Cellulases molecular weight

Three laccase preparations (I, II and III) were isolated from the racellular culture medium of Coriolus versicolor by consecutive fractionation through Sephadex G50 and DEAE Sephadex A25 (84). The laccase III preparation at pH 4.0 reduced the apparent molecular weight of a lignin-derived fraction that had been obtained by eluting the water-soluble extract of a cellulase treated ezomatsu wood residue through... 

Figure 14. Change (solid line) in apparent molecular weight distribution of water-soluble extract from a cellulase-treated ezomatsu wood residue (dotted line) at pH 4.0 brought about by the laccase HI preparation from C. versicolor, Sephadex GIO/H2O elution profiles adapted and redrawn from reference 85.

Elimination of Cellulases from Xylanases. Classical methods of protein fractionation can be used for to separate cellulases and xylanases on a large scale only when they differ considerably in molecular weight or isoelectric point. The Tricho-derma harzianum enzymes were separated by ultrafiltration because the xylanase was smaller and passed through the membrane into the ultrafiltrate 18). Fractional precipitation with organic solvents is another possibility (7). 

An indication of the validity of the extrapolation method could be given by light-scattering analysis of an intermediate-molecular-weight hydrolysate due to a very low cellulase activity. Indeed, at a relatively high HEC concentration of 3.152 X 10 3 g/mL, a limo-o Kc/Re value of... 

Fractionation Data and Distribution Analysis of HEC After One Hour of Cellulase Attack. The results of the gel chromatographic separation of HEC after one hour of enzymic hydrolysis are given in Table II. These fractionation data did not correspond to any of the distribution functions mentioned by Peebles (41) and by Tung (42). In the middle of the distribution it corresponded to the Lansing-Kraemer distribution functions, but deviations occurred at the low- and high-molecular-weight ends. 

The development of the sequential elution methods makes it possible not only to cleanly fractionate the three cellulase components, but to do the fractionation with very little loss of enzyme. The total recovery of major enzyme components, summarized in Table III, is considerably higher than those reported previously by other researchers (8,9). Table III also gives the molecular weights of the three enzyme components. 

Most of the mannan in the sprucewood holocellulose was hydrolyzed by the cellulase-mannanase treatments, and only about one-third of the xylan was dissolved and partly hydrolyzed into low-molecular-weight sugars. 

Physical Properties. All of the cellulase (CMCase) activity which develops in auxin-treated pea apices dissolves in salt solutions (e.g., phosphate buffer, 20mM, pH 6.2, containing 1M NaCl). Gel chromatography of such extracts indicates the presence of two cellulase components with similar levels of activity and elution volumes corresponding to molecular weights of about 20,000 and 70,000 (Figure 1). If the tissue is extracted with buffer alone, only the smaller cellulase dissolves (referred to as buffer-soluble or BS cellulase). The larger buffer-insoluble (BI) cellulase can then be extracted from the residue by salt solutions. This simple extraction procedure effectively separates the two cellulases, and can be used as an initial step for their estimation or purification. 

Figure 1. Elution profiles of cellulase activity from Sephadex G-100 gel chromatographs of crude extracts of auxin-treated pea apices. BS cellulose activity has an elution volume corresponding to a molecular weight of 20,000. BI cellulase activity dissolves in 1M NaCl and elutes with a molecular weight of 70,000. These values correspond to those observed for purified cellulases (3), indicating that the enzymes were not altered in molecular weight during purification, and could be effectively separated by differential extraction.

According to the International Union of Biochemistry, one enzyme international unit (IU) was defined as the enzyme strength which can catalyze 1 jumole of substrate per minute. In describing the activity of the cellulase, one IU is equivalent to the strength to release 1 /rmoles of glucose per minute, because the molecular weight of the substrate, cellulose polymer, is not well defined. 

An important parameter influencing the mode of action of cellulases is the accessibility of the cellulose to the enzymes. The molecular weights of cellulases range between 30 and 80 kDa. A comparison of the size of cellulase (3-8 nm) and the pore size of cotton swollen in water (1-7 nm) shows very clearly that cellulases can penetrate the cellulose to a limited extent only. In addition, the enzyme reaction takes place preferentially on amorphous cellulose because the more compact, crystalline cellulose structures do not offer any space for such macromolecules. Thus - provided of enzyme and process parameters have been selected correctly -cellulases act mainly on the textile surface. In this way interesting effects on cellu-losic fibers can be achieved. 

The cellulase enzyme was a commercial preparation made specifically for the National Renewable Energy Laboratory by Iogen (Ottawa, Ontario, Canada). It is supplied as a liquid solution, reported to contain 205 g/L of total soluble protein, of which 158 g/L is high molecular weight proteins retained by a 30,000-Dalton mol wt cutoff ultrafiltration membrane (20). 

Water-soluble polysaccharide species with higher molecular weights can be readily degraded by enzymic hydrolysis with a mixture of pectinase and hemi-cellulase to release the dRG-II complex [45]. The same mixture was found to be efFcient to extract the dRG-II D metal complexes from water-insoluble residues of vegetables, owing to the destruction of the pectic structure [45]. 

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