Crystalline cellulose substrate

For a given cellulase, AvicePs ( ) was less than PASC s (j), suggesting less productive cellulase adsorption on more crystalline cellulose surface. With crystalline cellulose substrate, it could be more difficult for the cellulase to thread a cellulose chain into its active site clefl/tunnel, since more inter-chain H bonds would need to be broken in a highly cr3 talline region than in an amorphous/chain end region. 

Poly(HASCL) depolymerases are able to bind to poly(3HB)-granules. This ability is specific because poly(3HB) depolymerases do not bind to chitin or to (crystalline) cellulose [56,57]. The poly(3HB)-binding ability is lost in truncated proteins which lack the C-terminal domain of about 60 amino acids, and these modified enzymes do not hydrolyze poly(3HB). However, the catalytic domain is unaffected since the activity with water-soluble oligomers of 3-hy-droxybutyrate or with artificial water-soluble substrates such as p-nitrophenyl-esters is unaffected [55, 56, 58, 59]. Obviously, the C-terminal domain of poly(3HB) depolymerases is responsible and sufficient for poly(3HB)-binding [poly(3HB)-binding domain]. These results are in agreement ... 

Although its two domains could function independently, removal of the substrate-binding domain of ngCenA reduced enzymatic activity against microcrystalline cellulose but not against CMC or amorphous cellulose (12). This suggested that the substrate-binding domain played a critical role in the hydrolysis of crystalline cellulose. 

The problem of modeling the hydrolysis kinetics is complicated by the fact that cellulose is a solid substrate consequently, the reaction can be surface limited (5,12). Furthermore, some sites are more susceptible to hydrolysis—e.g., the amorphous regions as well as specific regions of the crystalline cellulose such as edges, corners, and dislocations. Several investigators (17,20,36) have suggested that the kinetic model should be based on a shrinking site model in which the number of susceptible... 

The relative influence of vibratory milling on the course of enzymatic and dilute acid hydrolysis of four cellulosic substrates was investigated. The four substrates—cotton linters, newsprint, Douglas fir, and red oak— were vacuum-dried and then milled for various time periods ranging up to 240 min. Assays were then made of rate and extent of hydrolysis, maximum yield of reducing sugar, and cellulose crystallinity. 

Fungal cellulase enzyme systems capable of efficiently catalyzing the hydrolytic degradation of crystalline cellulose are typically composed of endo-acting cellulases (EGs), exo-acting cellulases (CBHs), and at least one cellobiase (1-6). The CBHs are typically the predominant enzymes, on a mole fraction basis, in such systems (7). Consequently, the CBHs have been the focus of many studies (8). The three-dimensional structure of prototypical CBHs is known (9-12) and their specificities are, in general, well characterized (13,14). However, mechanism-based kinetic analyses of CBH-catalyzed cellulose saccharification are rather limited (15,16). Studies of this latter type are particularly difficult owing to the inherent complexity of native cellulose substrates. 

Cellulose is a partly crystalline and highly hydrogen-bonded substrate. Consequently it is much less accessible to grafting than starch (2). The present paper describes grafting experiments with the Mn -initiator applied to never-dried pulp fibers from wood and to fibers from cellulose derivatives of low degrees of substitution, i.e. cellulosic substrates known to be more accessible to chemical reactions than other cellulose fibers after drying. 

High concentrations of stabilizing salts such as NaCl and Na2S04 were reported to increase, and chaotropic salts to decrease, the affinity of bacterial and fungal cellulases to crystalline cellulose. Denaturing agents are capable of eluting cellulases from the cellulosic substrate (Otter et al., 1989). 

Glycol cleavage. The initial periodate oxidation of cellulose, like other chemical reactions, was largely limited to the readily accessible component and has also been used to indicate the accessibility of cellulose substrates [151] (Table 1). Rowland and Cousins [232], based on the influence of periodate oxidation in the crystallinity of cotton, observed about 40% of the component being noncrystalline. Since the m-diol unit is generally more reactive than the /ran.9-diol, the cleavage of the mannose residues would proceed faster than that of the glucose or xylose residues. 

The information which is available now regarding the nature of highly purified enzymes capable of degrading native cellulose or its derivatives is reviewed in this section. In purifying enzymes from others which have similar or overlapping specificities, extreme care must be taken to ensure the chemical and physical identity of each enzyme component to assess its role accurately in the multistep process leading from crystalline cellulose to glucose. This entails verification of physical purity by different techniques and use of an appropriate variety of substrates to define enzymatic function. 

As understanding of the components of the cellulase complex has increased, we can better define the mode of action by which native cellulose is depolymerized. The Ci-Cx model has directed attention to the specific steps in the crystalline cellulose breakdown. The use of nomenclature describing the catalytic function of each specific enzyme should encourage higher standards of purity. The ubiquity of the substrate invites imaginative applications of cellulases. 

The cellulase complex diffuses through the pore system to the microfibrils, attacks the cellulose chains and hydrolyses each chain to the end. The diflerences in the efficacy of cellulases on various fibres are dependent on number of factors such as the amounts of non-cellulosic wood pulp-derived matter, the degree ol polymerisation, the type and degree of crystallinity, and the type and number of chemical substitutions to the cellulose [27-30]. Key features for the cellulose substrate are crystallinity, accessible surface area and pore dimensions [31 ]. Variation of any of these factors, e.g., structural changes of cellulose substrate by pre-treatments, will influence the course of the entire degradation process [32, 33]. 

At the molecular level, portions of cellulose assume a highly structured crystalline form, while other parts are amorphous. Amorphous cellulose is more easily digested by enzymes than the crystalline parts. The fraction of crystalline cellulose is called the relative crystallinity, an index of digestibility. The crystallinity of commercially available substrates lies between 85 and 90%. Even so-called non-crystalline cellulose has a relative crystallinity of 65%. 

---