Screening and Identification of Cellulase Producers
Five microlitres of overnight grown culture was spot plated on CMC agar (0.2% NaNO3, 0.1% K2HPO4, 0.05% MgSO4, 0.05% KCl, 0.2% carboxymethylcellulose (CMC) sodium salt, 0.02% peptone, and 1.7% agar) (HiMedia, India). Plates incubated at 28ºC for 48 hours were flooded with 1% hexadecyltrimethyl ammonium bromide (HAB) for 30 to 40 minutes [6]. From cellulase-producing microorganisms, two potential bacterial strains RK3 (Gram +ve) and MN34 (Gram –ve) and one fungal strain RS2 were selected for identification and further studies. Genomic DNA from bacterial strains was isolated and amplified by using universal 16S rRNA primers [9], and fungal DNA was isolated and amplified by using internal transcribed spacer 1 (ITS 1) and internal transcribed spacer 4 (ITS 4) primers [15]. The amplified gene products were purified from agarose gel using Qiaquick Gel Extraction Kit (Qiagen, Germany). Nucleotide sequencing of the genes was done by using Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems) and 3130xl Genetic Analyzer (Applied Biosystems). The BLASTN program (http://www.ncbi.nlm.nih.gov/BLAST/; National Center for Biotechnology Information, Bethesda, MD) was used for homology searches with the standard program default.