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 degrees C, pH 9.5 and 2 M NaCl. PulD7 had good thermal stability, with a half-life of 693 min at 50 degrees C. PulD7 is an amylopullulanase, hydrolyzing both alpha 1,4 and alpha 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 (beta-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. PulD7 Delta C had higher specific activity and halotolerance. The N-terminally truncated PulD7 Delta N 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. (C) 2018 Elsevier B.V. All rights reserved.