Design of Hyperthermophilic Lipase Chimeras by Key Motif-Directed Recombination

• Published in: Chembiochem : a European journal of chemical biology 2015-02, Vol.16 (3), p.455-462
• Main Authors: Zhou, Xiaoli, Gao, Le, Yang, Guangyu, Liu, Donglai, Bai, Aixi, Li, Binchun, Deng, Zixin, Feng, Yan
• Format: Article
• Language: English
• Subjects: alpha/beta hydrolase fold; Amino Acid Motifs; Bacteriology; Computer Simulation; domain recombination; enzymes; key motifs; Lipase - chemistry; Lipase - genetics; Lipase - metabolism; Mutagenesis, Site-Directed; Phylogeny; protein engineering; Protein Engineering - methods; Protein Folding; Protein Stability; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Software; Substrate Specificity
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.201402456

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. Best of both worlds: Lipase‐like chimeras were engineered through recombination of domains at the key motif regions from a mesophilic lipase and a hyperthermophilic esterase/peptidase with sequence identity lower than 21 %. The chimeras retained the preference for long‐chain substrates of their mesophilic parent and the thermostability of their hyperthermophilic parent.