Screening and molecular characterization of microorganisms producing hydrolytic enzymes for cassava (manihot esculenta crantz) starch degradation

Abstract

Corn starch is a traditional source of glucose in the food industry, but its use is limited due to competition with corn as a staple food. Cassava (Manihot esculenta Crantz), which has a high starch content, is a potential alternative starch source. However, the complex structure of cassava starch hinders its industrial application. This study aimed to isolate and identify microorganisms capable of producing amylase enzymes from diverse and unexplored environments in Kenya. The initial screening involved evaluating the ability of microorganisms to grow on minimal media supplemented with starch. Based on this screening, 70 microbial amylolytic isolates were selected. These isolates underwent a primary screening based on starch hydrolysis ratio (SHR), determined by halo formation on starch agar plates after flooding the plates with Lugol's iodine solution. This narrowed down the number of isolates to 17, which were presumed to be excellent amylase producers (SHR > 1.5) and were further used in subsequent experiments. The 17 selected isolates were cultivated in M9 starch broth media at an optimum temperature of 30°C and pH 7.0 to produce amylase enzymes. Crude amylases were extracted from the supernatant, quantified at 280 nm, and assessed for amylase activity using the 3,5-dinitrosalicylic acid (DNSA) method. Enzyme stability was tested through incubation at 30°C for 18 hours, while the effect of temperature and starch loading was evaluated by measuring activity at 45°C, 50°C, and 55°C, and with starch concentrations of 20%, 50%, and 60%, respectively. A commercial amylase was used as a control. The specific enzyme activities of the crude enzymes from the 17 isolates ranged from 0.0079 U g-1 to 0.0629 U g-1, which were lower compared to the specific activity (1.7188 U g-1) of the control enzyme. The top 5 enzymes with the highest specific enzyme activity were selected for cassava hydrolysis. These crude enzymes exhibited higher specific enzyme activity (0.0707–0.1853 U g-1) than the control enzyme (0.0052 U g-1) in hydrolyzing cassava starch. Among the isolates, those with the highest specific enzyme activity were identified through DNA sequencing and belonged to Lysinibacillus spp. (4 isolates), Alternaria spp. (1 isolate), and Bacillus spp. (2 isolates). Alkaline lakes (Lake Elementaita and Lake Bogoria) had the highest number (4) of isolates with enzymes showing the highest specific enzyme activity, followed by the gut contents of black soldier flies. The specific enzyme activity increased with temperature, and starch loading had an impact on enzyme activity. While most enzymes showed reduced activity over time at 30°C, enzymes from one isolate retained full activity at 18 hours of incubation, demonstrating thermostability. This study highlights the presence of microorganisms with amylase activity in tropical environments that can be scaled and optimized for practical applications