Enzymes are biological catalysts that accelerate specific biochemical reactions within living organisms by lowering the activation energy required. Their catalytic specificity arises from the unique three-dimensional structure of their active sites, which allows selective binding to their corresponding substrates. Amylase is an enzyme that catalyzes the hydrolysis of starch into smaller carbohydrates such as dextrins and simple sugars, including glucose. Amylase plays a critical catalytic role in the degradation of starch by breaking down its glycosidic bonds, ultimately yielding glucose as the monomeric end product [1]. Amylases represent a group of extracellular enzymes, which are α-amylase, β-amylase, and glucoamylase, that act on starch or oligosaccharide molecules randomly and hydrolyse starchy products into progressively smaller polymers composed of glucose units. Amylases are one of the most important enzymes and account for about 30% of the world’s enzyme production [2]. Amylases break down starch into fermentable sugars and have a wide range of applications across various industries, including food, fermentation, textiles, paper, detergents, and sugar production [3]. They also play an important role in biotechnology by helping to remove environmental pollutants, converting starch into desired substrates with the aid of various microorganisms, and processing waste that contains starch [4]. Amylases have been reported to occur in microorganisms (fungi, yeast, bacteria, and actinomycetes), although they are found in plants and animals [5]. The soil harbours various microbes which have advantages over other sources for industrial use [6][7]. Soil is a rich reservoir of diverse microbial communities, with commonly isolated bacterial genera including Bacillus, Pseudomonas, Enterobacter, Staphylococcus, and Klebsiella, among others [8]. Cassava production sites, in particular, have been reported to harbor a variety of microorganisms, some of which possess industrially relevant metabolic capabilities [9]. The present study aimed to identify amylase-producing bacteria from soil samples collected at cassava cultivation sites using molecular techniques.

Soil Collection and Isolation of Bacteria

Random soil samples were collected from the cassava production site (Gwagwalada area). Samples were placed in a clean polythene bag, transported to Sheda Science and Technology, Complex Abuja, and stored in the refrigerator for further analysis. One gram of the soil sample was suspended in 9 mL of sterile distilled water and thoroughly mixed in a test tube. Serial dilutions of the suspension were then prepared up to 10⁻⁸ and 10⁻⁵, following the method described by Bhatt and Singh [10]. In this method, 1 mL of the soil suspension is transferred into a new tube containing 9 mL of sterile diluent, achieving a 10-fold (1:10) dilution. This process was repeated sequentially across multiple tubes to obtain progressively lower concentrations of the original sample, facilitating the isolation of individual microbial colonies. One millilitre (1 ml) of the sample was poured plated separately on a starch medium prepared by using 1% starch slurry in a Nutrient agar as described by Kanimozhi et al. [11]. It was then incubated at 37˚C for 24 h for the isolation of bacteria. The bacterial isolates on the starch medium were further purified to obtain discrete representative colonies on a new plate containing the starch medium. The plates were incubated at 37˚C for 24 hours to obtain pure cultures, which were transferred to a slant and stored in a refrigerator at 4˚C [12].

Screening of Isolates for Amylase ActivityUsingLugol’sIodine Method

The Lugol’s iodine method is used as a qualitative assay to detect the activity of amylase-producing bacteria. Bacterial isolates were cultured on starch agar plates and incubated under optimal conditions. After incubation, the plates were flooded with Lugol’s iodine solution. Clear zones surrounding the bacterial colony indicate starch hydrolysis due to amylase activity [13].

