Overexpression, purification and preliminary X-ray analysis of pullulanase from Bacillus subtilis strain 168

The AmyX gene encoding pullulanase from the common spore-forming bacterium Bacillus subtilis strain 168 was cloned, overexpressed in Escherichia coli, purified and crystallized. The recombinant pullulanase was purified to homogeneity using ammonium sulfate precipitation, hydrophobic chromatography and anion-exchange chromatography, resulting in a specific activity of 24.10 U per milligram of protein. SDS–PAGE analysis showed that the molecular weight of the protein is approximately 81.0 kDa, which is similar to the calculated molecular weight, 81.1 kDa, from its translated cDNA sequence. The k_cat and K_m of the purified enzyme with pullulan as substrate were approximately 79 s⁻¹ and 1.284 mg ml⁻¹, respectively. X-ray crystallographic analysis of the pullulanase crystal showed that the crystal belongs to the orthorhombic space group P2₁2₁2₁, with unit-cell parameters a = 70.568, b = 127.68, c = 189.25 Å. The crystal contains two molecules of pullulanase in the asymmetric unit, with a solvent content of 53.15%. The crystal diffracted to 2.1 Å resolution at a synchrotron and is suitable for structure determination.