3-Cellobioside

C33H38Oi4 Methyl 2,3,6-Tri-0-acetyl-2,3 4,6-di-0-benzylidene-7(R)-/ -cellobioside (MACLBS)124... 

CJ3H24O11 CH4O Methyl 4-0-/J-D-gIucopyranosyl-/ -D-glucopyranoside, methanolate (methyl -cellobioside, methanolate) MCELOB 30 454... 

Fig. 57. Tris(2-ethylamino)amine (8) used as central core for the build up of poly-L-lysine cellobioside dendrimers. ... 

Phenyl l-thio-/S-cellobioside heptaacetate to 1,5-anhydro-4- (d-D-gluco-pyranosyl)-D-glucitol heptaacetate... 

Fig. 38.—13C-N.m.r. Spectrum of A, Partly Depolymerized O-Methylcellulose (d.s. 2.8) in CDC13 at 30° (R, signal due to reducing-end residue Me, O-methyl inset lines represent chemical shifts of corresponding carbon atoms in methyl hepta-O-methyl-jS-cellobioside) and of B, O-Methylcellulose (d.s. 0.7), Partially Degraded by Cellulase, in D20 at 30°. (S represents a 13C nucleus bonded to an OMe group inset lines give the chemical shifts of corresponding carbon atoms in methyl /3-cellobioside.)...

Clostridium thermocellum xylanase Z active on 4-methylumbelliferyl p-D-cellobioside 52... 

The MM2 model resides very near the minimum 2 in the cellobiose energy map (cf. Fig. 9). (Among others, the crystal structure of methyl cellobioside-methanol complex is found in that minimum (15)). On the other hand, the PS79 model resides on the shallow saddle point between minima 2 and 3. 

Figure 7. (Upper) C-1 signal of methyl fi-cellobioside-da (in DgO solution). The multiplet to the left that overlaps it partially is the C-1 signal. Computer-simulated C-1 signal is shown on the right. (Lower left), C-4 signal of methyl p-cello-bioside-ds and (lower right), computer-simulated C-4 signal.

Activity Measurements in Solution. The 2 -chloro, 4 -nitrophenyl / -D-glycosides offer an attractive alternative to classical reductometric methods. The substrate is sufficiently stable (pH 5.5, 50°C) and the favorable absorption characteristics of the liberated phenol (pK = 5.5, a/9000 M 1cm 1, pH 5.5 cm 16000 at pH 6.5) allow sensitive, continuous measurements. Kinetic parameters for some of these substrates and enzymes were determined K values were in the mM range for the lactoside (CBH I, EG I, EGD) and were at least 10 times lower for the cellobioside turnover numbers ranged from 1 (CBH I, cellobioside) to 300 min-1 (EG D, cellobioside) (25°C). 

Binding Experiments. Some of these chromophoric glycosides proved also to be valuable in cases where no hydrolysis by the cellulases occurred. This was shown in the case where the 4-methylumbelliferyl glucoside and cellobioside were not hydrolyzed by the CBH II from Trichoderma (see above) but could be used as reporter ligands in a series of binding experiments. Typically the fluorescence of the cellobioside was quenched in the presence of CBH II and was restored by the addition of excess amounts of non-chromophoric ligands, i.e., cellobiose (Fig. 3). Thus association constants... 

Coupling of an aryl 1-S-cellobioside to an affinity carrier was therefore expected to be useful in the chromatographic fractionation of endo and exo enzymes, e.g., from Tr. r. Preliminary tests indicated that CBH I and CBH II (prepurified by ion-exchange chromatography) were completely retained by the affinity support (4 -aminobenzyl 1-S-cellobioside coupled to Affigel-10 from Biorad). Desorption was achieved differentially by 0.1M lactose (elutes CBH I) and 0.01M cellobiose (elutes CBH I and CBH II). Attempts to elute the enzymes with 1M KC1, ethyleneglycol or glucose solutions were unsuccessful (11). 

Figure 4. Determination of binding constants of 2, 4 -dinitrophenyl 1-S-/ -cellobioside for CBH II by diafiltration (4°C).

Figure 6. Affinity chromatography of EGD from Clostridium thermocellum. Nucleic acid preparation, heat treatment and ammonium sulfate precipitation (0-70%, 70-100%) were carried out as described (10). The final precipitate ( 50 mg protein), dissolved in 50 mM sodium acetate, pH 5.0, was applied (after centrifugation) on the affinity column (2 x 25 cm) (4 -aminobenzyl l-thio-/ -cellobioside coupled to Sepharose 4B) (11). Protein was monitored at 280 nm and the activity of the fractions (2 ml) determined using 2 -chloro-4 -nitrophenyl / -cellobioside (pH 6.5, 25°C) as described in the text. Elution with 10 mM G2 was started as indicated.

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