Design of hyperthermophilic lipase chimeras by key motif-directed recombination

Abstract

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.

Keywords: alpha/beta hydrolase fold; domain recombination; enzymes; key motifs; protein engineering.