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Molecular Biology Reagents> | Protein expression and purification. Codon-optimized and his-tagged (C-terminus) genes for HG-series Kemp eliminases (Supplementary Table 1) cloned into the pET-11a vector (Novagen) via NdeI and BamHI were obtained from Genscript. | Get A Quote |
The creation of artificial enzymes is a key objective of computational protein design. Although de novo enzymes have been successfully designed, these exhibit low catalytic efficiencies, requiring directed evolution to improve activity. Here, we used room-temperature X-ray crystallography to study changes in the conformational ensemble during evolution of the designed Kemp eliminase HG3 (kcat/KM 160 M−1s−1). We observed that catalytic residues were increasingly rigidified, the active site became better pre-organized, and its entrance was widened. Based on these observations, we engineered HG4, an efficient biocatalyst (kcat/KM 120,000 M−1s−1) containing active-site mutations found during evolution b... More