Cellobiose measurement

Fig. 6. Cellulose conversion over time for the same experiments as shown in Fig. 5. The data represent cellulose conversions based on glucose production alone. Cellobiose measurements taken at the end of the experiments account for an additional conversion of about 4%. Error bars represent averages 1 SD for three repeated experiments.

Interproton distances of 0-ceIIobiose (see Ref. 49) error 0.01 A. Interproton distances of 1,6-anhydro- -D-glucopyranose (see Ref. 49) error 0.01 A. Interproton distances of -cellobiose octaacetate (see Ref. 49) error 0.05 A. Interproton distances of 2,3,4-tri-0-acetyl-l,6-anhydro- -D-glucopyranose (see Ref. 49) error 0.05 A. Error calculations based on the errors of the measured quantities in Eqs. 18 and 21. Interproton distances calculated from the relaxation parameters of the methylene protons. 

A different kind of host consisting of a peptide-based bicyclic structure has been described.118 In this case, the chemical shifts changes were followed by HSQC spectra in deuterated acetic acid and in water, when titrated with cellobiose. In any case, a low but measurable binding affinity constant was found. 

The torsion angles predicted by conformational analysis agree closely with those of crystalline cellobiose as measured by X-ray diffraction, the conformation of which is restricted by two chain-stabilising intramolecular hydrogen bonds between 0(3 )-H and 0(5) and also between 0(2 )-H and 0(6) (Figure 4.3). These are also found in cellulose and they assist in maintaining the highly extended conformation which allows it to function as a structural polymer. 

Figure 6 depicts the relationship between the specific enthalpy of denaturation measured for CBH I at each of the pH values used in this study, and the of the principal peak at that pH. The strai t line represents a least>squares best fit to the four experimental data points and the empirically derived intersection point (Reference 2, see Discussion) in the upper right comer. All the values determined in the absence of cellobiose, both those at pH values at which the denaturation exhibits a substantial degree of overall reversibility, and those at which the overall process is completefy irreversible, are in reasonably good agreement with the linear relationship. 

Figure 6. Specific enthalpy of denaturation for native CBH I, plotted as a function of the overall observed as the enzyme molecule is progressively destabilized by increasing the pH. Dot-centered circles represent the specific enthalpy in the absence of cellobiose the straight line is a linear least-squares best fit to these data points, plus the empirically derived intersection point (reference 2, see Discussion) represented by the crossed circle at upper right. The squares represent enthalpies measured at pH 4.80 and pH 8.34 in the presence of the indicated concentrations of cellobiose.

TGA analysis shows that polymer degradation starts at about 235°C which corresponds to the temperature of decomposition of the cellobiose monomer (m.p. 239°C with decom.). Torsion Braid analysis and differential scanning calorimetry measurements show that this polymer is very rigid and does not exhibit any transition in the range of -100 to +250 C, e.g. the polymer decomposition occurs below any transition temperature. This result is expected since both of the monomers, cellobiose and MDI, have rigid molecules and because cellobiose units of the polymer form intermolecular hydrogen bondings. Cellobiose polyurethanes based on aliphatic diisocyanates, e.g. HMDI, are expected to be more flexible. 

Figure 3. Quenching of the fluorescence spectrum of MeUmbG2 by CBH II from Trichoderma reesei (5). Curve A represents the MeUmbGj (2 /iM) spectrum in the absence of CBH II. Curves B, C and D show the spectra after the addition of several aliquots of 137.7 /iM CBH II. Spectrum D changes to E when solid cellobiose ( 2 mg) is added. When correction is made for dilution, spectrum E is equivalent to spectrum A. Curves F said G represent buffer and protein blanks, respectively. Spectra were measured at pH 5.0 and 6.6°C.

Figure 5. (A) Protein-difference spectrum for the binding of cellobiose onto CBH I (7.6°C). The baseline (a) was recorded (double beam spectrophotometer) with 0.720 mM cellobiose in the measuring cuvette and 0.720 mM sucrose in the reference cuvette. The difference spectrum (b) was recorded after addition of 9.3 /iM CBH I to both cuvettes.

There are two reasons for the measurable cellobiose concentration in the T. viride cellulase hydrolyzed syrups. The most likely is that T. viride has rather poor / -glucosidase activity so that cellobiose accumulates. Evidence of this is that additions of / -glucosidase to the T. viride cellulase improves its activity. A second reason is that the / -glucosidase enzyme is strongly glucose inhibited. Hence the rate of cellobiose hydrolysis slows down as the glucose concentration rises, allowing cellobiose to accumulate. 

Cellobiase can be measured by following the glucose production from cellobiose and cellodextrins, the saligenin from salicin, and the p-nitrophenol formation from its / -glucoside (2). 

During purification procedures cellobiase activity was monitored by measuring nitrophenol (at A42onm) release for p-nitrophenyl-/ -D-glucoside (JO). Kinetic studies and enzyme characterization were carried out using / -D-cellobiose as substrate with the product, glucose, measured with a Beckman Glucose Analyzer (JO). Assay conditions were pH 4.8 and 50°C. 

In order to measure the enzyme activity and the initial rate of reaction, 5 mL of cellobiose (lOOmumol/mL) and 44 mL of buffer solution were placed in a stirred vessel. The reaction was initiated by adding 1 mL of enzyme (beta-glucosidase) solution which contained 0.1 mg of protein per mL. At 1,5,10,15, and 30 minutes, O.lmL of sample was removed from the reaction mixture and its glucose content was measured. The results were as follows ... 

To measure kinetic parameters, hydrolysis rates were determined by varying the concentrations of pNPG (0.05-10 mM) and cellobiose (0.04-16 mM). The inhibition by glucose was evaluated with only pNPG (10 mM) as substrate, whereas the inhibitory effect of glucono-1,5-lactone was verified with both pNPG (10 mM) and cellobiose (1.0 mM) as substrates. Km, Vmax, and fC, values were calculated from Lineweaver-Burk plots. 

Fig. 5. Lineweaver-Burk plot of the Orpinomyces BglA on hydrolysis of cellobiose (0.04-16 mM). The release of glucose was measured. Reciprocal initial velocities (mg/U) are plotted against the reciprocal concentrations of substrates (1 mM). The inset is a plot of initial velocities (U/mg) against the log cellobiose concentrations (mM).

For yield calculations, the measured amount of xylose and arabinose was used as a measure of the hemicellulose fraction, while the cellulose fraction was calculated from the amount of glucose and cellobiose. 

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