Directed Evolution of an Esterase from Pseudomonas fluorescens Yields a Mutant with Excellent Enantioselectivity and Activity for the Kinetic Resolution of a Chiral Building Block

Abstract

A triple mutant of an esterase from Pseudomonas fluorescens (PFE) that was created by directed evolution exhibited high enantioselectivity (E=89) in a kinetic resolution and yielded the building block (S)-but-3-yn-2-ol. Surprisingly, a mutation close to the active site caused the formation of inclusion bodies, but remote mutations were found to be responsible for the high selectivity. Back mutations gave a variant (double mutant PFE Ile76Val/Val175Ala) that showed excellent selectivity (E=96) and activity (20 min for 50% conversion, which corresponds to 1.25 U per mg of protein).

From Commercial Enzymes to Biocatalysts Designed by Protein Engineering

“…27 After screening approximately 7000 esterase mutants, a variant was identified with good enantioselectivity (E = 89), but low activity. 28 Further analysis revealed that this variant contained three mutations (Ile76Val/Gly98Ala/Val175Ala), and, in contrast to the wild-type esterase, expression in E. coli resulted mostly in inclusion bodies (inactive protein aggregates).…”

Conversion of a Cyclodextrin Glucanotransferase into an α-Amylase: Assessment of Directed Evolution Strategies

“…In an effort to evolve the cyclization specificity of the Thermoanerobacterium thermosulfurigenes strain EM1 (Tabium) CGTase into R-amylase like hydrolytic activity on starch, we separately applied error-prone PCR (epPCR) and saturated mutagenesis, also evaluating the effectiveness of directed evolution techniques for interconversion of reaction specificities. Directed evolution has emerged as a powerful method for the creation of proteins with altered properties, e.g., tighter binding, resistance to denaturing conditions, and modified and/or improved reaction specificities (8)(9)(10)(11)(12)(13). Several directed evolution methods are commonly used nowadays, epPCR, DNA shuffling, site-saturation mutagenesis, etc., in the generation of genetic diversity, as reviewed (14,15).…”

Directed Evolution of Industrial Biocatalysts

“…If enantiomerically pure chiral ( R ) -and ( S ) -acetates are used in separate experiments but using the same enzyme variant, the method allows the determination of enantioselectivity. This assay could thus be successfully used to identify an esterase variant with high enantioselectivity in the synthesis of ( S ) -butyn -2 -ol [79] .…”

“…Our group used the mutator strain E. coli XL -1 Red, to develop an enantiopreference in the hydrolysis of methyl 3 -phenylbutyrate [123] . The same enzyme was also evolved by epPCR and the acetate assay for its application in the enantioselective hydrolysis of the very important building block ( S ) -3 -butyn -2 -ol with enantiomeric excess ( ee ) > 99% [79] . Here only a single round of epPCR was necessary to obtain a variant containing three point mutations that showed increased enantioselectivity without overhydrolysis.…”