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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.


Standard curves for xylose (X), xylobiose (●), xylotriose (□) and xylotetraose (▲) in the presence of beechwood xylan (9 mg/mL) obtained using the NS reducing sugar method
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Fig3: Standard curves for xylose (X), xylobiose (●), xylotriose (□) and xylotetraose (▲) in the presence of beechwood xylan (9 mg/mL) obtained using the NS reducing sugar method

Mentions: Standard curves relating the concentration of xylose, xylobiose, xylotriose and xylotetraose (in the presence of beechwood xylan) to the determined absorbance value (520 nm) using the NS reducing sugar method are shown in Fig. 3. Clearly, in contrast to the results obtained with the DNS assay, the molar responses from xylose and xylo-oligosaccharides of DP 2–4 are almost identical. This contrasts with the results of Jeffries et al. [13] who reported that the molar response factor for xylobiose was just 65 % of that for xylose and that for xylotriose was 62 %. The reason for this difference is not clear, but may be due to incorrect standardization of the xylo-oligosaccharides used. When the current experiments were performed in the presence of birchwood xylan or wheat arabinoxylan, the same results were obtained, i.e. essentially the same standard curves for xylose, xylobiose, xylotriose and xylotetraose were obtained. These results are in agreement with those reported by Breul and Saddler [15] for glucose and cellobiose using the NS reducing method.Fig. 3


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)

Standard curves for xylose (X), xylobiose (●), xylotriose (□) and xylotetraose (▲) in the presence of beechwood xylan (9 mg/mL) obtained using the NS reducing sugar method
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4633439&req=5

Fig3: Standard curves for xylose (X), xylobiose (●), xylotriose (□) and xylotetraose (▲) in the presence of beechwood xylan (9 mg/mL) obtained using the NS reducing sugar method
Mentions: Standard curves relating the concentration of xylose, xylobiose, xylotriose and xylotetraose (in the presence of beechwood xylan) to the determined absorbance value (520 nm) using the NS reducing sugar method are shown in Fig. 3. Clearly, in contrast to the results obtained with the DNS assay, the molar responses from xylose and xylo-oligosaccharides of DP 2–4 are almost identical. This contrasts with the results of Jeffries et al. [13] who reported that the molar response factor for xylobiose was just 65 % of that for xylose and that for xylotriose was 62 %. The reason for this difference is not clear, but may be due to incorrect standardization of the xylo-oligosaccharides used. When the current experiments were performed in the presence of birchwood xylan or wheat arabinoxylan, the same results were obtained, i.e. essentially the same standard curves for xylose, xylobiose, xylotriose and xylotetraose were obtained. These results are in agreement with those reported by Breul and Saddler [15] for glucose and cellobiose using the NS reducing method.Fig. 3

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.