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 <i>Rh</i>Est1 for the synthesis of a cilastatin precursor. The mutant <i>Rh</i>Est1<sub>M1</sub> (=<i>Rh</i>Est1<sub>A147I/V148F/G254A</sub>) was identified from a large library consisting of 1.5 × 10<sup>4</sup> 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 × 10<sup>3</sup>) focused on the cap domain of <i>Rh</i>Est1 was constructed to improve its catalytic performance comprehensively. As a result, a variant designated as <i>Rh</i>Est1<sub>M2</sub> (=<i>Rh</i>Est1<sub>M1‑A143T</sub>), 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 <i>Rh</i>Est1<sub>M2</sub> 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 <i>Rh</i>Est1<sub>M2</sub>. 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 × 10<sup>5</sup>.