Colorimetric determination of reducing sugars in soils

Department of Agronomy, Iowa State University, Ames, IA 50011, U.S.A.
Soil Biology and Biochemistry (Impact Factor: 3.93). 04/1994; 26(4):473-477. DOI: 10.1016/0038-0717(94)90179-1


Reducing sugars are the end products of many biological processes and enzymatic reactions in soils. They are determined in assay of several soil enzymes, including cellulase activity. Five colorimetric methods [phenol-sulfuric acid, anthrone-sulfuric acid, dinitrosalicylic acid (DNS), reaction with potassium ferric hexa-cyanide reagent (Prussian blue), and the Somogyi-Nelson (molybdenum blue) methods] were evaluated for determination of reducing sugars and total saccharides extracted from soils. Results showed that the Prussian blue and the molybdenum blue methods were the most sensitive and accurate for determination of reducing sugars in soils. Metals extracted from soils interfered with molybdenum blue color development. These metals, however, could be removed by K-saturated resin before analysis. The trace amount of metals extracted from soils did not interfere with the Prussian blue color development, but this method is too sensitive to be useful for determination of reducing sugars in soil extracts. Unlike the Prussian blue method, which is very sensitive and has 1 h color stability, the molybdenum blue method has color stability of at least 24 h. Reducing sugar values in soils increased significantly upon air-drying of field-moist soils or incubation of soils with acetate buffer (50 mM, pH 5.5) at 30°C for 24 h, suggesting enzymatic hydrolysis of the native substrates. Calibration graphs showed that the phenol-sulfuric acid, DNS, and anthrone-sulfuric acid methods are not as sensitive as the other two methods.

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    • "Eivazi and Tabatabai (1988), using p-nitrophenyl a-D-glucopyranoside as substrate in modified universal buffer (MUB) reported a pH optimum at 6.0. The optimum pH value (5.0) obtained in this study is close to the optima pH of other reported enzymes involved in C mineralization in soils (Eivazi and Tabatabai 1988; Deng and Tabatabai 1994). Various buffers have been used to assay for maltase activity from microbes isolated from soils and soil extracts. "
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    ABSTRACT: The enzyme maltase (glucoinvertase; glucosidosucrase; maltase-glucoamylase; α-glucopyranosidase; glucosidoinvertase; α-d-glucosidase; α-glucoside hydrolase; α-1,4-glucosidase EC, is involved in the exo-hydrolysis of 1,4-α-glucosidic linkages and certain oligosaccharides into glucose which is an important energy source for soil microbes. This enzyme originates from different sources, which include plants, seaweeds, protozoa, fungi, bacteria, vertebrates, and invertebrates. The assay of soil maltase using maltose as substrate and the released glucose determined using a glucose oxidase–peroxidase system has not been explored or investigated to the best of our knowledge. A simple assay protocol using this system is proposed to evaluate and characterize maltase activity in soils. The protocol involves the release of glucose (determined using a glucose oxidase–peroxidase colorimetric approach) when 1 g soil is treated with toluene and incubated with 5 mM maltose in 67 mM sodium acetate buffer (pH 5.0) at 37 °C for 1 h. The optimal activity using this procedure was at pH 5.0 and decreased at temperatures above 70 °C. The calculated K m values ranged from 0.8 to 6.5 mM, and are comparable to those of enzymes purified from microorganisms. The Arrhenius equation plots for the activity in the four soils were linear between 20 and 70 °C. The activation energy values ranged from 34.1 to 57.2 kJ mol−1, the temperature coefficients (Q 10) ranged from 1.5 to 1.9 (avg. = 1.7), and the coefficients of variation (CV) of the proposed assay protocol for the soils used was <6%. While we recognize the availability of established assay protocols to determine soil α-glucosidase (referred in other literature as maltase) activity based on the p-nitrophenol (artificial product) released from p-nitrophenyl-α-d-glucopyranoside (artificial substrate), our interest was to assay its activity by determining the glucose (natural product) released from maltose (natural substrate).
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    • "In these assays, 1 U of enzyme activity was defined as the amount of enzyme realizing 1 μmol of reducing sugar per min. Released sugars were determined by Somogyi–Nelson method (Deng and Tabatabai 1994) using calibration on glucose (for cellulases ) or xylose (in case of xylanase). MnP activity was measured by oxidation of phenol red. "
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    ABSTRACT: In this work, capability of Fusarium solani F-552 of producing lignocellulose-degrading enzymes in submerged fermentation was investigated. The enzyme cocktail includes hydrolases (cellulases, xylanases, and proteinases) as well as ligninolytic enzymes: manganese-dependent peroxidase (MnP), lignin peroxidase (LiP), and laccase (Lac). To our knowledge, this is the first report on production of MnP, LiP, and Lac together by one F. solani strain. The enzyme productions were significantly influenced by application of either lignocellulosic material or chemical inducers into the fermentation medium. Among them, corn bran significantly enhanced especially productions of cellulases and xylanases (248 and 170 U/mL, respectively) as compared to control culture (11.7 and 29.2 U/mL, respectively). High MnP activity (9.43 U/mL, control 0.45 U/mL) was observed when (+)-catechin was applied into the medium, the yield of LiP was maximal (33.06 U/mL, control 2.69 U/mL) in gallic acid, and Lac was efficiently induced by, 2,2'-azino-bis-[3-ethyltiazoline-6-sulfonate] (6.74 U/mL, not detected in control). Finally, in order to maximize the ligninolytic enzymes yields, a novel strategy of introduction of mild oxidative stress conditions caused by hydrogen peroxide into the fermentation broth was tested. Hydrogen peroxide significantly increased activities of MnP, LiP, and Lac which may indicate that these enzymes could be partially involved in stress response against H(2)O(2). The concentration of H(2)O(2) and the time of the stress application were optimized; hence, when 10 mmol/L H(2)O(2) was applied at the second and sixth day of cultivation, the MnP, LiP, and Lac yields reached 21.67, 77.42, and 12.04 U/mL, respectively.
    Full-text · Article · Apr 2012 · Folia Microbiologica
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    • "− 1 , respectively (Deng and Tabatabai, 1994b "
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    ABSTRACT: A new method for extracting soil enzymes is described and a microplate method for assaying soil beta-1,4-glucanases (cellulases) and beta-1,3-glucanases (laminarinases). Soil samples were mechanically disrupted to produce crude enzyme extracts, and diluted preps incubated in microplates containing either carboxymethyl cellulose (CMC) to determine cellulase activity or laminarin substrate to determine laminarinase activity. The resulting glucose was measured using the fluorometric Amplex Red glucose assay. The method was reproducible, could be completed in 1 day and measured twice as much enzyme activity than the standard passive soil enzyme extraction procedure. The method described herein facilitates the development of high-throughput soil multiplex enzymatic assays from several soil samples at one time, and is well suited to the study of functional microbial ecology.
    Full-text · Article · Sep 2009 · Journal of microbiological methods
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