Crude Amylase Assay

The purified bacterial isolates were inoculated into starch broth and incubated for 24 hours at 37˚C. Following incubation, cultures were centrifuged at 4000 rpm for 20 minutes to obtain cell-free supernatants for enzyme analysis. Amylase activity was quantified using the dinitrosalicylic acid (DNSA) assay, as described by Sundarapandiyan and Jayalakshmi [14] and Al-khafaji et al. [15]. The reaction mixture consisted of 1 mL of 1% soluble starch solution, 1 mL of potassium phosphate buffer (pH 6.9), and 0.1 mL of the enzyme-containing supernatant. The mixture was incubated at room temperature for 15 minutes. To terminate the reaction, 2 mL of DNSA reagent was added, and the tubes were placed in a boiling water bath (100˚C) for 10 minutes. Reducing sugar released during the reaction was measured spectrophotometrically, indicating the amylase activity. One unit of enzyme activity was defined as the amount of enzyme that hydrolyses 1mg of starch per minute under assay conditions. The reducing sugar liberated was estimated using the dinitrosalicylic acid method (DNSA). One unit of amylase was defined as the amount of 1µmol glucose equivalent per minute under the assay conditions [1].

Molecular Identification of Amylase-Producing Bacteria

Genomic DNA extraction from the two bacterial isolates with the highest amylase activity was performed using the DNA extraction kit following the protocol provided by Zymo Research USA, the manufacturer. Further, DNA was amplified by Polymerase Chain Reaction (PCR) using universal bacterial primers, Forward primer (27 F-AGAGTTTGATCCTGGCTCAG) and Reverse primer (1492r GGTTACCTTGTTACGACTT). The PCR was carried out at an initial denaturation step at 94˚C for 2 min, followed by 30 cycles at 94˚C for 30 sec, 52˚C for 30 sec and 72˚C for 2 min, and a final extension step at 72˚C for 5 min. The RT‐PCR products were examined by electrophoresis on a 1% agarose gel [8][4][16].

DNASequencingandAnalysis

The genomic DNA of the bacterial isolates exhibiting the highest amylase activity was subjected to sequencing. The obtained nucleotide sequences were analyzed using the Basic Local Alignment Search Tool (BLAST) to perform similarity searches against the GenBank nucleotide database maintained by the National Center for Biotechnology Information (NCBI). Taxonomic identification was based on the highest sequence similarity with reference strains in the database [17].

Description of Study Area

Soil samples were collected from a cassava-producing site in the Gwagwalada Area of Abuja. Gwagwalada, Abuja, Northern Nigeria. Abuja experiences a tropical savanna climate with two distinct seasons, which are the rainy season and the dry season. The rainy season lasts from April to October, with the peak rainfall occurring from July to September, while the dry season occurs from November to March, and it is usually accompanied by Harmattan winds from December to February [18]. The Temperatures in this area range from 30˚C to 37˚C during the day and from 18˚C to 24˚C at night, with the hottest period from March to May. Gwagwalada Area of Abuja lies at an altitude of approximately 150 to 300 meters above sea level and has an average annual rainfall of about 1100 to 1600 millimetres.

Isolation of Amylase-Producing Bacteria

The Soil samples were serially diluted to isolate bacteria, spread on a starch agar medium, and incubated at 37˚C for 24 hours. A dense, thick population of mixed bacterial colonies originating from a dilution factor of 10⁻⁵ was observed on the starch agar plates. A pure culture of 5 bacterial isolates (B1 to B5) was obtained as shown in Figure 1Figure 1, which were randomly picked up from the mass bacterial population based on their colony morphology.

Figure1. Bacterial isolates from the cassava production site.

Screening for Amylase Activity

The bacterial isolates were assessed for amylolytic activity using starch agar medium. A distinct, clear zone surrounding the bacterial colonies, as shown in Figure 2Figure 2, indicated positive starch hydrolysis. This clear zone results from the enzymatic degradation of starch by amylase, producing a starch-free region. In contrast, the unhydrolyzed starch in the surrounding medium produced a characteristic dark blue to purple coloration upon iodine application. Over time, the diameter of the clear zones increased, suggesting continued amylase activity and progressive starch degradation by the bacterial isolates.

Figure2. Screening for amylase-producing microbes using Lugol’s iodine.

EnzymeActivity DeterminationofAmylase-Producing

The Enzyme activity was estimated using the dinitrosalicylic acid (DNSA) reagent method, and the results are presented in Table 1. Among the tested isolates, bacterial isolate B1 exhibited the highest amylase activity, indicating strong starch-degrading potential. Isolate B2 also demonstrated relatively high enzyme activity, though slightly lower than B1. In contrast, isolate B4 showed the lowest amylase activity, suggesting comparatively reduced amylolytic capacity among the evaluated samples.

