Cyanobacteria are a phylum of autotrophic photosynthetic microorganisms that inhabit both aquatic and terrestrial environments. They likely appeared on Earth over 3.5 billion years ago, playing a crucial role in shaping the planet's atmosphere [1]. Cyanobacteria are classified into eight orders based on a polyphyletic approach: Gloeobacteriales, Synechococcales, Spirulinales, Chroococcales, Pleurocapsales, Oscillatoriales, Croococcidiopsidales, and Nostocales [2]. These microorganisms possess metabolite-producing properties of significant interest to the biotechnology industry, with applications spanning biofuels, dyes, food supplements, biofertilizers, biopolymers, cosmetics, water treatment, nanobiotechnology, medical applications, and enzyme production [3]. The Oscillatoriales and Nostocales orders are the most extensively studied for bioactive metabolites and their metabolic pathways [4]. Consequently, cyanobacteria represent a promising field of study as photoautotrophic prokaryotes that do not require complex growth media, making them suitable for green biocatalysis in sustainable approaches [5].

Amylases are enzymes from the Glycoside Hydrolase family that catalyze the breakdown of starch, converting starch molecules into fermentable sugars. There are three main types: α-Amylase, which hydrolyzes α-1,4-glycosidic linkages and can bypass α-1,6-glycosidic linkages; β-Amylase, which hydrolyzes α-1,4-glycosidic linkages at the non-reducing ends of sugars; and γ-Amylase, which hydrolyzes α-1,6-glycosidic linkages and other non-reducing linkages [6]. Amylases are found in plants, animals, bacteria, fungi, and yeasts, but those of microbial origin are the most commercially utilized due to their stability and adaptability, making them ideal for various industrial applications [7].

α-Amylase (EC 3.2.1.1) is an enzyme that catalyzes the hydrolysis of α-1,4 glycosidic bonds in starch, utilizing calcium ions as cofactors and producing smaller sugars, such as maltose and glucose [8]. It consists of three domains: Domain A (the D-E-D catalytic domain), Domain B (the calcium-binding and loop region), and Domain C (the terminal portion of the protein). α-Amylases typically range in size from 40 to 70 kDa and are encoded by the amy1 gene. These enzymes have widespread industrial applications, including in the food, textile, beverage, detergent, and paper industries [9]. They also contain conserved regions near the C-terminal structure, such as the β3 and β4 sheets (with one of the D residues responsible for nucleophilic attack), the β5 sheet (which contains the E residue involved in proton donation), and the β7 sheet (where the second D residue stabilizes the transition state during catalysis). Additionally, the β2 and β8 sheets near the TIM-barrel are involved in maintaining the structural integrity of the enzyme. Most residues within these conserved regions are nonpolar and should not undergo mutations, as such changes could compromise the enzyme's catalytic stability [10].

In cyanobacteria, α-Amylase activity has been described in Nostoc sp. PCC7119, with its activity evaluated and characterized using traditional bioassay methods. The enzyme demonstrated an optimal pH range of 6.5–7.5 and an optimal temperature of 31 °C. This study was instrumental in confirming that cyanobacteria can produce α-Amylases with amino acid sequences like those found in other bacteria, displaying excellent kinetic constants despite lacking thermostability. Previously described thermostable α-Amylases have shown activity at temperatures up to 50 °C [11]. This current study represents the first in silico characterization of potentially thermostable α-Amylases produced by cyanobacteria, evaluating enzyme-substrate interactions and comparing them with two previously studied enzymes.