α‐Amylase From Penicillium brevicompactum: Enzymatic Properties and Saccharification

ABSTRACTAlthough α‐amylases are critical biocatalysts in starch processing, fungal α‐amylase diversity remains largely untapped. This study presents the first systematic characterization of an α‐amylase from Penicillium brevicompactum NRRL864. Maximum α‐amylase activity of 195 U/mL was obtained at pH 6.0 and 55°C under optimized fermentation conditions. These conditions consisted of 0.75% starch, 3% peptone, 0.5% NaCl, incubation at 20°C for 7 days. A novel α‐amylase gene (DF) was identified via phylogenetic analysis and heterologously expressed in Escherichia coli. The gene encodes a 551‐amino‐acid α‐amylase with an open reading frame of 1656 bp, showing 68.36% sequence identity to the α‐amylase from Penicillium expansum. The DF enzyme displayed a specific activity of 489.4 U/mg under optimal conditions of 50°C and pH 6.0, and a 124.2% activity enhancement with 1 mM Ca2⁺, indicating Ca2⁺‐dependence. The activity of DF was inhibited by a range of chemical reagents, including EDTA and urea. Substrate specificity assays confirmed that the DF enzyme exhibited optimal activity in hydrolyzing soluble starch. DF‐mediated saccharification of corn starch yielded a reducing sugar concentration of 317.9 µg/mL within 70 min, confirmed by thin‐layer chromatography. Beyond identifying P. brevicompactum as a novel α‐amylase source, this work demonstrates DF as a Ca2⁺‐responsive GH13 enzyme with significant potential for industrial applications in biofuel and sweetener production.