Increasing γ-CD conversion rates by improving thermostability of Bacillus sp. FJAT-44876 γ-CGTase

Poor thermostability is a limiting factor for applications of γ-CGTase that affects starch utilization and yield of γ-CD. Here thermostability of Bacillus sp. FJAT-44876 γ-CGTase (BFγ-CGTase) was improved by addition of Ca²⁺ and site-directed mutagenesis. Thus, 10 mM Ca²⁺ increased the half-life (t_1/2) at 55 and 60°C from 3.0 to 0.3 h to 17.4 and 2.0 h, respectively. Fluorescence spectra indicated that Ca²⁺ stabilized the tertiary structure of the BFγ-CGTase and hence improved the thermostability. Mutation to serine of a glycine residue in an α-helix related to thermostability also improved the stability especially at the higher temperature. Importantly, in 10 mM Ca²⁺ the G208S mutant further increased t_1/2 to 20.0 h and 4.0 h at 55 and 60°C, respectively. The G208S mutant in 10 mM Ca²⁺ produced γ-CD from tapioca starch with 40% increased yield of that from BFγ-CGTase. This work involving rational protein engineering provided a new tool for enzymatic γ-CD production.