Cell-bound cellulase components

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

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. 

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. 

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. 

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. 

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). 

The scheme proposed above requires microbial colonization of the material and excludes degradation by amylases and cellulases that are present in soils (28), but are not newly synthesized or associated with microbial cells. Active polysaccharide hydrolases are found in nearly all soils, but these enzymes are primarily bound to soil organic matter or mineral components attachment is firm enough to severely limit migration of the enzymes from surrounding soil to the film. 

Cell walls, total membrane-bound components, and ribosomes were separated and assayed for cellulase activity to study the subcellular localization of the enzymes as follows. Segments (approx. 5 g fresh wt) were ground in two volumes of extraction medium containing 0.4M sucrose (ribonuclease-free), 5mM Mg acetate, lOmM Tris-HCl (pH 7.5 at 22°C), 20mM KC1 and 5mM / -mercaptoethanol. The brei was filtered and the filtrate centrifuged at 500 Xg for 20 min. The post-500 Xg supernatant was fractionated essentially as previously described (28). Aliquots (7 mL) of the supernatant were layered on a discontinuous gradient composed of 2 mL 70% (w/v) sucrose and 3 mL 15% (w/v) sucrose both in lOmM Tris-HCl (pH 7.5 at 22°C), lOmM KC1, 2.5mM Mg acetate and ImM / -mercaptoethanol. The tubes were centrifuged at... 

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