Saccharification, cellulose

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. 

The combined data provide a ready means by which to compare and select appropriate assays for application in cellulase-catalyzed cellulose saccharification experiments. Products in such experiments are expected to include glucose cellobiose and, potentially, some cellooligosaccharides. Optimum reducing sugar assays would have equivalent molar color yields for these soluble products. As shown in Table 3, this optimum situation only applies to the two copper-based assays (Nelson, BCA). Because of their importance with respect to the analysis of insoluble cellulose (discussed next), calibration curves reflecting the molar color yields for the DNS and BCA assays are presented in Fig. 1. 

Fig. 1. Absorbance response of equimolar amounts of soluble cellulose saccharification products glucose (G), cellobiose (G2), and cellotriose (G3) by (A) DNS assay and (B) BCA assay.

In this article, we report the cloning in Escherichia coli and sequencing of an Orpinomyces P-glucosidase cDNA. The enzyme was overexpressed in and secreted from the yeast Saccharomyces cerevisiae. Physiochemical properties of the secreted enzyme were determined after it was purified. Its applicability for cellulose saccharification was also assessed. 

In heated and milled cellulose, we have a material which is by far the best substrate yet found for cellulose saccharification, where the major goal is to obtain high glucose concentrations at a rapid rate. 

From the data on pure and crude cellulose saccharification presented in the paper it is evidenced that the initial enzymatic saccharification of cellulose is of pseudo first order and is therefore represented by... 

In the preceding methods, the enzyme is confined to small droplets, capsules, or inert carrier. Enlargement of a capsule to the size of a fermenter is theoretically possible, and this principle has been applied with practical modifications. The enzyme is free in solution as in the batch procedures, but an ultrafilter serves to separate the reaction products from the enzyme and substrate. Substrate is continuously pumped into the system, and product is removed by ultrafiltration to provide a continuous system. This method is applicable only to systems where the substrate is a large or an insoluble substance, so that it - with the enzyme - are retained inside the membrane. Successful demonstration of experimental runs on cellulose saccharification (Chose and Kostick, 1969) and on starch hydrolysis (Butterworth et al., 1969) have been made. Success depends upon the availability of suitable membranes and practical application on their cost. 

Dadi, A. P. Varanasi, S. Schall, G. A., Enhancement of cellulose saccharification kinetics... 

Millet, M.A., Baker, A.J., Scatter, L.D., 1976. Physical and chemical pretreatment for enhancing cellulose saccharification. Biotechnology and Bioengineering Symposium 6, 125—153. 

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