Characterization of different isogenic mutants derived from improved strains of Bacillus naganoensis using Active Hydrogen Bond Network (AHBN) for pullulanase production

This study aimed to improve the thermal and pH stability of pullulanase enzymes through protein engineering. Pullulanase is crucial in various industries, including food, detergent, and textile, as it breaks down starch into simple sugars. Using the active hydrogen bond network (AHBN) method, researchers constructed isogenic mutants of the <i>Bacillus naganoensis</i> pullulanase enzyme. The mutants were evaluated for their enzyme activity, pH stability, and thermal stability. Results showed that two mutants, H543R and H543G, exhibited improved biochemical properties compared to the primary mutant E431A. All three mutants had optimal enzyme activity at pH 5.4 and 65 °C. They also showed stability at pH 4.2-5.8 for over 48 h. The T50 experiment revealed that H543R retained 63 % of its enzyme activity at 62.7 °C, while the primary mutant E431A retained 68 %. The TM value experiment showed that H543G and H543R were stable at 65 °C and 64.7 °C, respectively. These findings demonstrate the potential of protein engineering in improving the stability and activity of pullulanase enzymes, which can benefit various industries that rely on starch degradation.