Cellobiose spectra

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.

The binding of chromophoric 1-thioglycosides from lactose or cellobiose to CBH I (Tr. r.) was followed by difference spectrophotometry of the ligand, or by the diafiltration technique (Fig. 4). Alternatively, perturbation of the protein spectrum could be used in the case of non-chromophoric ligands (Fig. 5). 

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.

The visible absorption spectrum of oxidised cellobiose oxidase is typical of cytochrome b (Fig. 5-15). The flavin in cellobiose oxidase is weakly fluorescent, with emission maxima at 564 nm and excitation maxima at 380 and 444 nm. There are no obvious transient changes on reduction that can be readily ascribed to flavin semiquinone, but the strong absorbance of the cytochrome would make such changes difficult to detect. 

FIGURE 3.16 The effect of a tiny ferromagnetic particle on the proton resonance spectrum of cellobiose octaacetate. The top spectra are run with the particles present the bottom curves are the spectra with the particle removed. 

The structure of cellobiose and Its 13C-NMR spectrum are shown in Figure 3a. The spectra have been identified (18-20). Cellobiose is water soluble. Figure 3b shows the spectrum of reaction products with formaldehyde at different molar ratios obtained by 15 min reaction at 150°C, i.e. under conditions that correspond to those during the manufacture of UF-bonded wood products. As expected, formaldehyde can react with several different functional groups. Therefore, complex mixtures of products are formed. 

Cellobiose Oxidases.—An extracellular enzyme which utilizes molecular oxygen to oxidise cellodextrins to the corresponding aldonic acids has been isolated from culture filtrates of the white rot fungus Sporotrichum pulveru-lentum. This enzyme, named cellobiose oxidase, was purified by classical techniques and was shown to be a glycoprotein (mol. wt. 9.3 x 10 ). The u.v. spectrum of the enzyme was characteristic of a haemoprotein, there being approximately one flavin component per enzyme molecule. The possible role of this complex enzyme in cellulose degradation was discussed. 

The bottleneck for production was thus assigned to the supply of oxaloacetate [15]. The maximum cadaverine concentration obtained with the recombinant strain was 9.6gl with a yield of 0.12gg glucose [15]. Cell-surface display of -galactosidase extended the substrate spectrum toward cellobiose [69]. 

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