Isolation, Production, and Optimization of Extracellular Pullulanase Enzyme from Soil Microbial Isolates

Abstract

Pullulanase, a debranching enzyme, is essential in converting starches into simpler sugars, which has significant applications in the food and biofuel industries. This study focused on isolating, producing, and optimizing extracellular pullulanase enzyme from soil microbial isolates. Soil samples were collected from diverse environments, leading to the isolation of 150 microbial strains. Twenty of these strains exhibited pullulanase activity, identified through clear zones in iodine-stained pullulan agar plates. These strains were further identified using phenotypic and genotypic methods, revealing species within the genera Bacillus, Pseudomonas, and Streptomyces.Optimization of fermentation conditions was conducted to maximize enzyme production. The optimal conditions were determined to be an initial pH of 6.5, a temperature of 35°C, a substrate concentration of 15 g/L, and an incubation period of 72 hours. Under these conditions, pullulanase activity reached 150 U/mL. The enzyme showed optimal activity at pH 6.0 and 50°C and demonstrated stability across a pH range of 5.0 to 7.0 and at 50°C. The enzyme displayed high specificity for pullulan, with kinetic parameters indicating a high affinity and catalytic efficiency.Scale-up experiments were conducted in a 5-liter bioreactor, yielding an enzyme activity of 140 U/mL under optimized conditions. The purification process, involving ammonium sulfate precipitation, dialysis, and chromatographic techniques, resulted in a high-purity enzyme, confirmed by SDS-PAGE.The characterized pullulanase has promising applications in various industries. In the food industry, it can produce maltose and glucose syrups and enhance brewing processes. In the biofuel industry, it can improve the saccharification process for bioethanol production. Additionally, it is useful for starch desizing in the textile industry and has potential applications in the paper and pulp, waste management, and pharmaceutical sectors.This research highlights the potential of soil microbial isolates as a valuable source of industrial enzymes and provides a foundation for further studies to enhance enzyme yield and stability through genetic and synthetic biology approaches.