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 °C and pH 5.5. The recombinant PulPB1 showed great stability, its half-life at 50 °C was 137 h. PulPB1 can efficiently hydrolyze pullulan and amylopectin, with activities of 292 and 184 U/mg at 50 °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 °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 °C. This demonstrates that PulPB1 is promising for development as a superior candidate for cold amylopectin hydrolysis.

具有高催化活性和稳定性的冷适应支链淀粉酶因其在冷淀粉水解中的广泛应用而受到特别关注，但迄今为止，几乎没有报道在环境温度和酸性pH下显示优异特性的支链淀粉酶。在这里，从一个新的普鲁兰甲醇芽孢杆菌PB1中成功表达大肠杆菌BL21（DE3），被确定为冷适应的I型支链淀粉酶（PulPB1），在50°C和pH 5.5下具有最大活性。重组PulPB1具有很好的稳定性，在50°C下的半衰期为137 h。PulPB1可以有效地水解支链淀粉和支链淀粉，在50°C和pH 5.5下的活性分别为292和184 U / mg。此外，发现PulPB1的N末端结构域显着影响酶的性能。N末端域被截断后，对支链淀粉的活性从292 U / mg显着下降，在50°C下的半衰期从137到10 h下降。与单独使用支链淀粉葡糖苷酶的水解系统相比，在40°C下结合PulPB1和支链淀粉葡糖苷酶进行支链淀粉水解的催化效率提高了2.4倍。