Production, Characterization of Pectinase from Bacillus inaquosorum and its applications

Abstract

The study focuses on isolating, screening, and characterizing extracellular polysaccharide-degrading enzymes, with particular emphasis on pectinase production, which has vast applications across industries such as pharmaceuticals, food, dairy, textiles, and plant fiber processing. The primary aim was to identify novel bacterial strains capable of producing pectinase and to optimize various parameters for maximizing enzyme production. Natural resources like industrial waste, decayed fruit waste, fruit peels, and rhizospheric soil were used as sample sources for microbial isolation. In total, hundred bacterial isolates were obtained from twenty-three different samples collected from various regions of Gujarat, India. One particular bacterial strain, identified as Bacillus inaquosorum, showed significant potential for pectinase production and was selected for further study. Submerged fermentation was employed to enhance enzyme production, followed by optimize pectinase production, the study employed the one-factor-at-a-time (OFAT) approach, where in key physicochemical factors such as pH, inoculum size, temperature, and incubation time were varied to determine their impact on enzyme production. Subsequent statistical optimization of media components was conducted using the Plackett-Burman design, which identified significant factors influencing enzyme production. Among the media components, glucose and pectin were identified as the best carbon sources, while ammonium sulfate and potassium nitrate (KNO₃) were the best nitrogen sources. In the next phase, further optimization was performed using the Response Surface Methodology (RSM) through a Central Composite Design (CCD). This optimization yielded a significant improvement in enzyme production, with the highest pectinase activity recorded at 1.35 ± 0.04 U/mL/min, representing an increase of 0.545 ± 0.02 U/mL/min compared to the unoptimized medium. Enzyme purification through a three-step process involving ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration chromatography using a Sephadex G-75 column. The purification process achieved a 60.86% yield of pectinase. Characterization of the purified enzyme revealed that the highest pectinase activity occurred at a pH of 6 and a temperature of 50°C. Additionally, the presence of Fe²⁺ ions further enhanced the enzyme’s activity. The stability of the enzyme in the presence of surfactants and inhibitors suggests its potential for industrial applications, particularly in sectors requiring pectin degradation. The kinetic parameters of the enzyme were determined, with the Km value recorded as 166.53 M and the Vmax as 769.24 µM/ml, indicating the enzyme's efficiency in catalyzing reactions. The optimal conditions were found to be a pH of 6.0, inoculum size of 3 mL, temperature of 37°C, and an incubation time of 48 hours. The results demonstrate that optimizing medium components and fermentation conditions can significantly enhance pectinase production, with potential applications in industries such as food processing, particularly for juice clarification and plant fiber degradation.