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. 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.

FIG. 1

(A) Restriction map of the 8.1-kb insert. The 3 ORFs and their positions are shown. (B) Subcloning of pulA to yield the clones pFD748 and pFD758.

FIG. 2

(a) SDS-PAGE of the purified pullulanases from F. pennavorans Ven5 and E. coli PL2125. Proteins were detected with Coomassie blue (0.1%). Arrows indicate molecular markers. (b) Gel electrophorectic analysis (left panel) and zymogram (right panel) of samples from various purification steps of the recombinant pullulanase from E. coli FD748m. On the left panel proteins were detected with Coomassie blue (0.1%). The right panel shows a zymogram as described in Materials and Methods. Lanes 1, crude extract (10 μg); lanes 2, crude extract after heat treatment (10 μg); lanes 3, affinity chromatography pool (4 μg); lanes 4, Mono Q pool (0.6 μg).

FIG. 3

Influence of temperature on the activity of the cloned pullulanase from F. pennavorans Ven5. For determination of the temperature optimum, the purified enzyme (0.03 mg/ml) was incubated in Na acetate buffer (pH 6.0), and incubation was performed for 10 min at various temperatures (35 to 100°C). (Inset) Arrhenius plot showing linearity between 35 and 80°C.

FIG. 4

HPLC analysis of hydrolysis products formed after incubation of purified recombinant pullulanase in the presence of 0.5% pullulan (a), 0.5% starch (b), or 0.2% amylose (c). Samples were incubated at 80°C (a) or 65°C (b and c), and at different time intervals aliquots were withdrawn and analyzed on an Aminex HPX 42-A column for the oligosaccharides. The results of amylose hydrolysis by pullulanase type II from P. woesei is also shown (c). Dp, degree of polymerization (Dp1, glucose; Dp2, maltose; etc.).