Xylanase characterization

The enzyme is distinct from other sequenced xylanases and shows < 15% homology to others characterized including C. thermocellum. 

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. 

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 ... 

The culture filtrates of T. reesei contained a large number of both cellulolytic and hemicellulolytic enzymes, which could be partially separated by chromatofocussing (Fig. 1). Of the cellulolytic enzymes, several endoglu-canases and two cellobiohydrolases have already been isolated and characterized (30). Some of the endoglucanases isolated are nonspecific and have xylanase activity (31). The two major xylanase (Xyl) peaks in Figure 1 corresponded to pi-values of above 7.5 and 5.5. When the former enzyme was further purified (24), its isoelectric point was found to be about pi 9 (Table I). Previously, the presence of at least three electrophoretically different xylanases in T. reesei culture filtrates was reported, with the most acidic one shown to have broad substrate specificity (10). This is in agreement with the reported occurrence of three xylanases (pi > 7, pi 5.1, pi... 

Chithra, M. and Muralikrishnan, G. (2007). Characterization of purified xylanase from finger millet (Eleusine corcana-lndai 15) malt. Eur. Food Res. Technol. 227,587-597. 

Multifect GC from Genencor, Rochester, NY. The enzyme solutions were characterized by the following activities endo-l,4-(3-xylanase, (3-xylosidase, acetylesterase, a-L-arabinofuranosidase, and filter paper activity (FPase, which describes the overall cellulolytic activity). The dilutions were calculated to provide 100 U of xylanase/g of total xylose in the reaction medium. Hydrolysis reactions were stopped by boiling for 5 min to inactivate enzymes and clarified by centrifugation prior to analysis. A blank OCL sample was assayed identically to enzyme-treated OCL using water instead of the enzyme solution. Control experiments were carried out for each enzyme, in which buffer replaced the OCL. All the hydrolysis trials were performed in duplicate. 

Ruiz-Arribas, A., Fernandez-Abalos, J. M., Sanchez, P., Garda, A. L., and Santamaria, R. I., Overproduction, purification, and biochemical characterization of a xylanase (Xysl) from Streptomyces halstedii JM8. Appl Environ Microbiol 1995, 61 (6), 2414-9. 

Suzuki, T., Kitagawa, E., Sakakibara, F., Ibata, K., Usui, K., and Kawai, K., Cloning, expression, and characterization of a family 52 beta- xylosidase gene (xysB) of a multiple-xylanase-producing bacterium, Aeromonas caviae ME-1. Biosci Biotechnol Biochem 2001, 65 (3), 487-94. 

Berens, S., Kaspari, H., and Klemme, J. H., Purification and characterization of two different xylanases from the thermophilic actinomycete Microtetraspora flexuosa SIIX. Antonie Van Leeuwenhoek 1996,69 (3), 235-41. 

Laxalt, A.M., Ter Riet, B., Verdonk, J.C., Parigi, L., Tameling, W.I.L., Vossen, J., Haring, M., Musgrave, A. and Munnik, T., 2001, Characterization of five tomato phospholipase D cDNAs rapid and specific expression of LePLDfil on elicitation with xylanase. Plant J. 26 237-247. 

J Wang, Z-H Jiang, DS Argyropoulos. Isolation and characterization of lignin extracted from softwood kraft pulp after xylanase treatment. J Pulp Pap Sci 23 J47-J51, 1997. 

Giesbert S, Lepping HB, Tenberge KB, Tudzynski P. The xylanolytic system of Claviceps purpurea cytological evidence for secretion of xylanases in infected rye tissue and molecular characterization of two xylanase genes. Phytopathology 88 1020-1030, 1998. 

Khasin, A., Alchanati, I., Shoham, Y. (1993). Purification and characterization of a thermostable xylanase from Bacillus stearothermophilus T-6. Applied and Environmental Microbiology, 59, 1725-1730. 

Srivastava, R., Srivastava, A. K. (1993). Characterization of a bacterial xylanase resistant to repression by glucose and xylose. Biotechnology Letter, 15(S), 847 52. 

Sigoillot, C., Lomascolo, A., Record, E., Robert, J. L., Asther, M., Sigoillot, J. C. (2002). Lignoeellulolytic and hemicellulolytic system of Pycnoporus cinnabarinus isolation and characterization of a cellobiose dehydrogenase and a new xylanase. Enzyme and Microbial Technology, 31, 876-883. 

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