Abstract: In this study, the enzymatic properties of TK-PUL, a thermoacidiphilic type III pullulan hydrolase produced by the extremely thermophilic archaea Thermococcus kodakarensis KOD1 were compared with those of its N-terminal truncated mutants, in order to determine the effect of the truncation of N-terminal structural modules on the enzymatic properties of TK-PUL. The truncation of the structural module N1 improved the catalytic characteristics of TK-PUL, resulting in a 1.11-fold increase in α-amylase specific activity, a 1.12-fold increase in pululanase specific activity and a 1.15-fold increase in half-life at 100 ℃. The truncation of the structural module N2 enhanced the thermal stability of the enzyme, and extended the half-life at 100 ℃ by 1.25 times, whilst it decreased the pH stability, substrate binding capacity and specific activity of TK-PUL. Moreover, the truncation of the structural module N2 changed the substrate specificity of TK-PUL, resulting in an increase in the ratio of α-amylase to pullulanase activity from 0.49 to 0.60. The results showed that neither the structural module N1 nor N2 of TK-PUL was the essential structural regions for enzymatic catalysis, but both of them had important effects on the substrate binding capacity, catalytic activity and stability of the enzyme.

Key words: type III pullulan hydrolase; N-terminal structural modules; truncated mutation; catalytic properties