Table1. Showing enzyme concentration in each sample of bacteria isolates (B1 - B5).

Molecular Identification of the Amylase-producing bacteria the earlier NCBI database. The result revealed B. cereus 99.41 % and S. saprophyticus99.88% nucleotide similarities.

Figure3. Agarose gel electrophoresis identifying the bacterial amplicons of amylase-producing bacteria.

(a)

(b)

Figure4.Genbank data for the Samples B1 and B3 showing nucleotide similarities (a) Gene bank data for B1 - B. cereus shows a 99.41% similarity score. (b) Gene bank data for B3 - S. saprophyticus shows a 99.88% similarity score.

Amylases are essential industrial enzymes widely utilized in the hydrolysis of starch into glucose, with significant applications in the food, pharmaceutical, and fermentation industries. In the present study, five bacterial isolates (designated B1 - B5) were recovered from soil samples collected at a cassava production site using starch agar medium (Figure 3Figure 3 & Figure 4Figure 4). To isolate potential amylase producers, soil samples were subjected to serial dilution and plated on starch agar, with nutrient agar used as the basal growth medium. Plates were incubated at 37˚C for 24 - 72 hours. Amylase activity was screened using a qualitative plate assay involving flooding with 1% Lugol’s iodine solution. The formation of distinct, clear zones around bacterial colonies indicated starch hydrolysis, while unaffected areas stained dark blue, confirming the presence of residual starch. This approach is consistent with the method described by Hossain [13], who utilized Lugol’s iodine flooding to identify amylase-producing bacteria from agricultural soils.

Quantitative estimation of enzymatic activity confirmed amylolytic potential in all isolates, with B1 and B3 exhibiting the highest levels of activity. 16S rRNA gene sequencing identified isolate B1 as Bacillus cereus with 99.41% nucleotide sequence similarity, while isolate B3 was identified as Staphylococcus saprophyticus with 99.88% similarity. The identification of B. cereus is consistent with previous studies highlighting the genus Bacillus as a prolific producer of industrially relevant enzymes [15][16]. Semira et al. [19] reported the isolation of thermostable amylase-producing Bacillus species from soil, while Abd-Elhalim et al. [20] confirmed the high α-amylase activity of B. cereus from similar environments.[21] further demonstrated the beneficial role of Bacillus thuringiensis in improving seed germination and plant growth. Although Staphylococcus saprophyticus is less commonly reported as an amylase producer, its identification in this study as a high-performing isolate expands the diversity of microbial sources for amylase production and offers new opportunities for biotechnological exploitation. These findings emphasize the potential of cassava cultivation environments as reservoirs of valuable enzyme-producing microorganisms. Further research should focus on optimizing culture conditions for maximum enzyme yield, scaling up fermentation processes, and characterizing the biochemical properties of the amylases. Such efforts will support the development of efficient and sustainable starch bioconversion strategies for industrial applications.

This study reveals the untapped potential of soil from cassava production environments as a rich reservoir of industrially relevant microorganisms, particularly amylase-producing bacteria. Among the isolates identified, Bacillus cereus and Staphylococcus saprophyticus exhibited the highest amylase activity, highlighting their strong potential for biotechnological exploitation. These strains represent promising candidates for the sustainable, large-scale production of amylase enzymes, which are integral to diverse industrial sectors including food processing, pharmaceuticals, biofuels, textiles, and paper manufacturing. Additionally, their use in enzyme-based detergent formulations offers an environmentally friendly alternative to conventional chemical surfactants.

Given the increasing global demand for sustainable and cost-effective enzyme sources, the identification of B. cereus and S. saprophyticus as efficient amylase producers presents a valuable opportunity for commercial enzyme development. Future work should focus on optimizing culture conditions, employing genetic or metabolic engineering strategies, and scaling up fermentation processes to maximize enzyme yield and industrial applicability.

All authors contributed to the concept and design of this work.