Sprucewood holocellulose from

The whole procedure normally takes about 1 hr. The acid is then evaporated, and the dry matter can be analyzed. This method can be applied to cellulose from wood, as a-cellulose or pulp, or to other celluloses (e.g., cotton) as well as to cellulosic materials with higher amounts of other polysaccharides (e.g., holocellulose). The chromatograms of the hydrolysates of a-cellulose from beechwood and of holocellulose from sprucewood (Figure 6) are examples of the application of this method. Compared with sulfuric acid hydrolysis, the total sugar yield from the spruce holocellulose is higher after the hydrolysis with concentrated TFA (Table II). Regarding the individual sugars, it can be seen that the... 

Figure 6. Chromatograms of the hydrolysates of a-cellulose from beech-wood and holocellulose from sprucewood...

Sprucewood holocellulose was treated with an endo-p-1,4-mannanase isolated from Aspergillus niger and an endo-/3-1,4-xylanase, two avicelases, and a cellobiohydrolase C isolated from Trichoderma viride. The mannanase hydrolyzed about a quarter of the mannan in 2-3 days without xylan or cellulose degradation. The xylanase hydrolyzed about half the xylan with 10% mannan solubilization. The three cellulases hydrolyzed up to 45% of the cellulose and 20% of the xylan, accompanied by 40-70% solubilization of the mannan. Combined xylanase-mannanase treatment hydrolyzed about half the xylan and mannan. Addition of mannanase to to cellulose-treated samples increased the degradation of the cellulose and mannan. Micromorphological studies of the variously treated specimens revealed a loss of substances in P/Slf T, and adjacent zones of S2 of the tracheid wall. 

Figure 2. Sugars from sprucewood holocellulose treated with mannanase for 80 hr. (For separation and detection method see Figure 1).

Xylanase. The sprucewood holocellulose treated with xylanase shows, in general, a mild attack especially in the wall region at the lumen-wall boundary. A progressive dissolution of substances can also be detected from Si into the adjacent S2 as well as from the lumen side into the secondary wall (Figure 8). This decrustation appears to be not very extensive in total, inasmuch as the affected wall areas are proportionally few. Compared with the loss of opacity in the lumen-wall boundary, the Si is not affected to such a great extent. The corners of the tracheids exhibit a high transparency, obviously due to removal of substances, as compared to the remainder of the middle lamella region. 

The experiments were repeated under similar conditions with the same and another mannanase fraction of Yamazaki s, with sprucewood holocellulose prepared in a slightly different way (19). After three weeks incubation, about 19% of the mannan was dissolved away, and the loci of hemicellulose removal were revealed by electron microscopy. In these experiments, the bulk of hemicelluloses was obviously removed by the buffer solution. In addition to mannose, glucose and galactose derived from mannan, xylose and glucose were found in the reaction solutions in appreciable amounts. 

The xylanase hydrolyzed about half the xylan in the sprucewood holocellulose. This is in the range of the xylan degradation obtained in former studies with delignified beechwood (7,10,22). Boutelje et al. (5) reported only 20-30% xylan hydrolysis of sprucewood holocellulose by a xylanase, even after repeated treatments. They used the same holocellulose and a xylanase isolated from the same commercial enzyme preparation as were used in the mannanase treatment referred to above. The holocellulose contained only a low xylan portion and very little arabinose this could be the reason for the inferior degradation rate, and hence it is not surprising that no free arabinose was detected, in contrast to what... 

The three cellulases decomposed about 25-45% of the cellulose accompanied by solubilization of about 40-70% of the mannan and, by partial hydrolysis, of about 20% of the xylan present in the untreated sprucewood holocellulose. Based on the degradation products (cf. Table III, Columns 13-15, and Table II), the catalytic actions of the three cellulases—all isolated from Trichoderma viride—are similar or identical. The lower absolute degradation values obtained with cellobiohydrolase C might merely be a result of enzyme concentration. 

The catalytic action of the two avicelases and the cellobiohydrolase C seems at least to be different from those of the xylanases isolated from Trichoderma viride i.e., the degradation products have a higher degree of polymerization even after prolonged incubation and, in the case of sprucewood holocellulose, no arabinose is liberated. 

The current observations confirm previous studies on beechwood and sprucewood holocellulose (7,10,19). The attack of the hemicellulose proceeds from the primary wall/Si as well as from the tertiary wall into S2 the pit chambers constitute preferred paths of enzyme diffusion into the walls. Also, substances of the middle lamella, especially in the cell corners, are removed by the xylanase and the mannanase treatments. Parallel to the removal of hemicelluloses, the fibrillar structure of the cellulose and its lamellar arrangement in transections of cell walls became obvious. In samples treated with cellulases, the cellulose fibrils were often completely hydrolyzed in the Si layer, occasionally accompanied by complete dissolution of cell-wall portions. This is also in conformity with the previous conclusion that the cellulases hydrolyze highly ordered zones of cellulose and remove hemicelluloses by hydrolysis or by detachment. 

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