Rational Engineering of α-CGTase from Paenibacillus macerans for Enhanced Thermostability and Product Specificity

Cyclodextrin glycosyltransferases are key biocatalysts for α-cyclodextrin (α-CD) production but are limited by insufficient thermostability and suboptimal product selectivity. Here, an α-CGTase from Paenibacillus macerans was modified via PROSS-aided design, Gibbs free-energy prediction and iterative combinatorial mutagenesis. M6 (G339D/I186L/N205P/E299 K/G268P/R146P) displayed improved thermostability and α-CD selectivity. Compared with the wild-type enzyme, M6 showed a 7.06 °C increase in melting temperature (Tm) and 9.41- and 4.90-fold longer half-lives (t1/2) at 50 and 55 °C respectively. Its optimal reaction temperature increased from 45 to 55 °C and the α-/β-CD activity ratio increased from 5.42 to 9.72, indicating a marked shift in product selectivity toward α-CD. In solvent process, M6 achieved an α-CD conversion of 58.48% and a total conversion rate of 72.00% at 55 °C. MD simulations suggested that reduced conformational flexibility and favorable electrostatic interactions underlay the improved thermostability. This work validates combining computational design and iterative engineering for enzyme optimization.