Cloning, overexpression, and structural characterization of a novel archaeal thermostable neopullulanase from DSM 2162.

The main purpose of the present study is to introduce the biochemical characteristics of the industrial valuable thermostable pullulan degrading enzyme from DSM2162. Recombinant protein was purified by a combination of thermal treatment and affinity chromatography, with a yield of 15.94% and 7.69-fold purity. Purified enzyme showed the molecular mass of 55,787 Da with optimum activity at 70 °C and a broad range of pH (5.0-9.0) with kcat of 2150 min and Km of 6.55 mg.mL, when using starch as substrate. The enzyme activity assay on various polysaccharide substrates revealed the substrate preference of pullulan > amylopectin > β cyclodextrin > starch > glycogen; therefore, it classified as a neopullulanase. The neopullulanase structural analysis by spectrofluorometer, FT-IR, and circular dichroism spectroscopy indicated the corporation of α-helix (47.3%) and β-sheet (31.6%) in its secondary structure. The melting temperature and specific heat capacity calculations using differential scanning calorimetry confirmed its extreme thermal stability. Further, salt-elevated concentrations resulted in oligomeric state dominancy without any significant influence on the starch-degrading ability. The newly cloned archaeal neopullulanase was with broad activity on polysaccharide substrates, with thermal and salt stability. Thus, the DSM2162 neopullulanase can be introduced as a good candidate to be used in carbohydrate industry.