Holocellulose mannanase

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. 

In the present work, one of the endo-/M,4-xylanase (E.C. 3.2.1.8), the endo-/ -l,4-mannanase (E.C. 3.2.1.78), and the avicelases used in the former experiments with beechwood holocellulose (10,11) were applied to sprucewood holocellulose in order to obtain a better understanding of the individual and combined actions of these enzymes on the complex carbohydrate skeleton of wood. The experiments could be conducted only with a limited number of samples therefore, the figures given in this chapter have to be considered as preliminary results. 

The galactoglucomannan of the holocellulose was degraded by the mannanase. The reaction was slow, but conversion showed a steady increase. After 80 hr of incubation, about 25% of the mannan was transformed into water-soluble products (Table I, Column 10). This figure was calculated on the basis of the mannose, glucose, and galactose present in the acid hydrolysate (cf. Experimental). 

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

Control samples of sprucewood holocellulose were treated as described before, but without mannanase. The solutions contained almost no reducing sugars. After additional acid hydrolysis, about 1% sugars were found (dry weight of holocellulose = 100). Mannose were predominant glucose, galactose, arabinose, and xylose were present in smaller amounts. 

Figure 3. Decomposition of sprucewood holocellulose by xylanase 2 + mannanase, monitored by quantitative sugar chromatography. Ordinate degradation in percent of dry holocellulose based on the amount of reducing sugars in the reaction solution. ( --total degradation products,...

Avicelases or Cellobiohydrolase C - - Mannanase. The action of mannanase on delignified sprucewood holocelluloses that were treated with avicelase 1, avicelase 2, or cellobiohydrolase C for 48 hr gave similar degradation patterns. The rate of total degradation increased rapidly when the mannanase was added the rate was most pronounced in the case of avicelase 1 (Figure 4). 

Avicelase or Cellobiohydrolase + Mannanase. The treatment of spruce holocellulose first with avicelase or cellobiohydrolase and subsequently with mannanase effected an overall intensive degradation of... 

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. 

In the present investigation, about eight times more mannanase per milligram of holocellulose was used with shorter incubation periods (up... 

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 combined action of xylanase and mannanase on sprucewood holocellulose increased the hydrolysis of hemicelluloses without any detectable attack of cellulose. At the end of the experiments—i.e., after 48 hr of xylanase incubation followed by 32 hr of combined xylanase-mannanase incubation—about half the hemicelluloses present in the starting material were selectively converted into low-molecular-weight sugars. The amount of mannan removed was two times higher than after 80 hr of incubation with mannanase only. Unexpectedly, the xylan dissolution was scarcely increased by the combined action of the two hemicellulases. 

Most of the mannan in the sprucewood holocellulose was hydrolyzed by the cellulase-mannanase treatments, and only about one-third of the xylan was dissolved and partly hydrolyzed into low-molecular-weight sugars. 

About a quarter of the mannan in the sprucewood holocellulose seems to be accessible to the mannanase. More mannan can be hydrolyzed only when the second hemicellulose becomes at least partly dissolved. The remaining mannan—less than half the initial amount— appears to be removable only together with cellulose degradation. In beechwood holocellulose, most of the main hemicellulose—i.e., the xylan —can be hydrolyzed rather selectively by the sole action of xylanase. This phenomenon could be due to the different molecular size of the two hemicellulases, mannanase about 24 A, xylanase about 18 A (JO), resulting in better diffusing conditions for the xylanase. However, the porosity of the... 

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. 

The loss of mannan caused by the mannanase treatment of sprucewood holocellulose seems to be similar in the Si and T layers. This has also been reported by Hoffmann and Parameswaran (19). 

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