A novel type III pullulan hydrolase from the bacterium Thermus scotoductus: Biochemical characterization and functional analysis of its carbohydrate-binding domains.
TL;DR
The results indicated that CBM48 was the major contributor to substrate binding and enzymatic activity, whereas CBM34 played a minor role in PulTS, which expands the knowledge of pullulan-hydrolyzing enzymes.
Abstract
A novel type III pullulan hydrolase (PulTS) was identified from the thermophilic bacterium Thermus scotoductus, representing the first bacterial member of this enzyme class. The recombinant PulTS exhibited optimal activity at pH 6.5 and 80 °C, with a specific activity of 46.3 ± 2.4 U/mg toward pullulan. The enzyme hydrolyzed pullulan into maltotriose, panose, maltose, and glucose, and also degraded soluble starch, amylose, amylopectin, dextran, glycogen, and γ-cyclodextrin. PulTS contains sequentially arranged carbohydrate-binding module family 48 (CBM48), CBM34, and glycoside hydrolase family 13 subfamily 20 (GH13_20) catalytic domains, an arrangement previously observed only in archaeal species. Deletion of CBM48 enhanced the thermostability of PulTS by approximately 3-fold, yet the enzyme still retained approximately 69% of its catalytic efficiency toward pullulan. Deletion of CBM34 resulted in no further changes. Affinity gel electrophoresis revealed that CBM48 bound pullulan and, more weakly, soluble starch, while CBM34 exhibited only weak binding ability toward pullulan. The results indicated that CBM48 was the major contributor to substrate binding and enzymatic activity, whereas CBM34 played a minor role in PulTS. This work expands our knowledge of pullulan-hydrolyzing enzymes.