Luan_2016_Ind.Eng.Chem.Res_55_12167

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 10 4 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 3) focused on the cap doman of Rh Est1 was constructed to improve its catalytic performance comprehensively. As a result, a variant designated as Rh Est1 M2 (=RhEst1M1 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 Rh Est1 M2was nearly 11C higher than that of the wild type. This work provides detailed insight into the vitalrole of alpha/beta 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 Rh Est1 M2. No obvious activity loss was observed when the immobilized enzyme was incubated at 30C 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 5