Cellulose enzymatic hydrolysis

Environment Canada, 18 542 23 120 Enzymatic cellulose hydrolysis, 26 359 Enzymatic hydrolysis, 10 503, 535-536 Enzymatic methods, in sugar analysis, 23 475-476... 

Further progress in understanding this effect, as well as others, of the physical structure of cellulose on enzymatic degradation may be expected from combining physicochemical and morphological techniques and from kinetic measurements in heterogeneous enzymatic hydrolysis, applying substrates of well-defined physical structure and isolated components of the enzyme systems. 

To illustrate the procedure for the development of kinetic models of the cellulose hydrolysis, let s examine the model proposed by Ryu et al. (1982). A kinetic model for the enzymatic hydrolysis of cellulose was derived based on the following assumptions ... 

A 72-h hydrolysis profile of a 10% acetic acid-pretreated softwood substrate (Fig. 1) represents a typical enzymatic cellulose hydrolysis course with the majority of the cellulose (up to 70%) broken down within the first 24 h. However, the conversion of the remaining cellulose ( 30%) was incomplete, even after another 2 d of incubation. The decrease in the hydrolysis rate in the latter phase is likely owing to accumulation of end products. To demonstrate that the end products played a major inhibitory role, we removed the produced sugar from the hydrolysate through ultrafiltration. Fresh buffer was then added to the retained protein and the residual substrate to attain the initial volume, and the hydrolysis was continued under the same condition. As shown in Fig. 1, significant increases in the hydrolysis rate were observed after the sugar removal at both 24 h and 48 h of incubation, with complete hydrolysis attained after 48 h and 60 h of incubation respectively. 

When cellulose is used as a raw material, the activity of cellulase (the enzyme catalyzing cellulose hydrolysis) is inhibited by glucose and short cellulose chains. One way to overcome this inhibition is to combine enzymatic hydrolysis with glucose fermentation to ethanol, as the accumulation of ethanol in fermenter does not inhibit cellulase. 

Figure 10. Data on the heterogeneous acid hydrolysis, enzymatic hydrolysis, and photochemical decomposition of regenerated cellulose. (Reproduced with permission from Ref. 16. Copyright 1966 TAPPI.)...

Gan Q, Allen SJ, and Taylor G. Design and operation of an integrated membrane reactor for enzymatic cellulose hydrolysis. Biochem Eng J, 2002 12(3) 223-229. 

Enzymatic action can be defined on three levels operational kinetics, molecular architecture, and chemical mechanism. Operational kinetic data have given indirect information about cellulolytic enzyme mode of action along with important information useful for modeling cellulose hydrolysis by specific cellulolytic enzyme systems. These data are based on measurement of initial rates of enzyme hydrolysis with respect to purified celluloses and their water soluble derivatives over a range of concentrations of both substrate and products. The resulting kinetic patterns facilitate definition of the enzyme s mode of action, kinetic equations, and concentration based binding constants. Since these enable the enzymes action to be defined with little direct knowledge of its mechanistic basis, the rate equations obtained are referred to as operational kinetics. The rate patterns have enabled mechanisms to be inferred, and these have often coincided with more direct observations of the enzyme s action on a molecular level [2-4]. 

Because free cellulose chain ends are so difficult to quantify, other measurable cellulose characteristics have been shown to be factors correlating to hydrolysis susceptibihty and rate [15,18]. These factors affect how well the cellulose-binding domain, CBD, adheres to the cellulose and how well the catalytic domain acts on the cellulose. Because of what can and cannot be measured easily, models of enzymatic cellulose hydrolysis combine adsorption behavior with kinetic action. 

The susceptibility of cellulose to enzymatic hydrolysis is determined largely by its accessibility to extracellular enzymes secreted by or bound on the surface of cellulolytic microorganisms. Direct physical contact between these enzymes and the cellulosic substrate molecules is an essential prerequisite to hydrolysis. Since cellulose is an insoluble and structurally complex substrate, this contact can be achieved only by diffusion of the enzymes from the organism into the complex structural matrix of the cellulose. Any structural feature that limits the accessibility of the cellulose to enzymes by diffusion within the fiber will diminish the susceptibility of the cellulose of that fiber to enzymatic degradation. In this review, the influence of eight such structural features have been discussed in detail. 

Size and Diffusibility of Cellulolytic Enzymes in Relation to the Capillary Structure of Cellulose. As discussed earlier, enzymatic degradation of cellulose requires that the cellulolytic and other extracellular enzymes of the organisms diffuse from the organism producing them to accessible surfaces on or in the walls of the fiber. This accessible surface is defined by the size, shape, and surface properties of the microscopic and submicroscopic capillaries within the fiber in relation to the size, shape, and diffusibility of the enzyme molecules themselves. The influence of these relationships on the susceptibility and resistance of cellulose to enzymatic hydrolysis has not been verified experimentally in natural fibers but the validity of the concepts that follow is demonstrated by the work of Stone, Scallan, Donefer, and Ahlgren (69). 

In addition to considerations of size and shape, the surface properties of the fiber capillaries and the diffusibility of the cellulolytic enzyme molecules within them can profoundly influence the susceptibility of cellulose to enzymatic hydrolysis. In contrast to inorganic catalysts, enzymes have a very strong and specific affinity for their specific substrate molecules. This affinity accounts for their susceptibility to competitive inhibitors. When the substrate exists as an insoluble polymer in a complex structural matrix, this specific affinity drastically reduces the rate of diffusion of the enzyme in the presence of the substrate. This retarded... 

Degree of Crystallinity. The influence of degree of crystallinity on the susceptibility of cellulose to enzymatic hydrolysis has been studied by Norkrans (48), Walseth (74, 75) and Reese, Segal, and Tripp (57), among others. Using several cellulose samples precipitated after swelling in phosphoric acid, Walseth (74) showed that the preparations with... 

Thus, DP of itself probably is of limited significance in determining the susceptibility of cellulose to enzymatic hydrolysis except in the relatively rare case of enzymes that act by an endwise mechanism. 

The Nature of the Substances with Which the Cellulose Is Associated and the Nature of That Association. As discussed earlier in this review, cellulose in cotton and wood is associated with a variety of other materials. These substances frequently have an influence on the susceptibility of the cellulose to enzymatic hydrolysis. The influence of several different types of material will be discussed in turn. 

Figure 2. Effect of various treatments on the susceptibility of cotton cellulose to enzymatic hydrolysis (16)...

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