Gene Cloning, Expression and Characterization of a Pullulanase from Paenibacillus puldeungensis LK18 Strain
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Abstract:
In this study, the pullulanase gene (pulA) was amplified from Paenibacillus puldeungensis LK18 strain based on the design and primer of its conserved region, and linked to the expression vector pET-28a (+). The recombinant plasmid pET-28a (+)-pulA was transformed into Escherichia coli BL21 (DE3) leading to the successful expression of the recombinant pullulanase. The results showed that the pulA gene had a full length of 1968bp encoded 655 amino acids. The recombinant pullulanase was purified by Ni affinity chromatography and had a specific activity of 508.8 U/mg and molecular weight of 76.95 ku. The optimal temperature of the recombinant enzyme was 45 ℃ and 60% of its enzyme activity was retained after an incubation at 35~40 ℃ for 120 min. This enzyme had an optimal pH of 6.0 and high stability at a pH in the range of 6.0~8.0. The enzyme was activated by K+ and Mg2+ at a concentration of 10 mmol/L, but inhibited by Zn2+, Mn2+, Ni2+, Fe2+, Cu2+, Co2+, and Ca2+ to different extents. In this study, a recombinant strain with efficiently expressed pullulanase was constructed, which had a potential in industrial applications.
