PURIFICATION OF CELLULOSE-BINDING PROTEINS 1 AND 2 FROM CELL LYSATE OF FIBROBACTER SUCCINOGENES S85

Abstract

Attempts were made to separate and purify cellulose-binding proteins (CBPs) from the lysate of cells of Fibrobacter succinogenes S85, utilizing their affinity to cellulose. After Avicel cellulose was incubated in the culture treated with Tween 20 at 37°C, cellulose was washed with buffer containing Softes 12 as a detergent to remove the cell debris and proteins incapable of adhering to cellulose. Proteins strongly adhering to cellulose after washing were eluted with buffer containing 10% cellobiose. The eluent contained mainly two major proteins and one minor protein. When the eluent, concentrated by ultrafiltration, was dialyzed against distilled water, small particles of aggregate appeared in it. The dialyzed eluent was separated into soluble and insoluble fractions by high speed centrifugation. Proteins present in both the soluble fraction and the solution of the insoluble fraction dissolved in buffer containing Softes 12 were purified by treatment with a saturated solution of ammonium sulfate. Protein purified from the soluble fraction was designated as CBP1 and protein from the insoluble fraction as CBP2. The molecular weight of CBP1 and CBP2 was approximately 120 and 225 kilodaltons (kDa), respectively. The culture containing a cell lysate of the bacterium exhibited such enzyme activities as β-glucosidase, cellobiosidase and CMCase, whereas both CBP1 and CBP2 did not show these enzyme activities. Both CBP1 and CBP2 displayed adhering ability to cellulose powder, but not to starch granules.