Cell-bound cellulases

Cellulolytic bacteria can be found which produce only cell-bound cellulase such as Cytophaga (12), only cell-free cellulase, such as CelMbrio vulgaris (21), Bacillus sp. (22), Clostridium sp. (23), Acetivibrio cellulofyticus (24), and Thermoactinomyces (25,26), and both cell-bound and cell-free cellulase such as Pseudomonas (27), Bacteroides succinogenes (28), and CelMbrio futvus (29). However, the location of cellulase in bacteria is also dependent upon the environments in which the bacteria are grown and the age of the culture (29,27). 

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

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. 

In regard to the cell-bound cellulase, however, no exact locality and function in the cell has been fully studied in general. The cells of Ps. fluorescens grown on 0.5% cellobiose were, therefore, fractionated principally according to the method of Burrous and Wood (5). At the same time, part of the same sample of cells was subjected to direct disintegration by sonic treatment and the sonicate was centrifuged to use as a standard for enzyme assays. The results are summarized in Table I. 

As clearly seen from Table I, most of the cell-bound cellulase was recovered in the fraction 4 designated as intrawall fraction. A low cellulase activity was found in cytoplasmic and membrane fractions, but it is uncertain whether these activities would be really derived from these fractions or caused by an incomplete resolution of these fractions. A similar experiment using cells grown on 0.5% sophorose indicated that about 80% of the cell-bound cellulase could be recovered in the intrawall fraction. 

The cell-bound cellulase of Ps. fluorescens could not be removed from the cell by several washings with the basal medium. Furthermore, cellulase released in the fluid obtained by cold osmotic shock of Heppel or cellulase in a fraction corresponding to the fraction 4 obtained by treatment with EDTA in 0.32M sucrose solution only amounted to about 20% of that released in the fraction obtained by lysozyme-EDTA-0.32M sucrose-treatment (fraction 4). Therefore, most of the cell-bound cellulase of this bacterium must reside in the cell-wall region and/or on the surface of cytoplasmic membrane and possibly adsorb thereon tighter... 

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. 

Figure 7. Purification steps of extracellular (Celluloses A and B) and cell-bound (Cellulase C) cellulases of Ps. fluorescens...

Two extracellular cellulases that act on carboxymethylcellulose have been isolated from the supernatant fluids of cultures of Sporocytophaga myxococcoid.es by gel-filtration and ion-exchange chromatography. More cellulase II (mol. wt. 5.2 X 10 by gel electrophoresis, p/ 4.75, pH optimum 5.5—7.5) was present than cellulase I (mol. wt. 4.6 x 10 , p/7.5, pH optimum 6.5—7.5). Both cellulases have very low contents of carbohydrate, possibly as impurities, and similar amino-acid compositions, and are endo-enzymes. A cell-bound cellulase was also partly purified. 

The release of cell-bound cellulase from mycelia of Rhizoctonia solani can be achieved, to various degrees, with salts and detergents, etc., although the enzyme was inactivated when sodium dodecyl sulphate was used. ... 

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. 

The simliar C-supply controlling culture was applied to Trichoderma viride, but no stimulated formation of cellulase was found in both cell-bound and extracellular fractions (unpublished data in our laboratory). 

Activities are represented as units per mg. of total protein in both extracellular and cell-bound enzyme preparations each figure is an average of values for three points selected from the maximal region of cellulase activity... 

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... 

Cell-surface BI cellulase is envisaged as the form which is active against cellulose in peas in vivo, with a function that may be constructive in that it can act synergistically with plasma membrane-bound / -glucan synthetase complexes to enhance the rate of cellulose deposition (7,8,9). BS cellulase never appears to reach the wall in vivo in a form recognized by a BS antiserum (II). BS cellulase does not even bind readily to wall material in homogenates (Table III) despite its ability to bind to cellulose (3) and hydrolyze it (Table I). It is possible that BS cellulase functions intracellularly to hydrolyze a noncellulosic organelle-bound polysac-... 

From a critical study of the metabolism of poly (A) in auxin-treated pea epicotyl, Verma and Maclachlan (73) showed that discreet classes of poly (A) (presumably part of mRNA s) are differentially associated with free and membrane-bound polysomes. The induction of specific mRNA s, the decline in the rate of synthesis of mRNA s, the polysome content per cell, and the formation of cellulase were all related to the membrane-bound polysomes. Although the rate of in vivo enzyme synthesis is... 

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