A combinatorial multi-site directed mutagenesis solution for improved thermal stability of <i>Lactobacillus plantarum</i> tannase

This study used a modified flapless (FLT) version of tannase from <i>Lactobacillus plantarum</i>, (<i>Lp</i>Tan) to explore the effects of "stacking" site mutations predicted by Protein Repair One Stop Shop (PROSS) to increase stability. Four different <i>Lp</i>Tan structural-state models (including apo, substrate- and product- bound as well as FLT) were comparatively applied, yielding 143 predicted mutations. Of these, eight mutations (including Q63T, A65D, A184Y, A257D, V276Y, T321G, G421D, and G439D (FLT numbering)) were selected to stack, based on conservation of the prediction across all four structural states. Combinatorial screening of the arising 256-member library yielded a selection of possible hits, of which four were further characterized. Variant P6H7 contained 7 of the 8 mutations (excluding V276Y) and showed the highest significant <i>k</i>_cat, 17% higher than FLT and 30% higher than <i>Lp</i>Tan, and a 4.5 °C increase in <i>T_m</i> . Variant P8E5 with 6 of 8 mutations (excluding A257D and G439D), yielded a 6.5 °C increase in <i>T_m</i> compared to FLT. The two other variants showed more moderate increases, albeit still greater than FLT or <i>Lp</i>Tan. Overall, the ability to design thermal stabilized versions of a tannase is emphasized. Putative mechanisms underlying the stabilization imparted by the highlighted variations are discussed.