Balancing α6/α6 barrel kinetic stability and loop flexibility in a thermophilic cellobiose 2-epimerase enhanced lower...

Cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus (CsCE) is a biocatalyst for lactulose synthesis effectively at 75 °C. However, its industrial usefulness is limited by high thermal requirements needed to meet such a high optimum temperature. From structural studies, we hypothesized that the enzyme's activity and lower temperature adaptability are governed by two structural elements; the α6/α6 barrel and flexible loops respectively. Targeted mutagenesis was employed, first by targeting high-RMSF residues, yielding single mutants all near the α6/α6 Barrel. These were K223H, K223Y, and G224N and had enhanced activities only when singly existing. To further facilitate bioconversion at temperatures lower than 75 °C, proline to glycine mutations in the flexible loops were explored for further increment in local flexibility, and this successfully led to P106G mutation which on combination with G224N mutations resulted into the best CsCE variant 82 %, 68.13 %, and 56.24 % conversions at 75 °C, 60 °C and 50 °C respectively. The significant biotransformation at 60 °C and 50 °C was a great achievement as undetectable biotransformation was obtained at the same temperatures with wild type CsCE. These results demonstrate how balancing stability and flexibility can be an effective strategy for improving CsCE's activity at lower than 75 °C, enabling more economical lactulose production.