Redesign the alpha/beta Fold to Enhance the Stability of Mannanase Man23 from Bacillus subtilis

In this work, we engineered the alpha/beta fold of mannanase Man23 based on its molecular structure analysis to obtain more stable variants. By introducing 31 single-site mutations in the alpha/beta fold and shuffling them, the incorporation of four mutations (K178R, K207R, N340R, and S354R) displayed a good balance between high activity and stability at higher temperature and broader pH. This quartet variant was characterized by an almost threefold increased activity and a sevenfold increased stability compared to native mannanase Man23. Our results suggest that such work is safe to increase our target protein stability with no loss of activity.