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Title: Cloning and characterization of a recombinant family 5 endoglucanase from Bacillus licheniformis strain B-41361

item Bischoff, Kenneth
item Liu, Siqing
item Hughes, Stephen

Submitted to: Process Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/3/2007
Publication Date: 5/1/2007
Citation: Bischoff, K.M., Liu, S., Hughes, S.R. 2007. Cloning and characterization of a recombinant family 5 endoglucanase from Bacillus licheniformis strain B-41361. Process Biochemistry. 42:1150-1154.

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, a gene for a cellulase enzyme was isolated from a thermophilic bacteria, expressed in a laboratory strain of bacteria, and the recombinant enzyme purified. The recombinant enzyme had some better properties than the native enzyme that was purified from the original thermophilic bacteria, including the ability to bind to cellulose and to partially degrade cellulose. Results will be valuable to researchers developing new enzymes to serve as biocatalysts in the conversion of agricultural residues to fermentable sugars.

Technical Abstract: The gene encoding a family 5 endoglucanase, cel5A, was cloned from the moderate thermophile Bacillus licheniformis strain B-41361. The primary structure of the translated cel5A gene predicts a 49 amino acid putative secretion signal and a 485 residue endoglucanase consisting of an N-terminal family 5 catalytic domain and C-terminal family 3 cellulose binding domain. The endoglucanase portion of the gene was expressed in Escherichia coli, but soluble activity in cell lysates was due to a truncated enzyme with an apparent mass of 42 kDa, the equivalent of the predicted catalytic domain. Insoluble protein renatured from inclusion bodies was protected against truncation, yielding an active holoenzyme (rCel5A) with apparent mass of 62 kDa. The recombinant rCel5A was optimally active at 65°C and pH 6.0, but retained only 10% activity after 1 h incubation at this temperature. At 55°C, rCel5A had a broad pH range for activity and stability, with greater than 75% relative activity from pH 4.5 – 7.0, and retaining greater than 80% relativity activity across the range pH 4.5 - 8.0 following one hour incubation at 55°C. It readily hydrolyzed pNPC, carboxymethylcellulose, barley beta-glucan, and lichenan, but despite strong binding to cellulose, had only weak activity against Avicel. Hydrolysis products from soluble polysaccharides included glucose, cellobiose, cellotriose, and cellotetraose. The catalytic properties, broad pH range and thermostability of the recombinant B. licheniformis endoglucanase may prove suitable for industrial applications.