Sprucewood

Here, another possible explanation for the origin of the acids XXIX and XXX must be mentioned. Sprucewood contains about 0.3%... 

Recently, small amounts of guaiacylglycerol 0-coniferyl ether have been isolated by Nimz (35) after percolating pre-extracted sprucewood for 8 days with 2% aqueous acetic acid at 100°C. This particular 0-aryl... 

Table II. Continuous Extraction of Black Sprucewood with Dioxane (0.2N HC1)...

Encouraged by the success of the hydrogenolysis of aspenwood meal this approach to lignin study w.as continued in these laboratories. Special attention was given to the effect of adding acid or base and to the use of palladium-charcoal as a catalyst on the products of hydrogenation of both aspen- and sprucewood meals. 

A third dimer was obtained from Norway sprucewood in somewhat impure condition (m.p., 117°-127°C.). However, its vapor phase retention time and ultraviolet spectrum 23) were identical with those of authentic 5,5 -diethyl-2,2 -dihydroxy-3,3 -dimethoxybiphenyl (VII), m.p. = 143°C. (P). Furthermore, the NMR spectrum and melting point of its purified diacetate were identical with those of the synthetic compound. The corresponding 5,5 -di-w-propyl derivative has already been isolated from neutral hydrogenation of softwood lignin in our laboratory 18). 

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...

Figure 8. Chromatograms of the hydrolysates of materials with low polysaccharide content milled wood lignin (MWL) from sprucewood and a 180-million-year-old protopinacea...

Degradation of Delignified Sprucewood by Purified Mannanase, Xylanase, and Cellulases... 

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. 

Figure J. Sulfuric acid hydrolysates of holocelluloses of sprucewood and beechwood. Separation on Durrum DA-X4 (column 0.4 X 30 cm) with 0.49M potassium-borate buffer at pH 9.2 and 60°C (16) detection with 2, 2-bicinchoninate (17).

Mannanase. Sprucewood delignified at room temperature was treated with mannanase as described by Yamazaki (15). However, a higher mannanase concentration was used, and the reaction was followed by means of quantitative, column chromatographic analysis of the reducing sugars in the reaction solutions. 

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. 

Avicelases or Cellobiohydrolase C. Treatment of sprucewood holocellulose with the three different cellulose-splitting enzymes gave very similar results. The cellulose was hydrolyzed to about 25-45% in 48 hr (Table I). Cellobiose was the predominant reaction product, but the amount of glucose increased considerably with incubation time (Table II). Acid hydrolysis of the reaction solutions showed that higher-... 

The main reaction products after the addition of mannanase were mannobiose, mannose, and glucose. Mannobiose was predominant its amount relative to mannose decreased with incubation time, as in the case of sprucewood treated solely with mannanase (Table II). The ratio of mannose glucose. -galactose in the acid hydrolysate of the reaction solution (80 hr) was 1 02 0.04. 

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). 

Control Sample. The holocellulose material of sprucewood evinces a rather homogeneous contrast across the width of the secondary wall after treatment with potassium permanganate the middle lamella/ primary wall region is, in general, more electron opaque than the other wall areas (Figure 5). In certain cases, a somewhat higher contrast of the tertiary wall region can be observed. 

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. 

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