Determination of reducing sugars using dns

The problems associated with the DNS assay have also been reported by other researchers [ 9 — 11 ]. Our data on CMCase activities of twelve enzyme preparations determined with two different RS assays Table 1 are very similar to those reported by Breuil and Saddler [ 11 ], who observed moderate overestimations of the endoglucanase CMCase activity of Trichoderma harzianum culture filtrates by the DNS assay in comparison with the NS assay.

Determination of reducing sugars using dns

This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract The Nelson-Somogyi NS and 3,5-dinitrosalicylic acid DNS assays for reducing sugars are widely used in measurements of carbohydrase activities against different polysaccharides.

Using twelve commercial enzyme preparations, the comparison of the NS and DNS assays in determination of cellulase, -glucanase, xylanase, and -mannanase activities was carried out. In the analysis of the xylanase, -mannanase, and -glucanase activities, the overestimations by the DNS assay were much more pronounced the observed differences in the activities were 3- to fold.

Reasons for preferential use of the NS assay for measuring activities of carbohydrases other than cellulases are discussed. Introduction Carbohydrases O-glycosidases, or glycoside hydrolases represent a large class of enzymes hydrolyzing polysaccharides and low-molecular-weight glycosides.

They belong to the EC 3.

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Carbohydrases are classified according to their specificity toward natural glycoside substrates, that is, they are called cellulases, xylanases, mannanases, pectinases, chitinases, and so forth. More recent classification of glycoside hydrolases in families based on amino acid sequence similarities has been proposed by Henrissat [ 1 ].

Many carbohydrases found extensive applications in biotechnology [ 2 ].

Determination of reducing sugars using dns

Most of the methods for determination of carbohydrase activity are based on the analysis of reducing sugars RSs formed as a result of the enzymatic scission of the glycosidic bond between two carbohydrates or between a carbohydrate and a noncarbohydrate moiety.

Different methods for assaying the RS have been applied in the carbohydrase activity measurements. The Nelson-Somogyi NS assay with copper and arsenomolybdate reagents [ 34 ] and the 3,5-dinitrosalicylic acid DNS assay described by Miller [ 5 ] are the most popular methods used by many researchers.

Other methods, such as those based on the use of sodium 2,2'-bicinchoninate [ 6 ], p-hydroxybenzoic acid hydrazide [ 7 ], or potassium ferricyanide [ 8 ], are less frequently used. Although the DNS assay is known to be approximately 10 times less sensitive than the NS assay and it does not provide stoichiometric data with oligosaccharides, giving significantly higher values of RS than the actual number of hemiacetal reducing groups [ 9 — 11 ], it has been recommended by the IUPAC commission on biotechnology for measuring standard cellulase activities against filter paper and carboxymethylcellulose CMC [ 12 ].

The method for determination of xylanase activity with the DNS reagent, reported by Bailey et al. Materials and Methods 2. Enzyme Activity Measurements The enzymatic hydrolysis of polysaccharides was carried out at pH 5. All assays were carried out in duplicates. NS Assay An aliquot of the substrate stock solution 0.

Then the enzyme reaction was initiated by adding 0. The tightly stoppered test tube was incubated in a boiling water bath for 40 min; then it was cooled to room temperature and 0. The solution was carefully mixed and incubated for 10 min at room temperature and then 1. After centrifugation at 13, rpm for 1 min, the absorbance of the supernatant at nm A was measured.

The A values for the substrate and enzyme blanks were subtracted from the A value for the analyzed sample. The substrate and enzyme blanks were prepared in the same way as the analyzed sample except that the necessary amount of the acetate buffer was added to the substrate enzyme solution instead of the enzyme substrate solution.

DNS Assay An aliquot of the substrate stock solution 0. After cooling to room temperature, the absorbance of the supernatant at nm was measured. The substrate and enzyme blanks were prepared in the same way as the analyzed sample except that 0.

When the enzyme activities against other polysaccharides under study were determined, the observed differences between the DNS and NS assays were much more pronounced.Reducing sugars concentrations were determined by spectrophotometry using the 3, 5 dinitrosalicylic acid (DNS) method.

The first sample was operated as blank for . The determination of lactose in dairy product is important and there are The DNS method for estimating the concentration of reducing sugars in a sample Reducing sugars contain free carbonyl group, have the property to reduce many of the reagents.

Introduction

Determination of Reducing Sugars Using Dns Essay INTRODUCTION Determining the sugar concentration of food samples is very important especially in industries where quality control is monitored. Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar - Free download as PDF File .pdf), Text File .txt) or read online for free.

Scribd is the 5/5(25). 3,5-Dinitrosalicylic acid (DNS) is used in colorimetric determination of reducing sugars and to analyze glycosidase (glycoside hydrolase) activity by quantitation of enzymatically released reducing sugar.

The dinitrosalicylic acid method has been compared to the Nelson-Somogi colorimetric method. May 26,  · The Nelson-Somogyi (NS) and 3,5-dinitrosalicylic acid (DNS) assays for reducing sugars are widely used in measurements of carbohydrase activities against different polysaccharides.

Using twelve commercial enzyme preparations, the comparison of the NS and DNS assays in determination of cellulase, β -glucanase, xylanase, and β -mannanase.

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