Isolation and Characterization of Amylase Producing Microbes and Its Sequential Strain Development for Industrial Enzyme Production

Abstract

Amylase enzymes are essential biocatalysts with wide-ranging industrial applications, including food processing, textile desizing, detergent formulation, paper production, and pharmaceuticals, owing to their ability to hydrolyze starch into simpler sugars. Despite the rising industrial demand, Bangladesh currently lacks domestic amylase production facilities, resulting in a dependence on total imports. To address this challenge, the present study aimed to isolate potent amylase-producing bacterial strains from natural sources, followed by their characterization, strain improvement through mutagenesis, optimization of production parameters, and evaluation of their industrial applicability. The ultimate goal was to develop a sustainable and indigenous source of amylase for local utilization. Following the harvesting period, soil samples were obtained from potato-growing areas in the Munshiganj and Rangpur districts, specifically from sites where remnants of decomposing potato matter remained in the fields. A total of 128 microbial isolates comprising 69 bacterial and 59 fungal strains were obtained using nutrient agar and potato dextrose agar media. Primary screening for amylase activity was conducted via starch agar assays followed by iodine staining, and hydrolysis zone-to-colony diameter ratios were determined. Among the bacterial isolates, S-1 and S-2 showed the highest hydrolytic potential, with the zone ratios of 3.44 and 4.98, respectively. Secondary screening using the dinitro salicylic acid (DNS) method revealed that isolate S-2 exhibited the highest crude amylase activity (7.98 ± 0.26 U/mL), compared to S-1 (5.21 ± 0.21 U/mL). Based on these findings, isolate S-2 was selected for further investigation. Protein concentration estimated by the Folin–Lowry method yielded 0.981 ± 0.03 mg/mL, with a corresponding specific activity of 8.14 ± 0.25 U/mg, confirming the isolates for industrial potential. Morphological analyses (Gram staining, spore staining, colony traits) indicated that isolate S-2 belonged to the Bacillus genus. This was supported by biochemical tests including catalase, Simmons’ citrate, starch hydrolysis, nitrate reduction, Voges-Proskauer and indole tests. The bacterial isolate was conclusively identified as Bacillus subtilis based on comprehensive molecular analysis involving 16S rRNA gene amplification, nucleotide sequencing, and subsequent phylogenetic evaluation showing over 99% similarity with reference sequences from the NCBI database. Amylase production from Bacillus subtilis S-2 was significantly enhanced through systematic strain improvement using physical (UV, gamma) and chemical (EMS) mutagenesis. The wild-type strain exhibited an initial amylase activity of 7.98 ± 0.26 U/mL, ii which significantly increased following mutagenic treatment. Among the conditions tested, UV exposure at 254 nm for 15 minutes resulted in the highest enzyme activity of 15.72 ± 0.32 U/mL, representing a 96.99% enhancement compared to the wild type. Gamma irradiation (1.0 kGy) and EMS (0.5%, 60 min) also significantly boosted production (15.11 ± 0.34 and 15.22 ± 0.48 U/mL, respectively). The UV-induced mutant (S2-UV3) was selected for further optimization. To maximize enzyme production, a dual-stage optimization strategy was employed, initially utilizing the conventional one-variable-at-a-time (OVAT) technique, followed by a more refined statistical optimization through Response Surface Methodology (RSM) based on the Box–Behnken Design (BBD). OVAT revealed optimal conditions pH 7.0, temperature at 45°C, 72 hours incubation and 1.5% starch achieving 24.21 ± 0.52 U/mL activity. Peptone (1.0%) was the best nitrogen source. Both commercial soluble starch and locally sourced potato starch yielded comparable enzyme activity (~24 U/mL), indicating agro-industrial applicability. RSM optimized the process further, with a robust model (R² = 98.96%, p < 0.0001). Predicted conditions (pH 7.02, 46.06°C and 1.56% starch) yielded 26.12± 0.46 U/mL in validation trials. Scaling-up in a 5 L fermenter with 1 vvm aeration and 130 rpm agitation improved production by 19.15%, reaching at 31.12± 0.62 U/mL attributed to enhanced oxygen transfer and temperature control. These results confirm process scalability and economic viability using locally available resources. Purification of amylase from UV-mutated (S2-UV3) was performed from RSM-optimized cultures. The preliminary crude enzyme preparation demonstrated an activity level of 26.12 ± 0.46 U/mL, accompanied by a protein content of 0.98 ± 0.04 mg/mL, thereby yielding a specific enzymatic activity of 26.65 U/mg protein. Following a three-step purification protocol comprising ammonium sulfate precipitation, dialysis, and gel filtration chromatography, the enzyme was recovered with an activity of 64.22 U/mL and a significantly enhanced specific activity of 133.79 U/mg. This process resulted in a 5.02-fold purification and a yield of 74.8%, indicating the effectiveness and reliability of the purification protocol in concentrating and refining the target enzyme. For long-term application, S2-UV3 was evaluated for strain preservation. Cryopreservation at –80°C in 20% glycerol retained ~80% activity after 12 months, while 4°C storage showed notable decline after 6 months. Thus, –80°C with glycerol is the optimal condition for long-term storage. Purified enzyme stability was assessed under varied storage conditions. While 4°C was maintained >70% (47.44 U/mL) activity for 6 months, significant losses occurred. Storage at iii –20°C and –80°C retained 75–78% activity after a year. Inclusion of 0.1% sodium azide and 20% glycerol enhanced stability up to 87%, while lyophilized enzyme at 4°C preserved over 92% activity. The lyophilization and freezing with glycerol are the most effective strategies for long-term enzyme stability. SDS-PAGE of the purified enzyme showed a single band (~56 kDa), confirming molecular purity and alignment with known Bacillus α-amylases. Absence of additional bands indicated structural homogeneity, essential for consistent industrial use. The purified amylase (64.22 U/mL) showed strong industrial applicability. In textile desizing, complete starch removal from cotton fabric was confirmed via iodine staining and reducing sugar release (4.20 ± 0.11 mg/mL). In detergent-assisted cleaning, enzyme-detergent synergy yielded maximum starch stain removal (4.86 ± 0.15 mg/mL). These findings validate the enzyme's eco-friendly, effective role in textile and detergent industries. In conclusion, this study successfully established a comprehensive work for the isolation, characterization, and mutagenesis-based improvement of a potent amylase-producing Bacillus subtilis strain sourced from locally collected soil samples. The use of low-cost agricultural waste, such as potato peels, as an alternative carbon source combined with systematic optimization and multistep purification led to the development of a highly active and stable enzyme. The purified amylase exhibited excellent performance in eco-friendly industrial applications, including textile desizing and detergent-based starch stain removal. Altogether, the optimized production process and improved bacterial strain offer a valuable biotechnological resource for sustainable, indigenous enzyme production in Bangladesh and hold promising potential for future industrial scale-up and commercialization.