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

Chembiochem : a European Journal of Chemical Biology
Jianfeng WangZhemin Zhou

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 (Km ) 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.

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