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A Comparison of Polysaccharide Substrates and Reducing Sugar Methods for the Measurement of endo-1,4-β-Xylanase.

McCleary BV, McGeough P - Appl. Biochem. Biotechnol. (2015)

Bottom Line: The most commonly used method for the measurement of the level of endo-xylanase in commercial enzyme preparations is the 3,5-dinitrosalicylic acid (DNS) reducing sugar method with birchwood xylan as substrate.It is well known that with the DNS method, much higher enzyme activity values are obtained than with the Nelson-Somogyi (NS) reducing sugar method.Purified beechwood xylan or wheat arabinoxylan is shown to be a suitable replacement for birchwood xylan which is no longer commercially available, and it is clearly demonstrated that the DNS method grossly overestimates endo-xylanase activity.

View Article: PubMed Central - PubMed

Affiliation: Megazyme International Ireland, Bray, County Wicklow, Ireland. barrymccleary@me.com.

ABSTRACT
The most commonly used method for the measurement of the level of endo-xylanase in commercial enzyme preparations is the 3,5-dinitrosalicylic acid (DNS) reducing sugar method with birchwood xylan as substrate. It is well known that with the DNS method, much higher enzyme activity values are obtained than with the Nelson-Somogyi (NS) reducing sugar method. In this paper, we have compared the DNS and NS reducing sugar assays using a range of xylan-type substrates and accurately compared the molar response factors for xylose and a range of xylo-oligosaccharides. Purified beechwood xylan or wheat arabinoxylan is shown to be a suitable replacement for birchwood xylan which is no longer commercially available, and it is clearly demonstrated that the DNS method grossly overestimates endo-xylanase activity. Unlike the DNS assay, the NS assay gave the equivalent colour response with equimolar amounts of xylose, xylobiose, xylotriose and xylotetraose demonstrating that it accurately measures the quantity of glycosidic bonds cleaved by the endo-xylanase. The authors strongly recommend cessation of the use of the DNS assay for measurement of endo-xylanase due to the fact that the values obtained are grossly overestimated due to secondary reactions in colour development.

No MeSH data available.


Relative rates of hydrolysis of beechwood glucuronoxylan (), wheat flour arabinoxylan (■) and acid debranched wheat flour arabinoxylan  at 9 mg/mL in the final incubation mixture by a) T. viride endo-xylanase, b) A. niger endo-xylanase, c) N. patriciarum endo-xylanase and d) C. mixtus endo-xylanase using the NS reducing sugar method
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Fig5: Relative rates of hydrolysis of beechwood glucuronoxylan (), wheat flour arabinoxylan (■) and acid debranched wheat flour arabinoxylan at 9 mg/mL in the final incubation mixture by a) T. viride endo-xylanase, b) A. niger endo-xylanase, c) N. patriciarum endo-xylanase and d) C. mixtus endo-xylanase using the NS reducing sugar method

