC 3.2.1.21) [5]. The endoglucanase randomly hydrolyzes the β-1,4 bonds in the cellulose molecule and the exocellobiohydrolases in most cases release a cellobiose unit showing a recurrent reaction from chain extremity. Lastly, the cellobiose is converted to glucose by glucosidase [6]. Some of the present applications of these complex cellulases are used in preparation of the minute rice by macerozyme, pharmaceutical formulations, detergent preparation, and fashion design in textile industry/fabric modification, food processing, brewing and paper and pulp industry. Although cellulase Research Article, Biotechnol. Bioinf. Bioeng. 2011, 1(1):33-37 © 2011 Society for Applied Biotechnology. Printed in India.