Abstract
BackgroundThere is still considerable debate regarding the actual mechanism by which a “cellulase mixture” deconstructs cellulosic materials, with accessibility to the substrate at the microscopic level being one of the major restrictions that limits fast, complete cellulose hydrolysis. In the work reported here we tried to determine the predominant mode of action, at the fiber level, of how a cellulase mixture deconstructs pretreated softwood and hardwood pulp fibers. Quantitative changes in the pulp fibers derived from different pretreated biomass substrates were monitored throughout the course of enzymatic hydrolysis to see if the dominant mechanisms involved either the fragmentation/cutting of longer fibers to shorter fibers or their “peeling/delamination/erosion,” or if both cutting and peeling mechanisms occurred simultaneously.ResultsRegardless of the source of biomass, the type of pretreatment and the chemical composition of the substrate, under typical hydrolysis conditions (50°C, pH 4.8, mixing) longer pulp fibers (fiber length >200 μm) were rapidly broken down until a relatively constant fiber length of 130 to 160 μm was reached. In contrast, shorter fibers with an initial average fiber length of 130 to 160 μm showed no significant change in length despite their substantial hydrolysis. The fragmentation/cutting mode of deconstruction was only observed on longer fibers at early stages of hydrolysis. Although the fiber fragmentation mode of deconstruction was not greatly influenced by enzyme loading, it was significantly inhibited by glucose and was mainly observed during initial mixing of the enzyme and substrate. In contrast, significant changes in the fiber width occurred throughout the course of hydrolysis for all of the substrates, suggesting that fiber width may limit the rate and extent of cellulose hydrolysis.ConclusionIt appears that, at the fiber level, pretreated pulp fibers are hydrolyzed through a two-step mode of action involving an initial rapid fragmentation followed by simultaneous swelling and peeling/erosion of the fragmented fibers. This latter mechanism is the predominant mode of action involved in effectively hydrolyzing the cellulose present in pretreated wood substrates.
Highlights
There is still considerable debate regarding the actual mechanism by which a “cellulase mixture” deconstructs cellulosic materials, with accessibility to the substrate at the microscopic level being one of the major restrictions that limits fast, complete cellulose hydrolysis
Despite the discovery and addition of auxiliary enzymes and proteins such as lytic polysaccharide monooxygenase and Swollenin, effective cellulose hydrolysis is still primarily achieved by the cooperative interaction of beta-1,4-endoglucanases, beta-1,4-exoglucanases/cellobiohydrolases, and beta-glucosidases [2]
A range of pretreated pulp fiber fractions was first created in order to determine the mechanism by which a “cellulase mixture” hydrolyzes pretreated pulp fibers at the macromolecular level and how dependent the predominant mechanisms are on the type of substrate and its physicochemical characteristics
Summary
There is still considerable debate regarding the actual mechanism by which a “cellulase mixture” deconstructs cellulosic materials, with accessibility to the substrate at the microscopic level being one of the major restrictions that limits fast, complete cellulose hydrolysis. Some workers using “model/pure” cellulose substrates have suggested that the initial mode of enzymatic attack takes place on the outer layer of the cellulose surface where the constituent fibers are peeled along their length, layer by layer, in an “onion peeling” fashion [4,5,6,7,8]. It has been suggested that cellulose deconstruction is a two-step process where the cellulose-rich fibers are initially fragmented or disaggregated into shorter fibers, resulting in a greater overall surface area for the enzymes to subsequently attack [9,10,11,12] This “cutting” mode of deconstruction has been termed a “fragmentation” or “scissoring” mode of action. Other work that has used “model/pure” cellulosic substrates has suggested [13,14] that the dominant mode of deconstruction was strongly influenced by the fiber dimensions of the initial substrate, with the larger particles first fragmented/cut into smaller particles (“cutting”) while the smaller particles seemed to be hydrolyzed by a “peeling/erosion” type of mechanism
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