Extracellular cellulase components

Purification and Physical and Chemical Properties. Extracellular cellulase components A and B and cell-bound cellulase component C were purified through the steps summarized in Figure 7 from the cultures of Ps. fluorescens on 0.5% Avicel and on 0.5% cellobiose, respectively. The purified cellulase components (Cellulases A, B, and C) thus obtained showed a single peak in zone electrophoresis on cellulose acetate film and starch bed. 

The point to be emphasized in relation to reports of multiple cellulases in plants or microorganisms, is that not all of these are necessarily functional components of an extracellular "cellulase complex that are needed for optimal or complete cellulose breakdown. Though all of the forms may show a capacity for hydrolyzing 3-1,4-linkages in vitro, in vivo they could function in different intra- or extracellular loci on different substrates, and some could represent processed forms of inactive precursors. In general, not enough is known about the mechanisms whereby these enzymes are synthesized and excreted to enable an informed decision to be made on the roles that they perform. 

Extracellular and Cell-bound Cellulase Components of Bacteria... 

Pseudomonas fluorescens produced two extracellular (A and B) and one cell-bound (C) cellulase components, the latter being released by treatment with EDTA-lysozyme in isotonic sucrose. Culture with 0.5% glucose formed little cellulase. Cellobiose stimulated only the synthesis of C. The formation of A and B was strikingly enhanced in cultures with cellulose, sophorose, or continuous low concentration of cellobiose. The absence of extracellular cellulase synthesis in 0.5% cellobiose culture may be caused by catabolite repression. The three cellulases were purified and characterized. None of them split cellobiose, but all hydrolyzed various cellodextrins and celluloses. C easily attacked cellotriose and cellotriosyl sorbitol, but A and B had no effect. When pure B was incubated with broken spheroplasts of sophorose-grown cells, a cellulase component indistinguishable from A was formed. 

Oince cellulose is insoluble high polymers under physiological condi- tions, cellulase which is destined to attack it has been expected to be an extracellular enzyme. In fact, most of cellulolytic microorganisms secrete some cellulase components into the culture medium, and almost all work on the cellulase have been performed using these extracellular components. In the cultures of cellulolytic bacteria, cellulases are not only found in their culture filtrates, but also are generally obtainable from the cells by treatment with autolytic agents—e.g., toluene (9, 19, 30). These facts indicate that at least certain components are existent within the bacterial cells and that their physiological function may be distinct from that of the extracellular components. 

Influences of glucose, cellobiose, sophorose, and cellulose, when they were each used as a C-source, upon the formation of both cell-bound and extracellular cellulases during the growth of this pseudomonad are shown in Figure 1. Glucose supported the bacterium for an excellent growth, but only slightly stimulated the formation of cellulases, and the enzymes produced were distributed almost equally to the cell and the culture medium. In the cellulose and sophorose cultures, the formation of cellulases, particularly that of extracellular component, was enhanced prominently (exo-type synthesis), whereas cellobiose which was a main end-product of enzymatic cellulolysis stimulated the formation of cell-bound component (endo-type synthesis). Thus, an apparent difference in the distribution of extracellular and cell-bound cellulases was noticed between the cultures with cellobiose and sophorose or cellulose. 

Electrophoretic properties of typical cellulase preparations, an extracellular cellulase from a culture on 0.5% cellulose and a cell-bound cellulase from that on 0.5% cellobiose, were compared in respect to their behavior in zone electrophoresis on cellulose acetate film. As shown in Figure 2, the former was separated into two components, A (fast moving to the cathode) and B (almost no moving). With the latter, a single component was detected under the same conditions. This fast moving component was in approximate agreement with component A in regard to its mobility, but as will be mentioned later, there was considerable difference in substrate specificity and other properties. Therefore, it seems to be a different component, and is referred to as component C. 

Recent observations by Carpenter and Barnett (7) have shown that membrane-bound ribosomes of Cellvibrio gilvus contained slightly higher cellulase activity than that which occurred in cytoplasmic ribosomes while the reverse relation was seen with the -glucosidase activities, thereby suggesting consideration of the biogenesis of these enzymes. To elucidate this problem, the exact characterization not only of extracellular and cell-bound cellulase components but also of the membrane-bound ribosomal cellulase appears to be most important, although much remains to be studied. 

Cellulase activities of Ps. fluorescens were shown to be located mainly in the extracellular and intrawall fractions, to which at least three cellulase components A, B, and C were distributed as already stated (Table I). They were located for different cultures using the discrimination procedures shown in Table IV. The results are summarized in Table V. 

All the extracellular cellulase preparations from various cultures contained only cellulase components A and B. On the other hand, the... 

Table V. Identification by the Discrimination Tests of Cellulase Components in Extracellular and Intrawall Fractions from Different Cultures of Ps. fluorescens...

The conversion of cellulase component B into A may be a result of some enzymatic modification of the enzyme molecule. Similar type of in vitro conversion has also been reported, for example, for the extracellular cellulase of Trichoderma viride (38) and the cell-bound invertase of bakers yeast (15). The occurrence of another type of conversion where the reversible association and dissociation of active subunits are operative, has been proven on the intrawall and extracellular invertases of Neurospora crassa (25). 

This bacterium produced three cellulase components A, B, and C. Of these, the components A and B occurred mainly in extracellular fraction or culture medium, and the component C was usually localized in... 

Fig. 4 Effects of the water soluble and insoluble components of wheat bran on growth and production of extracellular cellulase and xylanase by P. decumbens. The carbon sources were A 1% MCC plus 2% wheat bran B 1% MCC plus 2% wheat bran liquor C 1 % MCC plus 2% wheat bran residues D 3% MCC E 3% wheat bran F 3% wheat bran liquor G 3% wheat bran residues...

Biodeterioration of natural structural polymers has long been a significant problem regarding preservation of many commercial products. Depolymerization of cellulose and lignocellulose are well known processes found to be mediated by extracellular cellulases and "lignases". The former is well studied and exploited commercially for cellulose modification. The lignin structural component is less well defined, since its structure varies as a function of biological source. 

The anaerobic biological conversion of the major polymeric components of MSW identified require appropriate microorganisms and hydrolytic enzyme systems. Extracellular hydrolytic enzymes, such as cellulases and lipases, have been shown to be effective in the post hydrolysis of anaerobic digester efQuent solids 34) or pretreatment of complex organic polymers before the digestion process 48),... 

Component enzymes of the cellulase system have been purified from several microbial species (1-13), among which mutants of the imperfect fungus Trichoderma provide the highest levels of extracellular enzyme activity (14). From this organism have been purified / -glucosi-dases (EC 3.2.1.21), endo-l,4-/ -D-glucanases (EC 3.2.1.4) and 1,4-/ -d-... 

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