A Sustainable Alternative for the Food Industry: Production of α-Amylase by Coprinus comatus Using Agro-Industrial By-Products

The demand for sustainable, low-cost bioprocesses has encouraged the development of alternative enzyme production strategies. This study investigated the purification and characterization of a crude enzymatic extract (CEE) rich in α-amylase from Coprinus comatus, using wheat milling by-products as substrate. The CEE was obtained by submerged culture, followed by biomass removal, centrifugation, and ultrafiltration to yield a partially purified enzyme (PE). CEE and PE were evaluated for chemical composition, antioxidant and antibacterial activity, toxicity (Artemia salina assay), and enzymatic performance. Toxicity assays confirmed that CEE was non-toxic. Antioxidant activity reached 213.34 µmol TE·gDE−1 (DPPH assay), with a total phenolic content of 8.01 mg GAE·gDE−1. No antibacterial activity was detected. CEE hydrolyzed 96.31% of starch in 180 min, releasing 10.85 g·L−1 glucose, while PE achieved 98% hydrolysis and released 14.5 g·L−1. Optimal α-amylase activity occurred at 50 °C and pH 5.5 (CEE) or 5.0 (PE). Calcium ions improved the enzymatic activity and thermal stability. CEE retained over 60% activity after 721 days under refrigeration (4 °C) or freezing (0 °C). Although lyophilization enhanced enzyme concentration, it increased production costs. SDS-PAGE revealed bands of ~67 kDa (α-amylase) and ~35 kDa (glucoamylase). These findings support the use of CEE as a sustainable, low-cost biocatalyst for industrial use.