More is not always better. Multiple carbohydrate-binding domains for efficient starch hydrolysis

Lactobacillus amylovorus α-amylase is an endoenzyme with a peculiar starch-binding domain containing five identical family 26 carbohydrate-binding modules (CBM26). To investigate the impact of CBMs on catalytic activity, C-terminally truncated derivatives were constructed. The catalytic domain alone shows low affinity for the substrate and a very slow reaction rate, highlighting the importance of CBMs in maintaining the enzyme's optimal conformation and dynamics for efficient catalysis. CBMs enhance enzyme performance, as indicated by improved catalytic efficiency (kcat/Km). Interestingly, the enzyme variant La CD3CBM, with three CBMs, exhibits the best catalytic efficiency on soluble starch, outperforming the wild-type amylase, with five CBMs. In the case of insoluble starch, the catalytic domain alone could not hydrolyze it and even adding a CBM, the release of reducing sugars was very inefficient. However, this efficiency was significantly improved by two orders of magnitude for the three-CBM variant and the wild-type amylase. CBMs also played a crucial role in protein thermostability, contributing to a higher melting temperature of the catalytic domain with just a single CBM. Notably, thermostability did not increase with the number of CBMs. In conclusion, spatial arrangement and interactions between the catalytic domain and CBMs significantly influenced enzymatic efficiency with both soluble and insoluble substrates. These interactions optimize enzymatic activity and improve thermostability. • CBMs play a significant role in the productive adsorption on starch. • Adsorption is influenced by the number of CBMs. • CBMs can modulate the product pattern of amylase. • Presence of CBM improves amylase thermostability. • The most efficient form of the enzyme activity has a catalytic domain with 3CBMs.