%0 Journal Article
%T Comparison of Two Methods for Assaying Reducing Sugars in the Determination of Carbohydrase Activities
%A Alexander V. Gusakov
%A Elena G. Kondratyeva
%A Arkady P. Sinitsyn
%J International Journal of Analytical Chemistry
%D 2011
%I Hindawi Publishing Corporation
%R 10.1155/2011/283658
%X 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. When cellulase activities against CMC were measured, the DNS assay gave activity values, which were typically 40¨C50% higher than those obtained with the NS assay. 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 13-fold). Reasons for preferential use of the NS assay for measuring activities of carbohydrases other than cellulases are discussed. 1. 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 3.2.1.£¿) class of hydrolases. 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]. 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 [3, 4] 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¨C11], it has been recommended by the IUPAC commission on biotechnology for measuring standard cellulase activities
%U http://www.hindawi.com/journals/ijac/2011/283658/