D glucose

The DNS assay described by Miller (1959) was performed to monitor the enzymatic hydrolysis of carbohydrates through measuring the released reducing sugars [46 (link)] The DNS solution was prepared by dissolving 0.1 g of 98% 3,5-dinitrosalicylic acid (Loba Chemie, Mumbai, India) in 2 mL of 2 N NaOH (Qrec, Auckland, New Zealand). Then, 3 g of sodium potassium tartrate (Kemaus, Cherrybrook, Australia) was added to the solution at 50 °C. The solution was subsequently adjusted to 10 mL with deionized water. An aliquot of the sample (100 μL) was mixed with DNS reagent (100 μL). The mixture was incubated in a water bath at 95 °C for 10 min and was cooled down before adding 500 μL of deionized water. Then, the absorbance was read at 540 nm using a spectrophotometer (Spectra max plus 384, Molecular Devices, Ramsey, MN, USA), and the reducing sugar concentration was expressed in terms of D-glucose (MP Biomedicals, Strasbourg, France).

Both titanium diisopropoxide bis(acetylacetonate) and dicyandiamide were purchased from Sigma-Aldrich, India. Calcium nitrate (Ca(NO₃)₂·4H₂O), acrylamide and D-glucose were supplied by MP Biomedicals, India. Ammonia solution (NH₃ about 25%), nitric acid (HNO₃) and tartaric acid were supplied by Merck, India. All chemicals were used as received. Deionized (DI) water obtained from a double-stage water purifier (ELGA PURELAB Option-R7) was used for all experimental work.