Amyloglucosidase from a niger

The talipot flour was acquired from the stem pith of a matured talipot palm from Malappuram, South India, by following the method of Navaf et al. [2 (link)] and stored in a refrigerator (4 ± 2 °C). Phosphoryl chloride (POCl₃, >99% purity), potassium iodide, α-amylase from A. oryzae (∼30 U/mg), amyloglucosidase from A. niger (≥300 U/mL) were procured from Sigma Aldrich (St. Louis, MO, USA). Sodium hydroxide, hydrochloric acid, acetic acid, and iodine were purchased from Merck Ltd. (Mumbai, India). The chemicals availed for the study were of analytical grade, otherwise mentioned.

Soluble sugars were estimated as previously described (Payyavula et al., 2012 (link)), with minor modifications. Briefly, freeze-dried leaf and air-dried stem samples (20 mg) were each extracted three times with 80% ethanol at 80°C. The supernatants were then combined and re-extracted with 50 mg of activated charcoal. Next, the pigment-free extract was dried overnight at 50°C to eliminate ethanol, resuspended in either 800 or 250 μl of water (leaf or stem, respectively) and then employed in sucrose and glucose estimation assays using commercial kits (Sigma, St. Louis, MO, USA). Starch was estimated from the plant sample fraction leftover after ethanol extract (pellet remaining after soluble sugar extraction). The pellet was treated with 720 μl of 0.1 N NaOH at 50°C for 30 min and was neutralized in 900 μl of 0.1 N acetic acid. Pellet starch was digested using 1 U each of α-amylase (from Aspergillus oryzae, Sigma, St. Louis, MO, USA) and amyloglucosidase (from A. niger, Sigma), as described elsewhere (Chow and Landhäusser, 2004 (link); Payyavula et al., 2011 (link)). Starch quantities were calculated from the glucose standard calibration curve.