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.

A mechanistic model for rational design of optimal cellulase mixtures

“…BMCC is one of the most homogeneous cellulosic substrates available with a moderately high DP (Kleman-Leyer et al, 1994;Valjamae et al, 1999) and an open ribbon structure leading to high surface area (Hong et al, 2007). This system was chosen to demonstrate the rational design approach because it minimizes the impact of substrate surface heterogeneity on cellulase activity.…”

β-1,4-Glycanases of Cellulomonas fimi: Families, Mechanisms, and Kinetics

“…C.fimi enzymes exhibit different modes of attack on these substrates (59). CenA, CenB and CenD have similar activities on cotton and all three enzymes decrease the DP of the residual insoluble substrate progressively.…”

Toward an aggregated understanding of enzymatic hydrolysis of cellulose: Noncomplexed cellulase systems

“…During enzymatic hydrolysis, cellodextrins with DP > 4 are present in the solid phase associated with crystalline cellulose (Kleman-Leyer et al, 1994Srisodsuk et al, 1998;Stalbrand et al, 1998), and it has been suggested that this association impedes release of such cellodextrins to solution. However, cellodextrins with DP > 4 are not found associated with amorphous cellulose (Stalbrand et al, 1998).…”