ARS | Publication request: Purification and Characterization of a Family 5 Endoglucanase from a Moderately Thermophilic Strain of Bacillus Licheniformis

Submitted to: Biotechnology Letters
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 1, 2006
Publication Date: August 10, 2006
Citation: Bischoff, K.M., Rooney, A.P., Li, X., Liu, S., Hughes, S.R. 2006. Purification and characterization of a family 5 endoglucanase from a moderately thermophilic strain of Bacillus licheniformis. Biotechnology Letters. 28:1761-1765.

Interpretive Summary: New enzymes that function under harsh industrial conditions of extreme temperature and pH are needed to help overcome some of the technical barriers to using agricultural residues as feedstocks for fuel ethanol production. Thermophilic bacteria are microorganisms that grow at high temperatures and may possess robust enzymes useful to the fermentation industry. In the present study, isolates of thermophilic bacteria were screened for the production of cellulases, enzymes that degrade cellulose to sugars that ethanol-producing yeasts can use for growth. One strain was found to produce a cellulase enzyme, and the enzyme was purified and characterized. The enzyme was stable at high temperatures and was active over a broad range of pH, but its activity against natural cellulose was weak. This enzyme may serve, however, as a platform for future studies to improve its activity against natural cellulose while retaining its desirable temperature stability and pH properties. Results will be valuable to researchers developing new enzymes to serve as biocatalysts in the conversion of agricultural residues to fermentable sugars.

Technical Abstract: Strains of thermophilic bacilli were screened for cellulolytic activity by gel diffusion assay on selective medium at 55°C. Strain B-41361, identified as a strain of Bacillus licheniformis by rDNA analysis, displayed endoglucanase (EG) activity against carboxymethylcellulose (CMC). Zymogram analysis of cell-free culture supernatants demonstrated several catalytically active EG polypeptides with the most prominent species having a mass of 37 kDa. The 37 kDa EG was purified 59-fold with a 17% yield and specific activity of 183 U mg**-1 protein. The optimal temperature was 65°C, but the enzyme was most stable at 60°C. The EG had a broad pH range, with maximal activity at pH 6.0, 75% maximal activity at pH 4.5, and 40% at pH 10. The EG hydrolyzed p-nitrophenylcellobioside, barley beta-glucan, and lichenan, but no activity was detected against avicel or acid-swollen cellulose. The amino terminal sequence of the B-41361 enzyme was homologous to members of glycoside hydrolase family 5.