Thermostable pullulanase type I from new isolated Bacillus thermoleovorans US105: cloning, sequencing and expression of the gene in E. coli

A new thermophilic Gram-positive Bacillus strain named US105, producing high pullulanase activity, was isolated from soil of a Tunisian hot spring. US105 was identified as Bacillus thermoleovorans after determination of the nucleotide sequence of the 16S rRNA gene. A 4 kb HindIII DNA fragment from the genome of US105 strain, including a pullulanase gene, was cloned in Escherichia coli. Sequence analysis revealed a putative open reading frame (pulUS105) encoding a protein of 718 amino acids, which exhibits high homology with pullulanases type I. The recombinant PulUS105 activity is improved 15-fold after cloning of the open reading frame under control of the Lac promoter. This activity, having an optimal temperature of 75 °C, was classified as a type I pullulanase since it attacks specifically α-(1,6) glucosidic linkages in pullulan or starch.