Extracellular cellulases

RunUnococcus albus and Ruminococcus flavefadens. These bacteria are important cellulose-degraders found in the rumen of cattle and sheep (2). Most isolated strains ferment cellulose and xylan and all ferment cellobiose. Fermentation of glucose and some other carbohydrates depends on the particular strain. R flavefadens and B. succinogenes can ferment the highly ordered crystalline cellulosic su trates but R albus cannot. No evidence has been found for extracellular cellulase production by R albus, but Ohmiya et al. purified cellobiosidase from this culture 17). Laboratory growth of R albus has been conducted at pH 7.0 and 37 C. 

T. reesei is a useful experimental organism for studying regulation of extracellular protein biosynthesis. When grown in a medium in which an exogeneous inducer serves as the major or sole carbon source, T. reesei, synthesizes and secretes a cellololytic enzyme into the medium. Similarly, the extracellular cellulase is produced upon limitation of the carbon source and limitation of the utilization of the carbon source (31). Presently, there appears to be little data in the literature concerning regulation of cellulase biosynthesis. 

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. 

By using both enzymes in combination, the yield of crystalline D-glucose is increased.100 The use of cellulosic wastes for the production of glucose is of commercial interest, and in consequence much research work has been done on the characterization of cellulases. In particular, the cellulase system of the fungus T. ressei has the full complement of enzymes required to degrade crystalline cellulose. This and other fungal extracellular cellulases are commercially available.101... 

It should be emphasized that these phosphorolytic enzymes are intracellular. They are not found in the extracellular cellulase complex, and they are involved in the cellulolytic systems of relatively few microorganisms. 

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. 

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. 

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

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 experiments not described in this paper revealed that a relative amount of extracellular cellulase was increased with decreasing concen-... 

Fig. 1 Effects of starch and wheat bran on growth and production of extracellular cellulase, xylanase and amylase by P. decumbens. Cultures were grown and the levels of biomass and extracellular enzymes were measured as described in the Materials and Methods section. All cultures contained 1% MCC plus as additional carbon sources A 2% wheat bran B 0.7% wheat starch plus 1.4% wheat bran C 1% wheat starch plus 1% wheat bran D 1.4% wheat starch plus 0.7% wheat bran and E 2% wheat starch...

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...

Effects of Cello-oligosaccharides on Production of Extracellular Cellulase and Xylanase... 

Table 1 Effects of cellobiose and cello-oligosaccharides supplements on growth and production of extracellular cellulase and xylanase (enzyme activities per gram mycelia mass).

Cavedon, K., Leschine, S. B., and Parola, E. C. (1990). Characterization of the extracellular cellulases from a mesophlllc Clostridium (strain C7),... 

In direct microbial conversion of lignocellulosic biomass into ethanol that could simplify the ethanol production process from these materials and reduce ethanol production costs, Clostridium thermocellum, a thermoanaerobe was used for enzyme production, hydrolysis and glucose fermentation (755). Cofermentation with C thermosaccharolyticum simultaneously converted the hemicellulosic sugars to ethanol. However, the formations of by-products such as acetic acid and low ethanol tolerance are some drawbacks of the process. Neurospora crassa produces extracellular cellulase and xylanase and has the ability to ferment cellulose to ethanol 139). 

The synthesis of extracellular cellulase activity by three strains of Clostridium thermocellum growing on cellulose has been shown to be directly related to the degradation of cellulose and to the growth of the bacteria. Cellulase was not detected when the cells grow on cellobiose. The activities of the exo-D-glucanase (pH optimum 5.2, temperature optimum 65 °C) and e do-D-glucanase (pH optimum 5.4, temperature optimum 64 °C) were compared. o-Glucose and cellobiose were released when the crude cellulase acted on cellulose, cellotetraose, and cellotriose in vitro. 

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. 

Three extracellular cellulases have been purified from the cultures of a Cellulomonas species. One was found in solution in the cell-free supernatant and two others were found to be bound to the cellulose added as a carbon source. The free enzyme and one of the cellulose-bound enzymes exhibited binding for Sephadex . The two cellulose-bound enzymes are glycoproteins. All three enzymes behaved as ewffo-cellulases towards soluble carboxymethylcellulose and had little activity on cellulose powder. 

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