Functional Roles of N-Terminal Domains in Pullulanase from Human Gut Lactobacillus acidophilus .

Pullulanases are multidomain α-glucan debranching enzymes with one or more N-terminal domains (NTDs) including carbohydrate-binding modules (CBMs) and domains of unknown function (DUFs). To elucidate the roles of NTDs in Lactobacillus acidophilus NCFM pullulanase ( La Pul), two truncated variants, Δ41- La Pul (lacking CBM41) and Δ(41+DUFs)- La Pul (lacking CBM41 and two DUFs), were produced recombinantly. La Pul recognized 1.3- and 2.2-fold more enzyme attack-sites on starch granules compared to Δ41- La Pul and Δ(41+DUFs)- La Pul, respectively, as measured by interfacial kinetics. Δ41- La Pul displayed markedly lower affinity for starch granules and β-cyclodextrin (10- and >21-fold, respectively) in comparison to La Pul, showing substrate binding mainly stems from CBM41. Δ(41+DUFs)- La Pul exhibited a 12 °C lower melting temperature than La Pul and Δ41- La Pul, indicating that the DUFs are critical for La Pul stability. Notably, Δ41- La Pul exhibited a 14-fold higher turnover number ( k cat ) and 9-fold higher Michaelis constant ( K M ) compared to La Pul, while Δ(41+DUFs)- La Pul's values were close to those of La Pul, possibly due to the exposure of aromatic by truncation.