Mentions: The relative rates of increase in reducing sugar levels on the hydrolysis of beechwood xylan, birchwood xylan and wheat arabinoxylan by A. niger endo-xylanase as determined using the DNS assay are shown in Fig. 4a, and the relative rates as determined using the NS assay are shown in Fig. 4b. Using the DNS assay, similar relative rates of increase in reducing sugar levels were obtained on hydrolysis of beechwood and birchwood glucuronoxylans. This would be expected since both polysaccharides are 4-O-methyl glucuronoxylans with a similar content of 4-O-methyl glucuronic acid (approximately 10 %). The NS assay also gave similar rates with the two glucuronoxylans (Fig. 4b), but the determined endo-xylanase activities were much lower than those obtained with the DNS assay (see Table 1). With the DNS method, a much higher rate of increase in reducing sugar level was obtained with wheat arabinoxylan as substrate compared to beechwood glucuronoxylan. This may reflect a lower level of transglycosylation with the more highly branched arabinoxylan (39 % arabinose content, of which approximately half occurs on singly substituted d-xylosyl residues and half on doubly substituted d-xylosyl residues [21]). Beechwood glucuronoxylan contains just 10 % of 4-O-methyl glucuronic acid, meaning that approximately 90 % of the d-xylosyl residues are unsubstituted. In this case, a bulk of the hydrolysis products would be unsubstituted xylosaccharides which can readily serve as acceptors in transglycosylation reactions. With the NS reducing sugar procedure, the apparent rate of hydrolysis of wheat arabinoxylan (increase in reducing sugar level) by A. niger endo-xylanase is marginally less than that for the glucuronoxylans. The reasons for this are not clear. The relative rates of hydrolysis of beechwood glucuronoxylan, wheat flour arabinoxylan and acid debranched wheat flour arabinoxylan vary quite significantly between different endo-xylanases as shown in Fig. 5. The reasons for the differences cannot easily be attributed directly to the specific action pattern of the enzyme (CAZY family) as quite different relative rates of hydrolysis of the three substrates are observed with endo-xylanases from the same CAZY family, e.g. GH 11 endo-xylanases from A. niger, N. patriciarum and T. viride. With C. mixtus endo-xylanase (GH 10), significant differences were noted in the relative rates of hydrolysis of the three substrates. This appears to be related to the difference in the degrees of substitution of glucuronoxylan (10 % 4-O-methyl glucuronic acid content), wheat flour arabinoxylan (39 % α-linked l-arabinofuranose) and acid debranched wheat flour arabinoxylan (26 % α-l-arabinose). In most cases, acid debranched wheat flour arabinoxylan was hydrolysed slightly more rapidly than wheat flour arabinoxylan, but not as rapidly as beechwood glucuronoxylan (Fig. 5). Clearly, comparison of enzyme activities must involve the use of the same substrate and assay procedure. Substrate concentration is obviously also important, and the effect of the concentration of wheat flour arabinoxylan on the rates of hydrolysis by A. niger and N. patriciarum endo-xylanases is shown in Fig. 6a, b. The Km for A. niger endo-xylanase is 4.9 mg/mL while that for N. patriciarun endo-xylanase is much lower at 2.0 mg/mL. The most reliable assay results are obtained if the substrate concentration is approximately twice the Km value. With viscous polysaccharide substrates, this can be difficult to achieve, so in the studies described here, we have settled on a final substrate concentration in the assay mixture of 9 mg/mL.Fig. 4


A Comparison of Polysaccharide Substrates and Reducing Sugar Methods for the Measurement of endo-1,4-β-Xylanase.

McCleary BV, McGeough P - Appl. Biochem. Biotechnol. (2015)

