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 ) 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.

Roles of the N-terminal domain and remote substrate binding subsites in activity of the debranching barley limit dextrinase

“…Notably, HvLD-Ser14Arg/His108Arg and HvLD-His108Arg were both also significantly more prone to substrate inhibition by pullulan (Table 1). Overall, the enzymatic properties of the mutants in the CBM21-like domain indicate an involvement in maintaining HvLD activity, as generally envisaged for a multidomain enzyme [56][57][58]. The larger losses of activity towards pullulan, amylopectin and -limit dextrin compared to the oligosaccharide substrate is in agreement with polysaccharides more readily perceiving changes at a remote area of the protein surface.…”

Evolutionary coupling saturation mutagenesis: Coevolution‐guided identification of distant sites influencing Bacillus naganoensis pullulanase activity

“…Hence, the ECSM approach compares very favorably with site‐directed mutagenesis and regional truncation methods, which increased the catalytic efficiency only about two folds (Table S7).…”

“…Using ECSM, pullulanase mutants were created with significantly enhanced catalytic activity when compared with WT BnPUL ( k cat and k cat / K m were increased by of ~ 3 and ~ 6, respectively). These gains of catalytic activity are remarkable for a highly evolved naturally occurring enzyme, and they compare very favorably with previously engineered variants . Since high thermostability and high enzyme activity at comparatively low pH are key for industrial applications of pullulanases , it is important to observe that the mutant enzymes exhibit virtually the same dependence of activity on temperature and pH as the WT, making these mutants suitable for industrial applications.…”

A Hyperthermostable Type II Pullulanase from a Deep-Sea Microorganism Pyrococcus yayanosii CH1

“…As reported previously, functionally unknown or unnecessary loops and domains usually exist in the three-dimensional structure of enzyme; − deletion of these domains could simplify the whole structure and improve the catalytic efficiency and stability of pullulanase. − To increase the catalytic ability of pullulanase, we attempted to truncate the unnecessary loops and domains of Pul PY .…”