Enhancing the atypical esterase promiscuity of the γ-lactamase Sspg from Sulfolobus solfataricus by substrate screening.

Promiscuous enzymes can be modified by protein engineering, which enables the catalysis of non-native substrates. γ-lactamase Sspg from Sulfolobus solfataricus is an enzyme with high activity, high stability, and pronounced tolerance of high concentrations of the γ-lactam substrate. These characteristics suggest Sspg as a robust enzymatic catalyst for the preparation of optically pure γ-lactam. This study investigated the modification of this enzyme to expand its application toward resolving chiral esters. γ-Lactamase-esterase conversion was performed by employing a three-step method: initial sequence alignment, followed by substrate screening, and protein engineering based on the obtained substrate-enzyme docking results. This process of fine-tuning of chemical groups on substrates has been termed "substrate screening." Steric hindrance and chemical reactivity of the substrate are major concerns during this step, since both are determining factors for the enzyme-substrate interaction. By employing this three-step method, γ-lactamase Sspg was successfully converted into an esterase with high enantioselectivity towards phenylglycidate substrates (E value > 300). However, since both wild-type Sspg and Sspg mutants did not hydrolyze para-nitrophenyl substrates (pNPs), this esterase activity was termed "atypical esterase activity." The γ-lactamase activity and stability of the Sspg mutants were not severely compromised. The proposed method can be applied to find novel multi-functional enzyme catalysts within existing enzyme pools.