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 anaerobic bacterium Fervidobacterium pennavorans Ven5 was cloned and sequenced in Escherichia coli. The pulA gene from F. pennavorans Ven5 had 50.1% pairwise amino acid identity with pulA from the anaerobic hyperthermophile Thermotoga maritima and contained the four regions conserved among all amylolytic enzymes. The pullulanase gene (pulA) encodes a protein of 849 amino acids with a 28-residue signal peptide. The pulA gene was subcloned without its signal sequence and overexpressed in E. coli 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 (GGAGG) and TTG translation initiation codon was mutated to produce only the 93-kDa protein. The recombinant purified pullulanases (rPulAs) were optimally 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 debranching enzyme, pullulanase type I. The extraordinary high substrate specificity of rPulA together with its thermal stability makes this enzyme a good candidate for biotechnological applications in the starch-processing industry.
Bertoldo
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C
Department of Technical Microbiology, Institute of Biotechnology, Technical University Hamburg-Harburg, 21071 Hamburg, Germany.
Duffner
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F
Jorgensen
P L
PL
Antranikian
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G
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016965:Gram-Negative Anaerobic Straight, Curved, and Helical Rods / Q000201:enzymology* / Q000235:genetics*; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D017931:DNA Primers; D008670:Metals; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C100181:pullulanase