Heterologous expression and characterization of a recombinant thermostable amylopullulanases Coh4159 from Cohnella sp. A01

Document Type : Research Paper


1 Bioprocess Engineering Research Group, Institute of Industrial and Environmental Biotechnology, National Institute for Genetic Engineering and Biotechnology, Tehran, Iran

2 Bioprocess Engineering Research Group; Institute of Industrial and Environmental Biotechnology (IIEB); National Institute of. Genetic Engineering and. Biotechnology

3 Animal Biotechnology Department, Institute of Agricultural Biotechnology, National Institute for Genetic Engineering and Biotechnology, Tehran, Iran

4 Department of Plant Biology & Biotechnology, Faculty of Life Sciences & Biotechnology, Shahid Beheshti University G.C., Evin, Tehran, Iran

5 Departments of Physiology and Cellular Biophysics Columbia University Medical Center, USA


Starch debranching enzymes that merely hydrolyze α-(1→6) glycosidic linkages are classified into isoamylases (EC and pullulanases (EC An exception to this definition would be amylopullulanase, a type of pullulanase that is capable of cleaving both α-(1→4) and α-(1→6) linkages. Amylopullulanases are in demand in liquid sugar industries to generate glucose and some other starch derivatives. Pullulanases can be used in conjunction with amylases to improve sugar availability during sugar syrup production. Here, a thermophilic Cohnella sp. A01 amylopullulanase (EC gene, namely Coh4159, was PCR amplified and cloned in pET-26b(+) and transformed into BL21(DE3). Recombinant Coh4159 was heterologously expressed in the presence of 0.5 mM IPTG and purified via affinity chromatography, and further characterized. Enzyme activity was demonstrated via zymogram analysis in the presence of pullulan. The enzyme had a hydrolytic effect on pullulan with Vmax = 2.85 µmol.min-1 and Km = 0.5 mM. Temperature optima and pH were 60 ˚C and 6.0. In which the enzyme kept its activity at wide pH (4-9) and temperature (30-70 ˚C) ranges. The recombinant enzyme kept 50% of its activity for 60 min, 100 min and 120 min when incubated at 80, 70 and 60 ˚C, respectively. Amongst metal ions tested, Mn2+, and Ca2+ have improved the enzyme activity both at 5 and 10 mM. The results promise the capability of producing a commercial industrial enzyme, well-suited to liquid sugar syrup industry specification.