Enhancing the Thermostability of Phospholipase C by Structural-Based Proline Incorporation to Improve Its Degumming...

Thermostability can be improved by introducing prolines into targeted sites, enhancing enzyme performance in specific reactions. In our present study, a novel fungal phospholipase C derived from Talaromyces islandicus (TiPLC) was first heterologously expressed in Pichia pastoris and biochemically characterized. Given the poor thermal stability of TiPLC, a structure-based proline incorporation strategy was used to enhance its thermostability further. Two single-site (E92P and A375P) mutants were selected from seven designs, exhibiting improved stability while retaining wild-type's basic properties (optimum reaction pH and temperature). Compared to the wild-type, the t_1/2 of E92P and A375P under 40 °C extended by 1.62 and 1.27 times, respectively. Meanwhile, the E92P and A375P mutants exhibited a 20% increase in activity using p-NPPC as substrate. Moreover, double mutant E92P-A375P exhibited 2.43 times enhancement compared to the wild-type. Results of the oil degumming experiment further confirmed that the double mutant significantly improved the performance of TiPLC, with a reduction in residual phosphorus to 78 ppm, while for the wild-type, the residual phosphorus was 131 ppm under the same reaction. Molecular simulations indicated that proline incorporation into 92 and 375 sites significantly improved the rigidity of partial flexible regions, thus contributing to the increased thermostability.