Biochemical characterization of halophilic, alkalithermophilic amylopullulanase PulD7 and truncated amylopullulanases PulD7ΔN and PulD7ΔC.

A pullulanase, PulD7, was identified in the genome of the halophilic, alkalithermophilic isolate Alkalilimnicola sp. NM-DCM-1. PulD7 is 701 amino acids large with a carbohydrate binding module (CBM) 48 at the N-terminal. The full length PulD7 and N- and C-terminal truncated versions were cloned, heterologously expressed and functionally characterized. PulD7 displayed maximal activity at 55 °C, pH 9.5 and 2 M NaCl. PulD7 had good thermal stability, with a half-life of 693 min at 50 °C. PulD7 is an amylopullulanase, hydrolyzing both α‑1,4‑ and α‑1,6‑glycosidic bonds in soluble starch and pullulan, respectively. PulD7 was resistant to chemical reagents, including organic solvents (dimethyl sulfoxide, methanol, benzene, 20% v/v), reducing agents (β-mercaptoethanol, 5% v/v), surfactants (SDS and Tween 20, 5% v/v), the divalent chelator ethylene diamine tetra acetic acid (5 mM), and the chemical denaturant urea (8 M). PulD7 was not calcium-dependent. PulD7 was able to bind raw starch granules, reaching 52% binding in 3 h. The N-and C-terminal truncated forms of PulD7 had similar biochemical characteristics. PulD7ΔC had higher specific activity and halotolerance. The N-terminally truncated PulD7ΔN hydrolyzed amylose only, indicating that CBM48 is essential for binding branched substrates. PulD7 has unique characteristics giving it strong potential for application in biotechnological industries.