Relative rates of hydrolysis of beechwood glucuronoxylan (), wheat flour arabinoxylan (■) and acid debranched wheat flour arabinoxylan  at 9 mg/mL in the final incubation mixture by a) T. viride endo-xylanase, b) A. niger endo-xylanase, c) N. patriciarum endo-xylanase and d) C. mixtus endo-xylanase using the NS reducing sugar method
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig5: Relative rates of hydrolysis of beechwood glucuronoxylan (), wheat flour arabinoxylan (■) and acid debranched wheat flour arabinoxylan at 9 mg/mL in the final incubation mixture by a) T. viride endo-xylanase, b) A. niger endo-xylanase, c) N. patriciarum endo-xylanase and d) C. mixtus endo-xylanase using the NS reducing sugar method
Mentions: The relative rates of increase in reducing sugar levels on the hydrolysis of beechwood xylan, birchwood xylan and wheat arabinoxylan by A. niger endo-xylanase as determined using the DNS assay are shown in Fig. 4a, and the relative rates as determined using the NS assay are shown in Fig. 4b. Using the DNS assay, similar relative rates of increase in reducing sugar levels were obtained on hydrolysis of beechwood and birchwood glucuronoxylans. This would be expected since both polysaccharides are 4-O-methyl glucuronoxylans with a similar content of 4-O-methyl glucuronic acid (approximately 10 %). The NS assay also gave similar rates with the two glucuronoxylans (Fig. 4b), but the determined endo-xylanase activities were much lower than those obtained with the DNS assay (see Table 1). With the DNS method, a much higher rate of increase in reducing sugar level was obtained with wheat arabinoxylan as substrate compared to beechwood glucuronoxylan. This may reflect a lower level of transglycosylation with the more highly branched arabinoxylan (39 % arabinose content, of which approximately half occurs on singly substituted d-xylosyl residues and half on doubly substituted d-xylosyl residues [21]). Beechwood glucuronoxylan contains just 10 % of 4-O-methyl glucuronic acid, meaning that approximately 90 % of the d-xylosyl residues are unsubstituted. In this case, a bulk of the hydrolysis products would be unsubstituted xylosaccharides which can readily serve as acceptors in transglycosylation reactions. With the NS reducing sugar procedure, the apparent rate of hydrolysis of wheat arabinoxylan (increase in reducing sugar level) by A. niger endo-xylanase is marginally less than that for the glucuronoxylans. The reasons for this are not clear. The relative rates of hydrolysis of beechwood glucuronoxylan, wheat flour arabinoxylan and acid debranched wheat flour arabinoxylan vary quite significantly between different endo-xylanases as shown in Fig. 5. The reasons for the differences cannot easily be attributed directly to the specific action pattern of the enzyme (CAZY family) as quite different relative rates of hydrolysis of the three substrates are observed with endo-xylanases from the same CAZY family, e.g. GH 11 endo-xylanases from A. niger, N. patriciarum and T. viride. With C. mixtus endo-xylanase (GH 10), significant differences were noted in the relative rates of hydrolysis of the three substrates. This appears to be related to the difference in the degrees of substitution of glucuronoxylan (10 % 4-O-methyl glucuronic acid content), wheat flour arabinoxylan (39 % α-linked l-arabinofuranose) and acid debranched wheat flour arabinoxylan (26 % α-l-arabinose). In most cases, acid debranched wheat flour arabinoxylan was hydrolysed slightly more rapidly than wheat flour arabinoxylan, but not as rapidly as beechwood glucuronoxylan (Fig. 5). Clearly, comparison of enzyme activities must involve the use of the same substrate and assay procedure. Substrate concentration is obviously also important, and the effect of the concentration of wheat flour arabinoxylan on the rates of hydrolysis by A. niger and N. patriciarum endo-xylanases is shown in Fig. 6a, b. The Km for A. niger endo-xylanase is 4.9 mg/mL while that for N. patriciarun endo-xylanase is much lower at 2.0 mg/mL. The most reliable assay results are obtained if the substrate concentration is approximately twice the Km value. With viscous polysaccharide substrates, this can be difficult to achieve, so in the studies described here, we have settled on a final substrate concentration in the assay mixture of 9 mg/mL.Fig. 4

Bottom Line: The most commonly used method for the measurement of the level of endo-xylanase in commercial enzyme preparations is the 3,5-dinitrosalicylic acid (DNS) reducing sugar method with birchwood xylan as substrate.It is well known that with the DNS method, much higher enzyme activity values are obtained than with the Nelson-Somogyi (NS) reducing sugar method.Purified beechwood xylan or wheat arabinoxylan is shown to be a suitable replacement for birchwood xylan which is no longer commercially available, and it is clearly demonstrated that the DNS method grossly overestimates endo-xylanase activity.

View Article: PubMed Central - PubMed

Affiliation: Megazyme International Ireland, Bray, County Wicklow, Ireland. barrymccleary@me.com.

ABSTRACT
The most commonly used method for the measurement of the level of endo-xylanase in commercial enzyme preparations is the 3,5-dinitrosalicylic acid (DNS) reducing sugar method with birchwood xylan as substrate. It is well known that with the DNS method, much higher enzyme activity values are obtained than with the Nelson-Somogyi (NS) reducing sugar method. In this paper, we have compared the DNS and NS reducing sugar assays using a range of xylan-type substrates and accurately compared the molar response factors for xylose and a range of xylo-oligosaccharides. Purified beechwood xylan or wheat arabinoxylan is shown to be a suitable replacement for birchwood xylan which is no longer commercially available, and it is clearly demonstrated that the DNS method grossly overestimates endo-xylanase activity. Unlike the DNS assay, the NS assay gave the equivalent colour response with equimolar amounts of xylose, xylobiose, xylotriose and xylotetraose demonstrating that it accurately measures the quantity of glycosidic bonds cleaved by the endo-xylanase. The authors strongly recommend cessation of the use of the DNS assay for measurement of endo-xylanase due to the fact that the values obtained are grossly overestimated due to secondary reactions in colour development.

No MeSH data available.