Epicurian coli

Epothilones are a class of molecules that show anticancer activity. Production of a synthetic intermediate was investigated through the action of an esterase on various sterically hindered 3-hydroxy esters [76]. No initial activity was observed, so a Pseudomonasfluorescens esterase was transformed into a mutator strain Epicurian coli and screened using an indicator in the growth plates that would produce a red color if hydrolysis occurred. An ee of 25% was achieved from a variant containing two mutations. 

The host cell used in this experiment is Epicurian coli XL 1-Blue. This strain contains a deletion on the chromosome of the first 450 base pairs of its lacZ coding sequence (the LacZa peptide). The pUC19 plasmid will be able to restore /3-galac-tosidase activity in the host strain, since this plasmid contains an uninterrupted LacZa peptide coding sequence (the strain will be complemented for /3-galactosidase activity). The LacZa peptide expressed from the pUC19 plasmid will be able to... 

Epicurian coli XL 1-Blue competent cells—Stratagene, catalog 200130... 

Obtain a microcentrifuge tube from the instructor containing 160 fA of freshly thawed Epicurian coli XL 1-Blue competent cells (with /8-mercaptoethanol added as per the manufacturer s instructions). Keep these cells on ice at all times ... 

A brief report has appeared in which a wild-type esterase from Pseudomonas fluorescens (PFE), which shows no activity in the hydrolysis of the ester 16 (Fig. 11.21), was subjected to mutagenesis using the mutator strain Epicurian coli XL 1-Red [83], This resulted in a variant which catalyzes the reaction with an ee of 25 %. The absolute configuration of the major product ((R)- or (S)-17) was not determined. Sequencing of the esterase-variant revealed that two point mutations, A 209D and L181V, had occurred. Since the structure of the enzyme is unknown, a detailed interpretation was not possible, although reasonable speculations were made. 

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