Pullulanase Type I from Fervidobacterium pennavorans Ven5: Cloning, Sequencing, and Expression of the Gene and Biochemical Characterization of the Recombinant Enzyme

Abstract

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. pennavoransVen5 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°C and had a half-life of 2 h at 80°C. The rPulAs hydrolyzed α-1,6 glycosidic linkages of pullulan, starch, amylopectin, glycogen, α-β-limited dextrin. Interestingly, amylose, which contains only α-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.

Structural and biochemical properties of a novel pullulanase of Paenibacillus lautus DSM 3035

“…A few type I pullulanases have been investigated at the gene level, such as Bacillus flavocaldarius KP 1228 , Bacillus thermoleovorans US105 , Anaerobranca gottschalkii , Caldicellulosiruptor saccharolyticus , Bacillus sp. CICIM 263 , Thermotoga neapolitana , and Fervidobacterium pennavorans Ven5 . Paenibacillus species are widespread bacteria and have been isolated from different environments including aqueous, plant, insect and soil samples.…”

Cloning, expression and biochemical characterisation of a unique thermostable pullulan-hydrolysing enzyme from the hyperthermophilic archaeon Thermococcus aggregans

“…No evidence for proteolysis was provided since the appearance of two molecular species was independent of the addition of various protease inhibitors during puri¢cation. It can be speculated that the E. coli system recognises two start codons leading to the translation of two active pullulanases as has been observed with the pullulanase of Fervidobacterium pennivorans [3].…”

“…Four types of pullulan-hydrolysing enzymes, based on substrate speci¢cities and reaction products, have been described in the literature : (a) pullulan hydrolase type I (neopullulanase) attacks K-1,4-linkages in pullulan forming panose [1] ; (b) pullulan hydrolase type II (isopullulanase) attacks K-1,4-linkages in pullulan forming isopanose [2] ; (c) pullulanase type I speci¢cally hydrolyses K-1,6-glycosidic linkages in pullulan or branched substrates such as amylopectin forming maltotriose or longer linear products [3] ; and (d) pullulanase type II attacks K-1,6-linkages in pullulan and branched substrates in addition to K-1,4 links in polysaccharides other than pullulan [4,5].…”

Structure and expression of an amylopullulanase gene from Bacillus stearothermophilus TS‐23

“…Furthermore, it is noteworthy that, in addition to the Glu residue, the Trp residue is absolutely conserved in region III of all the eubacterial amylopullulanases compared. The Trp residue is also conserved absolutely in region III of all type-I pullulanases [43]. It is also present in region III of isoamylase from Pseudomonas amyloderamosa [44].…”