Recombinant expression and characterization of a novel cold-adapted type I pullulanase for efficient amylopectin hydrolysis

Cold-adapted pullulanase with high catalytic activity and stability is of special interest for its wide application in cold starch hydrolysis, but few pullulanases displaying excellent characteristics at ambient temperature and acidic pH have hitherto been reported. Here, a novel pullulanase from Bacillus methanolicus PB1 was successfully expressed in Escherichia coli BL21 (DE3) and determined to be a cold-adapted type I pullulanase (PulPB1) with maximum activity at 50 degrees C and pH 5.5. The recombinant PulPB1 showed great stability, its half-life at 50 degrees C was 137 h. PulPB1 can efficiently hydrolyze pullulan and amylopectin, with activities of 292 and 184 U/mg at 50 degrees C and pH 5.5, respectively. Moreover, the N-terminal domain of PulPB1 was found to significantly affect the enzymatic performance. Following truncation of the N-terminal domain, the activity towards pullulan decreased markedly from 292 to 141 U/mg and the half-life at 50 degrees C decreased from 137 to 10 h. Compared to the hydrolysis system with amyloglucosidase alone, the catalytic efficiency showed a 2.4-fold increase on combining PulPB1 with amyloglucosidase for amylopectin hydrolysis at 40 degrees C. This demonstrates that PulPB1 is promising for development as a superior candidate for cold amylopectin hydrolysis.