By Baico Biotech 丨 Textile Enzyme 丨 BIO-SCOURING ENZYME

When detecting cellulase activity using the sodium carboxymethyl cellulose (CMC-Na) method, cellulase takes CMC-Na as the substrate for catalytic reaction and hydrolyses macromolecules into small-molecule reducing sugars. The content of reducing sugars reflects the magnitude of enzyme activity. Nevertheless, the solution remains colorless and transparent throughout this process, making it impossible to visually observe the amount the amount of reducing sugar or even whether reducing sugars have been produced. Due to the reducibility of reducing sugars, an oxidizing agent may be added to trigger redox reaction and induce color variation in the solution.

Fehling’s reagent is commonly used in the biological field to detect reducing sugars. However, during the reaction, Cu 2+ is reduced to a brick-redprecipitate of Cu2O, making the reacted solution turbid and uneven, which makes accurate detection difficult using a spectrophotometer. Therefore, it is typically used for qualitative analysis. Furthermore, Fehling’s reagent must be prepared fresh and cannot be stored as a stock solution, making the process cumbersome. DNS reagent, on the other hand, is an excellent oxidizing agent, suitable for the quantitative detection of reducing sugars, and can be stored for a period of time after preparation. In the Chinese light industry standard QB/T 2583-2023, the method for detecting cellulase activity, whether using the filter-paper method or the CMC-Na method, uses DNS reagent to detect the reducing sugar content.

Although DNS reagent is a bright yellow solution formulated from multiple chemical substances, the substance that actually reacts with the reducing sugar is 3,5-dinitrosalicylic acid. Caustic soda provides a strongly alkaline environment, while the other substances maintain the stability of the DNS reagent. When DNS reagent reacts with reducing sugars under boiling water bath conditions, 3,5-dinitrosalicylic acid is reduced to brownish-red 3-amino-5-nitrosalicylic acid. Highercontent of reducing sugars yields deeper color of the reacted solution. No precipitation occurs throughout the entire reaction process, and the solution remains homogeneous.

After the solution cools down, dilute it to the specified volume and shake well. Since the maximum absorption wavelength of 3-amino-5-nitrosalicylic acid is around 540 nm, the detection wavelength of the spectrophotometer is set to this value. Pour the solution into a cuvette and place it inside the instrument for absorbance measurement. Substitute the absorbance value into the glucose-absorbance equation to obtain the corresponding glucose content, and then substitute the result into the enzyme activity calculation formula.

Baico Biotech BBT Enzymes is a textile enzyme and household detergent enzyme manufacturer in China. BBT Enzyme prepared its own DNS reagent according to the standard. BBT also prepared glucose solutions of different concentrations, reacted them with DNS reagent, plotted glucose-absorbance scatter points and fitted lines, and generated glucose-absorbance equations.

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