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Free keywords:
amino acids; directed svolution; enzymes; mutagenesis; solid-phase gene synthesis
Abstract:
Directed evolution of stereo‐ and regioselective enzymes as catalysts in organic chemistry and biotechnology constitutes a complementary alternative to selective transition‐metal catalysts and organocatalysts. Saturation mutagenesis at sites lining the binding pocket has emerged as a key method in this endeavor, but it suffers from amino acid bias, which reduces the quality of the library at the DNA level and, thus, at the protein level. Chemical solid‐phase gene synthesis for library construction offers a solution to this fundamental problem, and the Sloning and Twist platforms are two possible options. This concept article analyzes these approaches and compares them to traditional PCR‐based saturation mutagenesis; the superior commercial Twist technique shows almost no bias.
