Dramatically Improved Performance of an Esterase for Cilastatin Synthesis by Cap Domain Engineering

Whole-protein random mutation and substrate tunnel evolution have recently been applied to the pharmaceutically relevant esterase RhEst1 for the synthesis of a cilastatin precursor. The mutant RhEst1_M1 (=RhEst1_{A147I/V148F/G254A}) was identified from a large library consisting of 1.5 × 10⁴ variants. Though the activity of this mutant was improved 5-fold, the enantioselectivity for biohydrolysis decreased at the same time. Herein a smart library (3.0 × 10³) focused on the cap domain of RhEst1 was constructed to improve its catalytic performance comprehensively. As a result, a variant designated as RhEst1_M2 (=RhEst1_M1‑A143T), showed a 6-fold increase in specific activity compared with the wild type. Meanwhile, the decreased enantioselectivity for enzymatic resolution was recovered to the native enzyme level. The melting temperature of RhEst1_M2 was nearly 11 °C higher than that of the wild type. This work provides detailed insight into the vital role of α/β hydrolase cap domains in influencing all aspects of enzyme characteristics. Furthermore, the commercial resin ESR-1 with free amino groups was used for enzyme immobilization to enhance the operational performance of RhEst1_M2. No obvious activity loss was observed when the immobilized enzyme was incubated at 30 °C for 200 h. The immobilized enzyme could be repeatedly used for up to 20 batches, and the total turnover number (TTN) reached up to 8.0 × 10⁵.