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Structure-Guided Triple-Code Saturation Mutagenesis: Efficient Tuning of the Stereoselectivity of an Epoxide Hydrolase
- Source :
- ACS Catalysis. 6:1590-1597
- Publication Year :
- 2016
- Publisher :
- American Chemical Society (ACS), 2016.
-
Abstract
- The directed evolution of enzymes promises to eliminate the long-standing limitations of biocatalysis in organic chemistry and biotechnology—the often-observed limited substrate scope, insufficient activity, and poor regioselectivity or stereoselectivity. Saturation mutagenesis at sites lining the binding pocket with formation of focused libraries has emerged as the technique of choice, but choosing the optimal size of the randomization site and reduced amino acid alphabet for minimizing the labor-determining screening effort remains a challenge. Here, we introduce structure-guided triple-code saturation mutagenesis (TCSM) by encoding three rationally chosen amino acids as building blocks in the randomization of large multiresidue sites. In contrast to conventional NNK codon degeneracy encoding all 20 canonical amino acids and requiring the screening of more than 1015 transformants for 95% library coverage, TCSM requires only small libraries not exceeding 200–800 transformants in one library. The triple c...
- Subjects :
- chemistry.chemical_classification
010405 organic chemistry
Stereochemistry
Regioselectivity
General Chemistry
010402 general chemistry
Directed evolution
01 natural sciences
Catalysis
0104 chemical sciences
Amino acid
chemistry
Biocatalysis
Stereoselectivity
Codon degeneracy
Epoxide hydrolase
Saturated mutagenesis
Subjects
Details
- ISSN :
- 21555435
- Volume :
- 6
- Database :
- OpenAIRE
- Journal :
- ACS Catalysis
- Accession number :
- edsair.doi...........ea33116acc23d5e24d1a3544c56bd1f5