The esterase RhEst1 from Rhodococcus sp. ECU1013 has been reported for the enantioselective hydrolysis of ethyl (S)-(+)-2,2-dimethylcyclopropane carboxylate, producing the building block of cilastatin. In this work, error-prone PCR and site-directed saturation mutagenesis were applied to RhEst1 for activity improvement, with the pH-indicator assay as a high-throughput screening method. As a result, RhEst1A147I/V148F/G254A, with mutations surrounding the substrate access channel, showed a 5-fold increase in its specific activity compared with the native enzyme, as well as a 4-fold increase in protein solubility. Combined with the determination of protein structures and computational analysis, this work shows that the amino acids around the substrate channel play a more important role in the activity evolution of RhEst1 than those in the active site.
Luan ZJ, Li FL, Dou S, Chen Q, Kong XD, Zhou J, Yu HL, Xu JH (2015) Substrate channel evolution of an esterase for the synthesis of cilastatin Catal Sci Technol5: 2622-2629
Luan ZJ, Li FL, Dou S, Chen Q, Kong XD, Zhou J, Yu HL, Xu JH (2015) Catal Sci Technol5: 2622-2629