Specificities of xylanases

The hydrolysis of acetylated xylo-oligomers from the steaming extract of birchwood using an enzyme mixture containing only xylanase and / -xylosidase was very limited (Table III). Chemical deacetylation showed that the substrate specificities of xylanase and / -xylosidase with respect to substrate DP overlapped, and that /J-xylosidase also hydrolyzed longer oligosaccharides than xylobiose to xylose. The addition of acetyl esterase to the hydrolysis of acetylated xylo-oligomers enhanced xylose production (Table III), but deacetylation was still incomplete (the acetyl content of... 

In the treatment of cellulose pulps one essential criterion for a suitable enzyme preparation is that its cellulase activity should be as low as possible, or preferably absent completely. As even extremely low cellulase activities may ruin pulp quality, Trichoderma enzyme preparations are unlikely to be suitable for these applications. Many bacterial and fungal enzymes with low cellulase activity have been shown to be suitable for treatment of pulps 14, 15, 16,17), Regulation of the often synchronous production of cellulolytic and hemicellulolytic enzymes in micro-organisms is not well understood, and is further complicated by substrate cross-specificity of these enzymes. Enzymes with both endoglucanase and xylanase activity have been reported for bacteria 18, 19) and fungi 20, 21, 22), In addition to selection of strain and... 

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). 

Cellulases can also be eliminated fiom a mixture with xylanases by selective thermal inactivation. Cellulases are more thermolabile than xylanases in tiie cdlulolytic systems of the fungus Y-94 (79), T. harzianum 20), and Tkermoascus aurantiacus (77), but not in the Trickoderma reesei system (Biely, P. and Vrsanska, M., Slovak Academy of Sciences, Bratislava, unpublished results). Since cellulase thoixud inactivation causes a significant loss of xylanase also, a more convenient way to eliminate cellulase activity is by selective chemical or biological inhibition or inactivation. There appear, however, to be no reports on the existence of natural inhibitors that would be specific for cellulases. Such inhibitors of amylases and pectinases are known to occur in plants (27). 

Selective Production of Xylanases by Cellulolytic Microorganisms. Until recently there was little information on common or separate genetic control of cellulase and xylanase synthesis in microorganisms (60). Studies on this subject were complicated by the fact that numerous microbial ceUulases and xylanases are non-specific with respect to cellulose and xylan as substrates. As could be expected from a comparison of both polysaccharide structures, non-specificity is more frequently observed with cel-lulases, because their substrate binding sites can easily accommodate substrate using an unsubstituted p-(l 4)-linked chain of D-xylopyranosyl units. 

Low-Molecular Weight Xylanase Inducers. The response of cellulolytic fungi to low-molecular weight fragments of xylan and ceUulose confirmed the separate regulatory control of the formation of xylanases and ceUulases. Xylobiose [Xylp-p-(l- 4)-Xylp] was a specific inducer of xylanases in T. reesei (61) and Aspergillus terreus (67). Sophorose and otho glucobioses selectively induced the synthesis of... 

Feruloyl esterase activity was first detected in culture filtrates of Strepto-myces olivochromogenes (49), and has thereafter also been reported for some hemicellulolytic fungi (Table III). A partially purified feruloyl esterase from S. commune liberated hardly any ferulic acid without the presence of xylanase (65). Very recently a feruloyl esterase was purified from Aspergillus oryzae (Tenkanen, M. Schuseil, J. Puls, J. Poutanen, K., /. Biotechnol, in press). The enzyme is an acidic monomeric protein having an isoelectric point of 3.6 and a molecular weight of 30 kDa. It has wide substrate specificity, liberating ferulic, p-coumaric, and acetic acids from steam-extracted wheat straw arabinoxylan. 

The specificity of a xylanase preparation may be determined from the isolation and characterization of oligosaccharides formed from xylan hydrolysis. While the oligosaccharides released were mainly dependent on the specificity of the xylanase, the complex structure of the heteroxylan also determines the extent of hydrolysis and the site of cleavage. Two types of xylooligosaccharides are usually produced acidic (from glucuronoxylans) and neutral (from arabino- and arabinoglucurono-xylans). The specificity of the action of several purified xylanases on various heteroxylans is shown below ... 

Two broad areas of application for xylanolytic enzymes have been identified (1). The first involves the use of xylanases with other hydrolytic enzymes in the bioconversion of wastes such as those from the forest and agricultural industries, and in the clarification and liquification of juices, vegetables and fruits. For these purposes, the enzyme preparations need only to be filtered and concentrated as essentially no further purification is required. Several specific examples of applications involving crude xylanase preparations include bioconversion of cellulosic materials for subsequent fermentation (2) hydrolysis of pulp waste liquors and wood extractives to monomeric sugars for subsequent production of single cell protein (3-5). Xylose produced by the action of xylanases can be used for subsequent production of higher value compounds such as ethanol (6), xylulose (7) and xyIonic acid (8-9). 

Figure 1. Induction of xylanases in Trichoderma harzianum. The ratios of xylanase to endocellulase activities were determined in the culture filtrates of T. harzianum grown on steam treated aspenwood. Aspen chips were steam treated from 20 to 240 s at 240° C to produce a series of samples with a variable content of xylan and cellulose. The specific content of these carbohydrates were expressed as the ratio of xylan to glucan. Enzyme activities were determined on culture filtrates of 300 mL batch cultures of T. harzianum grown on these wood samples at a loading of 1% (w/v) as described by Saddler and Mes-Hartree (66). Enzyme activities were also determined in culture filtrates of T. harzianum grown on Avicel, Solka Floe and oat spelts xylan.

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). 

Fig. 1. Kinetic profile of xylanase production (— —) by T. lanuginosus IOC-4145 and its specific activity (—O—) using corncob as sbustrate on semisolid fermentation. The values correspond to the average of five and three measurements, respectively, and the error was -10%.

This Section will be concerned with (i) the specificities of D-xylanases of bacterial and fungal origin, and (it) the hemicellulase systems of rumen bacteria and protozoa, and it will include the xylobiases (/3-D-xylosidases), xylo-oligosaccharidases, a-L-arabino-furanosidases, and D-xylanases. 

Another application o f x ylanases is the use of this enzyme in poultry diets. Depression in weight gain and feed conversion efficiency in rye-fed broiler chicks has been associated with intestinal viscosity (24). The efficiency of xylanases in improving the quality of bread has also been demonstrated. The introduction of A. niger var. awamori xylanase into bread dough resulted in an increase in specific bread volume. This is further enhanced when amylase in combination with xylanase is used (25). 

Ding, M Teng, Y Yin, Q Zhao, J Zhao, F. The N-terminal cellulose-binding domain of EGXA increases thermal stability of xylanase and changes its specific activities on different substrates. Acta Biochimica etBiophysica Sinica, 2008,40(11), 949 - 54. 

The value and potential usefulness of a new enzyme depends on its properties and the extent to which it has been characterized. The initial characterization of an enzyme often involves the determination of its pH optimum, stability, gross physical properties, and substrates. The enzymes of L. edodes, typically show pH optima between 3.5 and 5.0, maximal activity at 50 to 60"C, little activity loss until over 70"C, and high relative specific activities (9,14). Below we will highlight some of the other characteristics determined for the major ligninase, p-(l,4)-D-xylanase, and a-(l,3)-L-arabinosidase purified from wood-grown cultures of L. edodes. 

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... 

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