Xylanase 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. 

Addition of mannanase resulted in high amounts of mannobiose and mannose. The amount of mannobiose relative to mannose was somewhat higher than in the case of the combined xylanase-mannanase treatment (Table II). The ratios of mannose. -galactose in the acid hydrolysates of the reaction solutions at the end of the experiment were 1 0.05 for the two avicelases and 1 0.03 for the cellobiohydrolase C. 

Xylanase + Mannanase. The combined action of xylanase and mannanase leads to a degradation of wall material, which resembles the mode of attack met with in the individual enzyme treatments only the degree of dissolution is rather more intense in the combined treatment. Of special interest is the decrustation of the cell corners, which appear to be attacked on a large scale (Figure 11). The Sx and tertiary wall zones also undergo a decomposition process (Figure 11). 

Figure 11. Xylanase + mannanase treatment. Decrustation of S. layer and cell corner (C) in an early wood tracheid. Scale = 1 fim.

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. 

Trichoderma reesei also produces a range of hemicellulases. Being a diverse group of heterogeneous polymers with various side groups, the hemi-cellulosic system is more complex and involves several hemicellulases, for example xylanases, mannanases, arabinanases and various esterases. Depending on the substrate and pre-treatment, hemicellulases can be crucial for efficient hydrolysis of the cell wall. ... 

Endoxylanase produced by a transformant strain of Escherichia coli has been shown to decrease the kappa number and increase the brightness of pulp in an enzymatic pretreatment followed by chemical extraction (27). Beta-xylosidase, on the other hand, had no effect in the treatment. The mannanase produced by Bacillus suhtilis was found to be equally effective as the xylanase. The specificities of mannanases produced by two different organisms, however, differed considerably 49). Enrichment of the culture broth of Streptomyces olivochromogenes, containing mainly endoxylanase, with different pure enzymes or enzyme mixtures acting on side chains and other hemicelluloses, resulted only in slight increases in brightness and in kappa number reduction (55). 

En me Treatment of LCCs. Purified Bacillus circulans xylanase (specific activity, 110 units/mg protein), purified Coriolus versicolor )9-mannanase (specific activity 179 units/mg protein), purified Thermoascus aurianticum /8-glucosidase (specific activity, 150 units/mg protein), and purified Schizophyllum commune acetyl xylan esterase (AXE specific activity, 180 units/mg protein) were used. Unit activity for xylanase and )9-mannanase was defined as the amount of enzyme catalyzing the release of 1 /xmol reducing power per minute at 50°C and pH 6.0 using oat spelts... 

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

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. 

Enzymes. The mannanase was isolated from Aspergillus niger (11) fraction 4 b was used throughout the experiments (cf. Table 1 in (11)). The xylanase 2, the avicelase 1, and the avicelase 2 were isolated from Trichoderma viride (10). The properties of these enzymes have also been described in the papers cited above. The cellobiohydrolase C was kindly supplied by Dr. R. D. Brown, Jr., and Dr. E. K. Gum, Jr. (Virginia Polytechnic Institute, Blacksburg). The isolation (from Trichoderma viride) and the properties of the cellobiohydrolase C are described in their 1974 paper (12). 

The xylan retained in the tissue after the xylanase treatment (first 48 hr) was left almost unaffected by the simultaneous action of xylanase and mannanase. Only xylobiose and higher-molecular xylan fragments, which were already present in the reaction solution (cf. Xylanase), were... 

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,...

Cellobiose or other cellulose fragments were not found in the reaction solution, neither after the xylanase nor after the additional action of mannanase. The glucose present probably was derived from the mannan see above and Mannanase). 

The properties of the enzymes used in this study have been described in former publications (10,11,15). Important for the following interpretation are their hydrolytic specificities. The xylanase did not hydrolyze either isolated mannans or celluloses—or only to a very small extent (10). The same is true for the mannanase with respect to xylans and celluloses (11,15). The avicelases, which were not purified to the same extent as the xylanase and mannanase, did not hydrolyze mannans, but they degraded xylans besides crystalline cellulose (10). Also, the highly purified cellobiohydrolase C (12) degraded xylan to some extent (Dr. E. K. Gum, Jr., personal communication). 

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 addition of mannanase after 48 hr of incubation with one of the three cellulases revived cellulose hydrolysis. The increase of the degradation rate was not as pronounced as in the corresponding treatments of delignified beechwood with cellulases and xylanase (JO), but the cellulose degradation reached about the same values at the end of the experiments (about 50-65% ). 

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 amino acid composition of many / -( l- 4) glycoside hydrolases, including cellulases (27-30), xylanases (15,31,32,33), and mannanases (34) have been determined. In general, the enzymes contain higher than... 

The enzymes cellulase, mannanase, and aryl-/ -glucosidase showed no significant decrease in activity as determined in the presence of up to 60% of ethanediol. Xylanase, / -glucosidase, and mannosidase lost approximately half of their initial activity at this ethanediol concentration while xylosidase lost most of its activity. The inhibition caused by ethanediol was found to be reversible. 

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