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Design of Hyperthermophilic Lipase Chimeras by Key Motif‐Directed Recombination

Recombination of diverse natural evolved domains within a superfamily offers greater opportunity for enzyme function leaps. How to recombine protein modules from distant parents with less disruption in cross‐interfaces is a challenging issue. Here, we identified the existence of a key motif, the sequence VVSVN(D)YR, within a structural motif ψ loop in the α/β‐hydrolase fold superfamily, by using a MEME server and the PROMOTIF program. To obtain thermostable lipase‐like enzymes, two chimeras were engineered at the key motif regions through recombination of domains from a mesophilic lipase and a hyperthermophilic esterase/peptidase with amino acid identity less than 21 %. The chimeras retained the desirable substrate preference of their mesophilic parent and exhibited more than 100‐fold increased thermostability at 50 °C. Through site‐directed mutation, we further improved activity of the chimera by 4.6‐fold. The recombination strategy presented here enables the creation of novel catalysts.