Cloing, Expression And Characterization Of Pullulanase From Paenibacillus Polymyxa Nws-pp2

Pullulanase is a kind of debranching enzyme,which can hydrolyze a-(1,6) glucosidic linkages in amylopectin.The enzyme is widely used in starch-processing industry,because it can promote the utility efficiency and productivity of starch on a largescale in order to reduce the production cost of food, feed and pharmaceutical,and that’s the reason why pullulanase get the attention of people.In this research paper, strain Nws-pp2was determined harboring the ability of secreting pululanase by observing clear halo on the pullulan enzyme screening plates. The strain was identified to be genus Paenibacillus polymyxa according to the analysis of the16S rDNA gene sequence.The type I pulullanase encoding gene named pulN was obtained by degenerate PCR, it has a ORF of2532bp, codes for843amino acids and belongs to GH13. By constructing recombinant plasmids, the encoding gene was cloned in to E-coli expression system and B. subtilis expression system.Finally all engineering bacteria successfully expressed the exogenous gene,and the highest enzyme activity measured was6.49U/mL obtained from BL21-pET28a-PulN. Pure enzyme was obtained by Ni-NTA Superflow column and show a single band at96kDa after SDS-PAGE analysis. Using Bradford method,specific activity of the enzyme was calculated to be13.1U/mg. Km and Fmax of it using pulullan as the substrate were15.25g/L and0.37g/(L·min),respectively. The optional temperature of the enzyme is35℃and stable at temperature below45℃. The highest activity was showed at pH6.0and the enzyme was relatively stable within the pH5.5to pH8.0. Metal ions Mn2+and Co2+to PulN has obvious activation effects, while10mm Cu2+can make all loss of enzyme activity. EDTA does not affect the activity.Furthermore, substrates that can be catalyzed by the restructuring pululanase were also studied. We calculate the relative enzyme activity of different substrates. HPLC analysis of the products once again prove that PulN hydrolyzes a-(1,6) glycosidic linkages specifically and it belongs to type I pullulanase.