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Design of Hyperthermophilic Lipase Chimeras by Key Motif‐Directed Recombination
Author(s) -
Zhou Xiaoli,
Gao Le,
Yang Guangyu,
Liu Donglai,
Bai Aixi,
Li Binchun,
Deng Zixin,
Feng Yan
Publication year - 2015
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201402456
Subject(s) - chimera (genetics) , thermostability , lipase , esterase , hydrolase , protein engineering , biology , biochemistry , protein design , homing endonuclease , sequence motif , directed evolution , homologous recombination , structural motif , recombination , genetics , enzyme , protein structure , mutant , gene , endonuclease
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.