Abstract
Nelson–Somogyi and 3,5-dinitrosalicylic acid (DNS) assays are the classical analytical methods for the determination of activity of starch-debranching enzymes, however, they have a narrow detection range and do not adapt to the quantitative measurement of linear polysaccharides. Herein, we developed a simple and accurate colorimetric assay for determining the activity of starch-debranching pullulanase through the modified Tollens’ reaction in combination with UV irradiation. Silver nanoparticles (AgNPs) were formed by reducing aldehyde groups in short-chain glucans (SCGs) generated by debranching of waxy maize starch using pullulanase through the modified Tollens’ reaction. In addition to providing a reducing moiety to the Tollens’ reaction, the debranching product, SCGs, also enhanced the colloidal stability of synthesized AgNPs, of which the amplitude of its surface plasmon resonance (SPR) absorbance peak was proportional to the concentration of SCGs ranging from 0.01–10 mg/mL. The detection limit of this system was 0.01 mg/mL, which was found to be 100 times higher than that of the conventional DNS assay. The purification of SCGs by recrystallization and gelatinization improved the selectivity of this colorimetric assay for debranching products, which provides a simple and accurate means of monitoring the debranching process and characterizing the activity of starch-debranching enzymes.
Highlights
Amylopectin, a highly branched polymer of α-glucose, is a major constituent of plant starch granules
Considering that glucose and fructose molecules are much smaller than short-chain glucans (SCGs), we assumed that these two monosaccharides adsorbed on the surface of nanoparticles do not act as an effective steric stabilizer to prevent the aggregation behavior of AgNPs [20]
We employed the principle of Tollens’ reaction to quantify the debranching product, SCG, of which the process was significantly facilitated by UV irradiation
Summary
Amylopectin, a highly branched polymer of α-glucose, is a major constituent of plant starch granules. The Nelson–Somogyi (NS) and 3,5-dinitrosalicylic acid (DNS) assays are the most widely used methods to measure the activity of DBEs [11] Both methods are based on the analysis of reducing sugars generated from the enzymatic cleavage of the glycosidic bond of highly branched amylopectin or glycogen by the specific catalytic action of DBEs. In particular, the DNS assay has been intensively employed due to its simple procedure, which requires a few minutes for the reaction and spectrophotometric measurement. This proposed analytical system is specific to the debranching products (short-chain glucan) and exhibited up to 100 times higher sensitivity in comparison to the DNS assay The mechanisms of this system were investigated through analyzing the AgNPs formed from the reaction, and its potential for sensitive and quantitative analysis of SCGs is presented
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