Pullulanase type I from Fervidobacterium pennavorans Ven5: Cloning, sequencing, and expression of the gene and biochemical characterization of the recombinant enzyme

The gene encoding the type I pullulanase from the extremely thermophilic an aerobic bacterium Fervidobacterium pennavorans Ven5 was cloned and sequence d in Escherichia coli, The pulA gene from F. pennavorans Ven5 had 50.1% pai rwise amino acid identity with pulA from the anaerobic hyperthermophile The rmotoga maritima and contained the four regions conserved among all amyloly tic enzymes. The pullulanase gene (pulA) encodes a protein of 849 amino aci ds with a 28-residue signal peptide. The pulA gene was subcloned without it s signal sequence and overexpressed in E. coil under the control of the trc promoter. This clone, E. coli FD748, produced two proteins (93 and 83 kDa) with pullulanase activity. A second start site, identified 118 amino acids downstream from the ATG start site, with a Shine-Dalgarno-like sequence (G GAGG) and TTG translation initiation codon was mutated to produce only the 93-kDa protein. The recombinant purified pullulanases (rPulAs) were optimal ly active at pH 6 and 80 degrees C and had a half-life of 2 h at 80 degrees C. The rPulAs hydrolyzed alpha-1,6 glycosidic linkages of pullulan, starch , amylopectin, glycogen, alpha-beta-limited dextrin. Interestingly, amylose , which contains only alpha-1,4 glycosidic linkages, was not hydrolyzed by rPulAs. According to these results, the enzyme is classified as a debranchi ng enzyme, pullulanase type I. The extraordinary high substrate specificity of rPulA together with its thermal stability makes this enzyme a good cand idate for biotechnological applications in the starch-processing industry.