Cellulase chromatography

Similar results were obtained with crude cellulase preparations from Penicillium pinophilum (12). The general applicability of this biospecific chromatography is illustrated by the isolation of the EGD from C. t., cloned in E. c. (Fig. 6). 

Our chromophoric substrates proved to be valuable in the study of several aspects of the enzymology of these cellulases. A rapid and specific method for purification (affinity chromatography) has been developed. Following our collaboration with several groups, new insights into the domain arrangement and tertiary structures of two cellulases were obtained. Contributions to the elucidation of the synergistic action (adsorption-hydrolysis) of these enzymes were achieved. 

The most intensive fractionation studies have been performed on the cellulase elaborated by T. koningii (5,6,9,16,26,27) and T. viride (8,10, 28-32). In a typical separation, normally by chromatography on DEAE-Sephadex, three protein peaks are obtained (Figure 1). The first and second contain Cx Ci is in the third. Cx and / -glucosidase are separated by chromatography on SE-Sephadex and Ci is purified by repeated chromatography on DEAE-Sephadex and by isoelectric focusing. 

As further evidence, we demonstrated by paper chromatography that hydrolysis products from cellooligosaccharides by Ex-1 are Gi and G2 from G3, and Gi, G2, and G3 from G5, but only G2 from G4, Ge, CMC, cellodextrine, and insoluble cellulose such as Avicel, swollen cellulose, absorbent cotton, and filter paper (Figures 13 and 14). However, G3 was formed from G6 when Ex-1 was incubated with a mixture of G6 and Gi. There is no indication that G6 was split by this cellulase into G3 plus G3, but rather that G2 produced from G6 was transferred immediately to Gi to form G3. The results are shown in Figure 15. 

The cellulase components that are synthesized in the presence of sophorose were investigated by the basic procedures previously described (1,2,4) for the isolation of cellulolytic components from commercial cellulase preparations. The purification to homogeneity of the proteins that yield the three predominant bands when the crude preparation is subjected to disc gel electrophoresis was accomplished by ion exchange chromatography. 

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. 

A number of relatively new methods are being investigated to improve the recovery of small molecules. These methods include elec-trokinetic separators with bipolar membranes, simulated moving-bed chromatography and supercritical fluid extraction. The latter is practiced for food components. It has also been described for proteins but has not yet found wide acceptance in this field. A fastgrowing field is the production of bioethanol via fermentation processes either from milled com or from recycled biomass. The fermentation and saccharification processes can occur simultaneously in the fermenting tank by means of saccharification enzymes (amylases, cellulases). 

Both the quantity and properties of cellulases produced by microorganisms depend on the culture conditions. Commonly, cellulases are produced by culture of the organism either (a) in a liquid medium, which may be stationary, shaken, or submerged with aeration, or (b) by a Koji process on a solid substrate such as wheat bran (7). The complexity of the crude cellulosic carbon source usually leads to the production of a mixture of hydrolytic enzymes which may include amylases, proteases, chitinases, etc., in addition to the cellulases. Separation of proteins from culture filtrates by high resolution techniques such as chromatography, electrophoresis, or electrofocusing often reveals a number of enzyme species which may differ in specificity toward cellulosic substrates. These forms may represent ... 

Cellobiase. Figure 2 outlines the general purification steps used to isolate a pure cellobiase (named for its function in the cellulase complex) and three forms of the hydrocellulase. In purifying cellobiase it was expedient to replace the adsorption or affinity column with a batch separation on DEAE-Sephadex A-50 and to complete the purification with cation exchange chromatography on SP-Sephadex (49, 50). Table IV is a summary of the purification of the cellobiase and co-purification of... 

Such recombination studies in our laboratory and others 17, 48, 54) have shown clearly the multi-enzymic character of the complete cellulase system. When recombined with fraction I enzymes, fraction II or fraction III enzymes from chromatography of the water fraction proteins on DEAE-Sephadex caused the degradation of hydrocellulose. 

Discrimination of Cellulase Components A, B, and C. The physical and chemical properties as well as substrate specificities of the highly purified cellulases of Ps. fluorescens have been characterized and are summarized in Table IV. Cellulase A is different from Cellulase B in the mobility on zone electrophoresis and in the pattern of Sephadex G-25 chromatography, but similar in substrate specificity toward several reduced and nonreduced cello-oligosaccharides. On the other hand, Cellulase C is different from A in the pattern of DEAE-Sephadex chromatography and the substrate specificity, and from B in all respects. These characteristics of each cellulase component are therefore different enough to be used as criteria to discriminate one from the other. 

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