Cell Cellobiose

Fig. 10. A. Chromatogram of a standard mixture of sugars analysed under conditions (b) Table III. Five nanomole of each sugar injected. Peak identity 2-d-GAL = 2-deoxygalac-tose 2-d-RIB = 2-deoxyribose CELL = cellobiose RHAM = rhamnose MAL = maltose LAC = lactose R/B = ribose AfAAf = mannose FRt/C = fructose ARA = arabinose FCC = fucose GAL = galactose XFL = xylose MAN-//RR = mannoheptulose GLU-HEP = glucoheptose GLU = glucose.

Study of the structure of cellulose (Figure 22.2) leads one to expect that the molecules would be essentially extended and linear and capable of existing in the crystalline state. This is confirmed by X-ray data which indicate that the cell repeating unit (10.25 A) corresponds to the cellobiose repeating unit of the molecule. 

A monoclinic unit-cell with a = 8.2 A (820 pm), b(fiber axis) = 10.30 A (1.030 nm), c — 7.90 A (790 pm), and /3 = 83.3° is used. The distance between the terminal oxygen atoms in the cellobiose unit is taken to be 10.3912 A (1.03912 nm). A left-handed, helical structure, with seven cellobiose residues in a pitch of 72.1 A (7.21 nm) was proposed. The packing arrangement involves the central reversed and comer chains, and a relative shift between them of 0.25 repeat length along the b axis. 

Hydrolysis of delignified wheat straw up to 90% was obtained in 96 hours with the cellulase system produced in SSF with wheat straw. It took only 72 hours to obtain over 90% hydrolysis of wheat straw with cellulase system produced with SSF on Pro-cell (Table VII). It is interesting to note that the quantity of cellobiose in the hydrolysate obtained with the cellulase system produced on Pro-cell was higher than that of the cellulase system produced on wheat straw. This is consistent with the observation that cellulase system produced on Pro-cell had a lower ratio of FP cellulase )3-glucosidase (1 0.77) as compared to that produced on wheat straw (1 1.2). However, this cellulase system had a faster hydrolysis rate it took only 72 hours to obtain over 90% hydrolysis. This might be related to cotton hydrolyzing activity of this cellulase system (Table V). 

R. flavefaciens cells, during growth in pure culture, released cellulase, endoglucanase, and xylanase into the culture fluid. This microorganism hydrolyzed cellulose to yield only cellobiose as a product 49). It had been reported that a ceUobiose phosphorylase and glucokinase were present in R flavefaciens (8). The Ruminococcus cellulase system was repressed by disaccharides such as cellobiose, sucrose, and lactose 50). 

The induction of this operon responds to the intracellular concentration of cAMP, which is determined by the carbon source available to the cell. When cells are grown on cellobiose or cellulose that do not inhibit adenylate cyclase, cAMP is made in sufficient quantities for induction of cellulase. On the contrary, when cells are grown on glucose or other readily metabolized carbohydrates that do inhibit adenylate cyclase. 

Figure 5.10. Accumulation of a radiolabelled LMWP in the lysosomes of the proximal tubular cell. Electron microscope autoradiography of renal proximal tubular cells from a rat injected i.v. with [1251]-tyramine-cellobiose-labelled cytochrome-c, 4 h prior to fixation throngh the abdominal aorta. An intense lysosomal accumulation of the protein is observed in three dark electron-dense lysosomes. A few grains are seen over the apical endocytic apparatus. Part of the luminal brush border is found in the upper right hand corner. Magnification, x 25 000. Unpublished data from E. I. Christensen, Arhus, Denmark, and M. Haas, Groningen, Netherlands.

Mannitol has often been used as an osmotic regulator in the external solutions, and has been presumed to be inert. It was found to be a respiratory substrate in 15 of 26 species representing 17 families of higher plants, some of which were capable of utilization of mannitol that was equal to that of n-glucose and D-fructose. Oat (Avena sativa), most often used for the cell-wall studies, showed only a slight output of carbon dioxide from labeled mannitol. About 10% of the carbon in the mannitol was converted, with time, into the hemicellulose and cellulose fractions. Only the glucose, and, perhaps, the cellobiose, was labeled.4 ... 

The stimulation of the synthesis of the cellulase system of T. reesei QM 9414 by sophorose was established as shown by the results of experiments summarized in Table IV. Other than sophorose, of the glycosides and oligosaccharides tested, only lactose caused even a limited production of the enzymes of the cellulase system. Lactose is not as closely related structurally to sophorose as is, for example, the disaccharide laminaribiose it is more closely related structurally to cellobiose, which, despite being the major product of cellulose breakdown, does not promote enzyme production under the conditions of this experiment. It was noted that both intra- and extracellular constitutive enzyme levels produced by cells growing on glucose (or by resting cells without inducer, Table IV) are less than 0.5% of the fully induced levels and thus are negligible. 

All naturally occurring fungal strains of Trichoderma require an inducer for cellulase synthesis. In the absence of an inducer such as cellulose, cellobiose (21,22), or sophorose (12,13,14,23), Trichoderma does not make any detectable cellulase complex enzymes. The true physiological inducer of cellulase is currently unknown. Insoluble cellulose is presumably not such an inducer since there is no way for the internal cell machinery to sense the presence of this insoluble material. However, a small transglycosylation product such as sophorose, 2-0-/ -glucopyranosyl-D-glucose, may well be the natural inducer. 

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