Fig 1.
Phylogenetic analysis of bacterial α-amylases and amylopullulanases, including both Coh01133 and Coh00831.
The tree illustrates that the two Cohnella amylopullulanases are well-grouped within GH57, residing close to other functionally characterized amylopullulanases. Phylogenetic tree was established using neighbor-joining with 1000× bootstrap value in MEGA5. The list of protein sequences and their accession numbers are provided in Table A in S1 File.
Table 1.
Conserved amino acid sequences in four motifs (I, II, III, IV) of pullulanases from this study and earlier reports.
Fig 2.
Polymerase chain reaction, heterologous gene expression and zymogram analysis of Coh01133.
a) Lane1 = DNA marker ladder, Lane 2 = PCR amplicon of Coh01133 separated on 1% agarose gel, Lane 3 = uninduced Coh01133 construct, Lane 4 = protein ladder, Lane 5 = induced Coh01133 construct, Lanes6 = Ni-NTA sepharose column washed with 250 mMImidazol, Lane 7 = Zymogram analysis of Coh01133 expressed and purified proteins in a gel containing starch as substarte b) Lane1 = DNA marker ladder, Lane 2 = PCR amplicon of Coh00831 separated on 1% agarose gel, Lane 3 = protein ladder, Lane 4 = uninduced Coh00831 construct, Lane 5 = induced Coh00831 construct, Lanes 6 = purified Coh00831 with ion exchange chromatography, Lane 7 = Zymogram analysis of Coh00831 expressed and purified proteins in a gel containing starch as substarte. c) chromatogram of Ni-NTA sepharose column (c1), chromatogram of ion exchange chromatography (c2).
Fig 3.
Temperature and pH characteristics of native amylopullulanases, namely Coh01133 (left panel) and Coh00831 (right panel), isolated from Cohnella sp.
a) pH profile, b) pH stability, c) temperature profile, d) temperature stability.
Table 2.
Purification of heterologously expressed Coh00831 and Coh01133.
Table 3.
Reaction products of Cohnella amylopullulanase on pullulan and soluble starch.
Solutions of 1% pullulan, and soluble starch were incubated at optimum temperature and pH with Coh01133 and Coh00831 enzymes. Reaction products were analyzed by HPLC for sugars.
Table 4.
The effects of cations (5 mM) and some chemical materials (a reducing agent, a chelator and detergents) on the relative activity of recombinant amylopullulanases.