title: 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.
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240 (P. R. China); Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, Jilin University, Changchun 130023 (P. R. China).
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Zhou
authors.forename: Le
authors.initials: L
authors.lastname: Gao
authors.forename: Guangyu
authors.initials: G
authors.lastname: Yang
authors.forename: Donglai
authors.initials: D
authors.lastname: Liu
authors.forename: Aixi
authors.initials: A
authors.lastname: Bai
authors.forename: Binchun
authors.initials: B
authors.lastname: Li
authors.forename: Zixin
authors.initials: Z
authors.lastname: Deng
authors.forename: Yan
authors.initials: Y
authors.lastname: Feng
mesh_terms: D020816:Amino Acid Motifs; D003198:Computer Simulation; D008049:Lipase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012984:Software; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D008049:Lipase
keywords: alpha/beta hydrolase fold; domain recombination; enzymes; key motifs; protein engineering