Cellulose structure, enzymic analysis

In an attempt to separate the domains from the cores, we used limited degradation with several proteases. CBH I (65 kda) and CBH II (58 kda) under native conditions could only be cleaved successfully with papain (15). The cores (56 and 45 kda) and terminal peptides (11 and 13 kda) were isolated by affinity chromatography (15,16) and the scission points were determined unequivocally. The effect on the activity of these enzymes was quite remarkable (Fig. 7). The cores remained perfectly active towards soluble substrates such as those described above. They exhibited, however, a considerably decreased activity towards native (microcrystalline) cellulose. These effects could be attributed to the loss of the terminal peptides, which were recognized as binding domains, whose role is to raise the relative concentration of the intact enzymes on the cellulose surface. This aspect is discussed further below. The tertiary structures of the intact CBH I and its core in solution were examined by small angle X-ray scattering (SAXS) analysis (17,18). The molecular parameters derived for the core (Rj = 2.09 mm, Dmax = 6.5 nm) and for the intact CBH I (R = 4.27 nm, Dmax = 18 nm) indicated very different shapes for both enzymes. Models constructed on the basis of these SAXS measurements showed a tadpole structure for the intact enzyme and an isotropic ellipsoid for the core (Fig. 8). The extended, flexible tail part of the tadpole should thus be identified with the C-terminal peptide of CBH I. 

The electron micrographs of the enzyme-treated sprucewood holo-cellulose revealed the loci of the removed substances when compared with untreated samples. The relative intensity of degradation at the ultra-structural level corresponded to the results obtained by chemical analysis of the dissolved carbohydrates. 

The following paragraphs Hi and iv) give a selection of pioneering work and more recent appheations of enzyme hydrolysis for structure analysis of starch and cellulose derivatives. 

Pioneer efforts in the apphcation of enzymes for chemical structure analysis of cellulose derivatives include the work of Husemann, Reese,Bhattachaijee et a/., F.rikson et and Wirick and Gelman. Techniques available at those... 

B. Saake, S. Homer, T. Krase, J. Puls, T. Liebert, and T. Heinze, Detailed investigation on the molecular structure of carboxymethyl cellulose with unusual substitution pattern by means of an enzyme-supported analysis, Macromol. Chem. Phys., 201 (2000) 1996-2002. 

Commercially available cellulose acetate sheets have a homogeneous micropore structure. Unlike paper, very little adsorption occurs. Cellulose acetate is less hydrophilic than paper and so less buffer is held in the medium, leading to shorter separation time and better resolution than paper and less sample required than with other mediums. The cellulose acetate sheet must be saturated with buffer prior to electrophoresis. This is done by floating the strip on the surface in a shallow tank, since rapid immersion can trap air bubbles in the medium. The relatively low amount of buffer held by the sheet can lead to excessive heat production and drying out. Cellulose acetate electrophoresis plays an important role in clinical diagnostics, for example, in the analysis of hemoglobin. It is also commonly used in the separation of other blood proteins, enzymes, mucopolysaccharides, urine, and other bodily fluids. 

Figure 8-4. Methylation analysis of in vitro P-D-glucans. (a) Gas chromatography of the permethyl-ated alditol acetate obtained from methylation analysis of the cellulose synthesized in vitro by the enzyme from blackberry. Peak 1, derivative characteristic of (1 4) linked glucosyl units. Peak 2, internal standard (mj o-inositol). The derivative characteristic of (1 3) linked glucosyl units usually elutes 1 min before the major derivative visible in the chromatogram (not shown see Bulone et al. 1995). (b) Structural characterization by electron impact mass spectrometry of the 1,4,5-tri-0-acetyl-2,3,6-tri-0-methyl-D-glucitol derivative corresponding to peak 1 in A and characteristic of (1 4) linked glucosyl units.

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