The N‐Terminal Domain of the Pullulanase from Anoxybacillus sp. WB42 Modulates Enzyme Specificity and Thermostability

Abstract Anoxybacillus sp. WB42 pullulanase (PulWB42) is a novel thermophilic amylopullulanase that was assigned to the glycoside hydrolase family 13 subfamily 14 (GH13_14) type I pullulanases in the carbohydrate‐active enzymes database. Its N‐terminal domain (Met1–Phe101) was identified as the carbohydrate‐binding module 68 (CBM68) by homology modeling. The N‐domain‐deleted PulWB42 exhibited an equivalent Michaelis constant ( K m ) for pullulan and significant decreases in pullulytic activity, amylose selectivity, and thermostability relative to PulWB42 having a high α‐amylase‐to‐pullulanase activity ratio. Furthermore, the replacement of Ala90 or Arg93 significantly changed the substrate specificity and catalytic efficiency of PulWB42, whereas Q87A, L173D, and H5A/R6A/T7A showed improvements in thermostability and changes in catalytic kinetics. Therefore, the N domain of PulWB42 is not essential for catalysis, but it does modulate enzyme catalysis, especially with respect to substrate specificity. The modulation was achieved mainly by the Leu86–Arg93 segment adjacent to the CBM48 domain and the catalytic A domain in the modeled structure of PulWB42.