Abstract：Pullulanase (EC 3.2.1.41) is one of the starch-debranching enzymes which is widely used in lots of areas such as food and pharmaceutical industries. It is necessary to explore pullulanases with high activities and good properties for cost cuttings of agricultural product's deep processing and starch industries. In previous work, an efficient pullulan-degrading bacterium Klebsiella variicola strain Z-13 had been isolated from the soil near a starch factory. In this study, the pullulanase from the fermentation broth was purified by 80% ammonium sulphate precipitation, Sephadex G-25 and Sephadex G-100 chromatography. The pure enzyme's activity was 51.15 U/mg and the recovery rate was 26.9%. SDS polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the molecular weight of this protein was 120 kD. The enzymatic properties were also studied. Thin layer chromatography (TLC) analysis showed that the enzymatic product of pullulan was maltotriose. The optimal temperature of the enzyme was 45 ℃ and the optimal pH was 5.6. The pure pullulanase was very stable at both optimal temperature (45 ℃) and optimal pH (pH5.6), and 80% activity still remained even after 120 min at 50 ℃. Zn2+, Na+ and Ca2+ promoted the enzyme's activity at the final concentration of 5 mmoL/L while K+, Mg2+, Al3+ and Co2+ inhibited the enzyme's activity at the same concentration. The activity was completely lost when the enzyme was incubated with Cu2+. Moreover, according to the pullulanase gene published on NCBI from Klebsiella variicola SHN-1 (Accession No. JX087429.1), the primers were designed and the pullulanase gene of Klebsiella variicola strain Z-13 was successfully cloned (Accession No. KJ146839). The similaritiy of the two genes reaches 98.88% while the amino acids sequence similarity was 98.18%. In addition, the amino acids sequence had a highly conserved region YNWGYDP of typeⅠpullulanase at the N terminal, and also 4 conserved domains of GH 13 family. This research purified a typeⅠpullulanase from Klebsiella variicola with relative good activity and stability, which has potential industrial uses. The cloning of the pullulanase gene of Klebsiella variicola strain Z-13 provides the possibility for further studies such as heterogeneous expression and molecular modification of the enzyme.
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