Biochemical Characterization of a Novel Thermostable Type I Pullulanase Produced Recombinantly in Bacillus subtilis

The pullulanase gene (pul(GK)), encoding a thermostable type I pullulanase (Pul(GK)), is obtained from the strain Geobacillus kaustophilus DSM7263. The gene has an open reading frame of 2157bp that encodes a 718-amino-acid pullulanase, and shows the highest identity with the pullulanase from Geobacillus thermoleovorans US105. The pul(GK) is expressed in Bacillus subtilis WB800N using the plasmid pHT43, and the recombinant protein is secreted using the amyQ signal peptide. The level of Pul(GK) produced in B. subtilis reaches 0.08mgmL(-1) after induction for 40h at 30 degrees C. The purified recombinant Pul(GK) can attack the -1,6 linkages specifically in pullulan to generate maltotriose as the major product. Its specific activity is observed to be 64.75Umg(-1) and the K(m)and V-max values of purified Pul(GK) are 11.7mgmL(-1) and 23.6molmin(-1). Purified Pul(GK) shows optimal activity at pH 6.0 and 65 degrees C. It also shows significant thermostability, with a T-1/2 of 60h at 65 degrees C. Recombinant Pul(GK) is immobilized and the thermostability of immobilized Pul(GK) (Im-Pul(GK)) is significantly improved (55-75 degrees C). Pul(GK) hydrolyzes pullulan, amylopectin, starch, and glycogen, but not amylose. Substrate specificity and product analysis proves that the purified pullulanase from Geobacillus kaustophilus DSM7263 belongs to a type I pullulanase. This is the first report of pullulanase from Geobacillus kaustophilus (which includes the wild strain and the recombinant production of the enzyme) with detailed enzymatic properties of heterologous expression. The significant thermostability and production of recombinant pullulanase by B. subtilis may also potentially prove to be valuable in industrial applications.