Characterization and structural analysis of a pullulanase from thermotoga maritima MSB8 using site-directed mutagenesis.

Pullulanase specifically hydrolyzes α-1,6-glycosidic bonds and is widely used in industry. In this study, a pullulanase PulA (GenBank: CAA04522.1) from Thermotoga maritima MSB8 was targeted with the aim of enhancing its thermostability by altering its hydrogen bond networks, hydrophobic packing, and disulfide bonds. Three mutants of PulA, created via site-directed mutagenesis, designated as M1 (E191G), M2 (G500M), and M3 (R727C), were constructed and expressed in Escherichia coli. The melting temperatures of these mutants, elucidated by differential scanning fluorimetry, were 77.4 °C, 77.9 °C, and 78.6 °C, respectively, in contrast to the original PulA's melting temperature of 76.5 °C. Mutants M2 and M3 retained more than 80% of their original activity after incubation at 80 °C for 1 h. The Km of these mutants, elucidated by double reciprocal mapping method, were 0.25,0.13,0.16, respectively, in contrast to the original PulA's Km of 0.14. The disulfide bond mutant M3 exhibited improved thermostability and specific activity. A spatial analysis revealed that the mutated residue favored disulfide geometry, reducing the electrostatic repulsion between charged amino acids and improving the structural integrity. This study demonstrated that the molecular structure modification of pullulanase effects the stability significantly.