Changes in the molecular-size distribution of insoluble celluloses by the action of recombinant Cellulomonas fimi cellulases

Abstract

Specific patterns of attacks of cotton, bacterial cellulose and bacterial microcrystalline cellulose (BMCC) by recombinant cellulases of Cellulomonas fimi were investigated. Molecular-size distributions of the celluloses were determined by high-performance size-exclusion chromatography. Chromatography of cotton and bacterial celluloses revealed single major peaks centered over progressively lower molecular-mass positions during attack by endoglucanase CenA. In advanced stages, a second peak appeared at very low average size (approx. 11 glucosyl units); ultimate weight losses were approximately 30%. The isolated catalytic domain of CenA, p30, gave results very similar to those with complete CenA. CenA did not effectively depolymerize or solubilize BMCC significantly. Molecular-size distributions of cotton and bacterial cellulose incubated with endoglucanases CenB or CenD exhibited one major peak regardless of incubation time; low-molecular-mass fragments did not accumulate. Weight losses were 40 and 35% respectively. The single peak shifted to lower-molecular-mass positions as incubation continued, but high-molecular-mass material persisted. CenB and CenD readily attacked and solubilized BMCC (approx. 70%). We conclude that CenA attacks cellulose by preferentially cleaving completely through the cellulose microfibrils at the amorphous sites, and much more slowly by degrading the crystalline surfaces. Conversely, CenB and CenD cleave the amorphous regions much less efficiently while vigorously degrading the surfaces of the crystalline regions of the microfibrils.