Cloning, molecular modelling, and docking analysis of GH-13 alpha-amylase from rumen metagenome for saccharification of starch rich biomass for greener future

α-Amylases are used in various biotechnological processes including the textile, paper, food, biofuels, detergents, and pharmaceutical industries. In this study, a unique starch-degrading enzyme (Alpha-Amy4) was isolated from the microbial diversity present in cattle rumen using functional metagenomic approach. A metagenomic library was constructed using fosmid vector (pCC1FOS) for high-throughput screening of enzymes through a multistage functional screening pipeline. Sequence analysis revealed an open reading frame of 1488 bp encoding a 496 amino acid long Alpha-Amy4 (GH13 family), sharing 92.73% similarity with an alpha amylase Escherichia coli (NCBI Reference Sequence: WP 001245695.1). The recombinant purified protein has a molecular weight of ∼54.0 kDa on SDS-PAGE and exhibited optimal activity at 45 °C and pH 5.5. The apparent Km and Vmax values were 0.368 mol mL−1 min−1 and 1092 µmol mL−1 min−1, respectively, according to the plot data in the absence of calcium activator. Homology modelling and molecular docking identified conserved catalytic triad residues (D172, E203, and D289) within the TIM (β/α)8-barrel motif involved in substrate hydrolysis. Finally, potato peel, a low-cost and high-starch agricultural waste was used to confirm the activity of Alpha-amy4. The enzyme demonstrated efficiency at a lower dosage (20 IU), resulting in enhanced saccharification (78.7%) of potato peels in a shorter time. Thus, Alpha-amy4 represents a novel member of the GH-13 glycoside hydrolase (GHs) family, distinguished by its enhanced enzymatic activity on raw agro-wastes, highlighting its potential for efficient and sustainable biomass valorisation.