Xylanase purification

This article describes the further purification of this endo-xylanase and the measurement of its isoelectric point, partial amino acid sequence, activity and stability at various pHs and tempCTatures, and characteristic products when it hydrolyzes linear xylo-oligosaccharides. 

Attempted purifications of Xylanase I, which has an apparent molecular weight four to six times that of Xylanase II by gel permeation chromatography, by various chromatographic procedures all failed, as more Xylanase II continued to form as the solution underwent dilution (Pestlin, S., Iowa State University, unpublished data). Xylanase I therefore appeared to be cluster of Xylanase II molecules. 

Enzyme Purification. The purification of the xylanolytic enzymes began with adsorption on a cation exchanger (CM-Sepharose FF) at pH 4.0. The final purification was accomplished by another ion exchange step as described previously for xylanase (24), / -xylosidase (25), a-arabinosidase (26) and acetyl esterase (27). 

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). 

Here we examine the recent progress which has been made in the production and purification of xylanases. 

Many examples of the purification of xylanase enzymes to homogeneity can be found in the reviews of Dekker and Richards (85), Woodward (86) and Reilly (87). Other xylanases which have been prepared recently to very high purity using traditional biochemical techniques include xylanases from Sporotrichum dimorphosporum (88) Streptomyces sp. (71) Trichoderma harzianum (5,55) Clostridium acetobuiylicum (30,89) mesophilic fungal strain Y-94 (80) Aspergillus nigcr (90-92) and several thermostable xylanases discussed above. 

Compound (44 g, NHAc form) (Scheme 14) was found to be a competitive inhibitor for CBHI cellulase (family 7) from Trichoderma reesei, when 4-methyl-umbelliferyl / -lactoside was used as substrate. Therefore (44 g, NHj form) was coupled to CH-Sepharose 4B, and the affinity gel was very effective for the purification of cellobiohydrolases from a crude commercial cellulolytic extract of T. reesei [40c]. Using the same approach aryl 1,4-dithioxylobioside and l,4,4 -trithioxylotrioside (44 h, NH2 form) were coupled to CH-Sepharose 4B to give affinity gels which were used for the purification of xylanases [40a,b]. 

Fractionation and Purification of Ex-1 Cellulase Component from Driselase. Driselase powder (50g) was extracted with several aliquots of water and the precipitate formed upon salting out with ammonium sulfate (on a saturation between 20% and 80%) was fractionated on a DEAE-Sephadex A-50 column. Each fraction was tested for -glucosi-dase, xylanase, CMCase, Avicelase activities, and protein content. The elution patterns are shown in Figures 1 and 2. 

Lin, J., Ndlovu, L. M., Singh, S., and Pillay, B., Purification and biochemical characteristics of beta-D-xylanase from a thermophilic fungus, Thermomyces lanuginosus-SSBP. Biotechnol Appl Biochem 1999, 30 (Pt 1), 73-9. 

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. 

Debeire, P., Priem, B., Strecker, G., and Vignon, M., Purification and properties of an endo-l,4-xylanase excreted by a hydrolytic thermophilic anaerobe, Clostridium thermolacticum. A proposal for its action mechanism on larchwood4-0-methylglucuronoxylan. Eur J Biochem 1990, 187 (3), 573-80. 

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. 

The (1 - 3)-/3-D-xylanase from Chaetomium globosum142 was purified by several fractionation steps employing ammonium sulfate and acetone, and the purified preparation resulting was shown to be free from a-amylase, cellulase, and (1 — 4)-/3-D-xylanase activities, and to have undergone a 106-fold purification. Whether this preparation was homogeneous was not indicated, but it has been used in study of mode of action on (1 — 3)-j8-D-xylan. 

Khanna, S., Gauri, P. (1993). Regulation, purification and properties of xylanase from Cellulomonas fimi. Enzyme and Microbial Technology, 15, 990-995. 

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. 

Fungal xylanases including A. oryzae xylanase have been reviewed recently by Haltrich et al (8). Xylanases produced by A. oryzae have been already studied fi om different aspects. Induction of xylanase production by a synthetic xyiobiose anidog, ft-metiiyl-D-xyloside, was dmonstrated by Bailey et al (9). Purification and characterization of xylanase components was studied in detail (10,11). Xylanase production in a 10-1 laboratory fermenter was also performed (12). 

Bakir U, Yavascaoglu S, Guvenc F, Ersayin A. (2(X)1). An endo-P-1,4-xylanase fmmRhizopus oryzae. production, partial purification and biochemical characterization. Enzyme Microb Technol, 29, 328-334. 

Chivero ET, Mutukumira AN, Zvauyu R. (2001). Partial purification and characterization of a xylanase enzyme produced by a microorganism isolated from selected indigenous fruits of Zimbabwe. Food Chem, 72, 179-185. 

---