Characterization of a Bacillus cereus strain and identification of its cellulolytic activity
A Bacillus cereus strain was confirmed to have cellulolytic activities on a cellulose substrate. The genome structure, the secreted proteomes from the bacteria and the biochemical activities in degrading cellulosic materials were analyzed. The whole genome was sequenced using next generation sequencing, and contigs were assembled using the Velvet program. The assembled DNA contigs were translated into proteins, and 3,500 proteins were predicted. By searching functional domains in NCBI database, interesting proteins were identified which include cell wall hydrolase, 6-phospho-beta-glucosidase, and xylose isomerase, and many others. For extracellular protein identification, bacteria were cultured in a M9 minimum broth supplemented with carboxymethyl cellulose sodium salt (CMC). Proteins secreted into the broth were purified and then separated on SDS-PAGE gels, which were stained with Bio-Safe Coomassie G250 stain. Protein bands containing highly abundant proteins were isolated from the gel followed by in-gel-trypsin-digestion. Proteins contained in those bands were identified using nano-mass spectrometry analysis. Gram and endospore stains were used to identify the cell structure and endospore morphology. Based on protein and genomic DNA sequences, Gram and Endospore staining reaction, the bacterium was confirmed to belong to Bacillus cereus. For cellulolytic activity analysis, bacterial stock was inoculated in M9 mineral medium, supplemented with soluble CMC or microcrystalline cellulose respectively. Gel permeation chromatography was used to determine the molecular mass changes in filtered supernatant. Results showed that there was a continuous decrease of high molecular mass in soluble CMC media with extended treatment time, which was not shown in control samples. These results indicate that the bacteria were able to degrade soluble cellulose into smaller molecules. For the microcrystalline cellulose, it did not show apparent decrease in high molecular mass, further work is still undergoing.
"Characterization of a Bacillus cereus strain and identification of its cellulolytic activity"
ETD Collection for Tennessee State University.