Xylanases assay

Pure xylanases can be useful in l ic research. They fmd application in analytical procedures designed to quantify specific xylan constituents 10) and to study xylan structure. They can be used to preparation of defined xylooligosacchaiides, eidia by hydrolysis of xylan 11-14) x by transglycosylation at high substrate concentrations 15,16). These products may serve as assay substrates and model compounds to study the mechanism of action of xylanolytic enzymes. Finally, xylanolytic eiuymes play an imptutant role in complex enzyme preparations used to the release of plant protoplasts. 

Specificity. Under Ae conAtions of the assay, Xylanase II had little or no activity on cellulose, carboxymeAylcellulose, Avicel, and starch. 

Koch-Light 1887-00 oat-spelt xylan (Lot 90560) was split into soluble and insoluble fractions by dissolving it in 0.05A/, pH 6.05 sodium citrate buffer and centrifuging out Ae unAssolved material. The msoluble portion was suspended in Ae buffer in roughly 1% concentration and was assayed wiA an equal volume of Xylanase II in... 

Effect of Temperature and pH on Activity and Stability. When Xylanase II was subjected to the standard assay at pH 5 but at several different temperatures, the highest activity was found after incubation for 30 min at 60°C. From 21 C to 45 C the energy of activation from a linear plot of In (activity) vs. T was 41.6 2.1 kJ/mol, where the range is the standard deviation. When the standard assay was conducted at various pHs and 50°C, the highest activity was at pH 6.05. Activities half the maximum were found at pHs 4.4 and 8.0. 

Figure 2. Heat inactivation profile of the xylanase in the culture filtrate from Thermoascus aurantiacus. Xylanase enzyme was prepared as described by Tan et al. (74). Xylanase and endocellulase activities were determined after the enzyme was incubated for the specified time at 70°C. Aliquots were removed and assayed at 50°C by the method described by Yu et al. (75). Activities were expressed as a percentage of the control stored at 4°C.

Xylanase was assayed using birchwood xylan as substrate. The solution of xylan and the enzyme at appropriated dilution were incubated at 75°C for 3 min, and the reducing sugar was determined by the dinitrosali-cylic acid procedure (12) with xylose as standard. The released color development was measured spectrophotometrically at 540 nm. One unit of enzyme activity was defined as 1 pmol of reducing sugar released 1 min under the described assay conditions. Protein concentration was measured by the Lowry method (13) using bovine serum albumin as standard. 

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. 

Table 1 shows the main activities present in the various reaction mixtures using Celluclast 1.5L, Novozym 342, Viscozyme L, Pentopan 500BG, Pulpzyme HC, Multifect Xylanase, and Multifect GC. Although most endoxylanases present in the commercial preparations exhibit optimal activity near 50°C, hydrolysis assays were carried out at a lower temperature (35°C) to increase enzyme stability and to reduce the possibility of inactivation of other enzymatic activities having unknown stability profiles. 

Biely, P., Mislovicova, D., and Toman, R., Soluble chromogenic substrates for the assay of endo-1,4-beta-xylanases and endo-l,4-beta-glucanases. Analytical Biochem 1985, 144(1), 142-146. 

Taguchi, H., Hamasaki. T., Akamatsu, T., and Okada, H., A simple assay for xylanase using o-nitrophenyl-beta-D-xylobioside. Biosci Biotechnol Biochem 1996, 60, (6) 983-5. 

Wang, G., Marquardt, R. R., Xiao, H., and Zhang, Z., Development of a 96-well enzyme-linked solid-phase assay for beta- glucanase and xylanase. JAgric Food Chem 1999, 47 (3), 1262-7. 

Substrates used in the procedure for the assay of (1— 3)-j3-d-xylanase activity have normally been (1 —> 3)-/3-D-xylans from marine... 

Most of these D-xylanase preparations, with the exception of that from Diplodia viticola, degraded D-xylan randomly, liberating xylose as well as oligosaccharides. The absence of D-xylose from the enzymic hydrolyzates from the latter source may be due to the fact that samples were only assayed in the early stages of hydrolysis (24 h). 

Xylanase activity was assayed by measuring reducing sugars released as xylose, using a dinitrosalycylic acid method [22]. The reaction mixture containing 0.9 mL of the substrate solution of birchwood xylan (5.0 g/L) in acetate buffer (pH 5.0, 0.1 M) and 0.1 mL of the crude enzyme solution was incubated at 60 °C for 10 min. One imit of enzyme activity was defined as the amount of enzyme releasing 1 pmol of xylose per minute, under the cited assay conditions. 

Fig. 6 Effects of the water soluble components of wheat bran supplements on growth and production of extracellular cellulase and xylanase by P. decumbens. Cultures were grown as described in the Materials and Methods and samples of the supernatant were collected assayed for (a) cellulase (FPA) and (b) xylanase over a 1 week period, (c) The maximum biomass accumulated was determined after 2 days. All cultures contained 2% MCC plus A through E additions of 0.5%, 1%, 2%, 5% or 10% soluble components of wheat bran, respectively...

A review of the occurrence, purification, properties, and modes of action of hemicellulases has dealt with L-arabinanases, D-galactanases, D-mannanase, and D-xylanases, while a comprehensive review of pectolytic enzymes has discussed their classification and the assay and properties of pectinesterases and poly-D-galacturonate hydrolases and lyases. ... 

Xylanases have been reported in two extremely thermophilic Bacillus strains growing at 65 °C [265, 266]. The enzyme from the B. stearothermophilus variant [265] was induced by xylan in the growth medium and degraded the substrate to about 45% in 5 h at 80 °C. The enzyme was maximally active at neutral pH and at 80 °C in 25 min assays. The second Bacillus xylanase was from an acidophilic variant [266] grown optimally at 65 °C and pH 3.5-4.0. The indudble enzyme gave maximum activity at 80 °C in 10 min assays at pH 4.0. Both enzymes are considerably more stable than that from a moderately thermophilic Thermo-monospora strain [267]. Work on the xylanase from a Thermotoga strain [227, 228, 268], however, indieates that this enzyme is the most thermostable xylanase reported to date. 

Ge, Y Antoulinakis, E. G. Gee, K. R. Johnson, I. An ultrasensitive, continuous assay for xylanase using the fluorogenic substrate 6,8-difluoro-4-methylumbelliferyl p-D-xylobioside. Anal. Biochem. 2007,362,63-68. 

---