Enhanced production of γ-cyclodextrin by optimization of reaction of γ-cyclodextrin glycosyltransferase as well as synchronous use of isoamylase

In this study, we examined the production of γ-cyclodextrin (γ-CD) using γ-cyclodextrin glycosyltransferase (γ-CGTase) from Bacillus clarkii 7364. We investigated a variety of factors that affect the yield of γ-CD, including key factors such as solvent, substrate, temperature, pH, enzyme dosage. After optimising the reaction parameters, a 50.4% (w/w) yield of γ-CD was obtained with 15% (w/v) potato starch and cyclododecanone (which could be easily removed). In order to further enhance the production of γ-CD, we constructed the γ-CGTase mutant A223K by site-directed mutagenesis, and used it synchronously with the isoamylase obtained from Thermobifida fusca. At pH 7.0 and 50 °C, 72.5% (w/w) γ-CD yield was achieved after 10 h, which was 22.1% higher than that obtained using γ-CGTase alone, and represents the highest yield reported to date. The substantially higher yield and selectivity of γ-CD obtained in the present study provides the basis for the industrial-scale production of γ-CD.